P97 FUSION PROTEINS

Abstract

Provided are p97 (melanotransferrin)-trastuzumab fusion proteins and related methods of use thereof, for instance, to facilitate delivery of trastuzumab across the blood-brain barrier (BBB) and/or improve tissue penetration of the antibody in CNS and peripheral tissues, and thereby treat and/or diagnose HER2-positive cancers, including those of the central nervous system (CNS).

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. .sctn.119(e) to U.S. Application No. 61/935,253, filed Feb. 3, 2014, which is incorporated by reference in its entirety.

SEQUENCE LISTING

[0002] The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is BIOA_008_01 WO_ST25.txt. The text file is about 340 KB, was created on Feb. 3, 2015, and is being submitted electronically via EFS-Web.

BACKGROUND

[0003] Technical Field

[0004] Embodiments of the present invention relate to p97 (melanotransferrin)-trastuzumab fusion proteins and antibody fusion proteins and related methods of use thereof, for instance, to facilitate delivery of trastuzumab across the blood-brain barrier (BBB) and/or improve tissue penetration of the antibody in CNS and peripheral tissues, and thereby treat and/or diagnose HER2-positive cancers, including those of the central nervous system (CNS).

[0005] Description of the Related Art

[0006] Overcoming the difficulties of delivering therapeutic or diagnostic agents to specific regions of the brain represents a major challenge to treatment or diagnosis of many central nervous system (CNS) disorders, including those of the brain. In its neuroprotective role, the blood-brain barrier (BBB) functions to hinder the delivery of many potentially important diagnostic and therapeutic agents to the brain.

[0007] Therapeutic molecules and genes that might otherwise be effective in diagnosis and therapy do not cross the BBB in adequate amounts and often have poor tissue penetration, even in peripheral tissues. It is reported that over 95% of all therapeutic molecules do not cross the blood-brain barrier.

[0008] Accordingly, there is a need for compositions and methods that facilitate the delivery of therapeutic agents and other molecules across the blood-brain-barrier, for instance, to effectively treat certain diseases of the central nervous system (CNS) such as cancers, particularly those that have metastasized to the CNS. The present invention addresses these needs and offers other related advantages.

BRIEF SUMMARY OF THE INVENTION

[0009] Embodiments of the present invention include p97 (melanotransferrin or MTf)-trastuzumab fusion proteins, comprising a trastuzumab heavy chain and/or light chain sequence fused to a p97 sequence and an optional linker in between. In some embodiments, the fusion protein comprises a trastuzumab heavy chain sequence fused to the N-terminus of the p97 sequence. In certain embodiments, the fusion protein comprises a trastuzumab heavy chain sequence fused to the C-terminus of the p97 sequence. In some embodiments, the fusion protein comprises a truncated trastuzumab heavy chain sequence fused to the C-terminus of the p97 sequence.

[0010] In some embodiments, the truncated trastuzumab heavy chain sequence consists essentially of the heavy chain constant region or a fragment thereof and substantially or entirely lacks the heavy chain variable region. In certain embodiments, the truncated trastuzumab heavy chain sequence consists essentially of the CH1 domain or a fragment thereof, the hinge region, the CH2 domain, and the CH3 domain. In certain embodiments, the truncated trastuzumab heavy chain sequence consists essentially of the hinge region or a fragment thereof, the CH2 domain, and the CH3 domain.

[0011] In some embodiments, the fusion protein comprises (a) a heavy chain amino acid sequence set forth in SEQ ID NOs:37-46 or 96-109; (b) a heavy chain amino acid sequence at least 90% identical to a sequence set forth in SEQ ID NOs:37-46 or 96-109; (c) or a heavy chain amino acid sequence that differs from SEQ ID NOs:37-46 or 96-109 by addition, substitution, insertion, or deletion of about 1-50 amino acids. In some embodiments, the fusion protein comprises a heavy chain amino acid sequence set forth in one or more of SEQ ID NOs:37-46 or 96-109.

[0012] In some embodiments, the fusion protein comprises a trastuzumab light chain sequence fused to the N-terminus of the p97 sequence. In certain embodiments, the fusion protein comprises a trastuzumab light chain sequence fused to the C-terminus of the p97 sequence. In some embodiments, the fusion protein comprises (a) a light amino acid sequence set forth in SEQ ID NOs:110-121; (b) a light chain amino acid sequence at least 90% identical to a sequence set forth in SEQ ID NOs: 110-121; (c) or a light chain amino acid sequence that differs from SEQ ID NOs: 110-121 by addition, substitution, insertion, or deletion of about 1-50 amino acids. In some embodiments, the fusion protein comprises a light chain amino acid sequence set forth in SEQ ID NOs: 110-121.

[0013] In specific embodiments, the p97 sequence comprises, consists, or consists essentially of SEQ ID NO:2 (soluble MTf) or SEQ ID NO:14 (MTfp or MTfpep).

[0014] Also included are isolated polynucleotides, which encode a p97 fusion protein described herein. In some aspects, the isolated polynucleotides are codon-optimized for expression in a host cell. In some embodiments, the host cell is a mammalian cell, an insect cell, a yeast cell, or a bacterial cell.

[0015] Also included are recombinant host cells, comprising an isolated polynucleotide described herein, optionally where the isolated polynucleotide is operably linked to one or more regulatory elements. In certain embodiments, the recombinant host cell comprises an isolated polynucleotide that encodes a (non-fusion) trastuzumab light chain sequence, which is operably linked to one or more regulatory elements. In certain embodiments, the recombinant host cell comprises an isolated polynucleotide that encodes a (non-fusion) trastuzumab heavy chain sequence, which is operably linked to one or more regulatory elements. In certain embodiments, the recombinant host cell comprises an isolated polynucleotide that encodes a (non-fusion) trastuzumab light chain sequence, and an isolated polynucleotide that encodes a (non-fusion) trastuzumab heavy chain sequence, which are operably linked to one or more regulatory elements.

[0016] Certain embodiments relate to vectors, comprising an isolated polynucleotide, which encodes a p97 fusion protein of any of the preceding claims, which is operably linked to one or more regulatory elements. In some embodiments, the vector comprises an isolated polynucleotide that encodes a (non-fusion) trastuzumab light chain sequence, which is operably linked to one or more regulatory elements. In some embodiments, the vector comprises an isolated polynucleotide that encodes a (non-fusion) trastuzumab heavy chain sequence, which is operably linked to one or more regulatory elements. In some embodiments, the vector comprises an isolated polynucleotide that encodes a (non-fusion) trastuzumab light chain sequence, and an isolated polynucleotide that encodes a (non-fusion) trastuzumab heavy chain sequence, which are operably linked to one or more regulatory elements. Also included are recombinant host cells, comprising one or more vectors as described herein.

[0017] Some embodiments relate to p97-antibody fusion proteins that comprise two (non-fusion) trastuzumab light chain sequences, and one or two p97-trastuzumab heavy chain fusion proteins described herein, where the one or two p97-trastuzumab heavy chain fusion protein(s) comprise a trastuzumab heavy chain sequence fused to the N-terminus of a p97 sequence and an optional linker in between (see, e.g., FIGS. 1A & 1B). In some embodiments, the p97-antibody fusion protein comprises two p97-trastuzumab heavy chain fusion proteins (see, e.g., FIGS. 1A and 1E). In certain embodiments, the p97-antibody fusion protein comprises one p97-trastuzumab heavy chain fusion protein and one (non-fusion) trastuzumab heavy chain sequence (see, e.g., FIG. 1B).

[0018] Also included are p97-antibody fusion proteins that comprise one trastuzumab light chain sequence, one trastuzumab heavy chain sequence, and one p97-trastuzumab heavy chain fusion protein of any of the preceding claims, where p97-trastuzumab heavy chain fusion protein comprises a truncated trastuzumab heavy chain fused to the C-terminus of a p97 sequence and an optional linker in between (see, e.g., FIG. 1C).

[0019] In some embodiments, the p97-antibody fusion protein comprises two trastuzumab light chain sequences, and two p97-trastuzumab heavy chain fusion proteins described herein, where the p97-trastuzumab heavy chain fusion proteins comprise a trastuzumab heavy chain fused to the C-terminus of a p97 sequence and an optional linker in between (see, e.g., FIG. 1D).

[0020] In some embodiments, the p97-antibody fusion protein comprises two p97-trastuzumab light chain fusion proteins described herein, and two p97-trastuzumab heavy chain fusion proteins described herein, where the p97-trastuzumab light chain fusion proteins comprise a trastuzumab light chain fused to the N-terminus of a p97 sequence and an optional linker in between, and where the p97-trastuzumab heavy chain fusion proteins comprise a trastuzumab heavy chain fused to the N-terminus of a p97 sequence and an optional linker in between (see, e.g., FIG. 1F).

[0021] Specific examples of p97-antibody fusion proteins include those that comprise two sets of heavy and light chains, where at least one set is selected from one or more of:

[0022] a) the heavy chain of SEQ ID NO:82 and the light chain of SEQ ID NO:83;

[0023] b) the heavy chain of SEQ ID NO:84 and the light chain of SEQ ID NO:85;

[0024] c) the heavy chain of SEQ ID NO:86 and the light chain of SEQ ID NO:87;

[0025] d) the heavy chain of SEQ ID NO:88 and the light chain of SEQ ID NO:89;

[0026] e) the heavy chain of SEQ ID NO:90 and the light chain of SEQ ID NO:91;

[0027] f) the heavy chain of SEQ ID NO:92 and the light chain of SEQ ID NO:93; and

[0028] g) the heavy chain of SEQ ID NO:94 and the light chain of SEQ ID NO:95; including fragments/variants thereof of any of the foregoing. In some embodiments, the p97-antibody fusion is a homodimer that comprises two sets of a), two sets of b), two sets of c), two sets of d), two sets of e), two sets of f), or two sets of g). In some embodiments, the p97-antibody fusion is a heterodimer that comprises any combination of a)-g) above. In particular embodiments, the p97-antibody fusion is a heterodimer that comprises a first set of sets of heavy and light chains selected from a)-g) above, and a second set of trastuzumab (non-fusion) heavy and light chains, for example, SEQ ID NOs: 29-35 or 122 (heavy chains) and 36 or 123 (light chains).

[0029] Certain embodiments relate to recombinant host cells that comprises a p97-antibody fusion protein of any of the preceding claims. In certain embodiments, the host cell is a mammalian cell, an insect cell, a yeast cell, or a bacterial cell. In certain embodiments, the mammalian cell is a Chinese hamster ovary (CHO) cell or a HEK-293 cell.

[0030] Also included are pharmaceutical compositions, comprising a pharmaceutically-acceptable carrier and a p97-antibody fusion protein of any of the preceding claims.

[0031] Some embodiments include methods for the treatment of a HER2-overexpressing cancer in a subject in need thereof, comprising administering to the subject a p97-antibody fusion protein or pharmaceutical composition described herein.

[0032] In certain embodiments, the HER2-overexpressing cancer is at risk for metastasizing to the CNS of the subject. In some embodiments, the HER2-overexpressing cancer has metastasized to the CNS of the subject. In certain embodiments, the HER2-overexpressing cancer is a breast cancer, ovarian cancer, gastric cancer, or uterine cancer.

[0033] In particular embodiments, the HER2-overexpressing cancer is a HER2-overexpressing metastatic breast cancer. In certain embodiments, the HER2-overexpressing metastatic breast cancer is at risk for metastasizing to the CNS of the subject. In some embodiments, the HER2-overexpressing breast cancer has metastasized to the CNS of the subject.

[0034] In certain embodiments, the HER2-overexpressing cancer is a HER2-overexpressing metastatic gastric or gastroesophageal junction adenocarcinoma. In certain embodiments, the HER2-overexpressing metastatic gastric or gastroesophageal junction adenocarcinoma is at risk for metastasizing to the CNS of the subject. In certain embodiments, the HER2-overexpressing metastatic gastric or gastroesophageal junction adenocarcinoma has metastasized to the CNS of the subject.

[0035] In certain embodiments, the HER2-overexpressing cancer is a HER2-overexpressing uterine serous carcinoma (USC). In certain embodiments, the HER2-overexpressing USC is at risk for metastasizing to the CNS of the subject. In certain embodiments, the HER2-overexpressing USC has metastasized to the CNS of the subject.

[0036] Certain methods include administering the p97-antibody fusion protein or pharmaceutical composition as part of an adjuvant treatment for a HER2-overexpressing breast cancer. In certain embodiments, the adjuvant treatment comprises doxorubicin, cyclophosphamide, and either paclitaxel or docetaxel. In certain embodiments, the adjuvant treatment comprises docetaxel and carboplatin.

[0037] Some methods include administering the p97-antibody fusion protein or pharmaceutical composition as a single agent following multi-modality anthracycline based therapy.

[0038] In certain embodiments, the subject is a female human.

[0039] Certain methods include administering the p97-antibody fusion protein or pharmaceutical composition by intravenous (IV) infusion.

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] FIGS. 1A-1G illustrate the general structure of exemplary p97-antibody fusion proteins (black circles represent p97). FIG. 1A illustrates a homodimeric antibody fusion protein composed of two trastuzumab light chains and two trastuzumab heavy chains each fused to the N-terminus of p97. FIG. 1B illustrates a heterodimeric antibody fusion protein composed of two trastuzumab light chains, one non-fused trastuzumab heavy chain, and one trastuzumab heavy chain fused to the N-terminus of p97. FIG. 1C illustrates a heterodimeric antibody fusion protein composed of one trastuzumab light chain, one non-fused trastuzumab heavy chain, and one trastuzumab heavy chain fused to the C-terminus of p97. FIGS. 1D-1E illustrate antibody fusions composed of two p97-trastuzumab heavy chain fusion proteins and two (non-fusion) trastuzumab light chains. FIG. 1F illustrates an antibody fusion composed of two p97-trastuzumab light chain fusion proteins and two 97-trastuzumab heavy chain fusion proteins. FIG. 1G illustrates an antibody fusion composed of two p97-trastuzumab light chain fusion proteins and two (non-fusion) trastuzumab heavy chain sequences.

[0041] FIGS. 2A-2D shows octet analysis demonstrating the affinity of antibody fusions for Her2 relative to IgG1 control. FIG. 2A shows the results for human IgG1, FIG. 2B shows the results for TZM HC-MTf, FIG. 2C shows the results for MTfp NH-TZM, and FIG. 2D shows the results for TZM HC-MTfp (see Example 1).

[0042] FIGS. 3A-3B show antibody-dependent cell-mediated cytotoxicity (ADCC) evaluation of antibody fusions in BT-474 breast cancer cells compared to Herceptin.RTM. and human IgG1 Fc controls (see Example 2). These data show that the p97-trastuzumab antibody fusions induced significant antibody-dependent cell-mediated cytotoxicity in breast cancer cells.

DETAILED DESCRIPTION

[0043] The practice of the present invention will employ, unless indicated specifically to the contrary, conventional methods of molecular biology and recombinant DNA techniques within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual (3.sup.rd Edition, 2000); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Oligonucleotide Synthesis: Methods and Applications (P. Herdewijn, ed., 2004); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Nucleic Acid Hybridization: Modern Applications (Buzdin and Lukyanov, eds., 2009); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Freshney, R. I. (2005) Culture of Animal Cells, a Manual of Basic Technique, 5.sup.th Ed. Hoboken N.J., John Wiley & Sons; B. Perbal, A Practical Guide to Molecular Cloning (3.sup.rd Edition 2010); Farrell, R., RNA Methodologies: A Laboratory Guide for Isolation and Characterization (3.sup.rd Edition 2005).

[0044] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

DEFINITIONS

[0045] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

[0046] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0047] By "about" is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0048] As used herein, the term "amino acid" is intended to mean both naturally occurring and non-naturally occurring amino acids as well as amino acid analogs and mimetics. Naturally occurring amino acids include the 20 (L)-amino acids utilized during protein biosynthesis as well as others such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline and ornithine, for example. Non-naturally occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine and the like, which are known to a person skilled in the art. Amino acid analogs include modified forms of naturally and non-naturally occurring amino acids. Such modifications can include, for example, substitution or replacement of chemical groups and moieties on the amino acid or by derivatization of the amino acid. Amino acid mimetics include, for example, organic structures which exhibit functionally similar properties such as charge and charge spacing characteristic of the reference amino acid. For example, an organic structure which mimics Arginine (Arg or R) would have a positive charge moiety located in similar molecular space and having the same degree of mobility as the e-amino group of the side chain of the naturally occurring Arg amino acid. Mimetics also include constrained structures so as to maintain optimal spacing and charge interactions of the amino acid or of the amino acid functional groups. Those skilled in the art know or can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics.

[0049] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises," and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By "consisting of" is meant including, and limited to, whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of" is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[0050] The term "conjugate" is intended to refer to the entity formed as a result of covalent or non-covalent attachment or linkage of an agent or other molecule, e.g., a biologically active molecule, to a p97 polypeptide or p97 sequence. One example of a conjugate polypeptide is a "fusion protein" or "fusion polypeptide," that is, a polypeptide that is created through the joining of two or more coding sequences, which originally coded for separate polypeptides; translation of the joined coding sequences results in a single, fusion polypeptide, typically with functional properties derived from each of the separate polypeptides. The terms "antibody fusion" and "antibody fusion protein" are used interchangeably herein to refer to an antibody or antibody-like molecule that comprises at least one fusion protein described herein.

[0051] As used herein, the terms "function" and "functional" and the like refer to a biological, enzymatic, or therapeutic function.

[0052] "Homology" refers to the percentage number of amino acids that are identical or constitute conservative substitutions. Homology may be determined using sequence comparison programs such as GAP (Deveraux et al., Nucleic Acids Research. 12, 387-395, 1984), which is incorporated herein by reference. In this way sequences of a similar or substantially different length to those cited herein could be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.

[0053] By "isolated" is meant material that is substantially or essentially free from components that normally accompany it in its native state. For example, an "isolated peptide" or an "isolated polypeptide" and the like, as used herein, includes the in vitro isolation and/or purification of a peptide or polypeptide molecule from its natural cellular environment, and from association with other components of the cell; i.e., it is not significantly associated with in vivo substances.

[0054] The term "linkage," "linker," "linker moiety," or "1" is used herein to refer to a linker that can be used to separate a p97 polypeptide from an agent of interest, or to separate a first agent from another agent, for instance where two or more agents are linked to form a p97 conjugate. The linker may be physiologically stable or may include a releasable linker such as an enzymatically degradable linker (e.g., proteolytically cleavable linkers). In certain aspects, the linker may be a peptide linker, for instance, as part of a p97 fusion protein. In some aspects, the linker may be a non-peptide linker or non-proteinaceous linker. In some aspects, the linker may be particle, such as a nanoparticle.

[0055] The terms "modulating" and "altering" include "increasing," "enhancing" or "stimulating," as well as "decreasing" or "reducing," typically in a statistically significant or a physiologically significant amount or degree relative to a control. An "increased," "stimulated" or "enhanced" amount is typically a "statistically significant" amount, and may include an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the amount produced by no composition (e.g., the absence of a fusion protein or antibody fusion of the invention) or a control composition, sample or test subject. A "decreased" or "reduced" amount is typically a "statistically significant" amount, and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease in the amount produced by no composition or a control composition, including all integers in between. As one non-limiting example, a control could compare the activity, such as the amount or rate of transport/delivery across the blood brain barrier, the rate and/or levels of distribution to central nervous system tissue, and/or the C.sub.max for plasma, central nervous system tissues, or any other systemic or peripheral non-central nervous system tissues, of a p97 fusion protein or antibody fusion relative to the agent/antibody alone. Other examples of comparisons and "statistically significant" amounts are described herein.

[0056] In certain embodiments, the "purity" of any given agent (e.g., a p97 conjugate such as a fusion protein or antibody fusion) in a composition may be specifically defined. For instance, certain compositions may comprise an agent that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure, including all decimals in between, as measured, for example and by no means limiting, by high pressure liquid chromatography (HPLC), a well-known form of column chromatography used frequently in biochemistry and analytical chemistry to separate, identify, and quantify compounds.

[0057] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers. The polypeptides described herein are not limited to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms may be used interchangeably herein unless specifically indicated otherwise. The polypeptides described herein may also comprise post-expression modifications, such as glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. A polypeptide may be an entire protein, or a subsequence, fragment, variant, or derivative thereof.

[0058] A "physiologically cleavable" or "hydrolyzable" or "degradable" bond is a bond that reacts with water (i.e., is hydrolyzed) under physiological conditions. The tendency of a bond to hydrolyze in water will depend not only on the general type of linkage connecting two central atoms but also on the substituents attached to these central atoms. Appropriate hydrolytically unstable or weak linkages include, but are not limited to: carboxylate ester, phosphate ester, anhydride, acetal, ketal, acyloxyalkyl ether, imine, orthoester, thio ester, thiol ester, carbonate, and hydrazone, peptides and oligonucleotides.

[0059] A "releasable linker" includes, but is not limited to, a physiologically cleavable linker and an enzymatically degradable linker. Thus, a "releasable linker" is a linker that may undergo either spontaneous hydrolysis, or cleavage by some other mechanism (e.g., enzyme-catalyzed, acid-catalyzed, base-catalyzed, and so forth) under physiological conditions. For example, a "releasable linker" can involve an elimination reaction that has a base abstraction of a proton, (e.g., an ionizable hydrogen atom, H.alpha.), as the driving force. For purposes herein, a "releasable linker" is synonymous with a "degradable linker." An "enzymatically degradable linkage" includes a linkage, e.g., amino acid sequence that is subject to degradation by one or more enzymes, e.g., peptidases or proteases. In particular embodiments, a releasable linker has a half life at pH 7.4, 25.degree. C., e.g., a physiological pH, human body temperature (e.g., in vivo), of about 30 minutes, about 1 hour, about 2 hour, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about 96 hours or less.

[0060] The term "reference sequence" refers generally to a nucleic acid coding sequence, or amino acid sequence, to which another sequence is being compared. All polypeptide and polynucleotide sequences described herein are included as references sequences, including those described by name and those described in the Sequence Listing.

[0061] The terms "sequence identity" or, for example, comprising a "sequence 50% identical to," as used herein, refer to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Included are nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to any of the reference sequences described herein (see, e.g., Sequence Listing), typically where the polypeptide variant maintains at least one biological activity of the reference polypeptide.

[0062] Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence," "comparison window," "sequence identity," "percentage of sequence identity," and "substantial identity." A "reference sequence" is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., Nucl. Acids Res. 25:3389, 1997. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al., "Current Protocols in Molecular Biology," John Wiley & Sons Inc, 1994-1998, Chapter 15.

[0063] By "statistically significant," it is meant that the result was unlikely to have occurred by chance. Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which is the frequency or probability with which the observed event would occur, if the null hypothesis were true. If the obtained p-value is smaller than the significance level, then the null hypothesis is rejected. In simple cases, the significance level is defined at a p-value of 0.05 or less.

[0064] The term "solubility" refers to the property of a protein to dissolve in a liquid solvent and form a homogeneous solution. Solubility is typically expressed as a concentration, either by mass of solute per unit volume of solvent (g of solute per kg of solvent, g per dL (100 mL), mg/ml, etc.), molarity, molality, mole fraction or other similar descriptions of concentration. The maximum equilibrium amount of solute that can dissolve per amount of solvent is the solubility of that solute in that solvent under the specified conditions, including temperature, pressure, pH, and the nature of the solvent. In certain embodiments, solubility is measured at physiological pH, or other pH, for example, at pH 5.0, pH 6.0, pH 7.0, or pH 7.4. In certain embodiments, solubility is measured in water or a physiological buffer such as PBS or NaCl (with or without NaP). In specific embodiments, solubility is measured at relatively lower pH (e.g., pH 6.0) and relatively higher salt (e.g., 500 mM NaCl and 10 mM NaP). In certain embodiments, solubility is measured in a biological fluid (solvent) such as blood or serum. In certain embodiments, the temperature can be about room temperature (e.g., about 20, 21, 22, 23, 24, 25.degree. C.) or about body temperature (.about.37.degree. C.). In certain embodiments, a p97 polypeptide or conjugate has a solubility of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 mg/ml at room temperature or at about 37.degree. C.

[0065] A "subject," as used herein, includes any animal that exhibits a symptom, or is at risk for exhibiting a symptom, which can be treated or diagnosed with a p97 fusion protein or related antibody fusion of the invention. Suitable subjects (patients) include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog). Non-human primates and, preferably, human patients, are included.

[0066] "Substantially" or "essentially" means nearly totally or completely, for instance, 95%, 96%, 97%, 98%, 99% or greater of some given quantity.

[0067] "Substantially free" refers to the nearly complete or complete absence of a given quantity for instance, less than about 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or less of some given quantity. For example, certain compositions may be "substantially free" of cell proteins, membranes, nucleic acids, endotoxins, or other contaminants.

[0068] "Treatment" or "treating," as used herein, includes any desirable effect on the symptoms or pathology of a disease or condition, and may include even minimal changes or improvements in one or more measurable markers of the disease or condition being treated. "Treatment" or "treating" does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof. The subject receiving this treatment is any subject in need thereof. Exemplary markers of clinical improvement will be apparent to persons skilled in the art.

[0069] The term "wild-type" refers to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally-occurring source. A wild type gene or gene product (e.g., a polypeptide) is that which is most frequently observed in a population and is thus arbitrarily designed the "normal" or "wild-type" form of the gene.

Fusion Proteins

[0070] Embodiments of the present invention relate generally to fusion proteins that comprise a human p97 (melanotransferrin; MTf) polypeptide sequence a trastuzumab sequence, or an antigen-binding fragment thereof, antibodies that comprise such fusion proteins (i.e., antibody fusions), polynucleotides encoding the fusion proteins, host cells and methods of producing fusion proteins/antibodies, and related compositions and methods of use thereof. Exemplary p97 polypeptide sequences and trastuzumab sequences are described below. Also described are exemplary methods and components, such as linker peptides, for coupling a p97 polypeptide sequence to a trastuzumab sequence.

[0071] p97 Sequences.

[0072] In certain embodiments, a p97 polypeptide sequence used in a composition and/or fusion protein of the invention comprises, consists essentially of, or consists of a human p97 reference sequence provided in Table 1 below. Also included are variants and fragments thereof.

TABLE-US-00001 TABLE 1 Exemplary p97 Sequences SEQ ID Description Sequence NO: FL Human p97 MRGPSGALWLLLALRTVLGGMEVRWCATSDPEQHKCGNMSEAFREAGIQ 1 PSLLCVRGTSADHCVQLIAAQEADAITLDGGAIYEAGKEHGLKPVVGEV YDQEVGTSYYAVAVVRRSSHVTIDTLKGVKSCHTGINRTVGWNVPVGYL VESGRLSVMGCDVLKAVSDYFGGSCVPGAGETSYSESLCRLCRGDSSGE GVCDKSPLERYYDYSGAFRCLAEGAGDVAFVKHSTVLENTDGKTLPSWG QALLSQDFELLCRDGSRADVTEWRQCHLARVPAHAVVVRADTDGGLIFR LLNEGQRLFSHEGSSFQMFSSEAYGQKDLLFKDSTSELVPIATQTYEAW LGHEYLHAMKGLLCDPNRLPPYLRWCVLSTPEIQKCGDMAVAFRRQRLK PEIQCVSAKSPQHCMERIQAEQVDAVTLSGEDIYTAGKTYGLVPAAGEH YAPEDSSNSYYVVAVVRRDSSHAFTLDELRGKRSCHAGFGSPAGWDVPV GALIQRGFIRPKDCDVLTAVSEFFNASCVPVNNPKNYPSSLCALCVGDE QGRNKCVGNSQERYYGYRGAFRCLVENAGDVAFVRHTTVFDNTNGHNSE PWAAELRSEDYELLCPNGARAEVSQFAACNLAQIPPHAVMVRPDTNIFT VYGLLDKAQDLFGDDHNKNGFKMFDSSNYHGQDLLFKDATVRAVPVGEK TTYRGWLGLDYVAALEGMSSQQCSGAAAPAPGAPLLPLLLPALAARLLP PAL Soluble GMEVRWCATSDPEQHKCGNMSEAFREAGIQPSLLCVRGTSADHCVQLIA 2 Human p97 AQEADAITLDGGAIYEAGKEHGLKPVVGEVYDQEVGTSYYAVAVVRRSS HVTIDTLKGVKSCHTGINRTVGWNVPVGYLVESGRLSVMGCDVLKAVSD YFGGSCVPGAGETSYSESLCRLCRGDSSGEGVCDKSPLERYYDYSGAFR CLAEGAGDVAFVKHSTVLENTDGKTLPSWGQALLSQDFELLCRDGSRAD VTEWRQCHLARVPAHAVVVRADTDGGLIFRLLNEGQRLFSHEGSSFQMF SSEAYGQKDLLFKDSTSELVPIATQTYEAWLGHEYLHAMKGLLCDPNRL PPYLRWCVLSTPEIQKCGDMAVAFRRQRLKPEIQCVSAKSPQHCMERIQ AEQVDAVTLSGEDIYTAGKTYGLVPAAGEHYAPEDSSNSYYVVAVVRRD SSHAFTLDELRGKRSCHAGFGSPAGWDVPVGALIQRGFIRPKDCDVLTA VSEFFNASCVPVNNPKNYPSSLCALCVGDEQGRNKCVGNSQERYYGYRG AFRCLVENAGDVAFVRHTTVFDNTNGHNSEPWAAELRSEDYELLCPNGA RAEVSQFAACNLAQIPPHAVMVRPDTNIFTVYGLLDKAQDLFGDDHNKN GFKMFDSSNYHGQDLLFKDATVRAVPVGEKTTYRGWLGLDYVAALEGMS SQQCSG P97 fragment WCATSDPEQHK 3 P97 fragment RSSHVTIDTLK 4 P97 fragment SSHVTIDTLKGVK 5 P97 fragment LCRGDSSGEGVCDK 6 P97 fragment GDSSGEGVCDKSPLER 7 P97 fragment YYDYSGAFR 8 P97 fragment ADVTEWR 9 P97 fragment VPAHAVVVR 10 P97 fragment ADTDGGLIFR 11 P97 fragment CGDMAVAFR 12 P97 fragment LKPEIQCVSAK 13 P97 fragment DSSHAFTLDELR 14 P97 fragment SEDYELLCPNGAR 15 P97 fragment AQDLFGDDHNKNGFK 16 P97 fragment FSSEAYGQKDLLFKDSTSELVPIATQTYEAWLGHEYLHAM 17 P97 fragment ERIQAEQVDAVTLSGEDIYTAGKTYGLVPAAGEHYAPEDSSNSYYVVAV 18 VRRDSSHAFTLDELRGKRSCHAGFGSPAGWDVPVGALIQRGFIRPKDCD VLTAVSEFFNASCVPVNNPKNYPSSLCALCVGDEQGRNKCVGNSQERYY GYRGAFRCLVENAGDVAFVRHTTVFDNTNGHNSEPWAAELRSEDYELLC PNGARAEVSQFAACNLAQIPPHAVM P97 fragment VRPDTNIFTVYGLLDKAQDLFGDDHNKNGFKM 19 P97 fragment GMEVRWCATSDPEQHKCGNMSEAFREAGIQPSLLCVRGTSADHCVQLIA 20 AQEADAITLDGGAIYEAGKEHGLKPVVGEVYDQEVGTSYYAVAVVRRSS HVTIDTLKGVKSCHTGINRTVGWNVPVGYLVESGRLSVMGCDVLKAVSD YFGGSCVPGAGETSYSESLCRLCRGDSSGEGVCDKSPLERYYDYSGAFR CLAEGAGDVAFVKHSTVLENTDGKTLPSWGQALLSQDFELLCRDGSRAD VTEWRQCHLARVPAHAVVVRADTDGGLIFRLLNEGQRLFSHEGSSFQMF SSEAYGQKDLLFKDSTSELVPIATQTYEAWLGHEYLHAMKGLLCDPNRL PPYLRWCVLSTPEIQKCGDMAVAFRRQRLKPEIQCVSAKSPQHCMERIQ AEQVDAVTLSGEDIYTAGKTYGLVPAAGEHYAPEDSSNSYYVVAVVRRD SSHAFTLDELRGKRSCHAGFGSPAGWDVPVGALIQRGFIRPKDCDVLTA VSEFFNASCVPVNNPKNYPSSLCALCVGDEQGRNKCVGNSQERYYGYRG AFRCLVENAGDVAFVRHTTVFDNTN P97 fragment GHNSEPWAAELRSEDYELLCPN 21 P97 fragment GARAEVSQFAACNLAQIPPHAVMVRPDTNIFTVYGLLDKAQDLFGDDHN 22 KN P97 fragment GFKMFDSSNYHGQDLLFKDATVRAVPVGEKTTYRGWLGLDYVAALEGMS 23 SQQC P97 fragment GMEVRWCATSDPEQHKCGNMSEAFREAGIQPSLLCVRGTSADHCVQLIA 24 AQEADAITLDGGAIYEAGKEHGLKPVVGEVYDQEVGTSYYAVAVVRRSS HVTIDTLKGVKSCHTGINRTVGWNVPVGYLVESGRLSVMGCDVLKAVSD YFGGSCVPGAGETSYSESLCRLCRGDSSGEGVCDKSPLERYYDYSGAFR CLAEGAGDVAFVKHSTVLENTDGKTLPSWGQALLSQDFELLCRDGSRAD VTEWRQCHLARVPAHAVVVRADTDGGLIFRLLNEGQRLFSHEGSSFQMF SSEAYGQKDLLFKDSTSELVPIATQTYEAWLGHEYLHAMKGLLCDPNRL PPYLRWCVLSTPEIQKCGDMAVAFRRQRLKPEIQCVSAKSPQHCMERIQ AEQVDAVTLSGEDIYTAGKTYGLVPAAGEHYAPEDSSNSYYVVAVVRRD SSHAFTLDELRGKRSCHAGFGSPAGWDVPVGALIQRGFIRPKDCDVLTA VSEFFNASCVPVNNPKNYPSSLCALCVGDEQGRNKCVGNSQERYYGYRG AFRCLVENAGDVAFVRHTTVFDNTNGHNSEPWAAELRSEDYELLCPN P97 fragment GMEVRWCATSDPEQHKCGNMSEAFREAGIQPSLLCVRGTSADHCVQLIA 25 AQEADAITLDGGAIYEAGKEHGLKPVVGEVYDQEVGTSYYAVAVVRRSS HVTIDTLKGVKSCHTGINRTVGWNVPVGYLVESGRLSVMGCDVLKAVSD YFGGSCVPGAGETSYSESLCRLCRGDSSGEGVCDKSPLERYYDYSGAFR CLAEGAGDVAFVKHSTVLENTDGKTLPSWGQALLSQDFELLCRDGSRAD VTEWRQCHLARVPAHAVVVRADTDGGLIFRLLNEGQRLFSHEGSSFQMF SSEAYGQKDLLFKDSTSELVPIATQTYEAWLGHEYLHAMKGLLCDPNRL PPYLRWCVLSTPEIQKCGDMAVAFRRQRLKPEIQCVSAKSPQHCMERIQ AEQVDAVTLSGEDIYTAGKTYGLVPAAGEHYAPEDSSNSYYVVAVVRRD SSHAFTLDELRGKRSCHAGFGSPAGWDVPVGALIQRGFIRPKDCDVLTA VSEFFNASCVPVNNPKNYPSSLCALCVGDEQGRNKCVGNSQERYYGYRG AFRCLVENAGDVAFVRHTTVFDNTNGHNSEPWAAELRSEDYELLCPNGA RAEVSQFAACNLAQIPPHAVMVRPDTNIFTVYGLLDKAQDLFGDDHNKN P97 fragment GHNSEPWAAELRSEDYELLCPNGARAEVSQFAACNLAQIPPHAVMVRPD 26 TNIFTVYGLLDKAQDLFGDDHNKN P97 fragment GHNSEPWAAELRSEDYELLCPNGARAEVSQFAACNLAQIPPHAVMVRPD 27 TNIFTVYGLLDKAQDLFGDDHNKNGFKMFDSSNYHGQDLLFKDATVRAV PVGEKTTYRGWLGLDYVAALEGMSSQQC P97 fragment GARAEVSQFAACNLAQIPPHAVMVRPDTNIFTVYGLLDKAQDLFGDDHN 28 KNGFKMFDSSNYHGQDLLFKDATVRAVPVGEKTTYRGWLGLDYVAALEG MSSQQC

[0073] In some embodiments, a p97 polypeptide sequence comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity or homology, along its length, to a human p97 sequence in Table 1, or a fragment thereof.

[0074] In particular embodiments, a p97 polypeptide sequence comprises a fragment of a human p97 sequence in Table 1. In certain embodiments, a p97 polypeptide fragment is about, at least about, or up to about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700. 700, 710, 720, 730 or more amino acids in length, including all integers and ranges in between, and which may comprise all or a portion of the sequence of a p97 reference sequence.

[0075] In certain embodiments, a p97 polypeptide fragment is about 5-700, 5-600, 5-500, 5-400, 5-300, 5-200, 5-100, 5-50, 5-40, 5-30, 5-25, 5-20, 5-15, 5-10, 10-700, 10-600, 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 10-40, 10-30, 10-25, 10-20, 10-15, 20-700, 20-600, 20-500, 20-400, 20-300, 20-200, 20-100, 20-50, 20-40, 20-30, 20-25, 30-700, 30-600, 30-500, 30-400, 30-300, 30-200, 30-100, 30-50, 30-40, 40-700, 40-600, 40-500, 40-400, 40-300, 40-200, 40-100, 40-50, 50-700, 50-600, 50-500, 50-400, 50-300, 50-200, 50-100, 60-700, 60-600, 60-500, 60-400, 60-300, 60-200, 60-100, 60-70, 70-700, 70-600, 70-500, 70-400, 70-300, 70-200, 70-100, 70-80, 80-700, 80-600, 80-500, 80-400, 80-300, 80-200, 80-100, 80-90, 90-700, 90-600, 90-500, 90-400, 90-300, 90-200, 90-100, 100-700, 100-600, 100-500, 100-400, 100-300, 100-250, 100-200, 100-150, 200-700, 200-600, 200-500, 200-400, 200-300, or 200-250 amino acids in length, and comprises all or a portion of a p97 reference sequence.

[0076] In certain embodiments, p97 polypeptide sequences of interest include p97 amino acid sequences, subsequences, and/or variants of p97 that are effective for transporting an agent of interest across the blood brain barrier and into the central nervous system (CNS). In particular embodiments, the variant or fragment comprises the N-lobe of human p97 (residues 20-361 of SEQ ID NO:1). In specific aspects, the variant or fragment comprises an intact and functional Fe.sup.3+-binding site.

[0077] In some embodiments, a p97 polypeptide sequence is a soluble form of a p97 polypeptide (see Yang et al., Prot Exp Purif. 34:28-48, 2004), or a fragment or variant thereof. In some aspects, the soluble p97 polypeptide has a deletion of the all or a portion of the hydrophobic domain (residues 710-738 of SEQ ID NO:1), alone or in combination with a deletion of all or a portion of the signal peptide (residues 1-19 of SEQ ID NO:1). In specific aspects, the soluble p97 polypeptide comprises or consists of SEQ ID NO:2 (residues 20-711 of SEQ ID NO:1), including variants and fragments thereof.

[0078] In some embodiments, the p97 polypeptide comprises, consists, or consists essentially of the sequence DSSHAFTLDELR (SEQ ID NO:14 or MTfp), including variants and fragments thereof. In some embodiments, the DSSHAFTLDELR (SEQ ID NO:14) peptide comprises a C-terminal tyrosine (Y).

[0079] In certain embodiments, for instance, those that employ liposomes, the p97 polypeptide sequence is a lipid soluble form of a p97 polypeptide. For instance, certain of these and related embodiments include a p97 polypeptide that comprises all or a portion of the hydrophobic domain, optionally with or without the signal peptide.

[0080] In certain other embodiments, the p97 fragment or variant is capable of specifically binding to a p97 receptor, an LRP1 receptor and/or an LRP1B receptor.

[0081] Variants and fragments of reference p97 polypeptides and other reference polypeptides are described in greater detail below.

[0082] Trastuzumab Sequences.

[0083] In certain embodiments, a trastuzumab antibody sequence used in a fusion protein of the invention comprises, consists essentially of, or consists of the trastuzumab light chain and/or heavy chain sequence(s) illustrated in Table 2 below.

TABLE-US-00002 TABLE 2 Exemplary Trastuzumab Sequences SEQ ID Description Sequence NO: FL heavy EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA 29 chain RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSR (hinge WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC underlined) LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK FL heavy EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA 30 chain RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSR (hinge WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC underlined) LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL with GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL substituted FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP amino acids REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK for ''hole'' GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN (bold NYKTWPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ underlined) KSLSTSPGK Heavy chain NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT 31 truncation PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV (hinge LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS underlined) RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Heavy chain DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE 32 truncation DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE (partial YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC hinge LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR underlined) WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Heavy chain NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT 33 truncation PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV (hinge LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS underlined) RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTWPPVLDSDGS with FFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSTSPGK substituted amino acids for ''hole'' (bold underlined) Heavy chain DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE 34 truncation DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE (partial YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC hinge LVKGFYPSDIAVEWESNGQPENNYKTWPPVLDSDGSFFLTSKLTVDKSR underlined) WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK with substituted amino acids for ''hole'' (bold underlined) FL heavy EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA 35 chain (hinge RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSR underlined) WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC with LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL substituted GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL amino acids FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP for ''knob'' REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (bold GQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPEN underlined) NYKTTPPVLDSDGSFALYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK FL heavy METDTLLLWVLLLWVPGSTGEVQLVESGGGLVQPGGSLRLSCAASGFNI 122 chain with KDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNT Signal AYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPS Sequence VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV (underlined) LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK FL Light DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIY 36 Chain SASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTF GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC FL Light METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCRASQDV 123 Chain with NTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSL Signal QPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKS Sequence GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS (underlined) STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0084] Also included are antigen-binding variants and fragments of the trastuzumab heavy and light chain sequences described herein. In certain embodiments, the trastuzumab antibody, antigen-binding fragment thereof, or related fusion protein or antibody fusion specifically binds to Her2/neu or an epitope or fragment thereof.

[0085] In particular embodiments, the trastuzumab heavy chain fragment(s), e.g., the Fc regions of the heavy chain fragments, are modified to increase a preferred chain combination, for example, by using knobs-into-holes (KiH) technology (see, e.g., Klein et al., mAbs. 4:6, 653-663, 2012) or other technologies, such as those described in U.S. Application No. 2012/0149876. As one example, to increase the formation of a heterodimeric antibody fusion (e.g., an antibody that comprises one p97-trastuzumab heavy chain fusion and one normal trastuzumab heavy chain; see, e.g., FIGS. 1B and 1C), one of the heavy chains can have amino acid modifications to generate the "knob" and the other heavy chain can have amino acid modifications to form the "hole". Specific, non-limiting examples of KiH modifications to trastuzumab heavy chain sequences are illustrated in Table 2 above. As another example, one heavy chain fragment could comprise a CH3 domain having amino acid modifications selected from one or more of T350V, L351Y, D399R, D399 (e.g., D399R, D399W, D399Y, D399K), 5400 (e.g., S400E, S400D, S400R, S400K), F405 (e.g., F4051, F405M, F405T, F405S, F405V, F405W), Y407A, Y4071, Y407V, including combinations thereof, and the other could comprise a CH3 domain having amino acid modifications selected from one or more of T350V, T366V, T3661, T366L, T366M, N390 (e.g., N390R, N390K, N390D), K392 (e.g., K392V, K392M, K392R, K392L, K392F, K392E), F405 (e.g., F4051, F405M, F405T, F405S, F405V, F405W), K409F, K409W, and T411 (e.g., T411N, T411R, T411Q, T411K, T411D, T411E, T411W) including combinations thereof (see U.S. Application No. 2012/0149876, hereby incorporated by reference in its entirety).

[0086] The term "antigen-binding fragment" as used herein refers to a polypeptide fragment that contains at least one CDR of an immunoglobulin heavy and/or light chains that bind to the antigen of interest. In this regard, an antigen-binding fragment of the herein described antibodies may comprise 1, 2, 3, 4, 5, or all 6 CDRs of a VH and VL sequence from antibodies that bind to a therapeutic or diagnostic target.

[0087] The term "antigen" refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody, and additionally capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen. An antigen may have one or more epitopes.

[0088] The term "epitope" includes any determinant, preferably a polypeptide determinant, capable of specific binding to an immunoglobulin or T-cell receptor. An epitope is a region of an antigen that is bound by an antibody. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl, and may in certain embodiments have specific three-dimensional structural characteristics, and/or specific charge characteristics. Epitopes can be contiguous or non-contiguous in relation to the primary structure of the antigen.

[0089] An antibody, antigen-binding fragment thereof, is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular cell or substance than it does with alternative cells or substances. An antibody "specifically binds" or "preferentially binds" to a target if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to a specific epitope is an antibody that binds that specific epitope with greater affinity, avidity, more readily, and/or with greater duration than it binds to other epitopes. It is also understood by reading this definition that, for example, an antibody (or moiety or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. As such, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding.

[0090] Immunological binding generally refers to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific, for example by way of illustration and not limitation, as a result of electrostatic, ionic, hydrophilic and/or hydrophobic attractions or repulsion, steric forces, hydrogen bonding, van der Waals forces, and other interactions. The strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (K.sub.d) of the interaction, wherein a smaller K.sub.d represents a greater affinity. Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigen-binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and on geometric parameters that equally influence the rate in both directions. Thus, both the "on rate constant" (K.sub.on) and the "off rate constant" (K.sub.off) can be determined by calculation of the concentrations and the actual rates of association and dissociation. The ratio of K.sub.off/K.sub.on enables cancellation of all parameters not related to affinity, and is thus equal to the dissociation constant K.sub.d.

[0091] Immunological binding properties of proteins such as trastuzumab, antigen-binding fragments thereof, and related fusion proteins and antibody fusions can be quantified using methods well known in the art (see Davies et al., Annual Rev. Biochem. 59:439-473, 1990). In some embodiments, a protein is said to specifically bind an antigen or epitope thereof when the equilibrium dissociation constant is about .ltoreq.10.sup.-7 or 10.sup.-8 M. In some embodiments, the equilibrium dissociation constant of a protein may be about .ltoreq.10.sup.-9 M or 10.sup.-10 M. In certain illustrative embodiments, a protein has an affinity (K.sub.d) for an antigen or target described herein (to which it specifically binds) of at least about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50 nM.

[0092] In certain embodiments, antibodies and antigen-binding fragments thereof as described herein include a heavy chain and a light chain CDR set, respectively interposed between a heavy chain and a light chain framework region (FR) set which provide support to the CDRs and define the spatial relationship of the CDRs relative to each other. As used herein, the term "CDR set" refers to the three hypervariable regions of a heavy or light chain V region. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted as "CDR1," "CDR2," and "CDR3" respectively. An antigen-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 or CDR3) is referred to herein as a "molecular recognition unit." Crystallographic analysis of a number of antigen-antibody complexes has demonstrated that the amino acid residues of CDRs form extensive contact with bound antigen, wherein the most extensive antigen contact is with the heavy chain CDR3. Thus, the molecular recognition units are primarily responsible for the specificity of an antigen-binding site.

[0093] As used herein, the term "FR set" refers to the four flanking amino acid sequences which frame the CDRs of a CDR set of a heavy or light chain V region. Some FR residues may contact bound antigen; however, FRs are primarily responsible for folding the V region into the antigen-binding site, particularly the FR residues directly adjacent to the CDRs. Within FRs, certain amino residues and certain structural features are very highly conserved. In this regard, all V region sequences contain an internal disulfide loop of around 90 amino acid residues. When the V regions fold into a binding-site, the CDRs are displayed as projecting loop motifs which form an antigen-binding surface. It is generally recognized that there are conserved structural regions of FRs which influence the folded shape of the CDR loops into certain "canonical" structures--regardless of the precise CDR amino acid sequence. Further, certain FR residues are known to participate in non-covalent interdomain contacts which stabilize the interaction of the antibody heavy and light chains.

[0094] The structures and locations of immunoglobulin variable domains may be determined by reference to Kabat, E. A. et al., Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987, and updates thereof.

[0095] A "monoclonal antibody" refers to a homogeneous antibody population wherein the monoclonal antibody is comprised of amino acids (naturally occurring and non-naturally occurring) that are involved in the selective binding of an epitope. Monoclonal antibodies are highly specific, being directed against a single epitope. The term "monoclonal antibody" encompasses not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab').sub.2, Fv), single chain (ScFv), variants thereof, fusion proteins comprising an antigen-binding portion, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen-binding fragment (epitope recognition site) of the required specificity and the ability to bind to an epitope. It is not intended to be limited as regards the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals). The term includes whole immunoglobulins as well as the fragments etc. described above under the definition of "antibody."

[0096] The proteolytic enzyme papain preferentially cleaves IgG molecules to yield several fragments, two of which (the F(ab) fragments) each comprise a covalent heterodimer that includes an intact antigen-binding site. The enzyme pepsin is able to cleave IgG molecules to provide several fragments, including the F(ab').sub.2 fragment which comprises both antigen-binding sites. An Fv fragment for use according to certain embodiments of the present invention can be produced by preferential proteolytic cleavage of an IgM, and on rare occasions of an IgG or IgA immunoglobulin molecule. Fv fragments are, however, more commonly derived using recombinant techniques known in the art. The Fv fragment includes a non-covalent V.sub.H::V.sub.L heterodimer including an antigen-binding site which retains much of the antigen recognition and binding capabilities of the native antibody molecule. See Inbar et al., PNAS USA. 69:2659-2662, 1972; Hochman et al., Biochem. 15:2706-2710, 1976; and Ehrlich et al., Biochem. 19:4091-4096, 1980.

[0097] In certain embodiments, single chain Fv or scFV antibodies are contemplated. For example, Kappa bodies (III et al., Prot. Eng. 10:949-57, 1997); minibodies (Martin et al., EMBO J 13:5305-9, 1994); diabodies (Holliger et al., PNAS 90: 6444-8, 1993); or Janusins (Traunecker et al., EMBO J 10: 3655-59, 1991; and Traunecker et al., Int. J. Cancer Suppl. 7:51-52, 1992), may be prepared using standard molecular biology techniques following the teachings of the present application with regard to selecting antibodies having the desired specificity.

[0098] A single chain Fv (sFv) polypeptide is a covalently linked V.sub.H::V.sub.L heterodimer which is expressed from a gene fusion including V.sub.H- and V.sub.L-encoding genes linked by a peptide-encoding linker. Huston et al. (PNAS USA. 85(16):5879-5883, 1988). A number of methods have been described to discern chemical structures for converting the naturally aggregated--but chemically separated--light and heavy polypeptide chains from an antibody V region into an sFv molecule which will fold into a three dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No. 4,946,778, to Ladner et al.

[0099] In certain embodiments, the antibodies or antigen-binding fragments thereof are humanized. These embodiments refer to a chimeric molecule, generally prepared using recombinant techniques, having an antigen-binding site derived from an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molecule based upon the structure and/or sequence of a human immunoglobulin. The antigen-binding site may comprise either complete variable domains fused onto constant domains or only the CDRs grafted onto appropriate framework regions in the variable domains. Epitope binding sites may be wild type or modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in human individuals, but the possibility of an immune response to the foreign variable region remains (LoBuglio et al., PNAS USA 86:4220-4224, 1989; Queen et al., PNAS USA. 86:10029-10033, 1988; Riechmann et al., Nature. 332:323-327, 1988). Illustrative methods for humanization of antibodies include the methods described in U.S. Pat. No. 7,462,697.

[0100] Another approach focuses not only on providing human-derived constant regions, but modifying the variable regions as well so as to reshape them as closely as possible to human form. It is known that the variable regions of both heavy and light chains contain three complementarity-determining regions (CDRs) which vary in response to the epitopes in question and determine binding capability, flanked by four framework regions (FRs) which are relatively conserved in a given species and which putatively provide a scaffolding for the CDRs. When nonhuman antibodies are prepared with respect to a particular epitope, the variable regions can be "reshaped" or "humanized" by grafting CDRs derived from nonhuman antibody on the FRs present in the human antibody to be modified. Application of this approach to various antibodies has been reported by Sato et al., Cancer Res. 53:851-856, 1993; Riechmann et al., Nature 332:323-327, 1988; Verhoeyen et al., Science 239:1534-1536, 1988; Kettleborough et al., Protein Engineering. 4:773-3783, 1991; Maeda et al., Human Antibodies Hybridoma 2:124-134, 1991; Gorman et al., PNAS USA. 88:4181-4185, 1991; Tempest et al., Bio/Technology 9:266-271, 1991; Co et al., PNAS USA. 88:2869-2873, 1991; Carter et al., PNAS USA. 89:4285-4289, 1992; and Co et al., J Immunol. 148:1149-1154, 1992. In some embodiments, humanized antibodies preserve all CDR sequences (for example, a humanized mouse antibody which contains all six CDRs from the mouse antibodies). In other embodiments, humanized antibodies have one or more CDRs (one, two, three, four, five, six) which are altered with respect to the original antibody, which are also termed one or more CDRs "derived from" one or more CDRs from the original antibody.

[0101] Linkers.

[0102] As noted above, certain fusion proteins may employ one or more linker groups, including peptide linkers. Such linkers can be stable linkers or releasable linkers.

[0103] For instance, for polypeptide-polypeptide conjugates, peptide linkers can separate the components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures. Such a peptide linker sequence may be incorporated into the fusion protein using standard techniques described herein and well-known in the art. Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180.

[0104] In certain illustrative embodiments, a peptide linker is between about 1 to 5 amino acids, between 5 to 10 amino acids, between 5 to 25 amino acids, between 5 to 50 amino acids, between 10 to 25 amino acids, between 10 to 50 amino acids, between 10 to 100 amino acids, or any intervening range of amino acids. In other illustrative embodiments, a peptide linker comprises about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids in length. Particular linkers can have an overall amino acid length of about 1-200 amino acids, 1-150 amino acids, 1-100 amino acids, 1-90 amino acids, 1-80 amino acids, 1-70 amino acids, 1-60 amino acids, 1-50 amino acids, 1-40 amino acids, 1-30 amino acids, 1-20 amino acids, 1-10 amino acids, 1-5 amino acids, 1-4 amino acids, 1-3 amino acids, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100 or more amino acids.

[0105] A peptide linker may employ any one or more naturally-occurring amino acids, non-naturally occurring amino acid(s), amino acid analogs, and/or amino acid mimetics as described elsewhere herein and known in the art. Certain amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., PNAS USA. 83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180. Particular peptide linker sequences contain Gly, Ser, and/or Asn residues. Other near neutral amino acids, such as Thr and Ala may also be employed in the peptide linker sequence, if desired.

[0106] Certain exemplary linkers include Gly, Ser and/or Asn-containing linkers, as follows: [G].sub.x, [S].sub.x, [N].sub.x, [GS].sub.x, [GGS].sub.x, [GSS].sub.x, [GSGS].sub.x (SEQ ID NO:47), [GGSG].sub.x (SEQ ID NO:48), [GGGS].sub.x (SEQ ID NO:49), [GGGGS].sub.x(SEQ ID NO:50), [GN].sub.x, [GGN].sub.x, [GNN].sub.x, [GNGN].sub.x(SEQ ID NO:51), [GGNG].sub.x(SEQ ID NO:52), [GGGN].sub.x (SEQ ID NO:53), [GGGGN].sub.x(SEQ ID NO:54) linkers, where .sub.x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more. Other combinations of these and related amino acids will be apparent to persons skilled in the art. In specific embodiments, the linker comprises or consists of a [GGGGS].sub.3 (SEQ ID NO:55) sequence, or GGGGSGGGGSGGGGS (SEQ ID NO:55).

[0107] In specific embodiments, the linker sequence comprises a Gly3 linker sequence, which includes three glycine residues. In particular embodiments, flexible linkers can be rationally designed using a computer program capable of modeling both DNA-binding sites and the peptides themselves (Desjarlais & Berg, PNAS. 90:2256-2260, 1993; and PNAS. 91:11099-11103, 1994) or by phage display methods.

[0108] The peptide linkers may be physiologically stable or may include a releasable linker such as a physiologically degradable or enzymatically degradable linker (e.g., proteolytically cleavable linker). In certain embodiments, one or more releasable linkers can result in a shorter half-life and more rapid clearance of the conjugate. These and related embodiments can be used, for example, to enhance the solubility and blood circulation lifetime of p97 conjugates in the bloodstream, while also delivering an agent into the bloodstream (or across the BBB) that, subsequent to linker degradation, is substantially free of the p97 sequence. These aspects are especially useful in those cases where polypeptides or other agents, when permanently conjugated to a p97 sequence, demonstrate reduced activity. By using the linkers as provided herein, such antibodies can maintain their therapeutic activity when in conjugated form. In these and other ways, the properties of the p97 conjugates can be more effectively tailored to balance the bioactivity and circulating half-life of the antibodies over time.

[0109] Enzymatically degradable linkages suitable for use in particular embodiments of the present invention include, but are not limited to: an amino acid sequence cleaved by a serine protease such as thrombin, chymotrypsin, trypsin, elastase, kallikrein, or substilisin. Illustrative examples of thrombin-cleavable amino acid sequences include, but are not limited to: -Gly-Arg-Gly-Asp-(SEQ ID NO:56), -Gly-Gly-Arg-, -Gly-Arg-Gly-Asp-Asn-Pro-(SEQ ID NO:57), -Gly-Arg-Gly-Asp-Ser-(SEQ ID NO:58), -Gly-Arg-Gly-Asp-Ser-Pro-Lys-(SEQ ID NO:59), -Gly-Pro-Arg-, -Val-Pro-Arg-, and -Phe-Val-Arg-. Illustrative examples of elastase-cleavable amino acid sequences include, but are not limited to: -Ala-Ala-Ala-, -Ala-Ala-Pro-Val-(SEQ ID NO:60), -Ala-Ala-Pro-Leu-(SEQ ID NO:61), -Ala-Ala-Pro-Phe-(SEQ ID NO:62), -Ala-Ala-Pro-Ala-(SEQ ID NO:63), and -Ala-Tyr-Leu-Val-(SEQ ID NO:64).

[0110] Enzymatically degradable linkages suitable for use in particular embodiments of the present invention also include amino acid sequences that can be cleaved by a matrix metalloproteinase such as collagenase, stromelysin, and gelatinase. Illustrative examples of matrix metalloproteinase-cleavable amino acid sequences include, but are not limited to: -Gly-Pro-Y-Gly-Pro-Z-(SEQ ID NO:65), -Gly-Pro-, Leu-Gly-Pro-Z-(SEQ ID NO:66), -Gly-Pro-Ile-Gly-Pro-Z-(SEQ ID NO:67), and -Ala-Pro-Gly-Leu-Z-(SEQ ID NO: 68), where Y and Z are amino acids. Illustrative examples of collagenase-cleavable amino acid sequences include, but are not limited to: -Pro-Leu-Gly-Pro-D-Arg-Z-(SEQ ID NO:69), -Pro-Leu-Gly-Leu-Leu-Gly-Z-(SEQ ID NO:70), -Pro-Gln-Gly-Ile-Ala-Gly-Trp-(SEQ ID NO:71), -Pro-Leu-Gly-Cys(Me)-His-(SEQ ID NO:72), -Pro-Leu-Gly-Leu-Tyr-Ala-(SEQ ID NO:73), -Pro-Leu-Ala-Leu-Trp-Ala-Arg-(SEQ ID NO:74), and -Pro-Leu-Ala-Tyr-Trp-Ala-Arg-(SEQ ID NO:75), where Z is an amino acid. An illustrative example of a stromelysin-cleavable amino acid sequence is -Pro-Tyr-Ala-Tyr-Tyr-Met-Arg-(SEQ ID NO:76); and an example of a gelatinase-cleavable amino acid sequence is -Pro-Leu-Gly-Met-Tyr-Ser-Arg-(SEQ ID NO:77).

[0111] Enzymatically degradable linkages suitable for use in particular embodiments of the present invention also include amino acid sequences that can be cleaved by an angiotensin converting enzyme, such as, for example, -Asp-Lys-Pro-, -Gly-Asp-Lys-Pro-(SEQ ID NO:78), and -Gly-Ser-Asp-Lys-Pro-(SEQ ID NO:79).

[0112] Enzymatically degradable linkages suitable for use in particular embodiments of the present invention also include amino acid sequences that can be degraded by cathepsin B, such as, for example, -Val-Cit-, -Ala-Leu-Ala-Leu- (SEQ ID NO:80), -Gly-Phe-Leu-Gly- (SEQ ID NO:81) and -Phe-Lys-.

[0113] In some embodiments, the linker comprises, consists, or consists essentially of 125 (SEQ ID NO:124), including fragments and variants thereof.

[0114] In certain embodiments, however, any one or more of the non-peptide or peptide linkers are optional. For instance, linker sequences may not be required in a fusion protein where the first and second polypeptides have non-essential N-terminal and/or C-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.

[0115] Fusion Proteins and Antibody Fusions.

[0116] Certain embodiments relate to fusion proteins, comprising a p97 polypeptide sequence fused to a trastuzumab polypeptide sequence such as a trastuzumab heavy or light chain sequence, and antibody fusions comprising the same. An "antibody fusion" refers to an antibody or antibody-like immunoglobulin molecule that comprises one or more p97-trastuzumab fusion proteins and optionally one or more non-fusion trastuzumab sequences, i.e., trastuzumab light chain or heavy chain sequences, or variants/fragments thereof, which are not fused to a p97 sequence. In some instances, an antibody fusion comprises two light chain sequences and two heavy chain sequences, which are individually selected from any of the light/heavy chain sequences and/or fusion protein sequences described herein. In some instances, an antibody fusion comprises one light chain sequence and two heavy chain sequences, which are individually selected from any of the light/heavy chain sequences and/or fusion protein sequences described herein.

[0117] Specific, non-limiting examples of p97-trastuzumab heavy chain fusion proteins are illustrated in Table 3 below, and in Table E1 (see Examples).

TABLE-US-00003 TABLE 3 Exemplary p97-trastuzmab fusion protein sequences SEQ ID Description Sequence NO: Trastuzumab EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA 37 FL heavy RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSR chain fused WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC to N- LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL terminus of GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL soluble FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP human p97 (no REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK linker) GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK/GMEVRWCATSDPEQHKCGNMSEAFREAGIQPSLLCVRGT SADHCVQLIAAQEADAITLDGGAIYEAGKEHGLKPVVGEVYDQEVGTSY YAVAVVRRSSHVTIDTLKGVKSCHTGINRTVGWNVPVGYLVESGRLSVM GCDVLKAVSDYFGGSCVPGAGETSYSESLCRLCRGDSSGEGVCDKSPLE RYYDYSGAFRCLAEGAGDVAFVKHSTVLENTDGKTLPSWGQALLSQDFE LLCRDGSRADVTEWRQCHLARVPAHAVVVRADTDGGLIFRLLNEGQRLF SHEGSSFQMFSSEAYGQKDLLFKDSTSELVPIATQTYEAWLGHEYLHAM KGLLCDPNRLPPYLRWCVLSTPEIQKCGDMAVAFRRQRLKPEIQCVSAK SPQHCMERIQAEQVDAVTLSGEDIYTAGKTYGLVPAAGEHYAPEDSSNS YYVVAVVRRDSSHAFTLDELRGKRSCHAGFGSPAGWDVPVGALIQRGFI RPKDCDVLTAVSEFFNASCVPVNNPKNYPSSLCALCVGDEQGRNKCVGN SQERYYGYRGAFRCLVENAGDVAFVRHTTVFDNTNGHNSEPWAAELRSE DYELLCPNGARAEVSQFAACNLAQIPPHAVMVRPDTNIFTVYGLLDKAQ DLFGDDHNKNGFKMFDSSNYHGQDLLFKDATVRAVPVGEKTTYRGWLGL DYVAALEGMSSQQCSG Trastuzumab EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA 38 FL heavy RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSR chain fused WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC to N- LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL terminus of GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL soluble FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP human p97 REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (linker GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN underlined) NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGKGGGGSGGGGSGGGGSGMEVRWCATSDPEQHKCGNMSEAFR EAGIQPSLLCVRGTSADHCVQLIAAQEADAITLDGGAIYEAGKEHGLKP VVGEVYDQEVGTSYYAVAVVRRSSHVTIDTLKGVKSCHTGINRTVGWNV PVGYLVESGRLSVMGCDVLKAVSDYFGGSCVPGAGETSYSESLCRLCRG DSSGEGVCDKSPLERYYDYSGAFRCLAEGAGDVAFVKHSTVLENTDGKT LPSWGQALLSQDFELLCRDGSRADVTEWRQCHLARVPAHAVVVRADTDG GLIFRLLNEGQRLFSHEGSSFQMFSSEAYGQKDLLFKDSTSELVPIATQ TYEAWLGHEYLHAMKGLLCDPNRLPPYLRWCVLSTPEIQKCGDMAVAFR RQRLKPEIQCVSAKSPQHCMERIQAEQVDAVTLSGEDIYTAGKTYGLVP AAGEHYAPEDSSNSYYVVAVVRRDSSHAFTLDELRGKRSCHAGFGSPAG WDVPVGALIQRGFIRPKDCDVLTAVSEFFNASCVPVNNPKNYPSSLCAL CVGDEQGRNKCVGNSQERYYGYRGAFRCLVENAGDVAFVRHTTVFDNTN GHNSEPWAAELRSEDYELLCPNGARAEVSQFAACNLAQIPPHAVMVRPD TNIFTVYGLLDKAQDLFGDDHNKNGFKMFDSSNYHGQDLLFKDATVRAV PVGEKTTYRGWLGLDYVAALEGMSSQQCSG Trastuzumab GMEVRWCATSDPEQHKCGNMSEAFREAGIQPSLLCVRGTSADHCVQLIA 39 truncated AQEADAITLDGGAIYEAGKEHGLKPVVGEVYDQEVGTSYYAVAVVRRSS heavy chain HVTIDTLKGVKSCHTGINRTVGWNVPVGYLVESGRLSVMGCDVLKAVSD fused to C- YFGGSCVPGAGETSYSESLCRLCRGDSSGEGVCDKSPLERYYDYSGAFR terminus of CLAEGAGDVAFVKHSTVLENTDGKTLPSWGQALLSQDFELLCRDGSRAD soluble VTEWRQCHLARVPAHAVVVRADTDGGLIFRLLNEGQRLFSHEGSSFQMF human p97 SSEAYGQKDLLFKDSTSELVPIATQTYEAWLGHEYLHAMKGLLCDPNRL (no linker) PPYLRWCVLSTPEIQKCGDMAVAFRRQRLKPEIQCVSAKSPQHCMERIQ AEQVDAVTLSGEDIYTAGKTYGLVPAAGEHYAPEDSSNSYYVVAVVRRD SSHAFTLDELRGKRSCHAGFGSPAGWDVPVGALIQRGFIRPKDCDVLTA VSEFFNASCVPVNNPKNYPSSLCALCVGDEQGRNKCVGNSQERYYGYRG AFRCLVENAGDVAFVRHTTVFDNTNGHNSEPWAAELRSEDYELLCPNGA RAEVSQFAACKLAQIPPHAVMVRPDTNIFTVYGLLDKAQDLFGDDHNKN GFKMFDSSNYHGQDLLFKDATVRAVPVGEKTTYRGWLGLDYVAALEGMS SQQCG/NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K Trastuzumab GMEVRWCATSDPEQHKCGNMSEAFREAGIQPSLLCVRGTSADHCVQLIA 40 truncated AQEADAITLDGGAIYEAGKEHGLKPVVGEVYDQEVGTSYYAVAVVRRSS heavy chain HVTIDTLKGVKSCHTGINRTVGWNVPVGYLVESGRLSVMGCDVLKAVSD fused to C- YFGGSCVPGAGETSYSESLCRLCRGDSSGEGVCDKSPLERYYDYSGAFR terminus of CLAEGAGDVAFVKHSTVLENTDGKTLPSWGQALLSQDFELLCRDGSRAD soluble VTEWRQCHLARVPAHAVVVRADTDGGLIFRLLNEGQRLFSHEGSSFQMF human p97 SSEAYGQKDLLFKDSTSELVPIATQTYEAWLGHEYLHAMKGLLCDPNRL (linker PPYLRWCVLSTPEIQKCGDMAVAFRRQRLKPEIQCVSAKSPQHCMERIQ underlined) AEQVDAVTLSGEDIYTAGKTYGLVPAAGEHYAPEDSSNSYYVVAVVRRD SSHAFTLDELRGKRSCHAGFGSPAGWDVPVGALIQRGFIRPKDCDVLTA VSEFFNASCVPVNNPKNYPSSLCALCVGDEQGRNKCVGNSQERYYGYRG AFRCLVENAGDVAFVRHTTVFDNTNGHNSEPWAAELRSEDYELLCPNGA RAEVSQFAACNLAQIPPHAVMVRPDTNIFTVYGLLDKAQDLFGDDHNKN GFKMFDSSNYHGQDLLFKDATVRAVPVGEKTTYRGWLGLDYVAALEGMS SQQCGGGGGSGGGGSGGGGSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK Trastuzumab GMEVRWCATSDPEQHKCGNMSEAFREAGIQPSLLCVRGTSADHCVQLIA 41 partial AQEADAITLDGGAIYEAGKEHGLKPVVGEVYDQEVGTSYYAVAVVRRSS hinge HVTIDTLKGVKSCHTGINRTVGWNVPVGYLVESGRLSVMGCDVLKAVSD truncated YFGGSCVPGAGETSYSESLCRLCRGDSSGEGVCDKSPLERYYDYSGAFR heavy chain CLAEGAGDVAFVKHSTVLENTDGKTLPSWGQALLSQDFELLCRDGSRAD fused to C- VTEWRQCHLARVPAHAVVVRADTDGGLIFRLLNEGQRLFSHEGSSFQMF terminus of SSEAYGQKDLLFKDSTSELVPIATQTYEAWLGHEYLHAMKGLLCDPNRL soluble PPYLRWCVLSTPEIQKCGDMAVAFRRQRLKPEIQCVSAKSPQHCMERIQ human p97 AEQVDAVTLSGEDIYTAGKTYGLVPAAGEHYAPEDSSNSYYVVAVVRRD (no linker) SSHAFTLDELRGKRSCHAGFGSPAGWDVPVGALIQRGFIRPKDCDVLTA VSEFFNASCVPVNNPKNYPSSLCALCVGDEQGRNKCVGNSQERYYGYRG AFRCLVENAGDVAFVRHTTVFDNTNGHNSEPWAAELRSEDYELLCPNGA RAEVSQFAACNLAQIPPHAVMVRPDTNIFTVYGLLDKAQDLFGDDHNKN GFKMFDSSNYHGQDLLFKDATVRAVPVGEKTTYRGWLGLDYVAALEGMS SQQCG/DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Trastuzumab GMEVRWCATSDPEQHKCGNMSEAFREAGIQPSLLCVRGTSADHCVQLIA 42 partial AQEADAITLDGGAIYEAGKEHGLKPVVGEVYDQEVGTSYYAVAVVRRSS hinge HVTIDTLKGVKSCHTGINRTVGWNVPVGYLVESGRLSVMGCDVLKAVSD truncated YFGGSCVPGAGETSYSESLCRLCRGDSSGEGVCDKSPLERYYDYSGAFR heavy chain CLAEGAGDVAFVKHSTVLENTDGKTLPSWGQALLSQDFELLCRDGSRAD fused to C- VTEWRQCHLARVPAHAVVVRADTDGGLIFRLLNEGQRLFSHEGSSFQMF terminus of SSEAYGQKDLLFKDSTSELVPIATQTYEAWLGHEYLHAMKGLLCDPNRL soluble PPYLRWCVLSTPEIQKCGDMAVAFRRQRLKPEIQCVSAKSPQHCMERIQ human p97 AEQVDAVTLSGEDIYTAGKTYGLVPAAGEHYAPEDSSNSYYVVAVVRRD (linker SSHAFTLDELRGKRSCHAGFGSPAGWDVPVGALIQRGFIRPKDCDVLTA underlined) VSEFFNASCVPVNNPKNYPSSLCALCVGDEQGRNKCVGNSQERYYGYRG AFRCLVENAGDVAFVRHTTVFDNTNGHNSEPWAAELRSEDYELLCPNGA RAEVSQFAACNLAQIPPHAVMVRPDTNIFTVYGLLDKAQDLFGDDHNKN GFKMFDSSNYHGQDLLFKDATVRAVPVGEKTTYRGWLGLDYVAALEGMS SQQCGGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK Trastuzumab GMEVRWCATSDPEQHKCGNMSEAFREAGIQPSLLCVRGTSADHCVQLIA 43 truncated AQEADAITLDGGAIYEAGKEHGLKPVVGEVYDQEVGTSYYAVAVVRRSS heavy chain HVTIDTLKGVKSCHTGINRTVGWNVPVGYLVESGRLSVMGCDVLKAVSD with KiH YFGGSCVPGAGETSYSESLCRLCRGDSSGEGVCDKSPLERYYDYSGAFR ''hole'' CLAEGAGDVAFVKHSTVLENTDGKTLPSWGQALLSQDFELLCRDGSRAD variation VTEWRQCHLARVPAHAVVVRADTDGGLIFRLLNEGQRLFSHEGSSFQMF fused to C- SSEAYGQKDLLFKDSTSELVPIATQTYEAWLGHEYLHAMKGLLCDPNRL terminus of PPYLRWCVLSTPEIQKCGDMAVAFRRQRLKPEIQCVSAKSPQHCMERIQ soluble AEQVDAVTLSGEDIYTAGKTYGLVPAAGEHYAPEDSSNSYYVVAVVRRD human p97 SSHAFTLDELRGKRSCHAGFGSPAGWDVPVGALIQRGFIRPKDCDVLTA (no linker) VSEFFNASCVPVNNPKNYPSSLCALCVGDEQGRNKCVGNSQERYYGYRG AFRCLVENAGDVAFVRHTTVFDNTNGHNSEPWAAELRSEDYELLCPNGA RAEVSQFAACNLAQIPPHAVMVRPDTNIFTVYGLLDKAQDLFGDDHNKN GFKMFDSSNYHGQDLLFKDATVRAVPVGEKTTYRGWLGLDYVAALEGMS SQQCSG/NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTWPP VLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK Trastuzumab GMEVRWCATSDPEQHKCGNMSEAFREAGIQPSLLCVRGTSADHCVQLIA 44 truncated AQEADAITLDGGAIYEAGKEHGLKPVVGEVYDQEVGTSYYAVAVVRRSS heavy chain HVTIDTLKGVKSCHTGINRTVGWNVPVGYLVESGRLSVMGCDVLKAVSD with KiH YFGGSCVPGAGETSYSESLCRLCRGDSSGEGVCDKSPLERYYDYSGAFR ''hole'' CLAEGAGDVAFVKHSTVLENTDGKTLPSWGQALLSQDFELLCRDGSRAD variation VTEWRQCHLARVPAHAVVVRADTDGGLIFRLLNEGQRLFSHEGSSFQMF fused to C- SSEAYGQKDLLFKDSTSELVPIATQTYEAWLGHEYLHAMKGLLCDPNRL terminus of PPYLRWCVLSTPEIQKCGDMAVAFRRQRLKPEIQCVSAKSPQHCMERIQ soluble AEQVDAVTLSGEDIYTAGKTYGLVPAAGEHYAPEDSSNSYYVVAVVRRD human p97 SSHAFTLDELRGKRSCHAGFGSPAGWDVPVGALIQRGFIRPKDCDVLTA (linker VSEFFNASCVPVNNPKNYPSSLCALCVGDEQGRNKCVGNSQERYYGYRG underlined) AFRCLVENAGDVAFVRHTTVFDNTNGHNSEPWAAELRSEDYELLCPNGA RAEVSQFAACNLAQIPPHAVMVRPDTNIFTVYGLLDKAQDLFGDDHNKN GFKMFDSSNYHGQDLLFKDATVRAVPVGEKTTYRGWLGLDYVAALEGMS SQQCSGGGGGSGGGGSGGGGSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTWPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK Trastuzumab GMEVRWCATSDPEQHKCGNMSEAFREAGIQPSLLCVRGTSADHCVQLIA 45 partial AQEADAITLDGGAIYEAGKEHGLKPVVGEVYDQEVGTSYYAVAVVRRSS hinge HVTIDTLKGVKSCHTGINRTVGWNVPVGYLVESGRLSVMGCDVLKAVSD truncated YFGGSCVPGAGETSYSESLCRLCRGDSSGEGVCDKSPLERYYDYSGAFR heavy chain CLAEGAGDVAFVKHSTVLENTDGKTLPSWGQALLSQDFELLCRDGSRAD with KiH VTEWRQCHLARVPAHAVVVRADTDGGLIFRLLNEGQRLFSHEGSSFQMF ''hole'' SSEAYGQKDLLFKDSTSELVPIATQTYEAWLGHEYLHAMKGLLCDPNRL variation PPYLRWCVLSTPEIQKCGDMAVAFRRQRLKPEIQCVSAKSPQHCMERIQ fused to C- AEQVDAVTLSGEDIYTAGKTYGLVPAAGEHYAPEDSSNSYYVVAVVRRD terminus of SSHAFTLDELRGKRSCHAGFGSPAGWDVPVGALIQRGFIRPKDCDVLTA soluble VSEFFNASCVPVNNPKNYPSSLCALCVGDEQGRNKCVGNSQERYYGYRG human p97 AFRCLVENAGDVAFVRHTTVFDNTNGHNSEPWAAELRSEDYELLCPNGA (no linker) RAEVSQFAACNLAQIPPHAVMVRPDTNIFTVYGLLDKAQDLFGDDHNKN GFKMFDSSNYHGQDLLFKDATVRAVPVGEKTTYRGWLGLDYVAALEGMS SQQCSG/DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTWPPVLDSDGSFFLTSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Trastuzumab GMEVRWCATSDPEQHKCGNMSEAFREAGIQPSLLCVRGTSADHCVQLIA 46 partial AQEADAITLDGGAIYEAGKEHGLKPVVGEVYDQEVGTSYYAVAVVRRSS hinge HVTIDTLKGVKSCHTGINRTVGWNVPVGYLVESGRLSVMGCDVLKAVSD truncated YFGGSCVPGAGETSYSESLCRLCRGDSSGEGVCDKSPLERYYDYSGAFR heavy chain CLAEGAGDVAFVKHSTVLENTDGKTLPSWGQALLSQDFELLCRDGSRAD with KiH VTEWRQCHLARVPAHAVVVRADTDGGLIFRLLNEGQRLFSHEGSSFQMF ''hole'' SSEAYGQKDLLFKDSTSELVPIATQTYEAWLGHEYLHAMKGLLCDPNRL variation PPYLRWCVLSTPEIQKCGDMAVAFRRQRLKPEIQCVSAKSPQHCMERIQ fused to C- AEQVDAVTLSGEDIYTAGKTYGLVPAAGEHYAPEDSSNSYYVVAVVRRD terminus of SSHAFTLDELRGKRSCHAGFGSPAGWDVPVGALIQRGFIRPKDCDVLTA soluble VSEFFNASCVPVNNPKNYPSSLCALCVGDEQGRNKCVGNSQERYYGYRG human p97 AFRCLVENAGDVAFVRHTTVFDNTNGHNSEPWAAELRSEDYELLCPNGA (linker RAEVSQFAACNLAQIPPHAVMVRPDTNIFTVYGLLDKAQDLFGDDHNKN underlined) GFKMFDSSNYHGQDLLFKDATVRAVPVGEKTTYRGWLGLDYVAALEGMS SQQCSGGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTWP PVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK TZM heavy EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA 96 chain: RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSR linker WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC (underlined): LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL MTf (bold) GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGKGGGGSGGGGSGMEVRWCATSDPEQHKCGNMSEAFREAGIQ PSLLCVRGTSADHCVQLIAAQEADAITLDGGAIYEAGKEHGLKPVVGEV YDQEVGTSYYAVAVVRRSSHVTIDTLKGVKSCHTGINRTVGWNVPVGYL VESGRLSVMGCDVLKAVSDYFGGSCVPGAGETSYSESLCRLCRGDSSGE GVCDKSPLERYYDYSGAFRCLAEGAGDVAFVKHSTVLENTDGKTLPSWG QALLSQDFELLCRDGSRADVTEWRQCHLARVPAHAVVVRADTDGGLIFR LLNEGQRLFSHEGSSFQMFSSEAYGQKDLLFKDSTSELVPIATQTYEAW LGHEYLHAMKGLLCDPNRLPPYLRWCVLSTPEIQKCGDMAVAFRRQRLK PEIQCVSAKSPQHCMERIQAEQVDAVTLSGEDIYTAGKTYGLVPAAGEH YAPEDSSNSYYVVAVVRRDSSHAFTLDELRGKRSCHAGFGSPAGWDVPV GALIQRGFIRPKDCDVLTAVSEFFNASCVPVNNPKNYPSSLCALCVGDE QGRNKCVGNSQERYYGYRGAFRCLVENAGDVAFVRHTTVFDNTNGHNSE PWAAELRSEDYELLCPNGARAEVSQFAACNLAQIPPHAVMVRPDTNIFT VYGLLDKAQDLFGDDHNKNGFKMFDSSNYHGQDLLFKDATVRAVPVGEK TTYRGWLGLDYVAALEGMSSQQCS TZM heavy EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA 97 chain: RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSR linker WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC

(underlined): LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL MTf (bold) GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGKGGGGSGGGGSGGGGSGMEVRWCATSDPEQHKCGNMSEAFR EAGIQPSLLCVRGTSADHCVQLIAAQEADAITLDGGAIYEAGKEHGLKP VVGEVYDQEVGTSYYAVAVVRRSSHVTIDTLKGVKSCHTGINRTVGWNV PVGYLVESGRLSVMGCDVLKAVSDYFGGSCVPGAGETSYSESLCRLCRG DSSGEGVCDKSPLERYYDYSGAFRCLAEGAGDVAFVKHSTVLENTDGKT LPSWGQALLSQDFELLCRDGSRADVTEWRQCHLARVPAHAVVVRADTDG GLIFRLLNEGQRLFSHEGSSFQMFSSEAYGQKDLLFKDSTSELVPIATQ TYEAWLGHEYLHAMKGLLCDPNRLPPYLRWCVLSTPEIQKCGDMAVAFR RQRLKPEIQCVSAKSPQHCMERIQAEQVDAVTLSGEDIYTAGKTYGLVP AAGEHYAPEDSSNSYYVVAVVRRDSSHAFTLDELRGKRSCHAGFGSPAG WDVPVGALIQRGFIRPKDCDVLTAVSEFFNASCVPVNNPKNYPSSLCAL CVGDEQGRNKCVGNSQERYYGYRGAFRCLVENAGDVAFVRHTTVFDNTN GHNSEPWAAELRSEDYELLCPNGARAEVSQFAACNLAQIPPHAVMVRPD TNIFTVYGLLDKAQDLFGDDHNKNGFKMFDSSNYHGQDLLFKDATVRAV PVGEKTTYRGWLGLDYVAALEGMSSQQCSG MTfp DSSHAFTLDELRYGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASG 98 sequence FNIKDTYIHWVRQAPGKGLEWVARTYPTNGYTRYADSVKGRFTISADTS (bold): KNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTK linker GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF (underlined): PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS TZM heavy CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH chain EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK MTfp DSSHAFTLDELRGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGF 99 sequence NIKDTYIHWVRQAPGKGLEWVARTYPTNGYTRYADSVKGRFTISADTSK (bold): NTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKG linker PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP (underlined): AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC TZM heavy DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE chain DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK MTfp DSSHAFTLDELRYEAAAKEAAAKEAAAKEVQLVESGGGLVQPGGSLRLS 100 sequence CAASGFNIKDTYIHWVRQAPGKGLEWVARTYPTNGYTRYADSVKGRFTI (bold) SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVS w/terminal Y SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS (bold): GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK linker VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV (underlined): VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD TZM heavy WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN chain QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK MTfp DSSHAFTLDELREAAAKEAAAKEAAAKEVQLVESGGGLVQPGGSLRLSC 101 sequence AASGFNIKDTYIHWVRQAPGKGLEWVARTYPTNGYTRYADSVKGRFTIS (bold) w/o ADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS terminal Y: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG linker VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (underlined) EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV : TZM heavy DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW chain LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK MTfp DSSHAFTLDELRYEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHW 102 sequence VRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNS (bold): TZM LRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPS heavy chain SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY w/terminal Y SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC (bold) PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK MTfp DSSHAFTLDELREVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWV 103 sequence RQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSL (bold) w/o RAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSS terminal Y: KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS TZM heavy LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP chain APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK TZM heavy EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA 104 chain : RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSR linker WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC (underlined): LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL MTfp GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL sequence FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP without C- REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK terminal Y GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN (bold) NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGKGGGGSGGGGSDSSHAFTLDELR TZM heavy EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA 105 chain linker RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSR (underlined): WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC MTfp LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL sequence GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL w/terminal Y FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP (bold) REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGKGGGGSGGGGSDSSHAFTLDELRY TZM heavy EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA 106 chain: RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSR linker WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC (underlined): LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL MTfp GTQTYICNVNHKPSNTKVDKKVEPPKSCDKTHTCPPCPAPELLGGPSVF w/terminal Y LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK (bold) PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGKEAAAKEAAAKEAAAKDSSHAFTLDELRY TZM heavy EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA 107 chain: RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSR linker WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC (underlined): LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL MTfp w/o GTQTYICNVNHKPSNTKVDKKVEPPKSCDKTHTCPPCPAPELLGGPSVF terminal LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK Y(bold PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGKEAAAKEAAAKEAAAKDSSHAFTLDELR TZM heavy EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA 108 chain: MTfp RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSR sequence WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC without C- LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL terminal Y GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL (bold) FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGKDSSHAFTLDELR TZM heavy EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA 109 chain: MTfp RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSR sequence WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC w/terminal Y LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL (bold) GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGKDSSHAFTLDELRY

[0118] In certain embodiments, the p97-trastuzumab fusion protein comprises, consists, or consists essentially of a trastuzumab heavy chain sequence fused to the N-terminus of a p97 sequence. In particular embodiments, the p97 sequence is human soluble p97, for example, comprising or consisting of SEQ ID NO:2, or a variant/fragment thereof. In some embodiments, the p97 sequence comprises or consists of SEQ ID NO:14, or a variant/fragment thereof. In some embodiments, the trastuzumab heavy chain sequence is selected from SEQ ID NO:29-35 or 122, or a variant/fragment thereof. Optionally, the fusion protein comprises a peptide linker in between the p97 and trastuzumab sequences. In specific embodiments, the linker is a (GGGGS).sub.2 or (GGGGS).sub.3 linker or a EAAAKEAAAKEAAAK (SEQ ID NO:124) linker. In specific embodiments, the p97-trastuzumab fusion protein comprises, consists, or consists essentially of SEQ ID NO:37 (trastuzumab heavy chain fused to the N-terminus of soluble p97) or a variant/fragment thereof. In specific embodiments, the linker is a (GGGGS).sub.3 linker, and the fusion protein optionally comprises, consists, or consists essentially of SEQ ID NO:38 (trastuzumab heavy chain fused to the N-terminus of soluble human p97, and separated by a (GGGGS).sub.3 linker), or a variant/fragment thereof. Other combinations will be apparent to persons skilled in the art.

[0119] In some embodiments, the p97-trastuzumab fusion protein comprises, consists, or consists essentially of a trastuzumab heavy chain sequence fused to the C-terminus of a p97 sequence. In particular embodiments, the p97 sequence is human soluble p97, for example, comprising or consisting of SEQ ID NO:2, or a variant/fragment thereof. In some embodiments, the p97 sequence comprises or consists of SEQ ID NO:14, or a variant/fragment thereof. In some embodiments, the trastuzumab heavy chain sequence is selected from SEQ ID NO:29-35 or 122, or a variant/fragment thereof. In some embodiments, the trastuzumab heavy chain sequence is a truncated sequence that comprises the polypeptide of SEQ ID NO:31-33 or 34, or a variant/fragment thereof. In specific embodiments, the p97-trastuzumab fusion protein comprises, consists, or consists essentially of SEQ ID NO:39, 41, 43, or 45 (truncated trastuzumab heavy chain fused to the C-terminus of soluble human p97), or a variant/fragment thereof. Optionally, the fusion protein comprises a peptide linker in between the p97 and trastuzumab sequences. In specific embodiments, the linker is a (GGGGS).sub.2 or (GGGGS).sub.3 linker or a EAAAKEAAAKEAAAK (SEQ ID NO:124) linker. In specific embodiments, the linker is a (GGGGS).sub.3 linker, and the fusion protein optionally comprises, consists, or consists essentially of SEQ ID NO:40, 42, 44 or 46 (truncated trastuzumab heavy chain fused to the C-terminus of soluble human p97, and separated by a (GGGGS).sub.3 linker), or a variant/fragment thereof.

[0120] In some embodiments, the p97-trastuzumab heavy chain fusion protein comprises, consists, or consists essentially of a polypeptide sequence selected from SEQ ID NOs:37-46 and 96-109, or a variant/fragment thereof.

[0121] Other combinations will be apparent to persons skilled in the art.

[0122] Also included are p97-trastuzumab light chain fusion proteins. Specific, non-limiting examples of p97-trastuzumab light chain fusion proteins are illustrated in Table 4 below, and in Table E1 (see Examples).

TABLE-US-00004 TABLE 4 Exemplary p97-trastuzmab fusion protein sequences SEQ ID Sequence NO: Trastuzumab DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIY 110 FL light SASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTF chain fused GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ to N- WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV terminus of THQGLSSPVTKSFNRGECDSSHAFTLDELRY MTfp(no linker) Trastuzumab DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIY 111 FL light SASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTF chain fused GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ to N- WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV terminus of THQGLSSPVTKSFNRGECEAAAKEAAAKEAAAKDSSHAFTLDELRY MTfp(linker underlined) Trastuzumab DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIY 112 FL light SASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTF chain fused GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ to N- WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV terminus of THQGLSSPVTKSFNRGECGGGGSGGGGSDSSHAFTLDELRY MTfp(linker underlined) Trastuzumab DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIY 113 FL light SASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTF chain fused GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ to N- WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV terminus of THQGLSSPVTKSFNRGECDSSHAFTLDELR MTfp(no linker) Trastuzumab DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIY 114 FL light SASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTF chain fused GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ to N- WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV terminus of THQGLSSPVTKSFNRGECEAAAKEAAAKEAAAKDSSHAFTLDELR MTfp(linker underlined) Trastuzumab DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIY 115 FL light SASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTF chain fused GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ to N- WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV terminus of THQGLSSPVTKSFNRGECGGGGSGGGGSDSSHAFTLDELRY MTfp(linker underlined) Trastuzumab DSSHAFTLDELRYDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWY 116 FL light QQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFAT chain fused YYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC to C- LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK terminus of ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC MTfp(no linker) Trastuzumab DSSHAFTLDELRYEAAAKEAAAKEAAAKDIQMTQSPSSLSASVGDRVTI 117 FL light TCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD chain fused FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFP to C- PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS terminus of KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC MTfp(linker underlined) Trastuzumab DSSHAFTLDELRYGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS 118 FL light QDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTI chain fused SSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQ to C- LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY terminus of SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC MTfp(linker underlined) Trastuzumab DSSHAFTLDELRDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQ 119 FL light QKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATY chain fused YCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL to C- LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA terminus of DYEKHKVYACEVTHQGLSSPVTKSFNRGEC MTfp(no linker) Trastuzumab DSSHAFTLDELREAAAKEAAAKEAAAKDIQMTQSPSSLSASVGDRVTIT 120 FL light CRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDF chain fused TLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPP to C- SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK terminus of DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC MTfp(linker underlined) Trastuzumab DSSHAFTLDELRGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ 121 FL light DVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTIS chain fused SLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQL to C- KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS terminus of LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC MTfp(linker underlined)

[0123] In some embodiments, the p97-trastuzumab fusion protein comprises, consists, or consists essentially of a trastuzumab light chain sequence fused to the N-terminus of a p97 sequence. In particular embodiments, the p97 sequence is human soluble p97, for example, comprising or consisting of SEQ ID NO:2, or a variant/fragment thereof. In some embodiments, the p97 sequence comprises or consists of SEQ ID NO:14, or a variant/fragment thereof. In some embodiments, the trastuzumab light chain sequence comprises or consists of SEQ ID NO:36 or 123, or a variant/fragment thereof. In specific embodiments, the p97-trastuzumab fusion protein comprises, consists, or consists essentially of a sequence selected from SEQ ID NOs:110-115 (trastuzumab light chain fused to the N-terminus of p97pep), or a variant/fragment thereof. Optionally, the fusion protein comprises a peptide linker in between the p97 and trastuzumab sequence, for example, as illustrated in SEQ ID NO:111-112 or 114-115. In specific embodiments, the linker is a (GGGGS).sub.2 or (GGGGS).sub.3 linker or a EAAAKEAAAKEAAAK (SEQ ID NO:124) linker. Other combinations will be apparent to persons skilled in the art.

[0124] In some embodiments, the p97-trastuzumab fusion protein comprises, consists, or consists essentially of a trastuzumab light chain sequence fused to the C-terminus of a p97 sequence. In particular embodiments, the p97 sequence is human soluble p97, for example, comprising or consisting of SEQ ID NO:2, or a variant/fragment thereof. In some embodiments, the p97 sequence comprises or consists of SEQ ID NO:14, or a variant/fragment thereof. In some embodiments, the trastuzumab light chain sequence comprises or consists of SEQ ID NO:36 or 123, or a variant/fragment thereof. In specific embodiments, the p97-trastuzumab fusion protein comprises, consists, or consists essentially of SEQ ID NO:116-121 (trastuzumab light chain fused to the C-terminus of p97p), or a variant/fragment thereof. Optionally, the fusion protein comprises a peptide linker in between the p97 and trastuzumab sequences, as illustrated, for example, in SEQ ID NOs:117-118 and 120-121. In specific embodiments, the linker is a (GGGGS).sub.2 or (GGGGS).sub.3 linker or a EAAAKEAAAKEAAAK (SEQ ID NO:124) linker. Other combinations will be apparent to persons skilled in the art.

[0125] Also included are p97-antibody fusion proteins, comprising one or more p97-trastuzumab heavy or light chain fusion proteins described herein. In particular embodiments, the p97-antibody fusion protein comprises two (non-fusion) trastuzumab light chain sequences, and one or two p97-trastuzumab heavy chain fusion proteins described herein, where the one or two p97-trastuzumab heavy chain fusion protein(s) comprise a trastuzumab heavy chain sequence fused to the N-terminus of a p97 sequence and an optional linker in between. In some embodiments, the p97-antibody fusion comprises two p97-trastuzumab heavy chain fusion proteins (see, e.g., FIGS. 1A and 1E), including homodimeric antibody fusions that comprise the same p97-trastuzumab heavy chain fusion proteins. In some embodiments, the p97-antibody fusion comprises one p97-trastuzumab heavy chain fusion protein (see, e.g., FIG. 1B).

[0126] In some embodiments, the p97-antibody fusion protein comprises one or two trastuzumab light chain sequences, one trastuzumab heavy chain sequence, and one p97-trastuzumab heavy chain fusion protein described herein, where p97-trastuzumab heavy chain fusion protein comprises a trastuzumab heavy chain fused to the C-terminus of a p97 sequence and an optional linker in between. In some embodiments, the p97-antibody fusion protein comprises one trastuzumab light chain sequence, one trastuzumab heavy chain sequence, and one p97-trastuzumab heavy chain fusion protein that has a truncated trastuzumab heavy chain (see, e.g., FIG. 1C).

[0127] In some embodiments, the p97-antibody fusion protein comprises two trastuzumab light chain sequences, and two p97-trastuzumab heavy chain fusion proteins described herein, where the p97-trastuzumab heavy chain fusion proteins comprise a trastuzumab heavy chain fused to the C-terminus of a p97 sequence and an optional linker in between (see, e.g., FIG. 1D).

[0128] In some embodiments, the p97-antibody fusion protein comprises two p97-trastuzumab light chain fusion proteins described herein, and two p97-trastuzumab heavy chain fusion proteins described herein, where the p97-trastuzumab light chain fusion proteins comprise a trastuzumab light chain fused to the N-terminus of a p97 sequence and an optional linker in between, and where the p97-trastuzumab heavy chain fusion proteins comprise a trastuzumab heavy chain fused to the N-terminus of a p97 sequence and an optional linker in between (see, e.g., FIG. 1F).

[0129] In some embodiments, the p97-antibody fusion protein comprises one or two p97-trastuzumab light chain fusion proteins described herein, and two trastuzumab heavy chain sequences, where the one or two p97-trastuzumab light chain fusion proteins comprise a trastuzumab light chain fused to the N-terminus of a p97 sequence and an optional linker in between. In some embodiments, the p97-antibody fusion protein comprises two p97-trastuzumab light chain fusion proteins described herein (see, e.g., FIG. 1G).

[0130] In specific embodiments, the p97-antibody fusion comprises one or two sets of heavy and light chains selected from one or more of the following:

[0131] a) the heavy chain of SEQ ID NO:82 and the light chain of SEQ ID NO:83;

[0132] b) the heavy chain of SEQ ID NO:84 and the light chain of SEQ ID NO:85;

[0133] c) the heavy chain of SEQ ID NO:86 and the light chain of SEQ ID NO:87;

[0134] d) the heavy chain of SEQ ID NO:88 and the light chain of SEQ ID NO:89;

[0135] e) the heavy chain of SEQ ID NO:90 and the light chain of SEQ ID NO:91;

[0136] f) the heavy chain of SEQ ID NO:92 and the light chain of SEQ ID NO:93;

[0137] g) the heavy chain of SEQ ID NO:94 and the light chain of SEQ ID NO:95;

[0138] including fragments/variants thereof. In some embodiments, the p97-antibody fusion is a homodimer that comprises two sets of a), two sets of b), two sets of c), two sets of d), two sets of e), two sets of f), or two sets of g). In particular embodiments, the p97-antibody fusion is a heterodimer that comprises a first set of sets of heavy and light chains selected from a)-g) above, and a second set of heavy and light chains composed of any combination of the p97-trastuzumab heavy or light chains described herein (for example, a)-g) above; SEQ ID NOS:37-46, 96-109, and 110-121), and/or any of the trastuzumab (non-fusion) heavy and light chains (for example, SEQ ID NOs: 29-35 or 122 (heavy chains) and 36 or 123 (light chains)).

[0139] Non-limiting examples of p97-antibody fusions are illustrated in FIGS. 1A-1G. FIG. 1A illustrates an antibody fusion composed of two p97-trastuzumab heavy chain fusion proteins, and two (non-fusion) trastuzumab light chains. FIG. 1B illustrates an antibody fusion composed of one p97-trastuzumab heavy chain fusion protein, one (non-fusion) trastuzumab heavy chain, and two (non-fusion) trastuzumab light chains. FIG. 1C illustrates an antibody fusion protein composed of one p97-trastuzumab heavy chain fusion protein (having a truncated trastuzumab heavy chain), one (non-fusion) trastuzumab light chain, and one (non-fusion) trastuzumab heavy chain. FIGS. 1D and 1E illustrate antibody fusions composed of two p97-trastuzumab heavy chain fusion proteins and two (non-fusion) trastuzumab light chains. FIG. 1F illustrates an antibody fusion composed of two p97-trastuzumab light chain fusion proteins and two 97-trastuzumab heavy chain fusion proteins. FIG. 1G illustrates an antibody fusion composed of two p97-trastuzumab light chain fusion proteins and two (non-fusion) trastuzumab heavy chain sequences. In any of the antibody fusions described herein, the first set of light/heavy chains can be the same as or different from the second set of light/heavy chains.

[0140] In some embodiments, the p97-antibody fusion is a homodimer, for example, which is composed of two identical sets of heavy and/or light chains, at least one of which is a p97-trastuzumab fusion protein. In some embodiments, the p97-antibody fusion is a heterodimer, for example, which is composed of a first set of heavy and/or light chains and a second set of heavy/light chains, where the first set comprises at least one heavy and/or light chain p97-trastuzumab fusion protein and the second set comprises only trastuzumab heavy and/or light chain sequences.

[0141] Other combinations will be apparent to persons skilled in the art. Thus, any of the p97 sequences described herein can be combined with any of the trastuzumab sequences described herein, to generate a desired p97-trastuzumab light chain or heavy chain fusion protein, and any such fusion proteins can be combined with the same or different fusion protein(s) or with any of the trastuzumab heavy chain or light chain sequences to generate a desired antibody fusion.

[0142] In certain embodiments, the p97-antibody fusion protein specifically binds to the human HER2/neu receptor. In specific embodiments, the p97-antibody fusion protein specifically binds to domain IV of the extracellular segment of the human HER2/neu receptor (see Cho et al., Nature. 421:756-760, 2003). In particular embodiments, the p97-antibody fusion protein is an HER2/neu receptor antagonist.

[0143] The functional properties of the fusion proteins and antibody fusions described herein may be assessed using a variety of methods known to the skilled person, including, e.g., affinity/binding assays (for example, surface plasmon resonance, competitive inhibition assays); cytotoxicity assays, cell viability assays, cell proliferation or differentiation assays, cancer cell and/or tumor growth inhibition using in vitro or in vivo models. For instance, the fusion proteins described herein may be tested for effects on receptor internalization, in vitro and in vivo efficacy, etc., including the rate of transport across the blood-brain barrier. Such assays may be performed using well-established protocols known to the skilled person (see e.g., Current Protocols in Molecular Biology (Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., NY, N.Y.); Current Protocols in Immunology (Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober 2001 John Wiley & Sons, NY, N.Y.); or commercially available kits.

[0144] Variant Sequences.

[0145] Certain embodiments include variants of the reference polypeptide and polynucleotide sequences described herein, whether described by name or by reference to a sequence identifier, including p97 sequences and trastuzumab sequences (see, e.g., the Sequence Listing). The wild-type or most prevalent sequences of these polypeptides are known in the art, and can be used as a comparison for the variants and fragments described herein.

[0146] A "variant" sequence, as the term is used herein, refers to a polypeptide or polynucleotide sequence that differs from a reference sequence disclosed herein by one or more substitutions, deletions (e.g., truncations), additions, and/or insertions. Certain variants thus include fragments of a reference sequence described herein. Variant polypeptides are biologically active, that is, they continue to possess the enzymatic or binding activity of a reference polypeptide. Such variants may result from, for example, genetic polymorphism and/or from human manipulation.

[0147] In many instances, a biologically active variant will contain one or more conservative substitutions. A "conservative substitution" is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. As described above, modifications may be made in the structure of the polynucleotides and polypeptides of the present invention and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics. When it is desired to alter the amino acid sequence of a polypeptide to create an equivalent, or even an improved, variant or portion of a polypeptide of the invention, one skilled in the art will typically change one or more of the codons of the encoding DNA sequence according to Table A below.

TABLE-US-00005 TABLE A Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

[0148] For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their utility.

[0149] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte & Doolittle, 1982, incorporated herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte & Doolittle, 1982). These values are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within .+-.2 is preferred, those within .+-.1 are particularly preferred, and those within .+-.0.5 are even more particularly preferred.

[0150] It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101 (specifically incorporated herein by reference in its entirety), states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within .+-.2 is preferred, those within .+-.1 are particularly preferred, and those within .+-.0.5 are even more particularly preferred.

[0151] As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0152] Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.

[0153] A variant may also, or alternatively, contain non-conservative changes. In a preferred embodiment, variant polypeptides differ from a native or reference sequence by substitution, deletion or addition of fewer than about 10, 9, 8, 7, 6, 5, 4, 3, 2 amino acids, or even 1 amino acid. Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure, enzymatic activity, and/or hydropathic nature of the polypeptide.

[0154] In certain embodiments, a polypeptide sequence is about, at least about, or up to about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700. 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800. 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 or more contiguous amino acids in length, including all integers in between, and which may comprise all or a portion of a reference sequence (see, e.g., Sequence Listing).

[0155] In other specific embodiments, a polypeptide sequence consists of about or no more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800. 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 or more contiguous amino acids, including all integers in between, and which may comprise all or a portion of a reference sequence (see, e.g., Sequence Listing).

[0156] In still other specific embodiments, a polypeptide sequence is about 10-1000, 10-900, 10-800, 10-700, 10-600, 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 10-40, 10-30, 10-20, 20-1000, 20-900, 20-800, 20-700, 20-600, 20-500, 20-400, 20-300, 20-200, 20-100, 20-50, 20-40, 20-30, 50-1000, 50-900, 50-800, 50-700, 50-600, 50-500, 50-400, 50-300, 50-200, 50-100, 100-1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200, 200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400, or 200-300 contiguous amino acids, including all ranges in between, and comprises all or a portion of a reference sequence. In certain embodiments, the C-terminal or N-terminal region of any reference polypeptide may be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 or more amino acids, or by about 10-50, 20-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800 or more amino acids, including all integers and ranges in between (e.g., 101, 102, 103, 104, 105), so long as the truncated polypeptide retains the binding properties and/or activity of the reference polypeptide. Typically, the biologically-active fragment has no less than about 1%, about 5%, about 10%, about 25%, or about 50% of an activity of the biologically-active reference polypeptide from which it is derived.

[0157] In general, variants will display at least about 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity or sequence identity or sequence homology to a reference polypeptide sequence. Moreover, sequences differing from the native or parent sequences by the addition (e.g., C-terminal addition, N-terminal addition, both), deletion, truncation, insertion, or substitution (e.g., conservative substitution) of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids (including all integers and ranges in between) but which retain the properties or activities of a parent or reference polypeptide sequence are contemplated.

[0158] In some embodiments, variant polypeptides differ from reference sequence by at least one but by less than 50, 40, 30, 20, 15, 10, 8, 6, 5, 4, 3 or 2 amino acid residue(s). In other embodiments, variant polypeptides differ from a reference sequence by at least 1% but less than 20%, 15%, 10% or 5% of the residues. (If this comparison requires alignment, the sequences should be aligned for maximum similarity. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.)

[0159] Calculations of sequence similarity or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In certain embodiments, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.

[0160] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0161] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch, (J. Mol. Biol. 48: 444-453, 1970) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used unless otherwise specified) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0162] The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller (Cabios. 4:11-17, 1989) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0163] The nucleic acid and protein sequences described herein can be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al., (1990, J. Mol. Biol, 215: 403-10). BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (Nucleic Acids Res. 25: 3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0164] In one embodiment, as noted above, polynucleotides and/or polypeptides can be evaluated using a BLAST alignment tool. A local alignment consists simply of a pair of sequence segments, one from each of the sequences being compared. A modification of Smith-Waterman or Sellers algorithms will find all segment pairs whose scores cannot be improved by extension or trimming, called high-scoring segment pairs (HSPs). The results of the BLAST alignments include statistical measures to indicate the likelihood that the BLAST score can be expected from chance alone.

[0165] The raw score, S, is calculated from the number of gaps and substitutions associated with each aligned sequence wherein higher similarity scores indicate a more significant alignment. Substitution scores are given by a look-up table (see PAM, BLOSUM).

[0166] Gap scores are typically calculated as the sum of G, the gap opening penalty and L, the gap extension penalty. For a gap of length n, the gap cost would be G+Ln. The choice of gap costs, G and L is empirical, but it is customary to choose a high value for G (10-15), e.g., 11, and a low value for L (1-2) e.g., 1.

[0167] The bit score, S', is derived from the raw alignment score S in which the statistical properties of the scoring system used have been taken into account. Bit scores are normalized with respect to the scoring system, therefore they can be used to compare alignment scores from different searches. The terms "bit score" and "similarity score" are used interchangeably. The bit score gives an indication of how good the alignment is; the higher the score, the better the alignment.

[0168] The E-Value, or expected value, describes the likelihood that a sequence with a similar score will occur in the database by chance. It is a prediction of the number of different alignments with scores equivalent to or better than S that are expected to occur in a database search by chance. The smaller the E-Value, the more significant the alignment. For example, an alignment having an E value of e.sup.-117 means that a sequence with a similar score is very unlikely to occur simply by chance. Additionally, the expected score for aligning a random pair of amino acids is required to be negative, otherwise long alignments would tend to have high score independently of whether the segments aligned were related. Additionally, the BLAST algorithm uses an appropriate substitution matrix, nucleotide or amino acid and for gapped alignments uses gap creation and extension penalties. For example, BLAST alignment and comparison of polypeptide sequences are typically done using the BLOSUM62 matrix, a gap existence penalty of 11 and a gap extension penalty of 1.

[0169] In one embodiment, sequence similarity scores are reported from BLAST analyses done using the BLOSUM62 matrix, a gap existence penalty of 11 and a gap extension penalty of 1.

[0170] In a particular embodiment, sequence identity/similarity scores provided herein refer to the value obtained using GAP Version 10 (GCG, Accelrys, San Diego, Calif.) using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix (Henikoff and Henikoff, PNAS USA. 89:10915-10919, 1992). GAP uses the algorithm of Needleman and Wunsch (J Mol Biol. 48:443-453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps.

[0171] In one particular embodiment, the variant polypeptide comprises an amino acid sequence that can be optimally aligned with a reference polypeptide sequence (see, e.g., Sequence Listing) to generate a BLAST bit scores or sequence similarity scores of at least about 50, 60, 70, 80, 90, 100, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, or more, including all integers and ranges in between, wherein the BLAST alignment used the BLOSUM62 matrix, a gap existence penalty of 11, and a gap extension penalty of 1.

[0172] As noted above, a reference polypeptide may be altered in various ways including amino acid substitutions, deletions, truncations, additions, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (PNAS USA. 82: 488-492, 1985); Kunkel et al., (Methods in Enzymol. 154: 367-382, 1987), U.S. Pat. No. 4,873,192, Watson, J. D. et al., ("Molecular Biology of the Gene," Fourth Edition, Benjamin/Cummings, Menlo Park, Calif., 1987) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.).

[0173] Methods for screening gene products of combinatorial libraries made by such modifications, and for screening cDNA libraries for gene products having a selected property are known in the art. Such methods are adaptable for rapid screening of the gene libraries generated by combinatorial mutagenesis of reference polypeptides. As one example, recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify polypeptide variants (Arkin and Yourvan, PNAS USA 89: 7811-7815, 1992; Delgrave et al., Protein Engineering. 6: 327-331, 1993).

[0174] Polynucleotides, Host Cells, and Methods of Production.

[0175] Certain embodiments relate to polynucleotides that encode the fusion proteins and antibody fusions described herein, and vectors that comprise such polynucleotides, for example, where the polynucleotides are operably linked to one or more regulatory elements. Also included are recombinant host cells that comprise such polynucleotides, vectors, fusion proteins, and antibody fusions, and methods of recombinant production of the foregoing.

[0176] Fusion proteins and antibody fusions may be prepared using standard techniques.

[0177] Preferably, however, a fusion protein is expressed as a recombinant protein in an expression system, as described herein and known in the art. Fusion proteins can contain one or multiple copies of a p97 sequence and one or multiple copies of a trastuzumab sequence, present in any desired arrangement.

[0178] Polynucleotides and fusion polynucleotides can contain one or multiple copies of a nucleic acid encoding a p97 polypeptide sequence, and/or may contain one or multiple copies of a nucleic acid encoding a trastuzumab sequence.

[0179] For fusion proteins, DNA sequences encoding the p97 polypeptide sequence, the trastuzumab sequence of interest, and optionally a peptide linker components may be assembled separately, and then ligated into an appropriate expression vector. The 3' end of the DNA sequence encoding one polypeptide component can be ligated, with or without a peptide linker, to the 5' end of a DNA sequence encoding the other polypeptide component(s) so that the reading frames of the sequences are in frame. The ligated DNA sequences are operably linked to suitable transcriptional and/or translational regulatory elements. The regulatory elements responsible for expression of DNA are usually located only 5' to the DNA sequence encoding the first polypeptides. Similarly, stop codons required to end translation and transcription termination signals are only present 3' to the DNA sequence encoding the most C-terminal polypeptide. This permits translation into a single fusion polypeptide that retains the biological activity of both component polypeptides.

[0180] Similar techniques, mainly the arrangement of regulatory elements such as promoters, stop codons, and transcription termination signals, can be applied to the recombinant production of non-fusion proteins, for instance, non-fusion trastuzumab sequences for the production of antibodies that comprise a fusion protein described herein.

[0181] Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992, or subsequent updates thereto.

[0182] As will be understood by those of skill in the art, it may be advantageous in some instances to produce polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce a recombinant RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence. Such polynucleotides are commonly referred to as "codon-optimized." Any of the polynucleotides described herein may be utilized in a codon-optimized form. In certain embodiments, a polynucleotide can be codon optimized for use in specific bacteria such as E. coli or yeast such as S. cerevisiae (see, e.g., Burgess-Brown et al., Protein Expr Purif. 59:94-102, 2008).

[0183] Exemplary polynucleotide sequences are provided in Table 5 below.

TABLE-US-00006 TABLE 5 Exemplary polynucleotide sequences SEQ ID Name Sequence NO: TZM HC- Heavy Chain 125 MTf ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCCACC GGAGAGGTGCAGCTGGTGGAGAGCGGCGGAGGCCTCGTGCAGCCCGGCGGATCTCTG CGGCTGAGCTGCGCCGCTAGCGGCTTCAACATCAAGGACACCTACATCCACTGGGTG CGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCCGGATCTACCCCACCAACGGC TACACCCGCTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCGCCGACACCTCC AAGAACACCGCCTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTAC TACTGCAGCCGGTGGGGCGGCGACGGATTCTACGCCATGGACTACTGGGGACAGGGC ACCCTGGTGACCGTGAGCAGCGCCTCTACCAAGGGCCCCAGCGTGTTCCCTCTGGCC CCCAGCAGCAAGAGCACCAGCGGCGGAACCGCCGCCCTGGGCTGCCTGGTGAAGGAC TACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGAGTG CACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTG ACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAG CCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCGACAAGACCCAC ACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTC CCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTG GTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGC GTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTAC CGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTAC AAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAG GCCAAGGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAG CTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGAC ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCT CCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAG TCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCAC AACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGAAAGGGTGGCGGAGGATCT GGCGGAGGCGGATCCGGCATGGAAGTGCGTTGGTGCGCCACCTCTGACCCCGAGCAG CACAAGTGCGGCAACATGTCCGAGGCCTTCAGAGAGGCCGGCATCCAGCCTTCTCTG CTGTGTGTGCGGGGCACCTCTGCCGACCATTGCGTGCAGCTGATCGCCGCCCAGGAA GCCGACGCTATCACACTGGATGGCGGCGCTATCTACGAGGCTGGCAAAGAGCACGGC CTGAAGCCCGTCGTGGGCGAGGTGTACGATCAGGAAGTGGGCACCTCCTACTACGCC GTGGCTGTCGTGCGGAGATCCTCCCACGTGACCATCGACACCCTGAAGGGCGTGAAG TCCTGCCACACCGGCATCAACAGAACCGTGGGCTGGAACGTGCCCGTGGGCTACCTG GTGGAATCCGGCAGACTGTCCGTGATGGGCTGCGACGTGCTGAAGGCCGTGTCCGAT TACTTCGGCGGCTCTTGTGTGCCTGGCGCTGGCGAGACATCCTACTCCGAGTCCCTG TGCAGACTGTGCAGGGGCGACTCTTCTGGCGAGGGCGTGTGCGACAAGTCCCCTCTG GAACGGTACTACGACTACTCCGGCGCCTTCAGATGCCTGGCTGAAGGTGCTGGCGAC GTGGCCTTCGTGAAGCACTCCACCGTGCTGGAAAACACCGACGGCAAGACCCTGCCT TCTTGGGGCCAGGCACTGCTGTCCCAGGACTTCGAGCTGCTGTGCCGGGATGGCTCC AGAGCCGATGTGACAGAGTGGCGGCAGTGCCACCTGGCCAGAGTGCCTGCCCATGCT GTGGTCGTGCGCGCCGATACAGATGGCGGCCTGATCTTCCGGCTGCTGAACGAGGGC CAGCGGCTGTTCTCTCACGAGGGCTCCAGCTTCCAGATGTTCTCCAGCGAGGCCTAC GGCCAGAAGGACCTGCTGTTCAAGGACTCCACCTCCGAGCTGGTGCCTATCGCCACC CAGACCTATGAGGCTTGGCTGGGCCACGAGTACCTGCACGCTATGAAGGGACTGCTG TGCGACCCCAACCGGCTGCCTCCTTATCTGAGGTGGTGCGTGCTGTCCACCCCCGAG ATCCAGAAATGCGGCGATATGGCCGTGGCCTTTCGGCGGCAGAGACTGAAGCCTGAG ATCCAGTGCGTGTCTGCCAAGAGCCCTCAGCACTGCATGGAACGGATCCAGGCCGAA CAGGTGGACGCCGTGACACTGTCCGGCGAGGATATCTACACCGCCGGAAAGACCTAC GGCCTGGTGCCAGCTGCTGGCGAGCATTACGCCCCTGAGGACTCCTCCAACAGCTAC TACGTGGTGGCAGTCGTGCGCCGGGACTCCTCTCACGCCTTTACCCTGGATGAGCTG CGGGGCAAGAGAAGCTGTCACGCCGGCTTTGGAAGCCCTGCCGGATGGGATGTGCCT GTGGGCGCTCTGATCCAGCGGGGCTTCATCAGACCCAAGGACTGTGATGTGCTGACC GCCGTGTCTGAGTTCTTCAACGCCTCCTGTGTGCCCGTGAACAACCCCAAGAACTAC CCCTCCAGCCTGTGCGCCCTGTGTGTGGGAGATGAGCAGGGCCGGAACAAATGCGTG GGCAACTCCCAGGAAAGATATTACGGCTACAGAGGCGCCTTCCGGTGTCTGGTGGAA AACGCCGGGGATGTGGCTTTTGTGCGGCACACCACCGTGTTCGACAACACCAATGGC CACAACTCCGAGCCTTGGGCCGCTGAGCTGAGATCCGAGGATTACGAACTGCTGTGT CCCAACGGCGCCAGGGCTGAGGTGTCCCAGTTTGCCGCCTGTAACCTGGCCCAGATC CCTCCCCACGCTGTGATGGTGCGACCCGACACCAACATCTTCACCGTGTACGGCCTG CTGGACAAGGCCCAGGATCTGTTCGGCGACGACCACAACAAGAACGGGTTCAAGATG TTCGACTCCAGCAACTACCACGGACAGGATCTGCTGTTTAAAGATGCCACCGTGCGG GCCGTGCCAGTGGGCGAAAAGACCACCTACAGAGGATGGCTGGGACTGGACTACGTG GCCGCCCTGGAAGGCATGTCCTCCCAGCAGTGTTCCGGCTAG Light Chain 126 ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCCACC GGAGACATCCAGATGACCCAGAGCCCTTCCAGCCTGAGCGCCAGCGTGGGCGACCGG GTGACCATCACCTGCCGCGCTAGCCAGGACGTGAACACCGCCGTGGCCTGGTACCAG CAGAAGCCCGGAAAGGCCCCCAAGCTGCTGATCTACTCTGCTAGCTTCCTGTACAGC GGCGTGCCCAGCCGGTTCAGCGGATCTCGCAGCGGCACCGACTTCACCCTGACCATC AGCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACG CCTCCCACCTTCGGACAGGGCACCAAGGTGGAGATCAAGCGGACCGTGGCCGCCCCC AGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTG GTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGAC AACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGAC AGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCAC AAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGC TTCAACCGGGGCGAGTGCTAA MTfp Heavy Chain 127 NH-TZM ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCCACC GGAGACTCCTCTCACGCCTTCACCCTGGACGAGCTGCGGTACGGTGGCGGAGGATCT GGCGGAGGCGGATCCGAGGTGCAGCTGGTGGAGAGCGGCGGAGGCCTCGTGCAGCCC GGCGGATCTCTGCGGCTGAGCTGCGCCGCTAGCGGCTTCAACATCAAGGACACCTAC ATCCACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCCGGATCTAC CCCACCAACGGCTACACCCGCTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGC GCCGACACCTCCAAGAACACCGCCTACCTGCAGATGAACAGCCTGCGCGCCGAGGAC ACCGCCGTGTACTACTGCAGCCGGTGGGGCGGCGACGGATTCTACGCCATGGACTAC TGGGGACAGGGCACCCTGGTGACCGTGAGCAGCGCCTCTACCAAGGGCCCCAGCGTG TTCCCTCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGAACCGCCGCCCTGGGCTGC CTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTG ACCAGCGGAGTGCACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTG AGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAAC GTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGC GACAAGACCCACACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTGGGCGGACCCAGC GTGTTCCTGTTCCCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAG GTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGG TACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTAC AACTCCACCTACCGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAAC GGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAG ACCATCAGCAAGGCCAAGGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCC AGCCGCGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTC TACCCCTCCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTAC AAGACCACCCCTCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTG ACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCAC GAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGAAAGTAA Light Chain 128 ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCCACC GGAGACATCCAGATGACCCAGAGCCCTTCCAGCCTGAGCGCCAGCGTGGGCGACCGG GTGACCATCACCTGCCGCGCTAGCCAGGACGTGAACACCGCCGTGGCCTGGTACCAG CAGAAGCCCGGAAAGGCCCCCAAGCTGCTGATCTACTCTGCTAGCTTCCTGTACAGC GGCGTGCCCAGCCGGTTCAGCGGATCTCGCAGCGGCACCGACTTCACCCTGACCATC AGCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACG CCTCCCACCTTCGGACAGGGCACCAAGGTGGAGATCAAGCGGACCGTGGCCGCCCCC AGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTG GTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGAC AACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGAC AGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCAC AAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGC TTCAACCGGGGCGAGTGCTAA TZM HC- Heavy Chain 129 MTfp ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCCACC GGAGAGGTGCAGCTGGTGGAGAGCGGCGGAGGCCTCGTGCAGCCCGGCGGATCTCTG CGGCTGAGCTGCGCCGCTAGCGGCTTCAACATCAAGGACACCTACATCCACTGGGTG CGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCCGGATCTACCCCACCAACGGC TACACCCGCTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCGCCGACACCTCC AAGAACACCGCCTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTAC TACTGCAGCCGGTGGGGCGGCGACGGATTCTACGCCATGGACTACTGGGGACAGGGC ACCCTGGTGACCGTGAGCAGCGCCTCTACCAAGGGCCCCAGCGTGTTCCCTCTGGCC CCCAGCAGCAAGAGCACCAGCGGCGGAACCGCCGCCCTGGGCTGCCTGGTGAAGGAC TACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGAGTG CACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTG ACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAG CCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCGACAAGACCCAC ACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTC CCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTG GTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGC GTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTAC CGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTAC AAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAG GCCAAGGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAG CTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGAC ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCT CCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAG TCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCAC AACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGAAAGGGTGGCGGAGGATCT GGCGGAGGCGGATCCGACTCCTCTCACGCCTTCACCCTGGACGAGCTGCGGTAG Light Chain 130 ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCCACC GGAGACATCCAGATGACCCAGAGCCCTTCCAGCCTGAGCGCCAGCGTGGGCGACCGG GTGACCATCACCTGCCGCGCTAGCCAGGACGTGAACACCGCCGTGGCCTGGTACCAG CAGAAGCCCGGAAAGGCCCCCAAGCTGCTGATCTACTCTGCTAGCTTCCTGTACAGC GGCGTGCCCAGCCGGTTCAGCGGATCTCGCAGCGGCACCGACTTCACCCTGACCATC AGCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACG CCTCCCACCTTCGGACAGGGCACCAAGGTGGAGATCAAGCGGACCGTGGCCGCCCCC AGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTG GTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGAC AACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGAC AGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCAC AAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGC TTCAACCGGGGCGAGTGCTAA TZM/MTf Heavy Chain 131 ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCCACC GGAGAGGTGCAGCTGGTGGAGAGCGGCGGAGGCCTCGTGCAGCCCGGCGGATCTCTG CGGCTGAGCTGCGCCGCTAGCGGCTTCAACATCAAGGACACCTACATCCACTGGGTG CGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCCGGATCTACCCCACCAACGGC TACACCCGCTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCGCCGACACCTCC AAGAACACCGCCTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTAC TACTGCAGCCGGTGGGGCGGCGACGGATTCTACGCCATGGACTACTGGGGACAGGGC ACCCTGGTGACCGTGAGCAGCGCCTCTACCAAGGGCCCCAGCGTGTTCCCTCTGGCC CCCAGCAGCAAGAGCACCAGCGGCGGAACCGCCGCCCTGGGCTGCCTGGTGAAGGAC TACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGAGTG CACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTG ACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAG CCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCGACAAGACCCAC ACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTC CCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTG GTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGC GTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTAC CGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTAC AAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAG GCCAAGGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAG CTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGAC ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCT CCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAG TCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCAC AACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGAAAGGGAGGTGGCGGTTCT GGTGGCGGAGGATCTGGCGGAGGCGGATCCGGCATGGAAGTGCGTTGGTGCGCCACC TCTGACCCCGAGCAGCACAAGTGCGGCAACATGTCCGAGGCCTTCAGAGAGGCCGGC ATCCAGCCTTCTCTGCTGTGTGTGCGGGGCACCTCTGCCGACCATTGCGTGCAGCTG ATCGCCGCCCAGGAAGCCGACGCTATCACACTGGATGGCGGCGCTATCTACGAGGCT GGCAAAGAGCACGGCCTGAAGCCCGTCGTGGGCGAGGTGTACGATCAGGAAGTGGGC ACCTCCTACTACGCCGTGGCTGTCGTGCGGAGATCCTCCCACGTGACCATCGACACC CTGAAGGGCGTGAAGTCCTGCCACACCGGCATCAACAGAACCGTGGGCTGGAACGTG CCCGTGGGCTACCTGGTGGAATCCGGCAGACTGTCCGTGATGGGCTGCGACGTGCTG AAGGCCGTGTCCGATTACTTCGGCGGCTCTTGTGTGCCTGGCGCTGGCGAGACATCC TACTCCGAGTCCCTGTGCAGACTGTGCAGGGGCGACTCTTCTGGCGAGGGCGTGTGC GACAAGTCCCCTCTGGAACGGTACTACGACTACTCCGGCGCCTTCAGATGCCTGGCT GAAGGTGCTGGCGACGTGGCCTTCGTGAAGCACTCCACCGTGCTGGAAAACACCGAC GGCAAGACCCTGCCTTCTTGGGGCCAGGCACTGCTGTCCCAGGACTTCGAGCTGCTG TGCCGGGATGGCTCCAGAGCCGATGTGACAGAGTGGCGGCAGTGCCACCTGGCCAGA GTGCCTGCTCATGCTGTGGTCGTGCGCGCCGATACAGATGGCGGCCTGATCTTCCGG CTGCTGAACGAGGGCCAGCGGCTGTTCTCTCACGAGGGCTCCAGCTTCCAGATGTTC TCCAGCGAGGCCTACGGCCAGAAGGACCTGCTGTTCAAGGACTCCACCTCCGAGCTG GTGCCTATCGCCACCCAGACCTATGAGGCTTGGCTGGGCCACGAGTACCTGCACGCT ATGAAGGGACTGCTGTGCGACCCCAACCGGCTGCCTCCTTATCTGAGGTGGTGCGTG CTGTCCACCCCCGAGATCCAGAAATGCGGCGATATGGCCGTGGCCTTTCGGCGGCAG AGACTGAAGCCTGAGATCCAGTGCGTGTCCGCCAAGAGCCCTCAGCACTGCATGGAA CGGATCCAGGCCGAACAGGTGGACGCCGTGACACTGTCCGGCGAGGATATCTACACC GCCGGAAAGACCTACGGCCTGGTGCCAGCTGCTGGCGAGCATTACGCCCCTGAGGAC TCCTCCAACAGCTACTACGTGGTGGCAGTCGTGCGCCGGGACTCCTCTCACGCCTTT ACCCTGGATGAGCTGCGGGGCAAGAGAAGCTGTCACGCCGGCTTTGGAAGCCCTGCC GGATGGGATGTGCCTGTGGGCGCTCTGATCCAGCGGGGCTTCATCAGACCCAAGGAC TGTGATGTGCTGACCGCCGTGTCTGAGTTCTTCAACGCCTCCTGTGTGCCCGTGAAC AACCCCAAGAACTACCCCTCCAGCCTGTGCGCCCTGTGTGTGGGAGATGAGCAGGGC CGGAACAAATGCGTGGGCAACTCCCAGGAAAGATATTACGGCTACAGAGGCGCCTTC CGGTGTCTGGTGGAAAACGCCGGGGATGTGGCTTTTGTGCGGCACACCACCGTGTTC GACAACACCAATGGCCACAACTCCGAGCCTTGGGCCGCTGAGCTGAGATCCGAGGAT TACGAACTGCTGTGTCCCAACGGCGCCAGGGCTGAGGTGTCCCAGTTTGCCGCCTGT AACCTGGCCCAGATCCCTCCCCACGCTGTGATGGTGCGACCCGACACCAACATCTTC ACCGTGTACGGCCTGCTGGACAAGGCCCAGGATCTGTTCGGCGACGACCACAACAAG AACGGGTTCAAGATGTTCGACTCCAGCAACTACCACGGACAGGATCTGCTGTTTAAA GATGCCACCGTGCGGGCCGTGCCAGTGGGCGAAAAGACCACCTACAGAGGATGGCTG GGACTGGACTACGTGGCCGCCCTGGAAGGCATGTCCTCCCAGCAGTGTTCCGGCTAG Light Chain 132 ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCCACC GGAGACATCCAGATGACCCAGAGCCCTTCCAGCCTGAGCGCCAGCGTGGGCGACCGG GTGACCATCACCTGCCGCGCTAGCCAGGACGTGAACACCGCCGTGGCCTGGTACCAG CAGAAGCCCGGAAAGGCCCCCAAGCTGCTGATCTACTCTGCTAGCTTCCTGTACAGC GGCGTGCCCAGCCGGTTCAGCGGATCTCGCAGCGGCACCGACTTCACCCTGACCATC AGCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACG CCTCCCACCTTCGGACAGGGCACCAAGGTGGAGATCAAGCGGACCGTGGCCGCCCCC AGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTG GTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGAC AACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGAC AGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCAC AAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGC TTCAACCGGGGCGAGTGCTAA LC- Heavy Chain 133 MTfp/Fc- ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCCACC MTfp GGAGAGGTGCAGCTGGTGGAGAGCGGCGGAGGCCTCGTGCAGCCCGGCGGATCTCTG CGGCTGAGCTGCGCCGCTAGCGGCTTCAACATCAAGGACACCTACATCCACTGGGTG

CGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCCGGATCTACCCCACCAACGGC TACACCCGCTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCGCCGACACCTCC AAGAACACCGCCTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTAC TACTGCAGCCGGTGGGGCGGCGACGGATTCTACGCCATGGACTACTGGGGACAGGGC ACCCTGGTGACCGTGAGCAGCGCCTCTACCAAGGGCCCCAGCGTGTTCCCTCTGGCC CCCAGCAGCAAGAGCACCAGCGGCGGAACCGCCGCCCTGGGCTGCCTGGTGAAGGAC TACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGAGTG CACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTG ACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAG CCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCGACAAGACCCAC ACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTC CCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTG GTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGC GTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTAC CGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTAC AAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAG GCCAAGGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAG CTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGAC ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCT CCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAG TCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCAC AACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGAAAGGAGGCCGCTGCTAAA GAGGCTGCCGCCAAAGAAGCCGCCGCTAAGGACTCCTCTCACGCCTTCACCCTGGAC GAGCTGCGGTACTAA Light Chain 134 ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCCACC GGAGACATCCAGATGACCCAGAGCCCTTCCAGCCTGAGCGCCAGCGTGGGCGACCGG GTGACCATCACCTGCCGCGCTAGCCAGGACGTGAACACCGCCGTGGCCTGGTACCAG CAGAAGCCCGGAAAGGCCCCCAAGCTGCTGATCTACTCTGCTAGCTTCCTGTACAGC GGCGTGCCCAGCCGGTTCAGCGGATCTCGCAGCGGCACCGACTTCACCCTGACCATC AGCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACG CCTCCCACCTTCGGACAGGGCACCAAGGTAGAGATCAAGCGGACCGTGGCCGCCCCC AGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTG GTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGAC AACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGAC AGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCAC AAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGC TTCAACCGGGGCGAGTGCGAGGCCGCTGCTAAAGAGGCTGCCGCCAAAGAAGCCGCC GCTAAGGACTCCTCTCACGCCTTCACCCTGGACGAGCTGCGGTACTAA LC/Fc- Heavy Chain 135 MTfp ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCCACC GGAGAGGTGCAGCTGGTGGAGAGCGGCGGAGGCCTCGTGCAGCCCGGCGGATCTCTG CGGCTGAGCTGCGCCGCTAGCGGCTTCAACATCAAGGACACCTACATCCACTGGGTG CGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCCGGATCTACCCCACCAACGGC TACACCCGCTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCGCCGACACCTCC AAGAACACCGCCTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTAC TACTGCAGCCGGTGGGGCGGCGACGGATTCTACGCCATGGACTACTGGGGACAGGGC ACCCTGGTGACCGTGAGCAGCGCCTCTACCAAGGGCCCCAGCGTGTTCCCTCTGGCC CCCAGCAGCAAGAGCACCAGCGGCGGAACCGCCGCCCTGGGCTGCCTGGTGAAGGAC TACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGAGTG CACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTG ACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAG CCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCGACAAGACCCAC ACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTC CCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTG GTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGC GTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTAC CGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTAC AAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAG GCCAAGGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAG CTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGAC ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCT CCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAG TCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCAC AACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGAAAGGAGGCCGCTGCTAAA GAGGCTGCCGCCAAAGAAGCCGCCGCTAAGGACTCCTCTCACGCCTTCACCCTGGAC GAGCTGCGGTACTAA Light Chain 136 ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCCACC GGAGACATCCAGATGACCCAGAGCCCTTCCAGCCTGAGCGCCAGCGTGGGCGACCGG GTGACCATCACCTGCCGCGCTAGCCAGGACGTGAACACCGCCGTGGCCTGGTACCAG CAGAAGCCCGGAAAGGCCCCCAAGCTGCTGATCTACTCTGCTAGCTTCCTGTACAGC GGCGTGCCCAGCCGGTTCAGCGGATCTCGCAGCGGCACCGACTTCACCCTGACCATC AGCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACG CCTCCCACCTTCGGACAGGGCACCAAGGTGGAGATCAAGCGGACCGTGGCCGCCCCC AGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTG GTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGAC AACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGAC AGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCAC AAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGC TTCAACCGGGGCGAGTGCTAA LC- Heavy Chain 137 MTfp/Fc ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCCACC GGAGAGGTGCAGCTGGTGGAGAGCGGCGGAGGCCTCGTGCAGCCCGGCGGATCTCTG CGGCTGAGCTGCGCCGCTAGCGGCTTCAACATCAAGGACACCTACATCCACTGGGTG CGCCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCCGGATCTACCCCACCAACGGC TACACCCGCTACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCGCCGACACCTCC AAGAACACCGCCTACCTGCAGATGAACAGCCTGCGCGCCGAGGACACCGCCGTGTAC TACTGCAGCCGGTGGGGCGGCGACGGATTCTACGCCATGGACTACTGGGGACAGGGC ACCCTGGTGACCGTGAGCAGCGCCTCTACCAAGGGCCCCAGCGTGTTCCCTCTGGCC CCCAGCAGCAAGAGCACCAGCGGCGGAACCGCCGCCCTGGGCTGCCTGGTGAAGGAC TACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGAGTG CACACCTTCCCTGCCGTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTG ACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAG CCCTCCAACACCAAGGTGGACAAGAAGGTGGAGCCTAAGAGCTGCGACAAGACCCAC ACCTGCCCTCCCTGCCCCGCCCCCGAGCTGCTGGGCGGACCCAGCGTGTTCCTGTTC CCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTG GTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGC GTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTACAACTCCACCTAC CGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTAC AAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCTCCCATCGAGAAGACCATCAGCAAG GCCAAGGGCCAGCCCCGGGAGCCTCAGGTGTACACCCTGCCCCCCAGCCGCGACGAG CTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCTCCGAC ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCT CCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAG TCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCAC AACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGAAAGTAA Light Chain 138 ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCCACC GGAGACATCCAGATGACCCAGAGCCCTTCCAGCCTGAGCGCCAGCGTGGGCGACCGG GTGACCATCACCTGCCGCGCTAGCCAGGACGTGAACACCGCCGTGGCCTGGTACCAG CAGAAGCCCGGAAAGGCCCCCAAGCTGCTGATCTACTCTGCTAGCTTCCTGTACAGC GGCGTGCCCAGCCGGTTCAGCGGATCTCGCAGCGGCACCGACTTCACCCTGACCATC AGCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACG CCTCCCACCTTCGGACAGGGCACCAAGGTAGAGATCAAGCGGACCGTGGCCGCCCCC AGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTG GTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGAC AACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGAC AGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCAC AAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGC TTCAACCGGGGCGAGTGCGAGGCCGCTGCTAAAGAGGCTGCCGCCAAAGAAGCCGCC GCTAAGGACTCCTCTCACGCCTTCACCCTGGACGAGCTGCGGTACTAA

[0184] Thus, in certain embodiments, a polynucleotide that encodes a fusion protein or antibody fusion described herein, or a portion thereof, comprises one or more polynucleotide sequences from Table 5 (e.g., SEQ ID NOS:125-138), or a fragment/variant thereof.

[0185] In some embodiments, one or more nucleic acids or vectors encoding a subject p97 polypeptide, a trastuzumab polypeptide (e.g., light chain polypeptide, heavy chain polypeptide), and/or a p97-trastuzumab fusion protein are introduced directly into a host cell, and the cell is incubated under conditions sufficient to induce expression of the encoded polypeptide(s). Therefore, according to certain related embodiments, there is provided a recombinant host cell which comprises a polynucleotide or a fusion polynucleotide that encodes one or more fusion proteins described herein, optionally in combination with other (non-fusion) components of an antibody, and which optionally comprises additional heterologous polynucleotide sequences.

[0186] Expression of a fusion protein or antibody fusion in the host cell may be achieved by culturing the recombinant host cells (containing the polynucleotide(s)) under appropriate conditions. Following production by expression, the polypeptide(s), fusion proteins, and/or antibody fusions may be isolated and/or purified using any suitable technique, and then used as desired. The term "host cell" is used to refer to a cell into which has been introduced, or which is capable of having introduced into it, a nucleic acid sequence encoding one or more of the polypeptides described herein, and which further expresses or is capable of expressing a selected gene of interest, such as a gene encoding any herein described polypeptide. The term includes the progeny of the parent cell, whether or not the progeny are identical in morphology or in genetic make-up to the original parent, so long as the selected gene is present. Host cells may be chosen for certain characteristics, for instance, the expression of aminoacyl tRNA synthetase(s) that can incorporate unnatural amino acids into the polypeptide.

[0187] Systems for cloning and expression of a protein in a variety of different host cells are well known. Suitable host cells include mammalian cells, bacteria, yeast, and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, HEK-293 cells, NSO mouse melanoma cells and many others. Additional examples of useful mammalian host cell lines include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells sub-cloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., PNAS USA 77:4216 (1980)); and myeloma cell lines such as NSO and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 255-268. Certain preferred mammalian cell expression systems include CHO and HEK293-cell based expression systems. Mammalian expression systems can utilize attached cell lines, for example, in T-flasks, roller bottles, or cell factories, or suspension cultures, for example, in 1 L and 5 L spinners, 5 L, 14 L, 40 L, 100 L and 200 L stir tank bioreactors, or 20/50 L and 100/200 L WAVE bioreactors, among others known in the art.

[0188] A common, preferred bacterial host is E. coli. The expression of proteins in prokaryotic cells such as E. coli is well established in the art. For a review, see for example Pluckthun, A. Bio/Technology. 9:545-551 (1991). Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for recombinant production of polypeptides (see Ref, Curr. Opinion Biotech. 4:573-576, 1993; and Trill et al., Curr. Opinion Biotech. 6:553-560, 1995). In specific embodiments, protein expression may be controlled by a T7 RNA polymerase (e.g., pET vector series). These and related embodiments may utilize the expression host strain BL21(DE3), a .lamda.DE3 lysogen of BL21 that supports T7-mediated expression and is deficient in Ion and ompT proteases for improved target protein stability. Also included are expression host strains carrying plasmids encoding tRNAs rarely used in E. coli, such as Rosetta.TM. (DE3) and Rosetta 2 (DE3) strains. Cell lysis and sample handling may also be improved using reagents such as Benzonase.RTM. nuclease and BugBuster.RTM. Protein Extraction Reagent. For cell culture, auto-inducing media can improve the efficiency of many expression systems, including high-throughput expression systems. Media of this type (e.g., Overnight Express.TM. Autoinduction System) gradually elicit protein expression through metabolic shift without the addition of artificial inducing agents such as IPTG. Particular embodiments employ hexahistidine tags (such as His.cndot.Tag.RTM. fusions), followed by immobilized metal affinity chromatography (IMAC) purification, or related techniques. In certain aspects, however, clinical grade proteins can be isolated from E. coli inclusion bodies, without or without the use of affinity tags (see, e.g., Shimp et al., Protein Expr Purif. 50:58-67, 2006). As a further example, certain embodiments may employ a cold-shock induced E. coli high-yield production system, because over-expression of proteins in Escherichia coli at low temperature improves their solubility and stability (see, e.g., Qing et al., Nature Biotechnology. 22:877-882, 2004).

[0189] In addition, a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, post-translational modifications such as acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing, which cleaves a "prepro" form of the protein may also be used to facilitate correct insertion, folding and/or function. Different host cells such as yeast, CHO, HeLa, MDCK, HEK293, and W138, in addition to bacterial cells, which have or even lack specific cellular machinery and characteristic mechanisms for such post-translational activities, may be chosen to ensure the correct modification and processing of the fusion protein or antibody fusion of interest.

[0190] For long-term, high-yield production of recombinant proteins, stable expression is generally preferred. For example, cell lines that stably express a polynucleotide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which, successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type. Transient production, such as by transient transfection or infection, can also be employed. Exemplary mammalian expression systems that are suitable for transient production include HEK293 and CHO-based systems.

[0191] Host cells transformed with a polynucleotide sequence of interest may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. Certain specific embodiments utilize serum free cell expression systems. Examples include HEK293 cells and CHO cells that can grow on serum free medium (see, e.g., Rosser et al., Protein Expr. Purif. 40:237-43, 2005; and U.S. Pat. No. 6,210,922).

[0192] The protein(s) produced by a recombinant cell can be purified and characterized according to a variety of techniques known in the art. Exemplary systems for performing protein purification and analyzing protein purity include fast protein liquid chromatography (FPLC) (e.g., AKTA and Bio-Rad FPLC systems), high-pressure liquid chromatography (HPLC) (e.g., Beckman and Waters HPLC). Exemplary chemistries for purification include ion exchange chromatography (e.g., Q, S), size exclusion chromatography, salt gradients, affinity purification (e.g., Ni, Co, FLAG, maltose, glutathione, protein A/G), gel filtration, reverse-phase, ceramic HyperD.RTM. ion exchange chromatography, and hydrophobic interaction columns (HIC), among others known in the art. Also included are analytical methods such as SDS-PAGE (e.g., coomassie, silver stain), immunoblot, Bradford, and ELISA, which may be utilized during any step of the production or purification process, typically to measure the purity of the protein composition.

[0193] Also included are methods of concentrating recombinantly produced proteins, e.g., antibodies. Examples include lyophilization, which is typically employed when the solution contains few soluble components other than the protein of interest. Lyophilization is often performed after HPLC run, and can remove most or all volatile components from the mixture. Also included are ultrafiltration techniques, which typically employ one or more selective permeable membranes to concentrate a protein solution. The membrane allows water and small molecules to pass through and retains the protein; the solution can be forced against the membrane by mechanical pump, gas pressure, or centrifugation, among other techniques.

[0194] In certain embodiments, the fusion proteins or antibody fusion proteins have a purity of at least about 90%, as measured according to routine techniques in the art. In certain embodiments, such as diagnostic compositions or certain therapeutic compositions, the fusion proteins or antibody fusions have a purity of at least about 95%. In specific embodiments, such as therapeutic or pharmaceutical compositions, the fusion proteins or antibody fusions have a purity of at least about 97% or 98% or 99%. In other embodiments, such as when being used as reference or research reagents, fusion proteins or antibody fusions can be of lesser purity, and may have a purity of at least about 50%, 60%, 70%, or 80%. Purity can be measured overall or in relation to selected components, such as other proteins, e.g., purity on a protein basis.

[0195] In certain embodiments, as noted above, the compositions described here are about substantially endotoxin free, including, for example, about 95% endotoxin free, preferably about 99% endotoxin free, and more preferably about 99.99% endotoxin free. The presence of endotoxins can be detected according to routine techniques in the art, as described herein. In specific embodiments, the fusion proteins or antibody fusions are made from a eukaryotic cell such as a mammalian or human cell in substantially serum free media.

[0196] Methods of Use Pharmaceutical Compositions

[0197] Certain embodiments relate to methods of using the p97-trastuzumab fusion proteins and/or related antibody fusion proteins described herein. Examples of such methods include methods of treatment and methods of diagnosis, the latter including, for instance, the medical imaging of certain organs/tissues, such as those of the central nervous system. Specific embodiments include methods of treating and/or diagnosing disorders or conditions of the central nervous system (CNS), or disorders or conditions having a CNS component. Also included are pharmaceutical compositions comprising the p97-trastuzumab fusion proteins and/or related antibody fusion proteins described herein.

[0198] Accordingly, certain embodiments include methods of treating a subject in need thereof, comprising administering a p97-trastuzumab fusion protein or antibody fusion protein described herein. In particular embodiments, the methods comprise administering a p97-antibody fusion protein, which comprises one or more p97-trastuzumab fusion proteins described herein (e.g., as at least one component of the antibody or antibody-like molecule), and optionally other non-fusion antibody components (e.g., non-fusion light chain(s), non-fusion heavy chain(s)). Also included are methods of delivering such molecules to the nervous system (e.g., central nervous system tissues) of a subject, comprising administering to the subject a p97-trastuzumab fusion protein or antibody fusion described herein.

[0199] In some embodiments, the methods increase the rate and/or amount of delivery of the trastuzumab antibody (or antigen-binding fragment thereof) to the central nervous system tissues of the subject, relative, for example, to delivery by a composition that comprises the trastuzumab antibody (or antigen-binding fragment thereof) alone. In certain embodiments, the methods reduce the distribution of trastuzumab antibody (or antigen-binding fragment thereof) to heart tissues of the subject, relative, for example, to distribution by a composition that comprises the trastuzumab antibody (or antigen-binding fragment thereof) alone, and thereby reduces the cardiotoxicity associated with trastuzumab.

[0200] In some instances, a subject has a disease, disorder, or condition that is associated with the central nervous system (CNS) or that has a CNS component, where increased delivery of the trastuzumab antibody (or antigen-binding fragment thereof) across the blood brain barrier to CNS tissues relative to peripheral tissues can improve treatment, for instance, by increasing the tissue concentration of the antibody in the CNS, and/or by reducing side-effects associated with exposure of the antibody to peripheral tissues/organs.

[0201] Certain embodiments include the treatment of various cancers. "Cancer" relates generally to a class of diseases or conditions in which a group of cells display one or more of uncontrolled growth (i.e., division beyond normal limits), invasion (i.e., intrusion on and destruction of adjacent tissues), and/or metastasis (i.e., spread to other locations in the body via lymph or blood). These malignant properties of cancers differentiate them from benign cancers, which are self-limited, and typically do not invade or metastasize. Included are cancers of the central nervous system (CNS), or neurological cancers, such as brain cancers.

[0202] In some instances, the neurological cancer is a metastatic brain cancer. Examples of cancers that can metastasize to the brain include, without limitation, breast cancers, lung cancers, genitourinary tract cancers, gastrointestinal tract cancers (e.g., colorectal cancers, pancreatic carcinomas), osteosarcomas, melanomas, head and neck cancers, prostate cancers (e.g., prostatic adenocarcinomas), and lymphomas. Certain embodiments thus include methods for treating, inhibiting or preventing metastasis of a cancer by administering to a patient a therapeutically effective amount of a fusion protein described herein (e.g., in an amount that, following administration, inhibits, prevents or delays metastasis of a cancer in a statistically significant manner, i.e., relative to an appropriate control as will be known to those skilled in the art). In particular embodiments, the subject has a cancer that is at risk for but has not yet metastasized to the central nervous system, including one or more of the above-described cancers, among others known in the art.

[0203] In some aspects, the subject has a cancer associated with expression of Her2/neu. In particular aspects, subject has a Her2/neu-expressing or Her2/neu-overexpressing cancer. Hence, certain embodiments include methods for the treatment of a HER2-overexpressing cancer in a subject in need thereof, comprising administering to the subject a (e.g., therapeutically-effective amount of a) p97-antibody fusion protein (described herein) or a pharmaceutical composition comprising the same. In some embodiments, the HER2-overexpressing cancer is at risk for metastasizing to the CNS of the subject. In particular embodiments, the HER2-overexpressing cancer has metastasized to the CNS of the subject. In some aspects, the antibody fusion is administered in an amount that inhibits, prevents, or delays the progression and/or metastasis of the cancer in a statistically significant manner (i.e., relative to an appropriate control as will be known to those skilled in the art). Included are amounts that inhibit, prevent, or delay the progression and/or metastasis into tissues of the CNS, and those that inhibit, prevent, or delay progression and/or metastasis within tissues of the CNS.

[0204] In certain aspects, the HER2-overexpressing cancer is a breast cancer, ovarian cancer, gastric cancer, or uterine cancer. In particular aspects, the HER2-overexpressing cancer is a metastatic breast cancer, metastatic ovarian cancer, metastatic gastric cancer, or metastatic or aggressive form of uterine cancer.

[0205] In some aspects, the HER2-overexpressing cancer is a HER2-overexpressing breast cancer, such as a HER2-overexpressing metastatic breast cancer. In certain instances, the HER2-overexpressing metastatic breast cancer is at risk for metastasizing to the CNS of the subject. In certain instances, the HER2-overexpressing metastatic breast cancer has already metastasized to the CNS of the subject. In some instances, the p97-antibody fusion is administered with paclitaxel for first-line treatment of HER2-overexpressing metastatic breast cancer. In particular instances, the p97-antibody fusion is administered as a single agent for treatment of HER2-overexpressing metastatic breast cancer in patients who have received one or more chemotherapy regimens for metastatic disease.

[0206] Certain embodiments include administering the p97-antibody fusion or pharmaceutical composition as part of an adjuvant treatment for HER2-overexpressing breast cancer. In some aspects, the adjuvant treatment comprises doxorubicin, cyclophosphamide, and either paclitaxel or docetaxel. In some aspects, the adjuvant treatment comprises docetaxel and carboplatin. Certain aspects include administering the p97-antibody fusion or pharmaceutical composition as a single agent following multi-modality anthracycline based therapy.

[0207] In some aspects, the HER2-overexpressing cancer is a HER2-overexpressing metastatic gastric or gastroesophageal junction adenocarcinoma. In some instances, the HER2-overexpressing metastatic gastric or gastroesophageal junction adenocarcinoma is at risk for metastasizing to the CNS of the subject. In certain instances, the HER2-overexpressing metastatic gastric or gastroesophageal junction adenocarcinoma has already metastasized to the CNS of the subject. In some instances, the p97-antibody fusion is administered in combination with cisplatin and capecitabine or 5-fluorouracil, optionally where the subject or patient has not received prior treatment for metastatic disease.

[0208] In certain aspects, the HER2-overexpressing uterine cancer is a HER2-overexpressing uterine serous carcinoma (USC) (see, e.g., Santin et al., Inti Gynaecol Obstet. 102:128-31, 2008). USC, also known as uterine papillary serous carcinoma (UPSC) and uterine serous adenocarcinoma, is a form of endometrial cancer that typically arises in postmenopausal women. In some instances, the HER2-overexpressing USC is at risk for metastasizing to the CNS of the subject. In certain instances, the HER2-overexpressing USC has already metastasized to the CNS of the subject.

[0209] Methods for identifying subjects with one or more of the diseases or conditions described herein are known in the art.

[0210] Also included are methods for imaging an organ or tissue component in a subject, comprising (a) administering to the subject a composition comprising a fusion protein or antibody fusion described herein, which is conjugated to a detectable entity, and (b) visualizing the detectable entity in the subject, organ, or tissue.

[0211] In particular embodiments, the organ or tissue compartment comprises the central nervous system (e.g., brain, brainstem, spinal cord). In specific embodiments, the organ or tissue compartment comprises the brain or a portion thereof, for instance, the parenchyma of the brain.

[0212] A variety of methods can be employed to visualize the detectable entity in the subject, organ, or tissue. Exemplary non-invasive methods include radiography, such as fluoroscopy and projectional radiographs, CT-scanning or CAT-scanning (computed tomography (CT) or computed axial tomography (CAT)), whether employing X-ray CT-scanning, positron emission tomography (PET), or single photon emission computed tomography (SPECT), and certain types of magnetic resonance imaging (MRI), especially those that utilize contrast agents, including combinations thereof.

[0213] Merely by way of example, PET can be performed with positron-emitting contrast agents or radioisotopes such as .sup.18F, SPECT can be performed with gamma-emitting contrast agents or radioisotopes such as .sup.201Tl, .sup.99mTC, .sup.123I, and .sup.67Ga, and MRI can be performed with contrast agents or radioisotopes such as .sup.3H, .sup.13C, .sup.19F, .sup.17O, .sup.23Na, .sup.31P, and .sup.129Xe, and Gd (gadolidinium; chelated organic Gd (III) complexes). Any one or more of these exemplary contrast agents or radioisotopes can be conjugated to or otherwise incorporated into a p97 polypeptide and administered to a subject for imaging purposes. For instance, p97 polypeptides can be directly labeled with one or more of these radioisotopes, or conjugated to molecules (e.g., small molecules) that comprise one or more of these radioisotopic contrast agents, or any others described herein.

[0214] For in vivo use, for instance, for the treatment of human disease, medical imaging, or testing, the fusion proteins or antibody fusions described herein are generally incorporated into a pharmaceutical composition prior to administration. A pharmaceutical composition comprises one or more of the fusion proteins or antibody fusions described herein in combination with a physiologically-acceptable, pharmaceutically-acceptable, or pharmaceutical grade carrier or excipient.

[0215] To prepare a pharmaceutical composition, an effective or desired amount of one or more fusion protein or antibody fusions is mixed with any pharmaceutical carrier(s) or excipient known to those skilled in the art to be suitable for the particular mode of administration. A pharmaceutical carrier may be liquid, semi-liquid or solid. Solutions or suspensions used for parenteral, intradermal, subcutaneous or topical application may include, for example, a sterile diluent (such as water), saline solution (e.g., phosphate buffered saline; PBS), fixed oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvent; antimicrobial agents (such as benzyl alcohol and methyl parabens); antioxidants (such as ascorbic acid and sodium bisulfite) and chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); buffers (such as acetates, citrates and phosphates). If administered intravenously (e.g., by IV infusion), suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, polypropylene glycol and mixtures thereof.

[0216] Administration of fusion proteins or antibody fusions described herein, in pure form or in an appropriate pharmaceutical composition, can be carried out via any of the accepted modes of administration of agents for serving similar utilities. The pharmaceutical compositions can be prepared by combining a fusion protein or antibody fusion-containing composition with an appropriate physiologically acceptable carrier, diluent or excipient, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. In addition, other pharmaceutically active ingredients (including other small molecules as described elsewhere herein) and/or suitable excipients such as salts, buffers and stabilizers may, but need not, be present within the composition.

[0217] Administration may be achieved by a variety of different routes, including oral, parenteral, nasal, intravenous, intradermal, subcutaneous or topical. Preferred modes of administration depend upon the nature of the condition to be treated or prevented. Particular embodiments include administration by IV infusion.

[0218] Carriers can include, for example, pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as polysorbate 20 (TWEEN.TM.) polyethylene glycol (PEG), and poloxamers (PLURONICS.TM.), and the like.

[0219] In certain aspects, a fusion protein or antibody fusion is bound to or encapsulated within a particle, e.g., a nanoparticle, bead, lipid formulation, lipid particle, or liposome, e.g., immunoliposome. The fusion proteins or antibody fusions may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate)microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980). The particle(s) or liposomes may further comprise other therapeutic or diagnostic agents, such as cytotoxic agents.

[0220] The precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by testing the compositions in model systems known in the art and extrapolating therefrom. Controlled clinical trials may also be performed. Dosages may also vary with the severity of the condition to be alleviated. A pharmaceutical composition is generally formulated and administered to exert a therapeutically useful effect while minimizing undesirable side effects. The composition may be administered one time, or may be divided into a number of smaller doses to be administered at intervals of time. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need.

[0221] Typical routes of administering these and related pharmaceutical compositions thus include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. Pharmaceutical compositions according to certain embodiments of the present invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Compositions that will be administered to a subject or patient may take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a herein described conjugate in aerosol form may hold a plurality of dosage units. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The composition to be administered will typically contain a therapeutically effective amount of a fusion protein or antibody fusion described herein, for treatment of a disease or condition of interest.

[0222] A pharmaceutical composition may be in the form of a solid or liquid. In one embodiment, the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) may be liquid, with the compositions being, for example, an oral oil, injectable liquid or an aerosol, which is useful in, for example, inhalatory administration. When intended for oral administration, the pharmaceutical composition is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.

[0223] As a solid composition for oral administration, the pharmaceutical composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like. Such a solid composition will typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent. When the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.

[0224] The pharmaceutical composition may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred composition contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.

[0225] The liquid pharmaceutical compositions, whether they be solutions, suspensions or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. An injectable pharmaceutical composition is preferably sterile.

[0226] A liquid pharmaceutical composition intended for either parenteral or oral administration should contain an amount of a fusion protein or antibody fusion such that a suitable dosage will be obtained. Typically, this amount is at least 0.01% of the agent of interest in the composition. When intended for oral administration, this amount may be varied to be between 0.1 and about 70% of the weight of the composition. Certain oral pharmaceutical compositions contain between about 4% and about 75% of the agent of interest. In certain embodiments, pharmaceutical compositions and preparations according to the present invention are prepared so that a parenteral dosage unit contains between 0.01 to 10% by weight of the agent of interest prior to dilution.

[0227] The pharmaceutical composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents may be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device.

[0228] The pharmaceutical composition may be intended for rectal administration, in the form, for example, of a suppository, which will melt in the rectum and release the drug. The composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter, and polyethylene glycol.

[0229] The pharmaceutical composition may include various materials, which modify the physical form of a solid or liquid dosage unit. For example, the composition may include materials that form a coating shell around the active ingredients. The materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredients may be encased in a gelatin capsule. The pharmaceutical composition in solid or liquid form may include an agent that binds to the conjugate or agent and thereby assists in the delivery of the compound. Suitable agents that may act in this capacity include monoclonal or polyclonal antibodies, one or more proteins or a liposome.

[0230] The pharmaceutical composition may consist essentially of dosage units that can be administered as an aerosol. The term aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols may be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like, which together may form a kit. One of ordinary skill in the art, without undue experimentation may determine preferred aerosols.

[0231] The compositions described herein may be prepared with carriers that protect the fusion proteins or antibody fusions against rapid elimination from the body, such as time release formulations or coatings. Such carriers include controlled release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid and others known to those of ordinary skill in the art.

[0232] The pharmaceutical compositions may be prepared by methodology well known in the pharmaceutical art. For example, a pharmaceutical composition intended to be administered by injection may comprise one or more of salts, buffers and/or stabilizers, with sterile, distilled water so as to form a solution. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with the conjugate so as to facilitate dissolution or homogeneous suspension of the conjugate in the aqueous delivery system.

[0233] The compositions may be administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific compound employed; the metabolic stability and length of action of the compound; the age, body weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy. Generally, a therapeutically effective daily dose is (for a 70 kg mammal) from about 0.001 mg/kg (i.e., .about.0.07 mg) to about 100 mg/kg (i.e., .about.7.0 g); preferably a therapeutically effective dose is (for a 70 kg mammal) from about 0.01 mg/kg (i.e., .about.0.7 mg) to about 50 mg/kg (i.e., .about.3.5 g); more preferably a therapeutically effective dose is (for a 70 kg mammal) from about 1 mg/kg (i.e., .about.70 mg) to about 25 mg/kg (i.e., .about.1.75 g).

[0234] Compositions described herein may also be administered simultaneously with, prior to, or after administration of one or more other therapeutic agents, as described herein. For instance, in one embodiment, the conjugate is administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal anti-inflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide and mycophenolate.

[0235] In certain embodiments, the compositions disclosed herein may be administered in conjunction with any number of chemotherapeutic or cytotoxic agents. Examples of chemotherapeutic or cytotoxic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN.TM.); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK.RTM.; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL.RTM., Bristol-Myers Squibb Oncology, Princeton, N.J.) and doxetaxel (TAXOTERE.RTM., Rhne-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid derivatives such as Targretin.TM. (bexarotene), Panretin.TM. (alitretinoin); ONTAK.TM. (denileukin diftitox); esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0236] Such combination therapy may include administration of a single pharmaceutical dosage formulation, which contains a compound of the invention (i.e., fusion protein, antibody fusion protein) and one or more additional active agents, as well as administration of compositions comprising conjugates of the invention and each active agent in its own separate pharmaceutical dosage formulation. For example, a fusion protein or antibody fusion as described herein and the other active agent can be administered to the patient together in a single oral dosage composition such as a tablet or capsule, or each agent administered in separate oral dosage formulations. Similarly, a fusion protein or antibody fusion as described herein and the other active agent can be administered to the patient together in a single parenteral dosage composition such as in a saline solution or other physiologically acceptable solution, or each agent administered in separate parenteral dosage formulations. Where separate dosage formulations are used, the compositions comprising fusion proteins or antibody fusions and one or more additional active agents can be administered at essentially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially and in any order; combination therapy is understood to include all these regimens.

[0237] The various embodiments described herein can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent application, foreign patents, foreign patent application and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, application and publications to provide yet further embodiments.

[0238] These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

EXAMPLES

Example 1

Binding of p97-Trastuzumab Fusion Proteins to Human Her2/Neu

[0239] Human p97 (MTf)-trastuzumab fusion constructs were prepared and tested for activity. The amino acid sequences of the MTf-trastuzumab fusion constructs are shown in Table E1 below.

TABLE-US-00007 TABLE E1 SEQ ID Name Heavy and Light Chain Sequences NO TZM HC-MTf Heavy Chain 82 Homodimer Signal sequence (italics): TZM heavy chain: linker (C-terminal (underlined): MTf (bold) TZM heavy METDTLLLWVLLLWVPGSTGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDT chain MTf; YIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMN See FIG. SLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSK 1A for STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV general VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG structure) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GKGGGGSGGGGSGMEVRWCATSDPEQHKCGNMSEAFREAGIQPSLLCVRGTS ADHCVQLIAAQEADAITLDGGAIYEAGKEHGLKPVVGEVYDQEVGTSYYAVA VVRRSSHVTIDTLKGVKSCHTGINRTVGWNVPVGYLVESGRLSVMGCDVLKA VSDYFGGSCVPGAGETSYSESLCRLCRGDSSGEGVCDKSPLERYYDYSGAFR CLAEGAGDVAFVKHSTVLENTDGKTLPSWGQALLSQDFELLCRDGSRADVTE WRQCHLARVPAHAVVVRADTDGGLIFRLLNEGQRLFSHEGSSFQMFSSEAYG QKDLLFKDSTSELVPIATQTYEAWLGHEYLHAMKGLLCDPNRLPPYLRWCVL STPEIQKCGDMAVAFRRQRLKPEIQCVSAKSPQHCMERIQAEQVDAVTLSGE DIYTAGKTYGLVPAAGEHYAPEDSSNSYYVVAVVRRDSSHAFTLDELRGKRS CHAGFGSPAGWDVPVGALIQRGFIRPKDCDVLTAVSEFFNASCVPVNNPKNY PSSLCALCVGDEQGRNKCVGNSQERYYGYRGAFRCLVENAGDVAFVRHTTVF DNTNGHNSEPWAAELRSEDYELLCPNGARAEVSQFAACNLAQIPPHAVMVRP DTNIFTVYGLLDKAQDLFGDDHNKNGFKMFDSSNYHGQDLLFKDATVRAVPV GEKTTYRGWLGLDYVAALEGMSSQQCS Light Chain 83 Signal sequence (italics): TZM light chain METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTA VAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFA TYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC MTfp NH-TZM Heavy Chain 84 (N-terminal Signal sequence(italics): MTfp sequence (bold): MTfp TZM linker (underlined): TZM heavy chain heavy chain; see METDTLLLWVLLLWVPGSTGDSSHAFTLDELRYGGGGSGGGGSEVQLVESGG FIG. 1D GLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYA for general DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQG structure) TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK Light Chain 85 Signal sequence(italics): TZM light chain METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTA VAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFA TYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC TZM HC-MTfp Heavy Chain 86 (C-terminal Signal sequence (italics): TZM heavy chain : linker TZM heavy (underlined): MTfp sequence without C-terminal Y chain MTfp; (bold) see FIG. METDTLLLWVLLLWVPGSTGEVQLVESGGGLVQPGGSLRLSCAASGENIKDT 1E for YIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMN general SLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSK structure) STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GKGGGGSGGGGSDSSHAFTLDELR Light Chain 87 Signal sequence (italics): TZM light chain METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTA VAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFA TYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC TZM/MTf Heavy Chain 88 Homodimer Signal sequence (italics): TZM heavy chain: linker (C-terminal (underlined): MTf (bold) TZM heavy METDTLLLWVLLLWVPGSTGEVQLVESGGGLVQPGGSLRLSCAASGENIKDT chain MTf; YIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMN see FIG. SLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSK 1A for STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV general VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG structure) PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GKGGGGSGGGGSGGGGSGMEVRWCATSDPEQHKCGNMSEAFREAGIQPSLLC VRGTSADHCVQLIAAQEADAITLDGGAIYEAGKEHGLKPVVGEVYDQEVGTS YYAVAVVRRSSHVTIDTLKGVKSCHTGINRTVGWNVPVGYLVESGRLSVMGC DVLKAVSDYFGGSCVPGAGETSYSESLCRLCRGDSSGEGVCDKSPLERYYDY SGAFRCLAEGAGDVAFVKHSTVLENTDGKTLPSWGQALLSQDFELLCRDGSR ADVTEWRQCHLARVPAHAVVVRADTDGGLIFRLLNEGQRLFSHEGSSFQMFS SEAYGQKDLLFKDSTSELVPIATQTYEAWLGHEYLHAMKGLLCDPNRLPPYL RWCVLSTPEIQKCGDMAVAFRRQRLKPEIQCVSAKSPQHCMERIQAEQVDAV TLSGEDIYTAGKTYGLVPAAGEHYAPEDSSNSYYVVAVVRRDSSHAFTLDEL RGKRSCHAGFGSPAGWDVPVGALIQRGFIRPKDCDVLTAVSEFFNASCVPVN NPKNYPSSLCALCVGDEQGRNKCVGNSQERYYGYRGAFRCLVENAGDVAFVR HTTVFDNTNGHNSEPWAAELRSEDYELLCPNGARAEVSQFAACNLAQIPPHA VMVRPDTNIFTVYGLLDKAQDLFGDDHNKNGFKMFDSSNYHGQDLLFKDATV RAVPVGEKTTYRGWLGLDYVAALEGMSSQQCSG Light Chain 89 Signal sequence: TZM light chain METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTA VAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFA TYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC LC-MTfp/Fc- Heavy Chain 90 MTfp Signal sequence (italics): TZM heavy chain: linker (C-terminal (underlined): Mtfp (bold) TZM light METDTLLLWVLLLWVPGSTGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDT chain YIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMN MTfp/C- SLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSK terminal STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV TZM heavy VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSCDKTHTCPPCPAPELLG chain MTfp; GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK see FIG. TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK 1F for GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK general TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS structure) PGKEAAAKEAAAKEAAAKDSSHAFTLDELRY Light Chain 91 Signal sequence (italics): TZM light chain: linker (underlined): Mtfp (bold) METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTA VAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFA TYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGECEAAAKEAAAKEAAAKDSSHAFTLDEL RY LC/Fc-MTfp Heavy Chain 92 (TZM light Signal sequence (italics): TZM heavy chain: linker chain/C- (underlined): Mtfp (bold) terminal METDTLLLWVLLLWVPGSTGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDT TZM heavy YIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMN chain MTfp; SLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSK see FIG. STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV 1E for VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSCDKTHTCPPCPAPELLG general GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK structure) TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGKEAAAKEAAAKEAAAKDSSHAFTLDELRY Light Chain 93 Signal sequence (italics): TZM heavy chain METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTA VAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFA TYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC LC-MTfp/Fc Heavy Chain 94 (C-terminal Signal sequence (italics): TZM heavy chain: linker: TZM light MTfp chain MTfp/ METDTLLLWVLLLWVPGSTGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDT TZM heavy YIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMN chain; see SLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSK FIG. 1G STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV for general VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSCDKTHTCPPCPAPELLG structure) GPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK PGK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS Light Chain 95 Signal sequence (italics): TZM light chain METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTA VAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFA TYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGECEAAAKEAAAKEAAAKDSSHAFTLDEL RY TZM-Trastuzumab MTf-p97 MTfp-p97 fragment HC-Heavy Chain LC-Light Chain

[0240] In vitro binding assays were performed to measure the binding of MTf-trastuzumab fusion constructs to His-tagged human Her2/Neu protein (Her2-His). Her2-His was loaded onto penta-His biosensor at a concentration of 20 .mu.g/mL and dipped into TZM HC-MTf, MTfp NH-TZM, and TZM HC-MTfp fusion proteins (see Table E1) and a human IgG1 control at varying concentrations.

[0241] The results of octet analysis in FIGS. 2A-2D and Table E2 (below) demonstrate the affinity of the MTf-trastuzumab fusions for Her2.

TABLE-US-00008 TABLE E2 Loading Full Sample ID Sample ID K.sub.D (M) K.sub.on (1/Ms) K.sub.off (1/s) Full R{circumflex over ( )}2 X{circumflex over ( )}2 Her2-His TZM HC-MTf <1.0E-12 4.94E+04 <1.0E-07 0.999074 0.002797 Her2-His MTfp NH-TZM <1.0E-12 9.06E+04 <1.0E-07 0.997464 0.014593 Her2-His TZM HC-MTfp <1.0E-12 1.21E+05 <1.0E-07 0.997177 0.019048 Her2-His Human IgG1 <1.0E-12 1.04E+06 <1.0E-07 0.997389 0.010269

[0242] MTf-trastuzumab fusion proteins demonstrated tight binding to human Her2, as shown by the equilibrium dissociation constant (K.sub.D), association rate constant (K.sub.on), and dissociation rate constant (K.sub.off).

Example 2

ADCC Cell-Killing Activity of p97-Trastuzumab Fusion Proteins

[0243] MTf-trastuzumab fusion constructs were tested for Antibody-dependent cell-mediated cytotoxicity (ADCC) in BT-474 breast cancer cells compared to Herceptin.RTM. (trastuzumab) and human IgG1 Fc as positive and negative controls, respectively.

[0244] BT-474 cells were purchased from ATCC and grown in Hybri-Care Medium supplemented with 1.5 g/L sodium bicarbonate and 10% fetal bovine serum. Human peripheral blood mononuclear cells (PBMCs) were freshly isolated by Histopaque centrifugation and incubated with carboxyfluorescein succinimidyl ester (CFSE)-labeled target BT-474 cells at a ratio of about 30:1.

[0245] Test samples were co-incubated with the effector:target cell mixture for four hours at ten different concentrations (up to 100 .mu.g/mL). Negative controls included effector:target cells only as no antibody control, and 100 .mu.g/mL IgG Fc. Each treatment was performed in duplicate. After the 4 hour co-incubation, cells were stained with propidium iodide (PI) and analyzed by flow cytometry (FACS). The percentage of PI/CFSE+ cells was quantitated as an indication of cytotoxicity. A dose-response curve was generated by plotting the mean.+-.range of the data points for all ten different concentrations, and the EC.sub.50 was calculated. The results are shown in FIGS. 3A-3B.

[0246] FIG. 3A shows the ADCC evaluation of TZM/MTf fusion in BT-474 breast cancer cells compared to Herceptin.RTM. and human IgG1 Fc. Here, the TZM/MTf homodimer fusion induced ADCC with an EC.sub.50 of 0.6 .mu.g/mL compared to that of Herceptin.RTM. at 0.2 pg/mL.

[0247] FIG. 3B shows the ADCC evaluation of LC-MTfp/Fc-MTfp, LC-MTfp/Fc, and LC/Fc-MTfp fusions in BT-474 breast cancer cells compared to Herceptin.RTM. and human IgG1 Fc. LC-MTfp/Fc-MTfp, LC-MTfp/Fc, and LC/Fc-MTfp induced ADCC with an EC.sub.50 of 0.216 .mu.g/mL, 0.171 .mu.g/mL, and 1.025 .mu.g/mL, respectively. These data show that MTf-trastuzumab fusion constructs induce significant antibody-dependent cell-mediated cytotoxicity in breast cancer cells.

Example 3

In Vivo Anti-Tumor Efficacy of p97-Trastuzumab Fusion Proteins

[0248] The p97-trastuzumab fusion proteins are tested for anti-tumor efficacy and effects on survival in mice that are intracranially injected with human BT-474 breast tumor cells. Specifically, the fusion constructs TZM HC-MTf and MTfp NH-TZM are tested relative to Herceptin.RTM. (Trastuzumab, AMM: 5621037, ROCHE).

[0249] Test Compounds.

[0250] The stock solutions of TZM HC-MTf and MTfp NH-TZM constructs are diluted in PBS and administered respectively at 60 mg/kg and 30 mg/kg (equivalent dose of 30 mg/kg Herceptin.RTM.). Herceptin.RTM. is prepared as follows: one vial containing 150 mg of Herceptin.RTM. is diluted in NaCl 0.9% (Aguettant, Lyon, France) to a final concentration of 20 mg/mL. The stock solution is stored at 4.degree. C. for the duration of the study. Each day of administration to mice, the stock solution is diluted in NaCl 0.9% to reach 3 mg/mL final concentration. Herceptin.RTM. is administrated at 30 mg/kg.

[0251] All compounds are administered by intravenous injection (IV, bolus) into the caudal vein of mice at a dose volume of 10 mL/kg/inj (i.e., for one mouse weighing 20 g, 200 .mu.L of test substance is administered).

[0252] Cancer Cell Line.

[0253] The BT-474 cell line is purchased from ATCC. It was originally isolated from a solid, invasive ductal carcinoma of the breast from a 60 year old Caucasian female patient (Lasfargues et al., J Natl Cancer Inst. 61:967-78, 1978). Tumor cells are grown as monolayer at 37.degree. C. in a humidified atmosphere (5% CO2, 95% air). The culture medium contains DMEM supplemented with 2 mM L glutamine (ref: BE12-604F, Lonza, Verviers, Belgium) and 10% fetal bovine serum (ref: 3302, Lonza). The cells are adherent to plastic flasks. For experimental use, tumor cells are detached from the culture flask by a 5 minute treatment with trypsin-versene (ref: BE02-007E, Lonza), in Hanks' medium without calcium or magnesium (ref: BE10-543F, Lonza) and neutralized by addition of complete culture medium. The cells are counted in a hemocytometer and their viability is assessed by 0.25% trypan blue exclusion assay.

[0254] Animals.

[0255] Sixty-one healthy female Balb/C nu/nu (CByJ.Cg-Foxn1nu/J) mice, 5-6 weeks old, are obtained from CHARLES RIVER (L'Arbresles). Animals are maintained in housing rooms under controlled environmental conditions: Temperature: 22.+-.2.degree. C., Humidity 55.+-.10%, Photoperiod (12 h light/12 h dark), HEPA filtered air, 15 air exchanges per hour with no recirculation.

[0256] Animal enclosures provide sterile and adequate space with bedding material, food and water, environmental and social enrichment (group housing) as described: Top filter polycarbonate Eurostandard Type III or IV cages, Corn cob bedding (ref: LAB COB 12, SERLAB, France), 25 kGy Irradiated diet (Ssniff.RTM. Soest, Germany), Complete food for immunodeficient rodents--NM Extrudate, Complete food for immunocompetent rodents--R/M-H Extrudate, Sterile, filtrated at 0.2 .mu.m water (supplemented with 2.5 .mu.g/mL estradiol), Environmental enrichment (SIZZLE-dri kraft-D20004 SERLAB, France).

[0257] Induction of BT-474 Tumors.

[0258] For stereotaxic injection of tumor cells, mice are anesthetized by an intraperitoneal injection of Ketamine 70 mg/kg (Ketamine500.RTM., Ref 043KET204, Centravet, France) and Xylazine 5 mg/kg (Rompun.RTM., Ref 002ROM001, Centravet, France) in 0.9% NaCl solution at 10 mL/kg/inj. Tumors are induced by stereotaxic injection of 1.times.10.sup.5 of BT-474 cells in 2 .mu.L of RPMI 1640 of 52 female animals. BT-474 tumor cell implantation is performed 24 to 72 hours after a whole body irradiation with a .gamma.-source (2 Gy, .sup.60Co, BioMep, Bretenieres, France).

[0259] The tumor cell suspension is injected into the caudate nucleus of the right cerebral hemisphere at a rate of 0.5 .mu.L/min. Five minutes after the end of the injection, the needle is slowly withdrawn by 1 mm every minute. Carprofen (dose: 5 mg/kg) is injected subcutaneously at the end of the surgery and 24 h post-surgery. The day of tumor cell implantation is considered as day zero (D.sub.0).

[0260] Treatment Schedule.

[0261] The treatment is started on day five (D.sub.5). Forty animals (40) out of fifty-two (52) are randomized according to their individual body weight into 4 groups each of 10 animals using Vivo Manager.RTM. software (Biosystemes, Couternon, France). A statistical test (analysis of variance) is performed to test for homogeneity between groups. The treatment schedule is as follows:

[0262] Group 1 mice receive two weekly IV administration of vehicle for 6 consecutive weeks.

[0263] Group 2 mice receive two weekly IV administrations of TZM HC-MTf at equivalent dose of 30 mg/kg Herceptin.RTM. for 6 consecutive weeks.

[0264] Group 3 mice receive two weekly IV administrations of MTfp NH-TZM at equivalent dose of 30 mg/kg Herceptin.RTM. for 6 consecutive weeks.

[0265] Group 4 mice receive two weekly IV administrations of Herceptin.RTM. at 30 mg/kg for 6 consecutive weeks.

[0266] The treatment schedule is summarized in Table E3 below:

TABLE-US-00009 TABLE E3 No. Dose Adm. Treatment Group animals Treatment (mg/kg/adm) Route schedule 1 10 Vehicle (PBS) -- IV TW .times. 6 2 10 TZM HC-MTf 60 IV TW .times. 6 3 10 MTfp NH-TZM 30 IV TW .times. 6 4 10 Herceptin .RTM. 30 IV TW .times. 6

[0267] Monitoring.

[0268] All study data, including animal body weight measurements, clinical and mortality records, and treatment are scheduled and recorded on Vivo Manager.RTM. database (Biosystemes, Dijon, France). The viability and behavior is recorded every day. Body weights are measured twice a week.

[0269] The following Human endpoints are measured (Workman et al., Br J Cancer. 102:1555-77, 2010).

[0270] Signs of pain, suffering or distress: pain posture, pain face mask, behavior

[0271] 20% body weight loss remaining for 3 consecutive days

[0272] Poor body condition, emaciation, cachexia, dehydration

[0273] Prolonged absence of voluntary responses to external stimuli

[0274] Rapid labored breathing, anemia, significant bleeding

[0275] Neurologic signs: circling, convulsion, paralysis

[0276] Sustained decrease in body temperature

[0277] Abdominal distension

[0278] Necropsy (macroscopic examination) is performed on all terminated animal in the study, and, if possible, on all euthanized moribund or found dead animal.

[0279] The following evaluation criteria of health are determined using Vivo Manager.RTM. software (Biosystemes, Couternon, France).

[0280] Individual and mean body weights of animals

[0281] Mean body weight change (MBWC): Average weight change of treated animals in percent (weight at day B minus weight at day A divided by weight at day A) is calculated. The intervals over which MBWC is calculated are chosen as a function of body weight curves and the days of body weight measurement

[0282] Magnetic Resonance Imaging (MRI).

[0283] Imaging experiments are performed on a 4.7T horizontal magnet (PharmaScan, Bruker Biospin GmbH, Germany) equipped with an actively shielded gradient system. For image analysis, mice are positioned prone in a dedicated mouse body cradle which is slid in a volume coil (38 mm internal diameter) within the Pharmascan and images are acquired under ParaVision (PV5.1, Bruker Biospin).

[0284] During all the image acquisitions, mice are continuously anaesthetized using isoflurane (Minerve, Bondoufle, France) in a mixture of air via a nose piece. Body temperature of the animals is maintained within physiological levels by a flow of warm air. Breathing rate is continuously monitored using a pressure sensor taped on its abdomen. Physiological signals are monitored via a laptop placed next to the MRI workstation and connected to the sensors by fiber optic cables (SA Instruments, USA).

[0285] The contrast agent Gadopentetate dimeglumine (Gd-DTPA, Magnevist, Bayer Healthcare Pharmaceuticals, Germany) is injected intravenously (IV) at 0.4 mmol/kg via the caudal vein of mice.

[0286] Images are transferred to a workstation to be analyzed under ImageJ (4). Regions of interest (ROIs) are drawn manually on anatomical images. Tumor volume is computed from the ROIs by multiplying the number of ROI voxels by the voxel volume (in mm.sup.3). Tumor volumes in mm.sup.3 are tabulated at each time point and for each animal.

[0287] Sample Collection.

[0288] All mice are euthanized fourteen days after the last treatment. Intracardiac blood collection is used in terminal procedures under deep gas anesthesia. Approximately 200 .mu.L of blood from ten animals per group is collected into blood collection tubes with clot activator. Tubes are centrifuged 30 minutes after sampling at 1300 g for 10 minutes at room temperature to obtain serum. The serum samples are stored in propylene tubes at 80.degree. C. until analysis. Immediately after termination, samples from the brain, heart, lung, liver and kidney of each animal are collected, weighed and stored for analysis.

[0289] Efficacy Parameters.

[0290] The treatment efficacy is assessed in terms of the effects of the test substance on the tumor volumes of treated animals relative to control animals, as measured by MRI (see below). The efficacy parameters are expressed as a percent treated over control survival (T/C %). T is the median of the survival times of animals treated with test substances and C is the median survival times of control animals treated with vehicle. Survival systems indicate a degree of success when T/C percent exceed 125% (7). T/C % is expressed as follows:

T / C % = T ( median survival time of treated animals ) C ( median survival time of vehicle - treated animals ) .times. 100 ##EQU00001##

[0291] Survival curves are drawn, and mean and median survival times are calculated.

[0292] Statistical Tests.

[0293] Statistical analyses are performed using Vivo Manager.RTM. software (Biosystemes, Couternon, France). Statistical analyses of mean body weights, MBWC, mean tumor volumes V, are performed using ANOVA and pairwise tests are performed using the Bonferroni/Dunn correction in case of significant ANOVA results. The log-Rank (Kaplan-Meier) test is used to compare the survival curves. A p value<0.05 is considered as significant.

Sequence CWU 1

1

1381738PRTHomo sapiens 1Met Arg Gly Pro Ser Gly Ala Leu Trp Leu Leu Leu Ala Leu Arg Thr 1 5 10 15 Val Leu Gly Gly Met Glu Val Arg Trp Cys Ala Thr Ser Asp Pro Glu 20 25 30 Gln His Lys Cys Gly Asn Met Ser Glu Ala Phe Arg Glu Ala Gly Ile 35 40 45 Gln Pro Ser Leu Leu Cys Val Arg Gly Thr Ser Ala Asp His Cys Val 50 55 60 Gln Leu Ile Ala Ala Gln Glu Ala Asp Ala Ile Thr Leu Asp Gly Gly 65 70 75 80 Ala Ile Tyr Glu Ala Gly Lys Glu His Gly Leu Lys Pro Val Val Gly 85 90 95 Glu Val Tyr Asp Gln Glu Val Gly Thr Ser Tyr Tyr Ala Val Ala Val 100 105 110 Val Arg Arg Ser Ser His Val Thr Ile Asp Thr Leu Lys Gly Val Lys 115 120 125 Ser Cys His Thr Gly Ile Asn Arg Thr Val Gly Trp Asn Val Pro Val 130 135 140 Gly Tyr Leu Val Glu Ser Gly Arg Leu Ser Val Met Gly Cys Asp Val 145 150 155 160 Leu Lys Ala Val Ser Asp Tyr Phe Gly Gly Ser Cys Val Pro Gly Ala 165 170 175 Gly Glu Thr Ser Tyr Ser Glu Ser Leu Cys Arg Leu Cys Arg Gly Asp 180 185 190 Ser Ser Gly Glu Gly Val Cys Asp Lys Ser Pro Leu Glu Arg Tyr Tyr 195 200 205 Asp Tyr Ser Gly Ala Phe Arg Cys Leu Ala Glu Gly Ala Gly Asp Val 210 215 220 Ala Phe Val Lys His Ser Thr Val Leu Glu Asn Thr Asp Gly Lys Thr 225 230 235 240 Leu Pro Ser Trp Gly Gln Ala Leu Leu Ser Gln Asp Phe Glu Leu Leu 245 250 255 Cys Arg Asp Gly Ser Arg Ala Asp Val Thr Glu Trp Arg Gln Cys His 260 265 270 Leu Ala Arg Val Pro Ala His Ala Val Val Val Arg Ala Asp Thr Asp 275 280 285 Gly Gly Leu Ile Phe Arg Leu Leu Asn Glu Gly Gln Arg Leu Phe Ser 290 295 300 His Glu Gly Ser Ser Phe Gln Met Phe Ser Ser Glu Ala Tyr Gly Gln 305 310 315 320 Lys Asp Leu Leu Phe Lys Asp Ser Thr Ser Glu Leu Val Pro Ile Ala 325 330 335 Thr Gln Thr Tyr Glu Ala Trp Leu Gly His Glu Tyr Leu His Ala Met 340 345 350 Lys Gly Leu Leu Cys Asp Pro Asn Arg Leu Pro Pro Tyr Leu Arg Trp 355 360 365 Cys Val Leu Ser Thr Pro Glu Ile Gln Lys Cys Gly Asp Met Ala Val 370 375 380 Ala Phe Arg Arg Gln Arg Leu Lys Pro Glu Ile Gln Cys Val Ser Ala 385 390 395 400 Lys Ser Pro Gln His Cys Met Glu Arg Ile Gln Ala Glu Gln Val Asp 405 410 415 Ala Val Thr Leu Ser Gly Glu Asp Ile Tyr Thr Ala Gly Lys Thr Tyr 420 425 430 Gly Leu Val Pro Ala Ala Gly Glu His Tyr Ala Pro Glu Asp Ser Ser 435 440 445 Asn Ser Tyr Tyr Val Val Ala Val Val Arg Arg Asp Ser Ser His Ala 450 455 460 Phe Thr Leu Asp Glu Leu Arg Gly Lys Arg Ser Cys His Ala Gly Phe 465 470 475 480 Gly Ser Pro Ala Gly Trp Asp Val Pro Val Gly Ala Leu Ile Gln Arg 485 490 495 Gly Phe Ile Arg Pro Lys Asp Cys Asp Val Leu Thr Ala Val Ser Glu 500 505 510 Phe Phe Asn Ala Ser Cys Val Pro Val Asn Asn Pro Lys Asn Tyr Pro 515 520 525 Ser Ser Leu Cys Ala Leu Cys Val Gly Asp Glu Gln Gly Arg Asn Lys 530 535 540 Cys Val Gly Asn Ser Gln Glu Arg Tyr Tyr Gly Tyr Arg Gly Ala Phe 545 550 555 560 Arg Cys Leu Val Glu Asn Ala Gly Asp Val Ala Phe Val Arg His Thr 565 570 575 Thr Val Phe Asp Asn Thr Asn Gly His Asn Ser Glu Pro Trp Ala Ala 580 585 590 Glu Leu Arg Ser Glu Asp Tyr Glu Leu Leu Cys Pro Asn Gly Ala Arg 595 600 605 Ala Glu Val Ser Gln Phe Ala Ala Cys Asn Leu Ala Gln Ile Pro Pro 610 615 620 His Ala Val Met Val Arg Pro Asp Thr Asn Ile Phe Thr Val Tyr Gly 625 630 635 640 Leu Leu Asp Lys Ala Gln Asp Leu Phe Gly Asp Asp His Asn Lys Asn 645 650 655 Gly Phe Lys Met Phe Asp Ser Ser Asn Tyr His Gly Gln Asp Leu Leu 660 665 670 Phe Lys Asp Ala Thr Val Arg Ala Val Pro Val Gly Glu Lys Thr Thr 675 680 685 Tyr Arg Gly Trp Leu Gly Leu Asp Tyr Val Ala Ala Leu Glu Gly Met 690 695 700 Ser Ser Gln Gln Cys Ser Gly Ala Ala Ala Pro Ala Pro Gly Ala Pro 705 710 715 720 Leu Leu Pro Leu Leu Leu Pro Ala Leu Ala Ala Arg Leu Leu Pro Pro 725 730 735 Ala Leu 2692PRTHomo sapiens 2Gly Met Glu Val Arg Trp Cys Ala Thr Ser Asp Pro Glu Gln His Lys 1 5 10 15 Cys Gly Asn Met Ser Glu Ala Phe Arg Glu Ala Gly Ile Gln Pro Ser 20 25 30 Leu Leu Cys Val Arg Gly Thr Ser Ala Asp His Cys Val Gln Leu Ile 35 40 45 Ala Ala Gln Glu Ala Asp Ala Ile Thr Leu Asp Gly Gly Ala Ile Tyr 50 55 60 Glu Ala Gly Lys Glu His Gly Leu Lys Pro Val Val Gly Glu Val Tyr 65 70 75 80 Asp Gln Glu Val Gly Thr Ser Tyr Tyr Ala Val Ala Val Val Arg Arg 85 90 95 Ser Ser His Val Thr Ile Asp Thr Leu Lys Gly Val Lys Ser Cys His 100 105 110 Thr Gly Ile Asn Arg Thr Val Gly Trp Asn Val Pro Val Gly Tyr Leu 115 120 125 Val Glu Ser Gly Arg Leu Ser Val Met Gly Cys Asp Val Leu Lys Ala 130 135 140 Val Ser Asp Tyr Phe Gly Gly Ser Cys Val Pro Gly Ala Gly Glu Thr 145 150 155 160 Ser Tyr Ser Glu Ser Leu Cys Arg Leu Cys Arg Gly Asp Ser Ser Gly 165 170 175 Glu Gly Val Cys Asp Lys Ser Pro Leu Glu Arg Tyr Tyr Asp Tyr Ser 180 185 190 Gly Ala Phe Arg Cys Leu Ala Glu Gly Ala Gly Asp Val Ala Phe Val 195 200 205 Lys His Ser Thr Val Leu Glu Asn Thr Asp Gly Lys Thr Leu Pro Ser 210 215 220 Trp Gly Gln Ala Leu Leu Ser Gln Asp Phe Glu Leu Leu Cys Arg Asp 225 230 235 240 Gly Ser Arg Ala Asp Val Thr Glu Trp Arg Gln Cys His Leu Ala Arg 245 250 255 Val Pro Ala His Ala Val Val Val Arg Ala Asp Thr Asp Gly Gly Leu 260 265 270 Ile Phe Arg Leu Leu Asn Glu Gly Gln Arg Leu Phe Ser His Glu Gly 275 280 285 Ser Ser Phe Gln Met Phe Ser Ser Glu Ala Tyr Gly Gln Lys Asp Leu 290 295 300 Leu Phe Lys Asp Ser Thr Ser Glu Leu Val Pro Ile Ala Thr Gln Thr 305 310 315 320 Tyr Glu Ala Trp Leu Gly His Glu Tyr Leu His Ala Met Lys Gly Leu 325 330 335 Leu Cys Asp Pro Asn Arg Leu Pro Pro Tyr Leu Arg Trp Cys Val Leu 340 345 350 Ser Thr Pro Glu Ile Gln Lys Cys Gly Asp Met Ala Val Ala Phe Arg 355 360 365 Arg Gln Arg Leu Lys Pro Glu Ile Gln Cys Val Ser Ala Lys Ser Pro 370 375 380 Gln His Cys Met Glu Arg Ile Gln Ala Glu Gln Val Asp Ala Val Thr 385 390 395 400 Leu Ser Gly Glu Asp Ile Tyr Thr Ala Gly Lys Thr Tyr Gly Leu Val 405 410 415 Pro Ala Ala Gly Glu His Tyr Ala Pro Glu Asp Ser Ser Asn Ser Tyr 420 425 430 Tyr Val Val Ala Val Val Arg Arg Asp Ser Ser His Ala Phe Thr Leu 435 440 445 Asp Glu Leu Arg Gly Lys Arg Ser Cys His Ala Gly Phe Gly Ser Pro 450 455 460 Ala Gly Trp Asp Val Pro Val Gly Ala Leu Ile Gln Arg Gly Phe Ile 465 470 475 480 Arg Pro Lys Asp Cys Asp Val Leu Thr Ala Val Ser Glu Phe Phe Asn 485 490 495 Ala Ser Cys Val Pro Val Asn Asn Pro Lys Asn Tyr Pro Ser Ser Leu 500 505 510 Cys Ala Leu Cys Val Gly Asp Glu Gln Gly Arg Asn Lys Cys Val Gly 515 520 525 Asn Ser Gln Glu Arg Tyr Tyr Gly Tyr Arg Gly Ala Phe Arg Cys Leu 530 535 540 Val Glu Asn Ala Gly Asp Val Ala Phe Val Arg His Thr Thr Val Phe 545 550 555 560 Asp Asn Thr Asn Gly His Asn Ser Glu Pro Trp Ala Ala Glu Leu Arg 565 570 575 Ser Glu Asp Tyr Glu Leu Leu Cys Pro Asn Gly Ala Arg Ala Glu Val 580 585 590 Ser Gln Phe Ala Ala Cys Asn Leu Ala Gln Ile Pro Pro His Ala Val 595 600 605 Met Val Arg Pro Asp Thr Asn Ile Phe Thr Val Tyr Gly Leu Leu Asp 610 615 620 Lys Ala Gln Asp Leu Phe Gly Asp Asp His Asn Lys Asn Gly Phe Lys 625 630 635 640 Met Phe Asp Ser Ser Asn Tyr His Gly Gln Asp Leu Leu Phe Lys Asp 645 650 655 Ala Thr Val Arg Ala Val Pro Val Gly Glu Lys Thr Thr Tyr Arg Gly 660 665 670 Trp Leu Gly Leu Asp Tyr Val Ala Ala Leu Glu Gly Met Ser Ser Gln 675 680 685 Gln Cys Ser Gly 690 311PRTHomo sapiens 3Trp Cys Ala Thr Ser Asp Pro Glu Gln His Lys 1 5 10 411PRTHomo sapiens 4Arg Ser Ser His Val Thr Ile Asp Thr Leu Lys 1 5 10 513PRTHomo sapiens 5Ser Ser His Val Thr Ile Asp Thr Leu Lys Gly Val Lys 1 5 10 614PRTHomo sapiens 6Leu Cys Arg Gly Asp Ser Ser Gly Glu Gly Val Cys Asp Lys 1 5 10 716PRTHomo sapiens 7Gly Asp Ser Ser Gly Glu Gly Val Cys Asp Lys Ser Pro Leu Glu Arg 1 5 10 15 89PRTHomo sapiens 8Tyr Tyr Asp Tyr Ser Gly Ala Phe Arg 1 5 97PRTHomo sapiens 9Ala Asp Val Thr Glu Trp Arg 1 5 109PRTHomo sapiens 10Val Pro Ala His Ala Val Val Val Arg 1 5 1110PRTHomo sapiens 11Ala Asp Thr Asp Gly Gly Leu Ile Phe Arg 1 5 10 129PRTHomo sapiens 12Cys Gly Asp Met Ala Val Ala Phe Arg 1 5 1311PRTHomo sapiens 13Leu Lys Pro Glu Ile Gln Cys Val Ser Ala Lys 1 5 10 1412PRTHomo sapiens 14Asp Ser Ser His Ala Phe Thr Leu Asp Glu Leu Arg 1 5 10 1513PRTHomo sapiens 15Ser Glu Asp Tyr Glu Leu Leu Cys Pro Asn Gly Ala Arg 1 5 10 1615PRTHomo sapiens 16Ala Gln Asp Leu Phe Gly Asp Asp His Asn Lys Asn Gly Phe Lys 1 5 10 15 1740PRTHomo sapiens 17Phe Ser Ser Glu Ala Tyr Gly Gln Lys Asp Leu Leu Phe Lys Asp Ser 1 5 10 15 Thr Ser Glu Leu Val Pro Ile Ala Thr Gln Thr Tyr Glu Ala Trp Leu 20 25 30 Gly His Glu Tyr Leu His Ala Met 35 40 18221PRTHomo sapiens 18Glu Arg Ile Gln Ala Glu Gln Val Asp Ala Val Thr Leu Ser Gly Glu 1 5 10 15 Asp Ile Tyr Thr Ala Gly Lys Thr Tyr Gly Leu Val Pro Ala Ala Gly 20 25 30 Glu His Tyr Ala Pro Glu Asp Ser Ser Asn Ser Tyr Tyr Val Val Ala 35 40 45 Val Val Arg Arg Asp Ser Ser His Ala Phe Thr Leu Asp Glu Leu Arg 50 55 60 Gly Lys Arg Ser Cys His Ala Gly Phe Gly Ser Pro Ala Gly Trp Asp 65 70 75 80 Val Pro Val Gly Ala Leu Ile Gln Arg Gly Phe Ile Arg Pro Lys Asp 85 90 95 Cys Asp Val Leu Thr Ala Val Ser Glu Phe Phe Asn Ala Ser Cys Val 100 105 110 Pro Val Asn Asn Pro Lys Asn Tyr Pro Ser Ser Leu Cys Ala Leu Cys 115 120 125 Val Gly Asp Glu Gln Gly Arg Asn Lys Cys Val Gly Asn Ser Gln Glu 130 135 140 Arg Tyr Tyr Gly Tyr Arg Gly Ala Phe Arg Cys Leu Val Glu Asn Ala 145 150 155 160 Gly Asp Val Ala Phe Val Arg His Thr Thr Val Phe Asp Asn Thr Asn 165 170 175 Gly His Asn Ser Glu Pro Trp Ala Ala Glu Leu Arg Ser Glu Asp Tyr 180 185 190 Glu Leu Leu Cys Pro Asn Gly Ala Arg Ala Glu Val Ser Gln Phe Ala 195 200 205 Ala Cys Asn Leu Ala Gln Ile Pro Pro His Ala Val Met 210 215 220 1932PRTHomo sapiens 19Val Arg Pro Asp Thr Asn Ile Phe Thr Val Tyr Gly Leu Leu Asp Lys 1 5 10 15 Ala Gln Asp Leu Phe Gly Asp Asp His Asn Lys Asn Gly Phe Lys Met 20 25 30 20564PRTHomo sapiens 20Gly Met Glu Val Arg Trp Cys Ala Thr Ser Asp Pro Glu Gln His Lys 1 5 10 15 Cys Gly Asn Met Ser Glu Ala Phe Arg Glu Ala Gly Ile Gln Pro Ser 20 25 30 Leu Leu Cys Val Arg Gly Thr Ser Ala Asp His Cys Val Gln Leu Ile 35 40 45 Ala Ala Gln Glu Ala Asp Ala Ile Thr Leu Asp Gly Gly Ala Ile Tyr 50 55 60 Glu Ala Gly Lys Glu His Gly Leu Lys Pro Val Val Gly Glu Val Tyr 65 70 75 80 Asp Gln Glu Val Gly Thr Ser Tyr Tyr Ala Val Ala Val Val Arg Arg 85 90 95 Ser Ser His Val Thr Ile Asp Thr Leu Lys Gly Val Lys Ser Cys His 100 105 110 Thr Gly Ile Asn Arg Thr Val Gly Trp Asn Val Pro Val Gly Tyr Leu 115 120 125 Val Glu Ser Gly Arg Leu Ser Val Met Gly Cys Asp Val Leu Lys Ala 130 135 140 Val Ser Asp Tyr Phe Gly Gly Ser Cys Val Pro Gly Ala Gly Glu Thr 145 150 155 160 Ser Tyr Ser Glu Ser Leu Cys Arg Leu Cys Arg Gly Asp Ser Ser Gly 165 170 175 Glu Gly Val Cys Asp Lys Ser Pro Leu Glu Arg Tyr Tyr Asp Tyr Ser 180 185 190 Gly Ala Phe Arg Cys Leu Ala Glu Gly Ala Gly Asp Val Ala Phe Val 195 200 205 Lys His Ser Thr Val Leu Glu Asn Thr Asp Gly Lys Thr Leu Pro Ser 210 215 220 Trp Gly Gln Ala Leu Leu Ser Gln Asp Phe Glu Leu Leu Cys Arg Asp 225 230 235 240 Gly Ser Arg Ala Asp Val Thr Glu Trp Arg Gln Cys His Leu Ala Arg 245 250 255 Val Pro Ala His Ala Val Val Val Arg Ala Asp Thr Asp Gly Gly Leu 260 265 270 Ile Phe Arg Leu Leu Asn Glu Gly Gln Arg Leu Phe Ser His Glu Gly 275 280 285 Ser Ser Phe Gln Met Phe Ser Ser Glu Ala Tyr Gly Gln Lys Asp Leu 290 295 300 Leu Phe Lys Asp Ser Thr Ser Glu Leu Val Pro Ile Ala Thr Gln Thr 305 310 315 320 Tyr Glu Ala Trp Leu Gly His Glu Tyr Leu His Ala Met Lys Gly Leu 325 330 335 Leu Cys Asp Pro Asn Arg Leu Pro Pro Tyr Leu Arg Trp Cys Val Leu 340 345 350 Ser Thr Pro Glu Ile Gln Lys Cys Gly Asp Met Ala Val Ala Phe Arg 355 360 365 Arg

Gln Arg Leu Lys Pro Glu Ile Gln Cys Val Ser Ala Lys Ser Pro 370 375 380 Gln His Cys Met Glu Arg Ile Gln Ala Glu Gln Val Asp Ala Val Thr 385 390 395 400 Leu Ser Gly Glu Asp Ile Tyr Thr Ala Gly Lys Thr Tyr Gly Leu Val 405 410 415 Pro Ala Ala Gly Glu His Tyr Ala Pro Glu Asp Ser Ser Asn Ser Tyr 420 425 430 Tyr Val Val Ala Val Val Arg Arg Asp Ser Ser His Ala Phe Thr Leu 435 440 445 Asp Glu Leu Arg Gly Lys Arg Ser Cys His Ala Gly Phe Gly Ser Pro 450 455 460 Ala Gly Trp Asp Val Pro Val Gly Ala Leu Ile Gln Arg Gly Phe Ile 465 470 475 480 Arg Pro Lys Asp Cys Asp Val Leu Thr Ala Val Ser Glu Phe Phe Asn 485 490 495 Ala Ser Cys Val Pro Val Asn Asn Pro Lys Asn Tyr Pro Ser Ser Leu 500 505 510 Cys Ala Leu Cys Val Gly Asp Glu Gln Gly Arg Asn Lys Cys Val Gly 515 520 525 Asn Ser Gln Glu Arg Tyr Tyr Gly Tyr Arg Gly Ala Phe Arg Cys Leu 530 535 540 Val Glu Asn Ala Gly Asp Val Ala Phe Val Arg His Thr Thr Val Phe 545 550 555 560 Asp Asn Thr Asn 2122PRTHomo sapiens 21Gly His Asn Ser Glu Pro Trp Ala Ala Glu Leu Arg Ser Glu Asp Tyr 1 5 10 15 Glu Leu Leu Cys Pro Asn 20 2251PRTHomo sapiens 22Gly Ala Arg Ala Glu Val Ser Gln Phe Ala Ala Cys Asn Leu Ala Gln 1 5 10 15 Ile Pro Pro His Ala Val Met Val Arg Pro Asp Thr Asn Ile Phe Thr 20 25 30 Val Tyr Gly Leu Leu Asp Lys Ala Gln Asp Leu Phe Gly Asp Asp His 35 40 45 Asn Lys Asn 50 2353PRTHomo sapiens 23Gly Phe Lys Met Phe Asp Ser Ser Asn Tyr His Gly Gln Asp Leu Leu 1 5 10 15 Phe Lys Asp Ala Thr Val Arg Ala Val Pro Val Gly Glu Lys Thr Thr 20 25 30 Tyr Arg Gly Trp Leu Gly Leu Asp Tyr Val Ala Ala Leu Glu Gly Met 35 40 45 Ser Ser Gln Gln Cys 50 24586PRTHomo sapiens 24Gly Met Glu Val Arg Trp Cys Ala Thr Ser Asp Pro Glu Gln His Lys 1 5 10 15 Cys Gly Asn Met Ser Glu Ala Phe Arg Glu Ala Gly Ile Gln Pro Ser 20 25 30 Leu Leu Cys Val Arg Gly Thr Ser Ala Asp His Cys Val Gln Leu Ile 35 40 45 Ala Ala Gln Glu Ala Asp Ala Ile Thr Leu Asp Gly Gly Ala Ile Tyr 50 55 60 Glu Ala Gly Lys Glu His Gly Leu Lys Pro Val Val Gly Glu Val Tyr 65 70 75 80 Asp Gln Glu Val Gly Thr Ser Tyr Tyr Ala Val Ala Val Val Arg Arg 85 90 95 Ser Ser His Val Thr Ile Asp Thr Leu Lys Gly Val Lys Ser Cys His 100 105 110 Thr Gly Ile Asn Arg Thr Val Gly Trp Asn Val Pro Val Gly Tyr Leu 115 120 125 Val Glu Ser Gly Arg Leu Ser Val Met Gly Cys Asp Val Leu Lys Ala 130 135 140 Val Ser Asp Tyr Phe Gly Gly Ser Cys Val Pro Gly Ala Gly Glu Thr 145 150 155 160 Ser Tyr Ser Glu Ser Leu Cys Arg Leu Cys Arg Gly Asp Ser Ser Gly 165 170 175 Glu Gly Val Cys Asp Lys Ser Pro Leu Glu Arg Tyr Tyr Asp Tyr Ser 180 185 190 Gly Ala Phe Arg Cys Leu Ala Glu Gly Ala Gly Asp Val Ala Phe Val 195 200 205 Lys His Ser Thr Val Leu Glu Asn Thr Asp Gly Lys Thr Leu Pro Ser 210 215 220 Trp Gly Gln Ala Leu Leu Ser Gln Asp Phe Glu Leu Leu Cys Arg Asp 225 230 235 240 Gly Ser Arg Ala Asp Val Thr Glu Trp Arg Gln Cys His Leu Ala Arg 245 250 255 Val Pro Ala His Ala Val Val Val Arg Ala Asp Thr Asp Gly Gly Leu 260 265 270 Ile Phe Arg Leu Leu Asn Glu Gly Gln Arg Leu Phe Ser His Glu Gly 275 280 285 Ser Ser Phe Gln Met Phe Ser Ser Glu Ala Tyr Gly Gln Lys Asp Leu 290 295 300 Leu Phe Lys Asp Ser Thr Ser Glu Leu Val Pro Ile Ala Thr Gln Thr 305 310 315 320 Tyr Glu Ala Trp Leu Gly His Glu Tyr Leu His Ala Met Lys Gly Leu 325 330 335 Leu Cys Asp Pro Asn Arg Leu Pro Pro Tyr Leu Arg Trp Cys Val Leu 340 345 350 Ser Thr Pro Glu Ile Gln Lys Cys Gly Asp Met Ala Val Ala Phe Arg 355 360 365 Arg Gln Arg Leu Lys Pro Glu Ile Gln Cys Val Ser Ala Lys Ser Pro 370 375 380 Gln His Cys Met Glu Arg Ile Gln Ala Glu Gln Val Asp Ala Val Thr 385 390 395 400 Leu Ser Gly Glu Asp Ile Tyr Thr Ala Gly Lys Thr Tyr Gly Leu Val 405 410 415 Pro Ala Ala Gly Glu His Tyr Ala Pro Glu Asp Ser Ser Asn Ser Tyr 420 425 430 Tyr Val Val Ala Val Val Arg Arg Asp Ser Ser His Ala Phe Thr Leu 435 440 445 Asp Glu Leu Arg Gly Lys Arg Ser Cys His Ala Gly Phe Gly Ser Pro 450 455 460 Ala Gly Trp Asp Val Pro Val Gly Ala Leu Ile Gln Arg Gly Phe Ile 465 470 475 480 Arg Pro Lys Asp Cys Asp Val Leu Thr Ala Val Ser Glu Phe Phe Asn 485 490 495 Ala Ser Cys Val Pro Val Asn Asn Pro Lys Asn Tyr Pro Ser Ser Leu 500 505 510 Cys Ala Leu Cys Val Gly Asp Glu Gln Gly Arg Asn Lys Cys Val Gly 515 520 525 Asn Ser Gln Glu Arg Tyr Tyr Gly Tyr Arg Gly Ala Phe Arg Cys Leu 530 535 540 Val Glu Asn Ala Gly Asp Val Ala Phe Val Arg His Thr Thr Val Phe 545 550 555 560 Asp Asn Thr Asn Gly His Asn Ser Glu Pro Trp Ala Ala Glu Leu Arg 565 570 575 Ser Glu Asp Tyr Glu Leu Leu Cys Pro Asn 580 585 25637PRTHomo sapiens 25Gly Met Glu Val Arg Trp Cys Ala Thr Ser Asp Pro Glu Gln His Lys 1 5 10 15 Cys Gly Asn Met Ser Glu Ala Phe Arg Glu Ala Gly Ile Gln Pro Ser 20 25 30 Leu Leu Cys Val Arg Gly Thr Ser Ala Asp His Cys Val Gln Leu Ile 35 40 45 Ala Ala Gln Glu Ala Asp Ala Ile Thr Leu Asp Gly Gly Ala Ile Tyr 50 55 60 Glu Ala Gly Lys Glu His Gly Leu Lys Pro Val Val Gly Glu Val Tyr 65 70 75 80 Asp Gln Glu Val Gly Thr Ser Tyr Tyr Ala Val Ala Val Val Arg Arg 85 90 95 Ser Ser His Val Thr Ile Asp Thr Leu Lys Gly Val Lys Ser Cys His 100 105 110 Thr Gly Ile Asn Arg Thr Val Gly Trp Asn Val Pro Val Gly Tyr Leu 115 120 125 Val Glu Ser Gly Arg Leu Ser Val Met Gly Cys Asp Val Leu Lys Ala 130 135 140 Val Ser Asp Tyr Phe Gly Gly Ser Cys Val Pro Gly Ala Gly Glu Thr 145 150 155 160 Ser Tyr Ser Glu Ser Leu Cys Arg Leu Cys Arg Gly Asp Ser Ser Gly 165 170 175 Glu Gly Val Cys Asp Lys Ser Pro Leu Glu Arg Tyr Tyr Asp Tyr Ser 180 185 190 Gly Ala Phe Arg Cys Leu Ala Glu Gly Ala Gly Asp Val Ala Phe Val 195 200 205 Lys His Ser Thr Val Leu Glu Asn Thr Asp Gly Lys Thr Leu Pro Ser 210 215 220 Trp Gly Gln Ala Leu Leu Ser Gln Asp Phe Glu Leu Leu Cys Arg Asp 225 230 235 240 Gly Ser Arg Ala Asp Val Thr Glu Trp Arg Gln Cys His Leu Ala Arg 245 250 255 Val Pro Ala His Ala Val Val Val Arg Ala Asp Thr Asp Gly Gly Leu 260 265 270 Ile Phe Arg Leu Leu Asn Glu Gly Gln Arg Leu Phe Ser His Glu Gly 275 280 285 Ser Ser Phe Gln Met Phe Ser Ser Glu Ala Tyr Gly Gln Lys Asp Leu 290 295 300 Leu Phe Lys Asp Ser Thr Ser Glu Leu Val Pro Ile Ala Thr Gln Thr 305 310 315 320 Tyr Glu Ala Trp Leu Gly His Glu Tyr Leu His Ala Met Lys Gly Leu 325 330 335 Leu Cys Asp Pro Asn Arg Leu Pro Pro Tyr Leu Arg Trp Cys Val Leu 340 345 350 Ser Thr Pro Glu Ile Gln Lys Cys Gly Asp Met Ala Val Ala Phe Arg 355 360 365 Arg Gln Arg Leu Lys Pro Glu Ile Gln Cys Val Ser Ala Lys Ser Pro 370 375 380 Gln His Cys Met Glu Arg Ile Gln Ala Glu Gln Val Asp Ala Val Thr 385 390 395 400 Leu Ser Gly Glu Asp Ile Tyr Thr Ala Gly Lys Thr Tyr Gly Leu Val 405 410 415 Pro Ala Ala Gly Glu His Tyr Ala Pro Glu Asp Ser Ser Asn Ser Tyr 420 425 430 Tyr Val Val Ala Val Val Arg Arg Asp Ser Ser His Ala Phe Thr Leu 435 440 445 Asp Glu Leu Arg Gly Lys Arg Ser Cys His Ala Gly Phe Gly Ser Pro 450 455 460 Ala Gly Trp Asp Val Pro Val Gly Ala Leu Ile Gln Arg Gly Phe Ile 465 470 475 480 Arg Pro Lys Asp Cys Asp Val Leu Thr Ala Val Ser Glu Phe Phe Asn 485 490 495 Ala Ser Cys Val Pro Val Asn Asn Pro Lys Asn Tyr Pro Ser Ser Leu 500 505 510 Cys Ala Leu Cys Val Gly Asp Glu Gln Gly Arg Asn Lys Cys Val Gly 515 520 525 Asn Ser Gln Glu Arg Tyr Tyr Gly Tyr Arg Gly Ala Phe Arg Cys Leu 530 535 540 Val Glu Asn Ala Gly Asp Val Ala Phe Val Arg His Thr Thr Val Phe 545 550 555 560 Asp Asn Thr Asn Gly His Asn Ser Glu Pro Trp Ala Ala Glu Leu Arg 565 570 575 Ser Glu Asp Tyr Glu Leu Leu Cys Pro Asn Gly Ala Arg Ala Glu Val 580 585 590 Ser Gln Phe Ala Ala Cys Asn Leu Ala Gln Ile Pro Pro His Ala Val 595 600 605 Met Val Arg Pro Asp Thr Asn Ile Phe Thr Val Tyr Gly Leu Leu Asp 610 615 620 Lys Ala Gln Asp Leu Phe Gly Asp Asp His Asn Lys Asn 625 630 635 2673PRTHomo sapiens 26Gly His Asn Ser Glu Pro Trp Ala Ala Glu Leu Arg Ser Glu Asp Tyr 1 5 10 15 Glu Leu Leu Cys Pro Asn Gly Ala Arg Ala Glu Val Ser Gln Phe Ala 20 25 30 Ala Cys Asn Leu Ala Gln Ile Pro Pro His Ala Val Met Val Arg Pro 35 40 45 Asp Thr Asn Ile Phe Thr Val Tyr Gly Leu Leu Asp Lys Ala Gln Asp 50 55 60 Leu Phe Gly Asp Asp His Asn Lys Asn 65 70 27126PRTHomo sapiens 27Gly His Asn Ser Glu Pro Trp Ala Ala Glu Leu Arg Ser Glu Asp Tyr 1 5 10 15 Glu Leu Leu Cys Pro Asn Gly Ala Arg Ala Glu Val Ser Gln Phe Ala 20 25 30 Ala Cys Asn Leu Ala Gln Ile Pro Pro His Ala Val Met Val Arg Pro 35 40 45 Asp Thr Asn Ile Phe Thr Val Tyr Gly Leu Leu Asp Lys Ala Gln Asp 50 55 60 Leu Phe Gly Asp Asp His Asn Lys Asn Gly Phe Lys Met Phe Asp Ser 65 70 75 80 Ser Asn Tyr His Gly Gln Asp Leu Leu Phe Lys Asp Ala Thr Val Arg 85 90 95 Ala Val Pro Val Gly Glu Lys Thr Thr Tyr Arg Gly Trp Leu Gly Leu 100 105 110 Asp Tyr Val Ala Ala Leu Glu Gly Met Ser Ser Gln Gln Cys 115 120 125 28104PRTHomo sapiens 28Gly Ala Arg Ala Glu Val Ser Gln Phe Ala Ala Cys Asn Leu Ala Gln 1 5 10 15 Ile Pro Pro His Ala Val Met Val Arg Pro Asp Thr Asn Ile Phe Thr 20 25 30 Val Tyr Gly Leu Leu Asp Lys Ala Gln Asp Leu Phe Gly Asp Asp His 35 40 45 Asn Lys Asn Gly Phe Lys Met Phe Asp Ser Ser Asn Tyr His Gly Gln 50 55 60 Asp Leu Leu Phe Lys Asp Ala Thr Val Arg Ala Val Pro Val Gly Glu 65 70 75 80 Lys Thr Thr Tyr Arg Gly Trp Leu Gly Leu Asp Tyr Val Ala Ala Leu 85 90 95 Glu Gly Met Ser Ser Gln Gln Cys 100 29450PRTHomo sapiens 29Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr

Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 30450PRTArtificial sequenceModified Trastuzumab Sequence 30Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Trp Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Thr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 31240PRTHomo sapiens 31Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 1 5 10 15 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 20 25 30 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 35 40 45 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 50 55 60 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 65 70 75 80 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 85 90 95 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 100 105 110 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 115 120 125 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 130 135 140 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 145 150 155 160 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 165 170 175 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 180 185 190 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 195 200 205 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 210 215 220 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 225 230 235 240 32227PRTHomo sapiens 32Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 33240PRTArtificial SequenceModified Trastuzumab Sequence 33Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 1 5 10 15 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 20 25 30 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 35 40 45 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 50 55 60 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 65 70 75 80 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 85 90 95 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 100 105 110 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 115 120 125 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 130 135 140 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 145 150 155 160 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 165 170 175 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Trp Pro Pro Val Leu Asp 180 185 190 Ser Asp Gly Ser Phe Phe Leu Thr Ser Lys Leu Thr Val Asp Lys Ser 195 200 205 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 210 215 220 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 225 230 235 240 34227PRTArtificial SequenceModified Trastuzumab Sequence 34Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Trp Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Thr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 35450PRTArtificial SequenceModified Trastuzumab Sequence 35Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Tyr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Ala Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 36214PRTHomo sapiens 36Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 371142PRTArtificial Sequencep97-trastuzumab heavy chain fusion protein 37Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185

190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys Gly Met Glu Val Arg Trp Cys Ala Thr Ser Asp Pro Glu Gln 450 455 460 His Lys Cys Gly Asn Met Ser Glu Ala Phe Arg Glu Ala Gly Ile Gln 465 470 475 480 Pro Ser Leu Leu Cys Val Arg Gly Thr Ser Ala Asp His Cys Val Gln 485 490 495 Leu Ile Ala Ala Gln Glu Ala Asp Ala Ile Thr Leu Asp Gly Gly Ala 500 505 510 Ile Tyr Glu Ala Gly Lys Glu His Gly Leu Lys Pro Val Val Gly Glu 515 520 525 Val Tyr Asp Gln Glu Val Gly Thr Ser Tyr Tyr Ala Val Ala Val Val 530 535 540 Arg Arg Ser Ser His Val Thr Ile Asp Thr Leu Lys Gly Val Lys Ser 545 550 555 560 Cys His Thr Gly Ile Asn Arg Thr Val Gly Trp Asn Val Pro Val Gly 565 570 575 Tyr Leu Val Glu Ser Gly Arg Leu Ser Val Met Gly Cys Asp Val Leu 580 585 590 Lys Ala Val Ser Asp Tyr Phe Gly Gly Ser Cys Val Pro Gly Ala Gly 595 600 605 Glu Thr Ser Tyr Ser Glu Ser Leu Cys Arg Leu Cys Arg Gly Asp Ser 610 615 620 Ser Gly Glu Gly Val Cys Asp Lys Ser Pro Leu Glu Arg Tyr Tyr Asp 625 630 635 640 Tyr Ser Gly Ala Phe Arg Cys Leu Ala Glu Gly Ala Gly Asp Val Ala 645 650 655 Phe Val Lys His Ser Thr Val Leu Glu Asn Thr Asp Gly Lys Thr Leu 660 665 670 Pro Ser Trp Gly Gln Ala Leu Leu Ser Gln Asp Phe Glu Leu Leu Cys 675 680 685 Arg Asp Gly Ser Arg Ala Asp Val Thr Glu Trp Arg Gln Cys His Leu 690 695 700 Ala Arg Val Pro Ala His Ala Val Val Val Arg Ala Asp Thr Asp Gly 705 710 715 720 Gly Leu Ile Phe Arg Leu Leu Asn Glu Gly Gln Arg Leu Phe Ser His 725 730 735 Glu Gly Ser Ser Phe Gln Met Phe Ser Ser Glu Ala Tyr Gly Gln Lys 740 745 750 Asp Leu Leu Phe Lys Asp Ser Thr Ser Glu Leu Val Pro Ile Ala Thr 755 760 765 Gln Thr Tyr Glu Ala Trp Leu Gly His Glu Tyr Leu His Ala Met Lys 770 775 780 Gly Leu Leu Cys Asp Pro Asn Arg Leu Pro Pro Tyr Leu Arg Trp Cys 785 790 795 800 Val Leu Ser Thr Pro Glu Ile Gln Lys Cys Gly Asp Met Ala Val Ala 805 810 815 Phe Arg Arg Gln Arg Leu Lys Pro Glu Ile Gln Cys Val Ser Ala Lys 820 825 830 Ser Pro Gln His Cys Met Glu Arg Ile Gln Ala Glu Gln Val Asp Ala 835 840 845 Val Thr Leu Ser Gly Glu Asp Ile Tyr Thr Ala Gly Lys Thr Tyr Gly 850 855 860 Leu Val Pro Ala Ala Gly Glu His Tyr Ala Pro Glu Asp Ser Ser Asn 865 870 875 880 Ser Tyr Tyr Val Val Ala Val Val Arg Arg Asp Ser Ser His Ala Phe 885 890 895 Thr Leu Asp Glu Leu Arg Gly Lys Arg Ser Cys His Ala Gly Phe Gly 900 905 910 Ser Pro Ala Gly Trp Asp Val Pro Val Gly Ala Leu Ile Gln Arg Gly 915 920 925 Phe Ile Arg Pro Lys Asp Cys Asp Val Leu Thr Ala Val Ser Glu Phe 930 935 940 Phe Asn Ala Ser Cys Val Pro Val Asn Asn Pro Lys Asn Tyr Pro Ser 945 950 955 960 Ser Leu Cys Ala Leu Cys Val Gly Asp Glu Gln Gly Arg Asn Lys Cys 965 970 975 Val Gly Asn Ser Gln Glu Arg Tyr Tyr Gly Tyr Arg Gly Ala Phe Arg 980 985 990 Cys Leu Val Glu Asn Ala Gly Asp Val Ala Phe Val Arg His Thr Thr 995 1000 1005 Val Phe Asp Asn Thr Asn Gly His Asn Ser Glu Pro Trp Ala Ala 1010 1015 1020 Glu Leu Arg Ser Glu Asp Tyr Glu Leu Leu Cys Pro Asn Gly Ala 1025 1030 1035 Arg Ala Glu Val Ser Gln Phe Ala Ala Cys Asn Leu Ala Gln Ile 1040 1045 1050 Pro Pro His Ala Val Met Val Arg Pro Asp Thr Asn Ile Phe Thr 1055 1060 1065 Val Tyr Gly Leu Leu Asp Lys Ala Gln Asp Leu Phe Gly Asp Asp 1070 1075 1080 His Asn Lys Asn Gly Phe Lys Met Phe Asp Ser Ser Asn Tyr His 1085 1090 1095 Gly Gln Asp Leu Leu Phe Lys Asp Ala Thr Val Arg Ala Val Pro 1100 1105 1110 Val Gly Glu Lys Thr Thr Tyr Arg Gly Trp Leu Gly Leu Asp Tyr 1115 1120 1125 Val Ala Ala Leu Glu Gly Met Ser Ser Gln Gln Cys Ser Gly 1130 1135 1140 381157PRTArtificial Sequencep97-trastuzumab heavy chain fusion protein 38Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 450 455 460 Ser Gly Met Glu Val Arg Trp Cys Ala Thr Ser Asp Pro Glu Gln His 465 470 475 480 Lys Cys Gly Asn Met Ser Glu Ala Phe Arg Glu Ala Gly Ile Gln Pro 485 490 495 Ser Leu Leu Cys Val Arg Gly Thr Ser Ala Asp His Cys Val Gln Leu 500 505 510 Ile Ala Ala Gln Glu Ala Asp Ala Ile Thr Leu Asp Gly Gly Ala Ile 515 520 525 Tyr Glu Ala Gly Lys Glu His Gly Leu Lys Pro Val Val Gly Glu Val 530 535 540 Tyr Asp Gln Glu Val Gly Thr Ser Tyr Tyr Ala Val Ala Val Val Arg 545 550 555 560 Arg Ser Ser His Val Thr Ile Asp Thr Leu Lys Gly Val Lys Ser Cys 565 570 575 His Thr Gly Ile Asn Arg Thr Val Gly Trp Asn Val Pro Val Gly Tyr 580 585 590 Leu Val Glu Ser Gly Arg Leu Ser Val Met Gly Cys Asp Val Leu Lys 595 600 605 Ala Val Ser Asp Tyr Phe Gly Gly Ser Cys Val Pro Gly Ala Gly Glu 610 615 620 Thr Ser Tyr Ser Glu Ser Leu Cys Arg Leu Cys Arg Gly Asp Ser Ser 625 630 635 640 Gly Glu Gly Val Cys Asp Lys Ser Pro Leu Glu Arg Tyr Tyr Asp Tyr 645 650 655 Ser Gly Ala Phe Arg Cys Leu Ala Glu Gly Ala Gly Asp Val Ala Phe 660 665 670 Val Lys His Ser Thr Val Leu Glu Asn Thr Asp Gly Lys Thr Leu Pro 675 680 685 Ser Trp Gly Gln Ala Leu Leu Ser Gln Asp Phe Glu Leu Leu Cys Arg 690 695 700 Asp Gly Ser Arg Ala Asp Val Thr Glu Trp Arg Gln Cys His Leu Ala 705 710 715 720 Arg Val Pro Ala His Ala Val Val Val Arg Ala Asp Thr Asp Gly Gly 725 730 735 Leu Ile Phe Arg Leu Leu Asn Glu Gly Gln Arg Leu Phe Ser His Glu 740 745 750 Gly Ser Ser Phe Gln Met Phe Ser Ser Glu Ala Tyr Gly Gln Lys Asp 755 760 765 Leu Leu Phe Lys Asp Ser Thr Ser Glu Leu Val Pro Ile Ala Thr Gln 770 775 780 Thr Tyr Glu Ala Trp Leu Gly His Glu Tyr Leu His Ala Met Lys Gly 785 790 795 800 Leu Leu Cys Asp Pro Asn Arg Leu Pro Pro Tyr Leu Arg Trp Cys Val 805 810 815 Leu Ser Thr Pro Glu Ile Gln Lys Cys Gly Asp Met Ala Val Ala Phe 820 825 830 Arg Arg Gln Arg Leu Lys Pro Glu Ile Gln Cys Val Ser Ala Lys Ser 835 840 845 Pro Gln His Cys Met Glu Arg Ile Gln Ala Glu Gln Val Asp Ala Val 850 855 860 Thr Leu Ser Gly Glu Asp Ile Tyr Thr Ala Gly Lys Thr Tyr Gly Leu 865 870 875 880 Val Pro Ala Ala Gly Glu His Tyr Ala Pro Glu Asp Ser Ser Asn Ser 885 890 895 Tyr Tyr Val Val Ala Val Val Arg Arg Asp Ser Ser His Ala Phe Thr 900 905 910 Leu Asp Glu Leu Arg Gly Lys Arg Ser Cys His Ala Gly Phe Gly Ser 915 920 925 Pro Ala Gly Trp Asp Val Pro Val Gly Ala Leu Ile Gln Arg Gly Phe 930 935 940 Ile Arg Pro Lys Asp Cys Asp Val Leu Thr Ala Val Ser Glu Phe Phe 945 950 955 960 Asn Ala Ser Cys Val Pro Val Asn Asn Pro Lys Asn Tyr Pro Ser Ser 965 970 975 Leu Cys Ala Leu Cys Val Gly Asp Glu Gln Gly Arg Asn Lys Cys Val 980 985 990 Gly Asn Ser Gln Glu Arg Tyr Tyr Gly Tyr Arg Gly Ala Phe Arg Cys 995 1000 1005 Leu Val Glu Asn Ala Gly Asp Val Ala Phe Val Arg His Thr Thr 1010 1015 1020 Val Phe Asp Asn Thr Asn Gly His Asn Ser Glu Pro Trp Ala Ala 1025 1030 1035 Glu Leu Arg Ser Glu Asp Tyr Glu Leu Leu Cys Pro Asn Gly Ala 1040 1045 1050 Arg Ala Glu Val Ser Gln Phe Ala Ala Cys Asn Leu Ala Gln Ile 1055 1060 1065 Pro Pro His Ala Val Met Val Arg Pro Asp Thr Asn Ile Phe Thr 1070 1075 1080 Val Tyr Gly Leu Leu Asp Lys Ala Gln Asp Leu Phe Gly Asp Asp 1085 1090 1095 His Asn Lys Asn Gly Phe Lys Met Phe Asp Ser Ser Asn Tyr His 1100 1105 1110 Gly Gln Asp Leu Leu Phe Lys Asp Ala Thr Val Arg Ala Val Pro 1115 1120 1125 Val Gly Glu Lys Thr Thr Tyr Arg Gly Trp Leu Gly Leu Asp Tyr 1130 1135 1140 Val Ala Ala Leu Glu Gly Met Ser Ser Gln Gln Cys Ser Gly 1145 1150 1155 39931PRTArtificial Sequencep97-trastuzumab heavy chain fusion protein 39Gly Met Glu Val Arg Trp Cys Ala Thr Ser Asp Pro Glu Gln His Lys 1 5 10 15 Cys Gly Asn Met Ser Glu Ala Phe Arg Glu Ala Gly Ile Gln Pro Ser 20 25 30 Leu Leu Cys Val Arg Gly Thr Ser Ala Asp His Cys Val Gln Leu Ile 35 40 45 Ala Ala Gln Glu Ala Asp Ala Ile Thr Leu Asp Gly Gly Ala Ile Tyr 50 55 60 Glu Ala Gly Lys Glu His Gly Leu Lys Pro Val Val Gly Glu Val Tyr 65 70 75 80 Asp Gln Glu Val Gly Thr Ser Tyr Tyr Ala Val Ala Val Val Arg Arg 85 90 95 Ser Ser His Val Thr Ile Asp Thr Leu Lys Gly Val Lys Ser Cys His 100 105 110 Thr Gly Ile Asn Arg Thr Val Gly Trp Asn Val Pro Val Gly Tyr Leu 115 120 125 Val Glu Ser Gly Arg Leu Ser Val Met Gly Cys Asp Val Leu Lys Ala 130 135 140 Val Ser Asp Tyr Phe Gly Gly Ser Cys Val Pro Gly Ala Gly Glu Thr 145 150 155 160 Ser Tyr Ser Glu Ser Leu Cys Arg Leu Cys Arg

Gly Asp Ser Ser Gly 165 170 175 Glu Gly Val Cys Asp Lys Ser Pro Leu Glu Arg Tyr Tyr Asp Tyr Ser 180 185 190 Gly Ala Phe Arg Cys Leu Ala Glu Gly Ala Gly Asp Val Ala Phe Val 195 200 205 Lys His Ser Thr Val Leu Glu Asn Thr Asp Gly Lys Thr Leu Pro Ser 210 215 220 Trp Gly Gln Ala Leu Leu Ser Gln Asp Phe Glu Leu Leu Cys Arg Asp 225 230 235 240 Gly Ser Arg Ala Asp Val Thr Glu Trp Arg Gln Cys His Leu Ala Arg 245 250 255 Val Pro Ala His Ala Val Val Val Arg Ala Asp Thr Asp Gly Gly Leu 260 265 270 Ile Phe Arg Leu Leu Asn Glu Gly Gln Arg Leu Phe Ser His Glu Gly 275 280 285 Ser Ser Phe Gln Met Phe Ser Ser Glu Ala Tyr Gly Gln Lys Asp Leu 290 295 300 Leu Phe Lys Asp Ser Thr Ser Glu Leu Val Pro Ile Ala Thr Gln Thr 305 310 315 320 Tyr Glu Ala Trp Leu Gly His Glu Tyr Leu His Ala Met Lys Gly Leu 325 330 335 Leu Cys Asp Pro Asn Arg Leu Pro Pro Tyr Leu Arg Trp Cys Val Leu 340 345 350 Ser Thr Pro Glu Ile Gln Lys Cys Gly Asp Met Ala Val Ala Phe Arg 355 360 365 Arg Gln Arg Leu Lys Pro Glu Ile Gln Cys Val Ser Ala Lys Ser Pro 370 375 380 Gln His Cys Met Glu Arg Ile Gln Ala Glu Gln Val Asp Ala Val Thr 385 390 395 400 Leu Ser Gly Glu Asp Ile Tyr Thr Ala Gly Lys Thr Tyr Gly Leu Val 405 410 415 Pro Ala Ala Gly Glu His Tyr Ala Pro Glu Asp Ser Ser Asn Ser Tyr 420 425 430 Tyr Val Val Ala Val Val Arg Arg Asp Ser Ser His Ala Phe Thr Leu 435 440 445 Asp Glu Leu Arg Gly Lys Arg Ser Cys His Ala Gly Phe Gly Ser Pro 450 455 460 Ala Gly Trp Asp Val Pro Val Gly Ala Leu Ile Gln Arg Gly Phe Ile 465 470 475 480 Arg Pro Lys Asp Cys Asp Val Leu Thr Ala Val Ser Glu Phe Phe Asn 485 490 495 Ala Ser Cys Val Pro Val Asn Asn Pro Lys Asn Tyr Pro Ser Ser Leu 500 505 510 Cys Ala Leu Cys Val Gly Asp Glu Gln Gly Arg Asn Lys Cys Val Gly 515 520 525 Asn Ser Gln Glu Arg Tyr Tyr Gly Tyr Arg Gly Ala Phe Arg Cys Leu 530 535 540 Val Glu Asn Ala Gly Asp Val Ala Phe Val Arg His Thr Thr Val Phe 545 550 555 560 Asp Asn Thr Asn Gly His Asn Ser Glu Pro Trp Ala Ala Glu Leu Arg 565 570 575 Ser Glu Asp Tyr Glu Leu Leu Cys Pro Asn Gly Ala Arg Ala Glu Val 580 585 590 Ser Gln Phe Ala Ala Cys Asn Leu Ala Gln Ile Pro Pro His Ala Val 595 600 605 Met Val Arg Pro Asp Thr Asn Ile Phe Thr Val Tyr Gly Leu Leu Asp 610 615 620 Lys Ala Gln Asp Leu Phe Gly Asp Asp His Asn Lys Asn Gly Phe Lys 625 630 635 640 Met Phe Asp Ser Ser Asn Tyr His Gly Gln Asp Leu Leu Phe Lys Asp 645 650 655 Ala Thr Val Arg Ala Val Pro Val Gly Glu Lys Thr Thr Tyr Arg Gly 660 665 670 Trp Leu Gly Leu Asp Tyr Val Ala Ala Leu Glu Gly Met Ser Ser Gln 675 680 685 Gln Cys Gly Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 690 695 700 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 705 710 715 720 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 725 730 735 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 740 745 750 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 755 760 765 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 770 775 780 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 785 790 795 800 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 805 810 815 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 820 825 830 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 835 840 845 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 850 855 860 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 865 870 875 880 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 885 890 895 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 900 905 910 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 915 920 925 Pro Gly Lys 930 40946PRTArtificial Sequencep97-trastuzumab heavy chain fusion protein 40Gly Met Glu Val Arg Trp Cys Ala Thr Ser Asp Pro Glu Gln His Lys 1 5 10 15 Cys Gly Asn Met Ser Glu Ala Phe Arg Glu Ala Gly Ile Gln Pro Ser 20 25 30 Leu Leu Cys Val Arg Gly Thr Ser Ala Asp His Cys Val Gln Leu Ile 35 40 45 Ala Ala Gln Glu Ala Asp Ala Ile Thr Leu Asp Gly Gly Ala Ile Tyr 50 55 60 Glu Ala Gly Lys Glu His Gly Leu Lys Pro Val Val Gly Glu Val Tyr 65 70 75 80 Asp Gln Glu Val Gly Thr Ser Tyr Tyr Ala Val Ala Val Val Arg Arg 85 90 95 Ser Ser His Val Thr Ile Asp Thr Leu Lys Gly Val Lys Ser Cys His 100 105 110 Thr Gly Ile Asn Arg Thr Val Gly Trp Asn Val Pro Val Gly Tyr Leu 115 120 125 Val Glu Ser Gly Arg Leu Ser Val Met Gly Cys Asp Val Leu Lys Ala 130 135 140 Val Ser Asp Tyr Phe Gly Gly Ser Cys Val Pro Gly Ala Gly Glu Thr 145 150 155 160 Ser Tyr Ser Glu Ser Leu Cys Arg Leu Cys Arg Gly Asp Ser Ser Gly 165 170 175 Glu Gly Val Cys Asp Lys Ser Pro Leu Glu Arg Tyr Tyr Asp Tyr Ser 180 185 190 Gly Ala Phe Arg Cys Leu Ala Glu Gly Ala Gly Asp Val Ala Phe Val 195 200 205 Lys His Ser Thr Val Leu Glu Asn Thr Asp Gly Lys Thr Leu Pro Ser 210 215 220 Trp Gly Gln Ala Leu Leu Ser Gln Asp Phe Glu Leu Leu Cys Arg Asp 225 230 235 240 Gly Ser Arg Ala Asp Val Thr Glu Trp Arg Gln Cys His Leu Ala Arg 245 250 255 Val Pro Ala His Ala Val Val Val Arg Ala Asp Thr Asp Gly Gly Leu 260 265 270 Ile Phe Arg Leu Leu Asn Glu Gly Gln Arg Leu Phe Ser His Glu Gly 275 280 285 Ser Ser Phe Gln Met Phe Ser Ser Glu Ala Tyr Gly Gln Lys Asp Leu 290 295 300 Leu Phe Lys Asp Ser Thr Ser Glu Leu Val Pro Ile Ala Thr Gln Thr 305 310 315 320 Tyr Glu Ala Trp Leu Gly His Glu Tyr Leu His Ala Met Lys Gly Leu 325 330 335 Leu Cys Asp Pro Asn Arg Leu Pro Pro Tyr Leu Arg Trp Cys Val Leu 340 345 350 Ser Thr Pro Glu Ile Gln Lys Cys Gly Asp Met Ala Val Ala Phe Arg 355 360 365 Arg Gln Arg Leu Lys Pro Glu Ile Gln Cys Val Ser Ala Lys Ser Pro 370 375 380 Gln His Cys Met Glu Arg Ile Gln Ala Glu Gln Val Asp Ala Val Thr 385 390 395 400 Leu Ser Gly Glu Asp Ile Tyr Thr Ala Gly Lys Thr Tyr Gly Leu Val 405 410 415 Pro Ala Ala Gly Glu His Tyr Ala Pro Glu Asp Ser Ser Asn Ser Tyr 420 425 430 Tyr Val Val Ala Val Val Arg Arg Asp Ser Ser His Ala Phe Thr Leu 435 440 445 Asp Glu Leu Arg Gly Lys Arg Ser Cys His Ala Gly Phe Gly Ser Pro 450 455 460 Ala Gly Trp Asp Val Pro Val Gly Ala Leu Ile Gln Arg Gly Phe Ile 465 470 475 480 Arg Pro Lys Asp Cys Asp Val Leu Thr Ala Val Ser Glu Phe Phe Asn 485 490 495 Ala Ser Cys Val Pro Val Asn Asn Pro Lys Asn Tyr Pro Ser Ser Leu 500 505 510 Cys Ala Leu Cys Val Gly Asp Glu Gln Gly Arg Asn Lys Cys Val Gly 515 520 525 Asn Ser Gln Glu Arg Tyr Tyr Gly Tyr Arg Gly Ala Phe Arg Cys Leu 530 535 540 Val Glu Asn Ala Gly Asp Val Ala Phe Val Arg His Thr Thr Val Phe 545 550 555 560 Asp Asn Thr Asn Gly His Asn Ser Glu Pro Trp Ala Ala Glu Leu Arg 565 570 575 Ser Glu Asp Tyr Glu Leu Leu Cys Pro Asn Gly Ala Arg Ala Glu Val 580 585 590 Ser Gln Phe Ala Ala Cys Asn Leu Ala Gln Ile Pro Pro His Ala Val 595 600 605 Met Val Arg Pro Asp Thr Asn Ile Phe Thr Val Tyr Gly Leu Leu Asp 610 615 620 Lys Ala Gln Asp Leu Phe Gly Asp Asp His Asn Lys Asn Gly Phe Lys 625 630 635 640 Met Phe Asp Ser Ser Asn Tyr His Gly Gln Asp Leu Leu Phe Lys Asp 645 650 655 Ala Thr Val Arg Ala Val Pro Val Gly Glu Lys Thr Thr Tyr Arg Gly 660 665 670 Trp Leu Gly Leu Asp Tyr Val Ala Ala Leu Glu Gly Met Ser Ser Gln 675 680 685 Gln Cys Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 690 695 700 Gly Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 705 710 715 720 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 725 730 735 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 740 745 750 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 755 760 765 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 770 775 780 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 785 790 795 800 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 805 810 815 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 820 825 830 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 835 840 845 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 850 855 860 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 865 870 875 880 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 885 890 895 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 900 905 910 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 915 920 925 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 930 935 940 Gly Lys 945 41918PRTArtificial Sequencep97-trastuzumab heavy chain fusion protein 41Gly Met Glu Val Arg Trp Cys Ala Thr Ser Asp Pro Glu Gln His Lys 1 5 10 15 Cys Gly Asn Met Ser Glu Ala Phe Arg Glu Ala Gly Ile Gln Pro Ser 20 25 30 Leu Leu Cys Val Arg Gly Thr Ser Ala Asp His Cys Val Gln Leu Ile 35 40 45 Ala Ala Gln Glu Ala Asp Ala Ile Thr Leu Asp Gly Gly Ala Ile Tyr 50 55 60 Glu Ala Gly Lys Glu His Gly Leu Lys Pro Val Val Gly Glu Val Tyr 65 70 75 80 Asp Gln Glu Val Gly Thr Ser Tyr Tyr Ala Val Ala Val Val Arg Arg 85 90 95 Ser Ser His Val Thr Ile Asp Thr Leu Lys Gly Val Lys Ser Cys His 100 105 110 Thr Gly Ile Asn Arg Thr Val Gly Trp Asn Val Pro Val Gly Tyr Leu 115 120 125 Val Glu Ser Gly Arg Leu Ser Val Met Gly Cys Asp Val Leu Lys Ala 130 135 140 Val Ser Asp Tyr Phe Gly Gly Ser Cys Val Pro Gly Ala Gly Glu Thr 145 150 155 160 Ser Tyr Ser Glu Ser Leu Cys Arg Leu Cys Arg Gly Asp Ser Ser Gly 165 170 175 Glu Gly Val Cys Asp Lys Ser Pro Leu Glu Arg Tyr Tyr Asp Tyr Ser 180 185 190 Gly Ala Phe Arg Cys Leu Ala Glu Gly Ala Gly Asp Val Ala Phe Val 195 200 205 Lys His Ser Thr Val Leu Glu Asn Thr Asp Gly Lys Thr Leu Pro Ser 210 215 220 Trp Gly Gln Ala Leu Leu Ser Gln Asp Phe Glu Leu Leu Cys Arg Asp 225 230 235 240 Gly Ser Arg Ala Asp Val Thr Glu Trp Arg Gln Cys His Leu Ala Arg 245 250 255 Val Pro Ala His Ala Val Val Val Arg Ala Asp Thr Asp Gly Gly Leu 260 265 270 Ile Phe Arg Leu Leu Asn Glu Gly Gln Arg Leu Phe Ser His Glu Gly 275 280 285 Ser Ser Phe Gln Met Phe Ser Ser Glu Ala Tyr Gly Gln Lys Asp Leu 290 295 300 Leu Phe Lys Asp Ser Thr Ser Glu Leu Val Pro Ile Ala Thr Gln Thr 305 310 315 320 Tyr Glu Ala Trp Leu Gly His Glu Tyr Leu His Ala Met Lys Gly Leu 325 330 335 Leu Cys Asp Pro Asn Arg Leu Pro Pro Tyr Leu Arg Trp Cys Val Leu 340 345 350 Ser Thr Pro Glu Ile Gln Lys Cys Gly Asp Met Ala Val Ala Phe Arg 355 360 365 Arg Gln Arg Leu Lys Pro Glu Ile Gln Cys Val Ser Ala Lys Ser Pro 370 375 380 Gln His Cys Met Glu Arg Ile Gln Ala Glu Gln Val Asp Ala Val Thr 385 390 395 400 Leu Ser Gly Glu Asp Ile Tyr Thr Ala Gly Lys Thr Tyr Gly Leu Val 405 410 415 Pro Ala Ala Gly Glu His Tyr Ala Pro Glu Asp Ser Ser Asn Ser Tyr 420 425 430 Tyr Val Val Ala Val Val Arg Arg Asp Ser Ser His Ala Phe Thr Leu 435 440 445 Asp Glu Leu Arg Gly Lys Arg Ser Cys His Ala Gly Phe Gly Ser Pro 450 455 460 Ala Gly Trp Asp Val Pro Val Gly Ala Leu Ile Gln Arg Gly Phe Ile 465 470 475 480 Arg Pro Lys Asp Cys Asp Val Leu Thr Ala Val Ser Glu Phe Phe Asn 485 490 495 Ala Ser Cys Val Pro Val Asn Asn Pro Lys Asn Tyr Pro Ser Ser Leu 500 505 510 Cys Ala Leu Cys Val Gly Asp Glu Gln Gly Arg Asn Lys Cys Val Gly 515 520 525 Asn Ser Gln Glu Arg Tyr Tyr Gly Tyr Arg Gly Ala Phe Arg Cys Leu 530 535 540 Val Glu Asn Ala Gly Asp Val Ala Phe Val Arg His Thr Thr Val Phe 545 550 555 560 Asp Asn Thr Asn Gly His Asn Ser Glu Pro Trp Ala Ala Glu Leu Arg 565 570 575 Ser Glu

Asp Tyr Glu Leu Leu Cys Pro Asn Gly Ala Arg Ala Glu Val 580 585 590 Ser Gln Phe Ala Ala Cys Asn Leu Ala Gln Ile Pro Pro His Ala Val 595 600 605 Met Val Arg Pro Asp Thr Asn Ile Phe Thr Val Tyr Gly Leu Leu Asp 610 615 620 Lys Ala Gln Asp Leu Phe Gly Asp Asp His Asn Lys Asn Gly Phe Lys 625 630 635 640 Met Phe Asp Ser Ser Asn Tyr His Gly Gln Asp Leu Leu Phe Lys Asp 645 650 655 Ala Thr Val Arg Ala Val Pro Val Gly Glu Lys Thr Thr Tyr Arg Gly 660 665 670 Trp Leu Gly Leu Asp Tyr Val Ala Ala Leu Glu Gly Met Ser Ser Gln 675 680 685 Gln Cys Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 690 695 700 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 705 710 715 720 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 725 730 735 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 740 745 750 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 755 760 765 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 770 775 780 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 785 790 795 800 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 805 810 815 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 820 825 830 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 835 840 845 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 850 855 860 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 865 870 875 880 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 885 890 895 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 900 905 910 Ser Leu Ser Pro Gly Lys 915 42933PRTArtificial Sequencep97-trastuzumab heavy chain fusion protein 42Gly Met Glu Val Arg Trp Cys Ala Thr Ser Asp Pro Glu Gln His Lys 1 5 10 15 Cys Gly Asn Met Ser Glu Ala Phe Arg Glu Ala Gly Ile Gln Pro Ser 20 25 30 Leu Leu Cys Val Arg Gly Thr Ser Ala Asp His Cys Val Gln Leu Ile 35 40 45 Ala Ala Gln Glu Ala Asp Ala Ile Thr Leu Asp Gly Gly Ala Ile Tyr 50 55 60 Glu Ala Gly Lys Glu His Gly Leu Lys Pro Val Val Gly Glu Val Tyr 65 70 75 80 Asp Gln Glu Val Gly Thr Ser Tyr Tyr Ala Val Ala Val Val Arg Arg 85 90 95 Ser Ser His Val Thr Ile Asp Thr Leu Lys Gly Val Lys Ser Cys His 100 105 110 Thr Gly Ile Asn Arg Thr Val Gly Trp Asn Val Pro Val Gly Tyr Leu 115 120 125 Val Glu Ser Gly Arg Leu Ser Val Met Gly Cys Asp Val Leu Lys Ala 130 135 140 Val Ser Asp Tyr Phe Gly Gly Ser Cys Val Pro Gly Ala Gly Glu Thr 145 150 155 160 Ser Tyr Ser Glu Ser Leu Cys Arg Leu Cys Arg Gly Asp Ser Ser Gly 165 170 175 Glu Gly Val Cys Asp Lys Ser Pro Leu Glu Arg Tyr Tyr Asp Tyr Ser 180 185 190 Gly Ala Phe Arg Cys Leu Ala Glu Gly Ala Gly Asp Val Ala Phe Val 195 200 205 Lys His Ser Thr Val Leu Glu Asn Thr Asp Gly Lys Thr Leu Pro Ser 210 215 220 Trp Gly Gln Ala Leu Leu Ser Gln Asp Phe Glu Leu Leu Cys Arg Asp 225 230 235 240 Gly Ser Arg Ala Asp Val Thr Glu Trp Arg Gln Cys His Leu Ala Arg 245 250 255 Val Pro Ala His Ala Val Val Val Arg Ala Asp Thr Asp Gly Gly Leu 260 265 270 Ile Phe Arg Leu Leu Asn Glu Gly Gln Arg Leu Phe Ser His Glu Gly 275 280 285 Ser Ser Phe Gln Met Phe Ser Ser Glu Ala Tyr Gly Gln Lys Asp Leu 290 295 300 Leu Phe Lys Asp Ser Thr Ser Glu Leu Val Pro Ile Ala Thr Gln Thr 305 310 315 320 Tyr Glu Ala Trp Leu Gly His Glu Tyr Leu His Ala Met Lys Gly Leu 325 330 335 Leu Cys Asp Pro Asn Arg Leu Pro Pro Tyr Leu Arg Trp Cys Val Leu 340 345 350 Ser Thr Pro Glu Ile Gln Lys Cys Gly Asp Met Ala Val Ala Phe Arg 355 360 365 Arg Gln Arg Leu Lys Pro Glu Ile Gln Cys Val Ser Ala Lys Ser Pro 370 375 380 Gln His Cys Met Glu Arg Ile Gln Ala Glu Gln Val Asp Ala Val Thr 385 390 395 400 Leu Ser Gly Glu Asp Ile Tyr Thr Ala Gly Lys Thr Tyr Gly Leu Val 405 410 415 Pro Ala Ala Gly Glu His Tyr Ala Pro Glu Asp Ser Ser Asn Ser Tyr 420 425 430 Tyr Val Val Ala Val Val Arg Arg Asp Ser Ser His Ala Phe Thr Leu 435 440 445 Asp Glu Leu Arg Gly Lys Arg Ser Cys His Ala Gly Phe Gly Ser Pro 450 455 460 Ala Gly Trp Asp Val Pro Val Gly Ala Leu Ile Gln Arg Gly Phe Ile 465 470 475 480 Arg Pro Lys Asp Cys Asp Val Leu Thr Ala Val Ser Glu Phe Phe Asn 485 490 495 Ala Ser Cys Val Pro Val Asn Asn Pro Lys Asn Tyr Pro Ser Ser Leu 500 505 510 Cys Ala Leu Cys Val Gly Asp Glu Gln Gly Arg Asn Lys Cys Val Gly 515 520 525 Asn Ser Gln Glu Arg Tyr Tyr Gly Tyr Arg Gly Ala Phe Arg Cys Leu 530 535 540 Val Glu Asn Ala Gly Asp Val Ala Phe Val Arg His Thr Thr Val Phe 545 550 555 560 Asp Asn Thr Asn Gly His Asn Ser Glu Pro Trp Ala Ala Glu Leu Arg 565 570 575 Ser Glu Asp Tyr Glu Leu Leu Cys Pro Asn Gly Ala Arg Ala Glu Val 580 585 590 Ser Gln Phe Ala Ala Cys Asn Leu Ala Gln Ile Pro Pro His Ala Val 595 600 605 Met Val Arg Pro Asp Thr Asn Ile Phe Thr Val Tyr Gly Leu Leu Asp 610 615 620 Lys Ala Gln Asp Leu Phe Gly Asp Asp His Asn Lys Asn Gly Phe Lys 625 630 635 640 Met Phe Asp Ser Ser Asn Tyr His Gly Gln Asp Leu Leu Phe Lys Asp 645 650 655 Ala Thr Val Arg Ala Val Pro Val Gly Glu Lys Thr Thr Tyr Arg Gly 660 665 670 Trp Leu Gly Leu Asp Tyr Val Ala Ala Leu Glu Gly Met Ser Ser Gln 675 680 685 Gln Cys Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 690 695 700 Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 705 710 715 720 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 725 730 735 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 740 745 750 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 755 760 765 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 770 775 780 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 785 790 795 800 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 805 810 815 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 820 825 830 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 835 840 845 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 850 855 860 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 865 870 875 880 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 885 890 895 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 900 905 910 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 915 920 925 Leu Ser Pro Gly Lys 930 43932PRTArtificial Sequencep97-trastuzumab heavy chain fusion protein 43Gly Met Glu Val Arg Trp Cys Ala Thr Ser Asp Pro Glu Gln His Lys 1 5 10 15 Cys Gly Asn Met Ser Glu Ala Phe Arg Glu Ala Gly Ile Gln Pro Ser 20 25 30 Leu Leu Cys Val Arg Gly Thr Ser Ala Asp His Cys Val Gln Leu Ile 35 40 45 Ala Ala Gln Glu Ala Asp Ala Ile Thr Leu Asp Gly Gly Ala Ile Tyr 50 55 60 Glu Ala Gly Lys Glu His Gly Leu Lys Pro Val Val Gly Glu Val Tyr 65 70 75 80 Asp Gln Glu Val Gly Thr Ser Tyr Tyr Ala Val Ala Val Val Arg Arg 85 90 95 Ser Ser His Val Thr Ile Asp Thr Leu Lys Gly Val Lys Ser Cys His 100 105 110 Thr Gly Ile Asn Arg Thr Val Gly Trp Asn Val Pro Val Gly Tyr Leu 115 120 125 Val Glu Ser Gly Arg Leu Ser Val Met Gly Cys Asp Val Leu Lys Ala 130 135 140 Val Ser Asp Tyr Phe Gly Gly Ser Cys Val Pro Gly Ala Gly Glu Thr 145 150 155 160 Ser Tyr Ser Glu Ser Leu Cys Arg Leu Cys Arg Gly Asp Ser Ser Gly 165 170 175 Glu Gly Val Cys Asp Lys Ser Pro Leu Glu Arg Tyr Tyr Asp Tyr Ser 180 185 190 Gly Ala Phe Arg Cys Leu Ala Glu Gly Ala Gly Asp Val Ala Phe Val 195 200 205 Lys His Ser Thr Val Leu Glu Asn Thr Asp Gly Lys Thr Leu Pro Ser 210 215 220 Trp Gly Gln Ala Leu Leu Ser Gln Asp Phe Glu Leu Leu Cys Arg Asp 225 230 235 240 Gly Ser Arg Ala Asp Val Thr Glu Trp Arg Gln Cys His Leu Ala Arg 245 250 255 Val Pro Ala His Ala Val Val Val Arg Ala Asp Thr Asp Gly Gly Leu 260 265 270 Ile Phe Arg Leu Leu Asn Glu Gly Gln Arg Leu Phe Ser His Glu Gly 275 280 285 Ser Ser Phe Gln Met Phe Ser Ser Glu Ala Tyr Gly Gln Lys Asp Leu 290 295 300 Leu Phe Lys Asp Ser Thr Ser Glu Leu Val Pro Ile Ala Thr Gln Thr 305 310 315 320 Tyr Glu Ala Trp Leu Gly His Glu Tyr Leu His Ala Met Lys Gly Leu 325 330 335 Leu Cys Asp Pro Asn Arg Leu Pro Pro Tyr Leu Arg Trp Cys Val Leu 340 345 350 Ser Thr Pro Glu Ile Gln Lys Cys Gly Asp Met Ala Val Ala Phe Arg 355 360 365 Arg Gln Arg Leu Lys Pro Glu Ile Gln Cys Val Ser Ala Lys Ser Pro 370 375 380 Gln His Cys Met Glu Arg Ile Gln Ala Glu Gln Val Asp Ala Val Thr 385 390 395 400 Leu Ser Gly Glu Asp Ile Tyr Thr Ala Gly Lys Thr Tyr Gly Leu Val 405 410 415 Pro Ala Ala Gly Glu His Tyr Ala Pro Glu Asp Ser Ser Asn Ser Tyr 420 425 430 Tyr Val Val Ala Val Val Arg Arg Asp Ser Ser His Ala Phe Thr Leu 435 440 445 Asp Glu Leu Arg Gly Lys Arg Ser Cys His Ala Gly Phe Gly Ser Pro 450 455 460 Ala Gly Trp Asp Val Pro Val Gly Ala Leu Ile Gln Arg Gly Phe Ile 465 470 475 480 Arg Pro Lys Asp Cys Asp Val Leu Thr Ala Val Ser Glu Phe Phe Asn 485 490 495 Ala Ser Cys Val Pro Val Asn Asn Pro Lys Asn Tyr Pro Ser Ser Leu 500 505 510 Cys Ala Leu Cys Val Gly Asp Glu Gln Gly Arg Asn Lys Cys Val Gly 515 520 525 Asn Ser Gln Glu Arg Tyr Tyr Gly Tyr Arg Gly Ala Phe Arg Cys Leu 530 535 540 Val Glu Asn Ala Gly Asp Val Ala Phe Val Arg His Thr Thr Val Phe 545 550 555 560 Asp Asn Thr Asn Gly His Asn Ser Glu Pro Trp Ala Ala Glu Leu Arg 565 570 575 Ser Glu Asp Tyr Glu Leu Leu Cys Pro Asn Gly Ala Arg Ala Glu Val 580 585 590 Ser Gln Phe Ala Ala Cys Asn Leu Ala Gln Ile Pro Pro His Ala Val 595 600 605 Met Val Arg Pro Asp Thr Asn Ile Phe Thr Val Tyr Gly Leu Leu Asp 610 615 620 Lys Ala Gln Asp Leu Phe Gly Asp Asp His Asn Lys Asn Gly Phe Lys 625 630 635 640 Met Phe Asp Ser Ser Asn Tyr His Gly Gln Asp Leu Leu Phe Lys Asp 645 650 655 Ala Thr Val Arg Ala Val Pro Val Gly Glu Lys Thr Thr Tyr Arg Gly 660 665 670 Trp Leu Gly Leu Asp Tyr Val Ala Ala Leu Glu Gly Met Ser Ser Gln 675 680 685 Gln Cys Ser Gly Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 690 695 700 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 705 710 715 720 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 725 730 735 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 740 745 750 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 755 760 765 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 770 775 780 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 785 790 795 800 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 805 810 815 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 820 825 830 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 835 840 845 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 850 855 860 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Trp Pro 865 870 875 880 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Thr Ser Lys Leu Thr 885 890 895 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 900 905 910 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 915 920 925 Ser Pro Gly Lys 930 44947PRTArtificial Sequencep97-trastuzumab heavy chain fusion protein 44Gly Met Glu Val Arg Trp Cys Ala Thr Ser Asp Pro Glu Gln His Lys 1 5 10 15 Cys Gly Asn Met Ser Glu Ala Phe Arg Glu Ala Gly Ile Gln Pro Ser 20 25 30 Leu Leu Cys Val Arg Gly Thr Ser Ala Asp His Cys Val Gln Leu Ile 35 40 45 Ala Ala Gln Glu Ala Asp Ala Ile Thr Leu Asp Gly Gly Ala Ile Tyr 50 55 60 Glu Ala Gly Lys Glu His Gly Leu Lys Pro Val Val Gly Glu Val Tyr 65

70 75 80 Asp Gln Glu Val Gly Thr Ser Tyr Tyr Ala Val Ala Val Val Arg Arg 85 90 95 Ser Ser His Val Thr Ile Asp Thr Leu Lys Gly Val Lys Ser Cys His 100 105 110 Thr Gly Ile Asn Arg Thr Val Gly Trp Asn Val Pro Val Gly Tyr Leu 115 120 125 Val Glu Ser Gly Arg Leu Ser Val Met Gly Cys Asp Val Leu Lys Ala 130 135 140 Val Ser Asp Tyr Phe Gly Gly Ser Cys Val Pro Gly Ala Gly Glu Thr 145 150 155 160 Ser Tyr Ser Glu Ser Leu Cys Arg Leu Cys Arg Gly Asp Ser Ser Gly 165 170 175 Glu Gly Val Cys Asp Lys Ser Pro Leu Glu Arg Tyr Tyr Asp Tyr Ser 180 185 190 Gly Ala Phe Arg Cys Leu Ala Glu Gly Ala Gly Asp Val Ala Phe Val 195 200 205 Lys His Ser Thr Val Leu Glu Asn Thr Asp Gly Lys Thr Leu Pro Ser 210 215 220 Trp Gly Gln Ala Leu Leu Ser Gln Asp Phe Glu Leu Leu Cys Arg Asp 225 230 235 240 Gly Ser Arg Ala Asp Val Thr Glu Trp Arg Gln Cys His Leu Ala Arg 245 250 255 Val Pro Ala His Ala Val Val Val Arg Ala Asp Thr Asp Gly Gly Leu 260 265 270 Ile Phe Arg Leu Leu Asn Glu Gly Gln Arg Leu Phe Ser His Glu Gly 275 280 285 Ser Ser Phe Gln Met Phe Ser Ser Glu Ala Tyr Gly Gln Lys Asp Leu 290 295 300 Leu Phe Lys Asp Ser Thr Ser Glu Leu Val Pro Ile Ala Thr Gln Thr 305 310 315 320 Tyr Glu Ala Trp Leu Gly His Glu Tyr Leu His Ala Met Lys Gly Leu 325 330 335 Leu Cys Asp Pro Asn Arg Leu Pro Pro Tyr Leu Arg Trp Cys Val Leu 340 345 350 Ser Thr Pro Glu Ile Gln Lys Cys Gly Asp Met Ala Val Ala Phe Arg 355 360 365 Arg Gln Arg Leu Lys Pro Glu Ile Gln Cys Val Ser Ala Lys Ser Pro 370 375 380 Gln His Cys Met Glu Arg Ile Gln Ala Glu Gln Val Asp Ala Val Thr 385 390 395 400 Leu Ser Gly Glu Asp Ile Tyr Thr Ala Gly Lys Thr Tyr Gly Leu Val 405 410 415 Pro Ala Ala Gly Glu His Tyr Ala Pro Glu Asp Ser Ser Asn Ser Tyr 420 425 430 Tyr Val Val Ala Val Val Arg Arg Asp Ser Ser His Ala Phe Thr Leu 435 440 445 Asp Glu Leu Arg Gly Lys Arg Ser Cys His Ala Gly Phe Gly Ser Pro 450 455 460 Ala Gly Trp Asp Val Pro Val Gly Ala Leu Ile Gln Arg Gly Phe Ile 465 470 475 480 Arg Pro Lys Asp Cys Asp Val Leu Thr Ala Val Ser Glu Phe Phe Asn 485 490 495 Ala Ser Cys Val Pro Val Asn Asn Pro Lys Asn Tyr Pro Ser Ser Leu 500 505 510 Cys Ala Leu Cys Val Gly Asp Glu Gln Gly Arg Asn Lys Cys Val Gly 515 520 525 Asn Ser Gln Glu Arg Tyr Tyr Gly Tyr Arg Gly Ala Phe Arg Cys Leu 530 535 540 Val Glu Asn Ala Gly Asp Val Ala Phe Val Arg His Thr Thr Val Phe 545 550 555 560 Asp Asn Thr Asn Gly His Asn Ser Glu Pro Trp Ala Ala Glu Leu Arg 565 570 575 Ser Glu Asp Tyr Glu Leu Leu Cys Pro Asn Gly Ala Arg Ala Glu Val 580 585 590 Ser Gln Phe Ala Ala Cys Asn Leu Ala Gln Ile Pro Pro His Ala Val 595 600 605 Met Val Arg Pro Asp Thr Asn Ile Phe Thr Val Tyr Gly Leu Leu Asp 610 615 620 Lys Ala Gln Asp Leu Phe Gly Asp Asp His Asn Lys Asn Gly Phe Lys 625 630 635 640 Met Phe Asp Ser Ser Asn Tyr His Gly Gln Asp Leu Leu Phe Lys Asp 645 650 655 Ala Thr Val Arg Ala Val Pro Val Gly Glu Lys Thr Thr Tyr Arg Gly 660 665 670 Trp Leu Gly Leu Asp Tyr Val Ala Ala Leu Glu Gly Met Ser Ser Gln 675 680 685 Gln Cys Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 690 695 700 Gly Gly Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 705 710 715 720 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 725 730 735 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 740 745 750 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 755 760 765 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 770 775 780 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 785 790 795 800 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 805 810 815 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 820 825 830 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 835 840 845 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 850 855 860 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 865 870 875 880 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Trp Pro Pro 885 890 895 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Thr Ser Lys Leu Thr Val 900 905 910 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 915 920 925 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 930 935 940 Pro Gly Lys 945 45919PRTArtificial Sequencep97-trastuzumab heavy chain fusion protein 45Gly Met Glu Val Arg Trp Cys Ala Thr Ser Asp Pro Glu Gln His Lys 1 5 10 15 Cys Gly Asn Met Ser Glu Ala Phe Arg Glu Ala Gly Ile Gln Pro Ser 20 25 30 Leu Leu Cys Val Arg Gly Thr Ser Ala Asp His Cys Val Gln Leu Ile 35 40 45 Ala Ala Gln Glu Ala Asp Ala Ile Thr Leu Asp Gly Gly Ala Ile Tyr 50 55 60 Glu Ala Gly Lys Glu His Gly Leu Lys Pro Val Val Gly Glu Val Tyr 65 70 75 80 Asp Gln Glu Val Gly Thr Ser Tyr Tyr Ala Val Ala Val Val Arg Arg 85 90 95 Ser Ser His Val Thr Ile Asp Thr Leu Lys Gly Val Lys Ser Cys His 100 105 110 Thr Gly Ile Asn Arg Thr Val Gly Trp Asn Val Pro Val Gly Tyr Leu 115 120 125 Val Glu Ser Gly Arg Leu Ser Val Met Gly Cys Asp Val Leu Lys Ala 130 135 140 Val Ser Asp Tyr Phe Gly Gly Ser Cys Val Pro Gly Ala Gly Glu Thr 145 150 155 160 Ser Tyr Ser Glu Ser Leu Cys Arg Leu Cys Arg Gly Asp Ser Ser Gly 165 170 175 Glu Gly Val Cys Asp Lys Ser Pro Leu Glu Arg Tyr Tyr Asp Tyr Ser 180 185 190 Gly Ala Phe Arg Cys Leu Ala Glu Gly Ala Gly Asp Val Ala Phe Val 195 200 205 Lys His Ser Thr Val Leu Glu Asn Thr Asp Gly Lys Thr Leu Pro Ser 210 215 220 Trp Gly Gln Ala Leu Leu Ser Gln Asp Phe Glu Leu Leu Cys Arg Asp 225 230 235 240 Gly Ser Arg Ala Asp Val Thr Glu Trp Arg Gln Cys His Leu Ala Arg 245 250 255 Val Pro Ala His Ala Val Val Val Arg Ala Asp Thr Asp Gly Gly Leu 260 265 270 Ile Phe Arg Leu Leu Asn Glu Gly Gln Arg Leu Phe Ser His Glu Gly 275 280 285 Ser Ser Phe Gln Met Phe Ser Ser Glu Ala Tyr Gly Gln Lys Asp Leu 290 295 300 Leu Phe Lys Asp Ser Thr Ser Glu Leu Val Pro Ile Ala Thr Gln Thr 305 310 315 320 Tyr Glu Ala Trp Leu Gly His Glu Tyr Leu His Ala Met Lys Gly Leu 325 330 335 Leu Cys Asp Pro Asn Arg Leu Pro Pro Tyr Leu Arg Trp Cys Val Leu 340 345 350 Ser Thr Pro Glu Ile Gln Lys Cys Gly Asp Met Ala Val Ala Phe Arg 355 360 365 Arg Gln Arg Leu Lys Pro Glu Ile Gln Cys Val Ser Ala Lys Ser Pro 370 375 380 Gln His Cys Met Glu Arg Ile Gln Ala Glu Gln Val Asp Ala Val Thr 385 390 395 400 Leu Ser Gly Glu Asp Ile Tyr Thr Ala Gly Lys Thr Tyr Gly Leu Val 405 410 415 Pro Ala Ala Gly Glu His Tyr Ala Pro Glu Asp Ser Ser Asn Ser Tyr 420 425 430 Tyr Val Val Ala Val Val Arg Arg Asp Ser Ser His Ala Phe Thr Leu 435 440 445 Asp Glu Leu Arg Gly Lys Arg Ser Cys His Ala Gly Phe Gly Ser Pro 450 455 460 Ala Gly Trp Asp Val Pro Val Gly Ala Leu Ile Gln Arg Gly Phe Ile 465 470 475 480 Arg Pro Lys Asp Cys Asp Val Leu Thr Ala Val Ser Glu Phe Phe Asn 485 490 495 Ala Ser Cys Val Pro Val Asn Asn Pro Lys Asn Tyr Pro Ser Ser Leu 500 505 510 Cys Ala Leu Cys Val Gly Asp Glu Gln Gly Arg Asn Lys Cys Val Gly 515 520 525 Asn Ser Gln Glu Arg Tyr Tyr Gly Tyr Arg Gly Ala Phe Arg Cys Leu 530 535 540 Val Glu Asn Ala Gly Asp Val Ala Phe Val Arg His Thr Thr Val Phe 545 550 555 560 Asp Asn Thr Asn Gly His Asn Ser Glu Pro Trp Ala Ala Glu Leu Arg 565 570 575 Ser Glu Asp Tyr Glu Leu Leu Cys Pro Asn Gly Ala Arg Ala Glu Val 580 585 590 Ser Gln Phe Ala Ala Cys Asn Leu Ala Gln Ile Pro Pro His Ala Val 595 600 605 Met Val Arg Pro Asp Thr Asn Ile Phe Thr Val Tyr Gly Leu Leu Asp 610 615 620 Lys Ala Gln Asp Leu Phe Gly Asp Asp His Asn Lys Asn Gly Phe Lys 625 630 635 640 Met Phe Asp Ser Ser Asn Tyr His Gly Gln Asp Leu Leu Phe Lys Asp 645 650 655 Ala Thr Val Arg Ala Val Pro Val Gly Glu Lys Thr Thr Tyr Arg Gly 660 665 670 Trp Leu Gly Leu Asp Tyr Val Ala Ala Leu Glu Gly Met Ser Ser Gln 675 680 685 Gln Cys Ser Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 690 695 700 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 705 710 715 720 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 725 730 735 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 740 745 750 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 755 760 765 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 770 775 780 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 785 790 795 800 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 805 810 815 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 820 825 830 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 835 840 845 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 850 855 860 Thr Trp Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Thr Ser 865 870 875 880 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 885 890 895 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 900 905 910 Leu Ser Leu Ser Pro Gly Lys 915 46934PRTArtificial Sequencep97-trastuzumab heavy chain fusion protein 46Gly Met Glu Val Arg Trp Cys Ala Thr Ser Asp Pro Glu Gln His Lys 1 5 10 15 Cys Gly Asn Met Ser Glu Ala Phe Arg Glu Ala Gly Ile Gln Pro Ser 20 25 30 Leu Leu Cys Val Arg Gly Thr Ser Ala Asp His Cys Val Gln Leu Ile 35 40 45 Ala Ala Gln Glu Ala Asp Ala Ile Thr Leu Asp Gly Gly Ala Ile Tyr 50 55 60 Glu Ala Gly Lys Glu His Gly Leu Lys Pro Val Val Gly Glu Val Tyr 65 70 75 80 Asp Gln Glu Val Gly Thr Ser Tyr Tyr Ala Val Ala Val Val Arg Arg 85 90 95 Ser Ser His Val Thr Ile Asp Thr Leu Lys Gly Val Lys Ser Cys His 100 105 110 Thr Gly Ile Asn Arg Thr Val Gly Trp Asn Val Pro Val Gly Tyr Leu 115 120 125 Val Glu Ser Gly Arg Leu Ser Val Met Gly Cys Asp Val Leu Lys Ala 130 135 140 Val Ser Asp Tyr Phe Gly Gly Ser Cys Val Pro Gly Ala Gly Glu Thr 145 150 155 160 Ser Tyr Ser Glu Ser Leu Cys Arg Leu Cys Arg Gly Asp Ser Ser Gly 165 170 175 Glu Gly Val Cys Asp Lys Ser Pro Leu Glu Arg Tyr Tyr Asp Tyr Ser 180 185 190 Gly Ala Phe Arg Cys Leu Ala Glu Gly Ala Gly Asp Val Ala Phe Val 195 200 205 Lys His Ser Thr Val Leu Glu Asn Thr Asp Gly Lys Thr Leu Pro Ser 210 215 220 Trp Gly Gln Ala Leu Leu Ser Gln Asp Phe Glu Leu Leu Cys Arg Asp 225 230 235 240 Gly Ser Arg Ala Asp Val Thr Glu Trp Arg Gln Cys His Leu Ala Arg 245 250 255 Val Pro Ala His Ala Val Val Val Arg Ala Asp Thr Asp Gly Gly Leu 260 265 270 Ile Phe Arg Leu Leu Asn Glu Gly Gln Arg Leu Phe Ser His Glu Gly 275 280 285 Ser Ser Phe Gln Met Phe Ser Ser Glu Ala Tyr Gly Gln Lys Asp Leu 290 295 300 Leu Phe Lys Asp Ser Thr Ser Glu Leu Val Pro Ile Ala Thr Gln Thr 305 310 315 320 Tyr Glu Ala Trp Leu Gly His Glu Tyr Leu His Ala Met Lys Gly Leu 325 330 335 Leu Cys Asp Pro Asn Arg Leu Pro Pro Tyr Leu Arg Trp Cys Val Leu 340 345 350 Ser Thr Pro Glu Ile Gln Lys Cys Gly Asp Met Ala Val Ala Phe Arg 355 360 365 Arg Gln Arg Leu Lys Pro Glu Ile Gln Cys Val Ser Ala Lys Ser Pro 370 375 380 Gln His Cys Met Glu Arg Ile Gln Ala Glu Gln Val Asp Ala Val Thr 385 390 395 400 Leu Ser Gly Glu Asp Ile Tyr Thr Ala Gly Lys Thr Tyr Gly Leu Val 405 410 415 Pro Ala Ala Gly Glu His Tyr Ala Pro Glu Asp Ser Ser Asn Ser Tyr 420 425 430 Tyr Val Val Ala Val Val Arg Arg Asp Ser Ser His Ala Phe Thr Leu 435 440 445 Asp Glu Leu Arg Gly Lys Arg Ser Cys His Ala Gly Phe Gly Ser Pro 450 455 460 Ala Gly Trp Asp Val Pro Val Gly Ala Leu Ile Gln Arg Gly Phe Ile 465 470 475 480 Arg Pro Lys Asp Cys Asp Val Leu Thr Ala Val Ser Glu Phe Phe Asn 485 490 495 Ala Ser

Cys Val Pro Val Asn Asn Pro Lys Asn Tyr Pro Ser Ser Leu 500 505 510 Cys Ala Leu Cys Val Gly Asp Glu Gln Gly Arg Asn Lys Cys Val Gly 515 520 525 Asn Ser Gln Glu Arg Tyr Tyr Gly Tyr Arg Gly Ala Phe Arg Cys Leu 530 535 540 Val Glu Asn Ala Gly Asp Val Ala Phe Val Arg His Thr Thr Val Phe 545 550 555 560 Asp Asn Thr Asn Gly His Asn Ser Glu Pro Trp Ala Ala Glu Leu Arg 565 570 575 Ser Glu Asp Tyr Glu Leu Leu Cys Pro Asn Gly Ala Arg Ala Glu Val 580 585 590 Ser Gln Phe Ala Ala Cys Asn Leu Ala Gln Ile Pro Pro His Ala Val 595 600 605 Met Val Arg Pro Asp Thr Asn Ile Phe Thr Val Tyr Gly Leu Leu Asp 610 615 620 Lys Ala Gln Asp Leu Phe Gly Asp Asp His Asn Lys Asn Gly Phe Lys 625 630 635 640 Met Phe Asp Ser Ser Asn Tyr His Gly Gln Asp Leu Leu Phe Lys Asp 645 650 655 Ala Thr Val Arg Ala Val Pro Val Gly Glu Lys Thr Thr Tyr Arg Gly 660 665 670 Trp Leu Gly Leu Asp Tyr Val Ala Ala Leu Glu Gly Met Ser Ser Gln 675 680 685 Gln Cys Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 690 695 700 Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 705 710 715 720 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 725 730 735 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 740 745 750 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 755 760 765 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 770 775 780 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 785 790 795 800 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 805 810 815 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 820 825 830 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 835 840 845 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 850 855 860 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 865 870 875 880 Trp Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Thr Ser Lys 885 890 895 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 900 905 910 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 915 920 925 Ser Leu Ser Pro Gly Lys 930 474PRTArtificial SequencePeptide linker 47Gly Ser Gly Ser 1 484PRTArtificial SequencePeptide linker 48Gly Gly Ser Gly 1 494PRTArtificial SequencePeptide linker 49Gly Gly Gly Ser 1 505PRTArtificial SequencePeptide Linker 50Gly Gly Gly Gly Ser 1 5 514PRTArtificial SequencePeptide linker 51Gly Asn Gly Asn 1 524PRTArtificial SequencePeptide linker 52Gly Gly Asn Gly 1 534PRTArtificial SequencePeptide linker 53Gly Gly Gly Asn 1 545PRTArtificial SequencePeptide linker 54Gly Gly Gly Gly Asn 1 5 5515PRTArtificial SequencePeptide linker 55Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 564PRTArtificial SequenceThrombin cleavable linker 56Gly Arg Gly Asp 1 576PRTArtificial SequenceThrombin cleavable linker 57Gly Arg Gly Asp Asn Pro 1 5 585PRTArtificial SequenceThrombin cleavable linker 58Gly Arg Gly Asp Ser 1 5 597PRTArtificial SequenceThrombin cleavable linker 59Gly Arg Gly Asp Ser Pro Lys 1 5 604PRTArtificial SequenceElastase cleavable linker 60Ala Ala Pro Val 1 614PRTArtificial SequenceElastase cleavable linker 61Ala Ala Pro Leu 1 624PRTArtificial SequenceElastase cleavable linker 62Ala Ala Pro Phe 1 634PRTArtificial SequenceElastase cleavable linker 63Ala Ala Pro Ala 1 644PRTArtificial SequenceElastase cleavable linker 64Ala Tyr Leu Val 1 656PRTArtificial SequenceMatrix metalloproteinase cleavable linker 65Gly Pro Xaa Gly Pro Xaa 1 5 664PRTArtificial SequenceMatrix metalloproteinase cleavable linker 66Leu Gly Pro Xaa 1 676PRTArtificial SequenceMatrix metalloproteinase cleavable linker 67Gly Pro Ile Gly Pro Xaa 1 5 685PRTArtificial SequenceMatrix metalloproteinase cleavable linker 68Ala Pro Gly Leu Xaa 1 5 697PRTArtificial SequenceCollagenase cleavable linker 69Pro Leu Gly Pro Asp Arg Xaa 1 5 707PRTArtificial SequenceCollagenase cleavable linker 70Pro Leu Gly Leu Leu Gly Xaa 1 5 717PRTArtificial SequenceCollagenase cleavable linker 71Pro Gln Gly Ile Ala Gly Trp 1 5 725PRTArtificial SequenceCollagenase cleavable linker 72Pro Leu Gly Cys His 1 5 736PRTArtificial SequenceCollagenase cleavable linker 73Pro Leu Gly Leu Tyr Ala 1 5 747PRTArtificial SequenceCollagenase cleavable linker 74Pro Leu Ala Leu Trp Ala Arg 1 5 757PRTArtificial SequenceCollagenase cleavable linker 75Pro Leu Ala Tyr Trp Ala Arg 1 5 767PRTArtificial SequenceStromelysin cleavable linker 76Pro Tyr Ala Tyr Tyr Met Arg 1 5 777PRTArtificial SequenceGelatinase cleavable linker 77Pro Leu Gly Met Tyr Ser Arg 1 5 784PRTArtificial SequenceAngiotensin converting enzyme cleavable linker 78Gly Asp Lys Pro 1 795PRTArtificial SequenceAngiotensin converting enzyme cleavable linker 79Gly Ser Asp Lys Pro 1 5 804PRTArtificial SequenceCathepsin B cleavable linker 80Ala Leu Ala Leu 1 814PRTArtificial SequenceCathepsin B cleavable linker 81Gly Phe Leu Gly 1 821171PRTArtificial SequenceTZM heavy chain - linker - p97 fusion protein 82Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 20 25 30 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn 35 40 45 Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly 50 55 60 Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr 65 70 75 80 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys 85 90 95 Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 225 230 235 240 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 355 360 365 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 370 375 380 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 465 470 475 480 Gly Met Glu Val Arg Trp Cys Ala Thr Ser Asp Pro Glu Gln His Lys 485 490 495 Cys Gly Asn Met Ser Glu Ala Phe Arg Glu Ala Gly Ile Gln Pro Ser 500 505 510 Leu Leu Cys Val Arg Gly Thr Ser Ala Asp His Cys Val Gln Leu Ile 515 520 525 Ala Ala Gln Glu Ala Asp Ala Ile Thr Leu Asp Gly Gly Ala Ile Tyr 530 535 540 Glu Ala Gly Lys Glu His Gly Leu Lys Pro Val Val Gly Glu Val Tyr 545 550 555 560 Asp Gln Glu Val Gly Thr Ser Tyr Tyr Ala Val Ala Val Val Arg Arg 565 570 575 Ser Ser His Val Thr Ile Asp Thr Leu Lys Gly Val Lys Ser Cys His 580 585 590 Thr Gly Ile Asn Arg Thr Val Gly Trp Asn Val Pro Val Gly Tyr Leu 595 600 605 Val Glu Ser Gly Arg Leu Ser Val Met Gly Cys Asp Val Leu Lys Ala 610 615 620 Val Ser Asp Tyr Phe Gly Gly Ser Cys Val Pro Gly Ala Gly Glu Thr 625 630 635 640 Ser Tyr Ser Glu Ser Leu Cys Arg Leu Cys Arg Gly Asp Ser Ser Gly 645 650 655 Glu Gly Val Cys Asp Lys Ser Pro Leu Glu Arg Tyr Tyr Asp Tyr Ser 660 665 670 Gly Ala Phe Arg Cys Leu Ala Glu Gly Ala Gly Asp Val Ala Phe Val 675 680 685 Lys His Ser Thr Val Leu Glu Asn Thr Asp Gly Lys Thr Leu Pro Ser 690 695 700 Trp Gly Gln Ala Leu Leu Ser Gln Asp Phe Glu Leu Leu Cys Arg Asp 705 710 715 720 Gly Ser Arg Ala Asp Val Thr Glu Trp Arg Gln Cys His Leu Ala Arg 725 730 735 Val Pro Ala His Ala Val Val Val Arg Ala Asp Thr Asp Gly Gly Leu 740 745 750 Ile Phe Arg Leu Leu Asn Glu Gly Gln Arg Leu Phe Ser His Glu Gly 755 760 765 Ser Ser Phe Gln Met Phe Ser Ser Glu Ala Tyr Gly Gln Lys Asp Leu 770 775 780 Leu Phe Lys Asp Ser Thr Ser Glu Leu Val Pro Ile Ala Thr Gln Thr 785 790 795 800 Tyr Glu Ala Trp Leu Gly His Glu Tyr Leu His Ala Met Lys Gly Leu 805 810 815 Leu Cys Asp Pro Asn Arg Leu Pro Pro Tyr Leu Arg Trp Cys Val Leu 820 825 830 Ser Thr Pro Glu Ile Gln Lys Cys Gly Asp Met Ala Val Ala Phe Arg 835 840 845 Arg Gln Arg Leu Lys Pro Glu Ile Gln Cys Val Ser Ala Lys Ser Pro 850 855 860 Gln His Cys Met Glu Arg Ile Gln Ala Glu Gln Val Asp Ala Val Thr 865 870 875 880 Leu Ser Gly Glu Asp Ile Tyr Thr Ala Gly Lys Thr Tyr Gly Leu Val 885 890 895 Pro Ala Ala Gly Glu His Tyr Ala Pro Glu Asp Ser Ser Asn Ser Tyr 900 905 910 Tyr Val Val Ala Val Val Arg Arg Asp Ser Ser His Ala Phe Thr Leu 915 920 925 Asp Glu Leu Arg Gly Lys Arg Ser Cys His Ala Gly Phe Gly Ser Pro 930 935 940 Ala Gly Trp Asp Val Pro Val Gly Ala Leu Ile Gln Arg Gly Phe Ile 945 950 955 960 Arg Pro Lys Asp Cys Asp Val Leu Thr Ala Val Ser Glu Phe Phe Asn 965 970 975 Ala Ser Cys Val Pro Val Asn Asn Pro Lys Asn Tyr Pro Ser Ser Leu 980 985 990 Cys Ala Leu Cys Val Gly Asp Glu Gln Gly Arg Asn Lys Cys Val Gly 995 1000 1005 Asn Ser Gln Glu Arg Tyr Tyr Gly Tyr Arg Gly Ala Phe Arg Cys 1010 1015 1020 Leu Val Glu Asn Ala Gly Asp Val Ala Phe Val Arg His Thr Thr 1025 1030 1035 Val Phe Asp Asn Thr Asn Gly His Asn Ser Glu Pro Trp Ala Ala 1040 1045 1050 Glu Leu Arg Ser Glu Asp Tyr Glu Leu Leu Cys Pro Asn Gly Ala 1055 1060 1065 Arg Ala Glu Val Ser Gln Phe Ala Ala Cys Asn Leu Ala Gln Ile 1070 1075 1080 Pro Pro His Ala Val Met Val Arg Pro Asp Thr Asn Ile Phe Thr 1085 1090 1095 Val Tyr Gly Leu Leu Asp Lys Ala Gln Asp Leu Phe Gly Asp Asp 1100 1105 1110 His Asn Lys Asn Gly Phe Lys Met Phe Asp Ser Ser Asn Tyr His 1115 1120 1125 Gly Gln Asp Leu Leu Phe Lys Asp Ala Thr Val Arg Ala Val Pro 1130 1135 1140 Val Gly Glu Lys Thr Thr Tyr Arg Gly Trp Leu Gly Leu Asp Tyr 1145 1150 1155 Val Ala Ala Leu Glu Gly Met Ser Ser Gln Gln Cys Ser 1160 1165 1170 83234PRTHomo sapiens 83Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp 35 40 45 Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60 Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr 100 105 110 Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220 Val Thr Lys Ser Phe Asn Arg Gly

Glu Cys 225 230 84493PRTArtificial Sequencep97 fragment - linker - TZM heavy chain fusion protein 84Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ser Ser His Ala Phe Thr Leu Asp Glu Leu Arg 20 25 30 Tyr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 35 40 45 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 50 55 60 Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val 65 70 75 80 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro 85 90 95 Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 100 105 110 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 115 120 125 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly 130 135 140 Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 145 150 155 160 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 165 170 175 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 180 185 190 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 195 200 205 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 210 215 220 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 225 230 235 240 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 245 250 255 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 260 265 270 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 275 280 285 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 290 295 300 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 305 310 315 320 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 325 330 335 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 340 345 350 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 355 360 365 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 370 375 380 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 385 390 395 400 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 405 410 415 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 420 425 430 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 435 440 445 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 450 455 460 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 465 470 475 480 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 485 490 85234PRTHomo sapiens 85Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp 35 40 45 Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60 Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr 100 105 110 Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 86492PRTArtificial SequenceTZM heavy chain - llinker - p97 fragment fusion protein 86Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 20 25 30 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn 35 40 45 Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly 50 55 60 Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr 65 70 75 80 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys 85 90 95 Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 225 230 235 240 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 355 360 365 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 370 375 380 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 465 470 475 480 Asp Ser Ser His Ala Phe Thr Leu Asp Glu Leu Arg 485 490 87234PRTHomo sapiens 87Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp 35 40 45 Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60 Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr 100 105 110 Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 881177PRTArtificial SequenceTZM heavy chain - linker - p97 fusion protein 88Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 20 25 30 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn 35 40 45 Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly 50 55 60 Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr 65 70 75 80 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys 85 90 95 Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 225 230 235 240 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 355 360 365 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 370 375 380 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 465 470 475 480 Gly Gly Gly Gly Ser Gly Met Glu Val Arg Trp Cys Ala Thr Ser Asp 485 490 495 Pro Glu Gln His Lys Cys Gly Asn Met Ser Glu Ala Phe Arg Glu Ala 500 505 510 Gly Ile Gln Pro Ser Leu Leu Cys Val Arg Gly Thr Ser Ala Asp His 515 520 525 Cys Val Gln Leu Ile Ala Ala Gln Glu Ala Asp Ala Ile Thr Leu Asp 530 535 540 Gly Gly Ala Ile Tyr Glu Ala Gly Lys Glu His Gly Leu Lys Pro Val 545 550 555 560 Val Gly Glu Val Tyr Asp Gln Glu Val Gly Thr Ser Tyr Tyr Ala Val 565 570 575 Ala Val Val Arg Arg Ser Ser His Val Thr Ile Asp Thr Leu Lys Gly 580 585 590 Val Lys Ser Cys His Thr Gly Ile Asn Arg Thr Val Gly Trp Asn Val 595 600 605 Pro Val Gly Tyr Leu Val Glu Ser Gly Arg Leu Ser Val Met Gly Cys 610 615 620 Asp Val Leu Lys Ala Val Ser Asp Tyr Phe Gly Gly Ser Cys Val Pro 625 630 635 640 Gly Ala Gly Glu Thr Ser Tyr Ser Glu Ser Leu Cys Arg Leu Cys Arg 645 650 655 Gly Asp Ser Ser Gly Glu Gly Val Cys Asp Lys Ser Pro Leu Glu Arg 660 665 670 Tyr Tyr Asp Tyr Ser Gly Ala Phe Arg Cys Leu Ala Glu Gly Ala Gly 675 680 685 Asp Val Ala Phe Val Lys His Ser Thr Val Leu Glu Asn Thr Asp Gly 690 695 700 Lys Thr Leu Pro Ser Trp Gly Gln Ala Leu Leu Ser Gln Asp Phe Glu 705 710 715 720 Leu Leu Cys Arg Asp Gly Ser Arg Ala Asp Val Thr Glu Trp Arg Gln 725 730 735 Cys His Leu Ala Arg Val Pro Ala His Ala Val Val Val Arg Ala Asp 740 745 750 Thr Asp Gly Gly Leu Ile Phe Arg Leu Leu Asn Glu Gly Gln Arg Leu 755 760 765 Phe Ser His Glu Gly Ser Ser Phe Gln Met Phe Ser Ser Glu Ala Tyr 770 775 780 Gly Gln Lys Asp Leu Leu Phe Lys Asp Ser Thr Ser Glu Leu Val Pro 785 790 795 800 Ile Ala Thr Gln Thr Tyr Glu Ala Trp Leu Gly His Glu Tyr Leu His

805 810 815 Ala Met Lys Gly Leu Leu Cys Asp Pro Asn Arg Leu Pro Pro Tyr Leu 820 825 830 Arg Trp Cys Val Leu Ser Thr Pro Glu Ile Gln Lys Cys Gly Asp Met 835 840 845 Ala Val Ala Phe Arg Arg Gln Arg Leu Lys Pro Glu Ile Gln Cys Val 850 855 860 Ser Ala Lys Ser Pro Gln His Cys Met Glu Arg Ile Gln Ala Glu Gln 865 870 875 880 Val Asp Ala Val Thr Leu Ser Gly Glu Asp Ile Tyr Thr Ala Gly Lys 885 890 895 Thr Tyr Gly Leu Val Pro Ala Ala Gly Glu His Tyr Ala Pro Glu Asp 900 905 910 Ser Ser Asn Ser Tyr Tyr Val Val Ala Val Val Arg Arg Asp Ser Ser 915 920 925 His Ala Phe Thr Leu Asp Glu Leu Arg Gly Lys Arg Ser Cys His Ala 930 935 940 Gly Phe Gly Ser Pro Ala Gly Trp Asp Val Pro Val Gly Ala Leu Ile 945 950 955 960 Gln Arg Gly Phe Ile Arg Pro Lys Asp Cys Asp Val Leu Thr Ala Val 965 970 975 Ser Glu Phe Phe Asn Ala Ser Cys Val Pro Val Asn Asn Pro Lys Asn 980 985 990 Tyr Pro Ser Ser Leu Cys Ala Leu Cys Val Gly Asp Glu Gln Gly Arg 995 1000 1005 Asn Lys Cys Val Gly Asn Ser Gln Glu Arg Tyr Tyr Gly Tyr Arg 1010 1015 1020 Gly Ala Phe Arg Cys Leu Val Glu Asn Ala Gly Asp Val Ala Phe 1025 1030 1035 Val Arg His Thr Thr Val Phe Asp Asn Thr Asn Gly His Asn Ser 1040 1045 1050 Glu Pro Trp Ala Ala Glu Leu Arg Ser Glu Asp Tyr Glu Leu Leu 1055 1060 1065 Cys Pro Asn Gly Ala Arg Ala Glu Val Ser Gln Phe Ala Ala Cys 1070 1075 1080 Asn Leu Ala Gln Ile Pro Pro His Ala Val Met Val Arg Pro Asp 1085 1090 1095 Thr Asn Ile Phe Thr Val Tyr Gly Leu Leu Asp Lys Ala Gln Asp 1100 1105 1110 Leu Phe Gly Asp Asp His Asn Lys Asn Gly Phe Lys Met Phe Asp 1115 1120 1125 Ser Ser Asn Tyr His Gly Gln Asp Leu Leu Phe Lys Asp Ala Thr 1130 1135 1140 Val Arg Ala Val Pro Val Gly Glu Lys Thr Thr Tyr Arg Gly Trp 1145 1150 1155 Leu Gly Leu Asp Tyr Val Ala Ala Leu Glu Gly Met Ser Ser Gln 1160 1165 1170 Gln Cys Ser Gly 1175 89234PRTHomo sapiens 89Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp 35 40 45 Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60 Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr 100 105 110 Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 90499PRTArtificial SequenceTZM heavy chain - linker - p97 fragment fusion protein 90Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 20 25 30 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn 35 40 45 Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly 50 55 60 Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr 65 70 75 80 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys 85 90 95 Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 225 230 235 240 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 370 375 380 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala 465 470 475 480 Lys Glu Ala Ala Ala Lys Asp Ser Ser His Ala Phe Thr Leu Asp Glu 485 490 495 Leu Arg Tyr 91262PRTArtificial SequenceTZM light chain - linker - p97 fragment fusion protein 91Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp 35 40 45 Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60 Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr 100 105 110 Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Glu Ala Ala Ala Lys Glu 225 230 235 240 Ala Ala Ala Lys Glu Ala Ala Ala Lys Asp Ser Ser His Ala Phe Thr 245 250 255 Leu Asp Glu Leu Arg Tyr 260 92499PRTArtificial SequenceTZM heavy chain - linker - p97 fragment 92Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 20 25 30 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn 35 40 45 Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly 50 55 60 Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr 65 70 75 80 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys 85 90 95 Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 225 230 235 240 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 370 375 380 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala 465 470 475 480 Lys Glu Ala Ala Ala Lys Asp Ser Ser His Ala Phe Thr Leu Asp Glu 485 490 495 Leu Arg Tyr 93234PRTHomo sapiens 93Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp 35 40 45 Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60 Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr 100 105 110 Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 94471PRTArtificial SequenceTZM heavy chain - linker - p97 fragment fusion protein 94Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 20 25 30 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn 35 40 45 Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly 50 55 60 Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr 65 70 75 80 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys 85 90 95 Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145

150 155 160 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 225 230 235 240 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 370 375 380 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 95262PRTArtificial SequenceTZM light chain - linker - p97 fragment fusion protein 95Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp 35 40 45 Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60 Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr 100 105 110 Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Glu Ala Ala Ala Lys Glu 225 230 235 240 Ala Ala Ala Lys Glu Ala Ala Ala Lys Asp Ser Ser His Ala Phe Thr 245 250 255 Leu Asp Glu Leu Arg Tyr 260 961151PRTArtificial SequenceTZM heavy chain - linker - p97 fusion protein 96Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Met Glu Val 450 455 460 Arg Trp Cys Ala Thr Ser Asp Pro Glu Gln His Lys Cys Gly Asn Met 465 470 475 480 Ser Glu Ala Phe Arg Glu Ala Gly Ile Gln Pro Ser Leu Leu Cys Val 485 490 495 Arg Gly Thr Ser Ala Asp His Cys Val Gln Leu Ile Ala Ala Gln Glu 500 505 510 Ala Asp Ala Ile Thr Leu Asp Gly Gly Ala Ile Tyr Glu Ala Gly Lys 515 520 525 Glu His Gly Leu Lys Pro Val Val Gly Glu Val Tyr Asp Gln Glu Val 530 535 540 Gly Thr Ser Tyr Tyr Ala Val Ala Val Val Arg Arg Ser Ser His Val 545 550 555 560 Thr Ile Asp Thr Leu Lys Gly Val Lys Ser Cys His Thr Gly Ile Asn 565 570 575 Arg Thr Val Gly Trp Asn Val Pro Val Gly Tyr Leu Val Glu Ser Gly 580 585 590 Arg Leu Ser Val Met Gly Cys Asp Val Leu Lys Ala Val Ser Asp Tyr 595 600 605 Phe Gly Gly Ser Cys Val Pro Gly Ala Gly Glu Thr Ser Tyr Ser Glu 610 615 620 Ser Leu Cys Arg Leu Cys Arg Gly Asp Ser Ser Gly Glu Gly Val Cys 625 630 635 640 Asp Lys Ser Pro Leu Glu Arg Tyr Tyr Asp Tyr Ser Gly Ala Phe Arg 645 650 655 Cys Leu Ala Glu Gly Ala Gly Asp Val Ala Phe Val Lys His Ser Thr 660 665 670 Val Leu Glu Asn Thr Asp Gly Lys Thr Leu Pro Ser Trp Gly Gln Ala 675 680 685 Leu Leu Ser Gln Asp Phe Glu Leu Leu Cys Arg Asp Gly Ser Arg Ala 690 695 700 Asp Val Thr Glu Trp Arg Gln Cys His Leu Ala Arg Val Pro Ala His 705 710 715 720 Ala Val Val Val Arg Ala Asp Thr Asp Gly Gly Leu Ile Phe Arg Leu 725 730 735 Leu Asn Glu Gly Gln Arg Leu Phe Ser His Glu Gly Ser Ser Phe Gln 740 745 750 Met Phe Ser Ser Glu Ala Tyr Gly Gln Lys Asp Leu Leu Phe Lys Asp 755 760 765 Ser Thr Ser Glu Leu Val Pro Ile Ala Thr Gln Thr Tyr Glu Ala Trp 770 775 780 Leu Gly His Glu Tyr Leu His Ala Met Lys Gly Leu Leu Cys Asp Pro 785 790 795 800 Asn Arg Leu Pro Pro Tyr Leu Arg Trp Cys Val Leu Ser Thr Pro Glu 805 810 815 Ile Gln Lys Cys Gly Asp Met Ala Val Ala Phe Arg Arg Gln Arg Leu 820 825 830 Lys Pro Glu Ile Gln Cys Val Ser Ala Lys Ser Pro Gln His Cys Met 835 840 845 Glu Arg Ile Gln Ala Glu Gln Val Asp Ala Val Thr Leu Ser Gly Glu 850 855 860 Asp Ile Tyr Thr Ala Gly Lys Thr Tyr Gly Leu Val Pro Ala Ala Gly 865 870 875 880 Glu His Tyr Ala Pro Glu Asp Ser Ser Asn Ser Tyr Tyr Val Val Ala 885 890 895 Val Val Arg Arg Asp Ser Ser His Ala Phe Thr Leu Asp Glu Leu Arg 900 905 910 Gly Lys Arg Ser Cys His Ala Gly Phe Gly Ser Pro Ala Gly Trp Asp 915 920 925 Val Pro Val Gly Ala Leu Ile Gln Arg Gly Phe Ile Arg Pro Lys Asp 930 935 940 Cys Asp Val Leu Thr Ala Val Ser Glu Phe Phe Asn Ala Ser Cys Val 945 950 955 960 Pro Val Asn Asn Pro Lys Asn Tyr Pro Ser Ser Leu Cys Ala Leu Cys 965 970 975 Val Gly Asp Glu Gln Gly Arg Asn Lys Cys Val Gly Asn Ser Gln Glu 980 985 990 Arg Tyr Tyr Gly Tyr Arg Gly Ala Phe Arg Cys Leu Val Glu Asn Ala 995 1000 1005 Gly Asp Val Ala Phe Val Arg His Thr Thr Val Phe Asp Asn Thr 1010 1015 1020 Asn Gly His Asn Ser Glu Pro Trp Ala Ala Glu Leu Arg Ser Glu 1025 1030 1035 Asp Tyr Glu Leu Leu Cys Pro Asn Gly Ala Arg Ala Glu Val Ser 1040 1045 1050 Gln Phe Ala Ala Cys Asn Leu Ala Gln Ile Pro Pro His Ala Val 1055 1060 1065 Met Val Arg Pro Asp Thr Asn Ile Phe Thr Val Tyr Gly Leu Leu 1070 1075 1080 Asp Lys Ala Gln Asp Leu Phe Gly Asp Asp His Asn Lys Asn Gly 1085 1090 1095 Phe Lys Met Phe Asp Ser Ser Asn Tyr His Gly Gln Asp Leu Leu 1100 1105 1110 Phe Lys Asp Ala Thr Val Arg Ala Val Pro Val Gly Glu Lys Thr 1115 1120 1125 Thr Tyr Arg Gly Trp Leu Gly Leu Asp Tyr Val Ala Ala Leu Glu 1130 1135 1140 Gly Met Ser Ser Gln Gln Cys Ser 1145 1150 971157PRTArtificial SequenceTZM heavy chain - linker - p97 fusion protein 97Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 450 455 460 Ser Gly Met Glu Val Arg Trp Cys Ala Thr Ser Asp Pro Glu Gln His 465 470 475 480 Lys Cys Gly Asn Met Ser Glu Ala Phe Arg Glu Ala Gly Ile Gln Pro 485 490 495 Ser Leu Leu Cys Val Arg Gly Thr Ser Ala Asp His Cys Val Gln Leu 500 505 510 Ile Ala Ala Gln Glu Ala Asp Ala Ile Thr Leu Asp Gly Gly Ala Ile 515 520 525 Tyr Glu Ala Gly Lys Glu

His Gly Leu Lys Pro Val Val Gly Glu Val 530 535 540 Tyr Asp Gln Glu Val Gly Thr Ser Tyr Tyr Ala Val Ala Val Val Arg 545 550 555 560 Arg Ser Ser His Val Thr Ile Asp Thr Leu Lys Gly Val Lys Ser Cys 565 570 575 His Thr Gly Ile Asn Arg Thr Val Gly Trp Asn Val Pro Val Gly Tyr 580 585 590 Leu Val Glu Ser Gly Arg Leu Ser Val Met Gly Cys Asp Val Leu Lys 595 600 605 Ala Val Ser Asp Tyr Phe Gly Gly Ser Cys Val Pro Gly Ala Gly Glu 610 615 620 Thr Ser Tyr Ser Glu Ser Leu Cys Arg Leu Cys Arg Gly Asp Ser Ser 625 630 635 640 Gly Glu Gly Val Cys Asp Lys Ser Pro Leu Glu Arg Tyr Tyr Asp Tyr 645 650 655 Ser Gly Ala Phe Arg Cys Leu Ala Glu Gly Ala Gly Asp Val Ala Phe 660 665 670 Val Lys His Ser Thr Val Leu Glu Asn Thr Asp Gly Lys Thr Leu Pro 675 680 685 Ser Trp Gly Gln Ala Leu Leu Ser Gln Asp Phe Glu Leu Leu Cys Arg 690 695 700 Asp Gly Ser Arg Ala Asp Val Thr Glu Trp Arg Gln Cys His Leu Ala 705 710 715 720 Arg Val Pro Ala His Ala Val Val Val Arg Ala Asp Thr Asp Gly Gly 725 730 735 Leu Ile Phe Arg Leu Leu Asn Glu Gly Gln Arg Leu Phe Ser His Glu 740 745 750 Gly Ser Ser Phe Gln Met Phe Ser Ser Glu Ala Tyr Gly Gln Lys Asp 755 760 765 Leu Leu Phe Lys Asp Ser Thr Ser Glu Leu Val Pro Ile Ala Thr Gln 770 775 780 Thr Tyr Glu Ala Trp Leu Gly His Glu Tyr Leu His Ala Met Lys Gly 785 790 795 800 Leu Leu Cys Asp Pro Asn Arg Leu Pro Pro Tyr Leu Arg Trp Cys Val 805 810 815 Leu Ser Thr Pro Glu Ile Gln Lys Cys Gly Asp Met Ala Val Ala Phe 820 825 830 Arg Arg Gln Arg Leu Lys Pro Glu Ile Gln Cys Val Ser Ala Lys Ser 835 840 845 Pro Gln His Cys Met Glu Arg Ile Gln Ala Glu Gln Val Asp Ala Val 850 855 860 Thr Leu Ser Gly Glu Asp Ile Tyr Thr Ala Gly Lys Thr Tyr Gly Leu 865 870 875 880 Val Pro Ala Ala Gly Glu His Tyr Ala Pro Glu Asp Ser Ser Asn Ser 885 890 895 Tyr Tyr Val Val Ala Val Val Arg Arg Asp Ser Ser His Ala Phe Thr 900 905 910 Leu Asp Glu Leu Arg Gly Lys Arg Ser Cys His Ala Gly Phe Gly Ser 915 920 925 Pro Ala Gly Trp Asp Val Pro Val Gly Ala Leu Ile Gln Arg Gly Phe 930 935 940 Ile Arg Pro Lys Asp Cys Asp Val Leu Thr Ala Val Ser Glu Phe Phe 945 950 955 960 Asn Ala Ser Cys Val Pro Val Asn Asn Pro Lys Asn Tyr Pro Ser Ser 965 970 975 Leu Cys Ala Leu Cys Val Gly Asp Glu Gln Gly Arg Asn Lys Cys Val 980 985 990 Gly Asn Ser Gln Glu Arg Tyr Tyr Gly Tyr Arg Gly Ala Phe Arg Cys 995 1000 1005 Leu Val Glu Asn Ala Gly Asp Val Ala Phe Val Arg His Thr Thr 1010 1015 1020 Val Phe Asp Asn Thr Asn Gly His Asn Ser Glu Pro Trp Ala Ala 1025 1030 1035 Glu Leu Arg Ser Glu Asp Tyr Glu Leu Leu Cys Pro Asn Gly Ala 1040 1045 1050 Arg Ala Glu Val Ser Gln Phe Ala Ala Cys Asn Leu Ala Gln Ile 1055 1060 1065 Pro Pro His Ala Val Met Val Arg Pro Asp Thr Asn Ile Phe Thr 1070 1075 1080 Val Tyr Gly Leu Leu Asp Lys Ala Gln Asp Leu Phe Gly Asp Asp 1085 1090 1095 His Asn Lys Asn Gly Phe Lys Met Phe Asp Ser Ser Asn Tyr His 1100 1105 1110 Gly Gln Asp Leu Leu Phe Lys Asp Ala Thr Val Arg Ala Val Pro 1115 1120 1125 Val Gly Glu Lys Thr Thr Tyr Arg Gly Trp Leu Gly Leu Asp Tyr 1130 1135 1140 Val Ala Ala Leu Glu Gly Met Ser Ser Gln Gln Cys Ser Gly 1145 1150 1155 98473PRTArtificial Sequencep97 fragment - linker - TZM heavy chain fusion protein 98Asp Ser Ser His Ala Phe Thr Leu Asp Glu Leu Arg Tyr Gly Gly Gly 1 5 10 15 Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly 20 25 30 Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser 35 40 45 Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro 50 55 60 Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr 65 70 75 80 Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp 85 90 95 Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu 100 105 110 Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr 115 120 125 Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 130 135 140 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser 145 150 155 160 Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 165 170 175 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 180 185 190 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 195 200 205 Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr 210 215 220 Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys 225 230 235 240 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 245 250 255 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 260 265 270 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 275 280 285 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 290 295 300 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 305 310 315 320 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 325 330 335 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 340 345 350 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 355 360 365 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 370 375 380 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 385 390 395 400 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 405 410 415 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 420 425 430 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 435 440 445 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 450 455 460 Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 99472PRTArtificial Sequencep97 fragment - linker - TZM heavy chain fusion protein 99Asp Ser Ser His Ala Phe Thr Leu Asp Glu Leu Arg Gly Gly Gly Gly 1 5 10 15 Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly 20 25 30 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr 65 70 75 80 Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr 85 90 95 Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala 115 120 125 Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser 130 135 140 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr 145 150 155 160 Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 165 170 175 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 180 185 190 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 195 200 205 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile 210 215 220 Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val 225 230 235 240 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 245 250 255 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 260 265 270 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 275 280 285 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 290 295 300 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 305 310 315 320 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 325 330 335 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 340 345 350 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 355 360 365 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 370 375 380 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 385 390 395 400 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 405 410 415 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 420 425 430 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 435 440 445 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 450 455 460 Ser Leu Ser Leu Ser Pro Gly Lys 465 470 100478PRTArtificial Sequencep97 fragment - linker - TZM heavy chain fusion protein 100Asp Ser Ser His Ala Phe Thr Leu Asp Glu Leu Arg Tyr Glu Ala Ala 1 5 10 15 Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Val Gln Leu 20 25 30 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 35 40 45 Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp 50 55 60 Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr 65 70 75 80 Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe 85 90 95 Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn 100 105 110 Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly 115 120 125 Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 130 135 140 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 145 150 155 160 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 165 170 175 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 180 185 190 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 195 200 205 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 210 215 220 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 225 230 235 240 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 245 250 255 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 260 265 270 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 275 280 285 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 290 295 300 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 305 310 315 320 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 325 330 335 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 340 345 350 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 355 360 365 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 370 375 380 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 385 390 395 400 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 405 410 415 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 420 425 430 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 435 440 445 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 450 455 460 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 475 101477PRTArtificial Sequencep97 fragment - linker - TZM heavy chain fusion protein 101Asp Ser Ser His Ala Phe Thr Leu Asp Glu Leu Arg Glu Ala Ala Ala 1 5 10 15 Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Val Gln Leu Val 20 25 30 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 35 40 45 Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val 50 55 60 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro 65 70 75 80 Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 85 90 95 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 100 105 110 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly 115 120 125 Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 130 135 140 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 145 150 155 160 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 165 170 175 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 180 185 190 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 195

200 205 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 210 215 220 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 225 230 235 240 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 245 250 255 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 260 265 270 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 275 280 285 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 290 295 300 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 305 310 315 320 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 325 330 335 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 340 345 350 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 355 360 365 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 370 375 380 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 385 390 395 400 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 405 410 415 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 420 425 430 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 435 440 445 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 450 455 460 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 475 102463PRTArtificial Sequencep97 fragment - TZM heavy chain fusion protein 102Asp Ser Ser His Ala Phe Thr Leu Asp Glu Leu Arg Tyr Glu Val Gln 1 5 10 15 Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg 20 25 30 Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His 35 40 45 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile 50 55 60 Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg 65 70 75 80 Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met 85 90 95 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp 100 105 110 Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu 115 120 125 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 130 135 140 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 145 150 155 160 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 165 170 175 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 180 185 190 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 195 200 205 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 210 215 220 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 225 230 235 240 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 245 250 255 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 260 265 270 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 275 280 285 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 290 295 300 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 305 310 315 320 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 325 330 335 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 340 345 350 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 355 360 365 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 370 375 380 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 385 390 395 400 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 405 410 415 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 420 425 430 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 435 440 445 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 460 103462PRTArtificial Sequencep97 fragment - TZM heavy chain fusion protein 103Asp Ser Ser His Ala Phe Thr Leu Asp Glu Leu Arg Glu Val Gln Leu 1 5 10 15 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 20 25 30 Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp 35 40 45 Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr 50 55 60 Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe 65 70 75 80 Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn 85 90 95 Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly 100 105 110 Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 115 120 125 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 130 135 140 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 145 150 155 160 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 165 170 175 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 180 185 190 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 195 200 205 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 210 215 220 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 225 230 235 240 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 245 250 255 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 260 265 270 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 275 280 285 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 290 295 300 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 305 310 315 320 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 325 330 335 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 340 345 350 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 355 360 365 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 370 375 380 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 385 390 395 400 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 405 410 415 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 420 425 430 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 435 440 445 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 460 104472PRTArtificial SequenceTZM heavy chain - linker - p97 fragment fusion protein 104Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ser Ser His 450 455 460 Ala Phe Thr Leu Asp Glu Leu Arg 465 470 105473PRTArtificial SequenceTZM heavy chain - linker - p97 fragment fusion protein 105Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ser Ser His 450 455 460 Ala Phe Thr Leu Asp Glu Leu Arg Tyr 465 470 106479PRTArtificial SequenceTZM heavy chain - linker - p97 fragment fusion protein 106Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100

105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala 450 455 460 Ala Lys Asp Ser Ser His Ala Phe Thr Leu Asp Glu Leu Arg Tyr 465 470 475 107478PRTArtificial SequenceTZM heavy chain - linker - p97 fragment fusion protein 107Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala 450 455 460 Ala Lys Asp Ser Ser His Ala Phe Thr Leu Asp Glu Leu Arg 465 470 475 108462PRTArtificial SequenceTZM heavy chain - p97 fragment fusion protein 108Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys Asp Ser Ser His Ala Phe Thr Leu Asp Glu Leu Arg 450 455 460 109463PRTArtificial SequenceTZM heavy chain - p97 fragment fusion protein 109Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys Asp Ser Ser His Ala Phe Thr Leu Asp Glu Leu Arg Tyr 450 455 460 110227PRTArtificial SequenceTrastuzumab FL light chain - p97 fragment fusion protein 110Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys Asp Ser Ser His Ala Phe Thr Leu Asp Glu 210 215 220 Leu Arg Tyr 225 111242PRTArtificial SequenceTrastuzumab FL light chain - linker - p97 fragment fusion protein 111Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys 210 215 220 Glu Ala Ala Ala Lys Asp Ser Ser His Ala Phe Thr Leu Asp Glu Leu 225 230 235 240 Arg Tyr 112237PRTArtificial

SequenceTrastuzumab FL light chain - linker - p97 fragment fusion protein 112Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 210 215 220 Asp Ser Ser His Ala Phe Thr Leu Asp Glu Leu Arg Tyr 225 230 235 113226PRTArtificial SequenceTrastuzumab FL light chain - p97 fragment fusion protein 113Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys Asp Ser Ser His Ala Phe Thr Leu Asp Glu 210 215 220 Leu Arg 225 114241PRTArtificial SequenceTrastuzumab FL light chain - linker - p97 fragment fusion protein 114Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys 210 215 220 Glu Ala Ala Ala Lys Asp Ser Ser His Ala Phe Thr Leu Asp Glu Leu 225 230 235 240 Arg 115237PRTArtificial SequenceTrastuzumab FL light chain - linker - p97 fragment fusion protein 115Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 210 215 220 Asp Ser Ser His Ala Phe Thr Leu Asp Glu Leu Arg Tyr 225 230 235 116227PRTArtificial Sequencep97 fragment - Trastuzumab FL light chain fusion protein 116Asp Ser Ser His Ala Phe Thr Leu Asp Glu Leu Arg Tyr Asp Ile Gln 1 5 10 15 Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val 20 25 30 Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp 35 40 45 Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala 50 55 60 Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser 65 70 75 80 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe 85 90 95 Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly 100 105 110 Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val 115 120 125 Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser 130 135 140 Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln 145 150 155 160 Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val 165 170 175 Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu 180 185 190 Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu 195 200 205 Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg 210 215 220 Gly Glu Cys 225 117242PRTArtificial Sequencep97 fragment - linker - Trastuzumab FL light chain fusion protein 117Asp Ser Ser His Ala Phe Thr Leu Asp Glu Leu Arg Tyr Glu Ala Ala 1 5 10 15 Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Asp Ile Gln Met 20 25 30 Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr 35 40 45 Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr 50 55 60 Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser 65 70 75 80 Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly 85 90 95 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 100 105 110 Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln 115 120 125 Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe 130 135 140 Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val 145 150 155 160 Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp 165 170 175 Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr 180 185 190 Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr 195 200 205 Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val 210 215 220 Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly 225 230 235 240 Glu Cys 118237PRTArtificial Sequencep97 fragment - linker - Trastuzumab FL light chain fusion protein 118Asp Ser Ser His Ala Phe Thr Leu Asp Glu Leu Arg Tyr Gly Gly Gly 1 5 10 15 Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser 20 25 30 Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala 35 40 45 Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly 50 55 60 Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly 65 70 75 80 Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu 85 90 95 Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln 100 105 110 Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu 115 120 125 Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser 130 135 140 Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn 145 150 155 160 Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala 165 170 175 Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys 180 185 190 Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp 195 200 205 Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu 210 215 220 Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 119226PRTArtificial Sequencep97 fragment - Trastuzumab FL light chain fusion protein 119Asp Ser Ser His Ala Phe Thr Leu Asp Glu Leu Arg Asp Ile Gln Met 1 5 10 15 Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr 20 25 30 Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr 35 40 45 Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser 50 55 60 Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly 65 70 75 80 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 85 90 95 Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln 100 105 110 Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe 115 120 125 Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val 130 135 140 Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp 145 150 155 160 Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr 165 170 175 Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr 180 185 190 Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val 195 200 205 Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly 210 215 220 Glu Cys 225 120241PRTArtificial Sequencep97 fragment - linker - Trastuzumab FL light chain fusion protein 120Asp Ser Ser His Ala Phe Thr Leu Asp Glu Leu Arg Glu Ala Ala Ala 1 5 10 15 Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Asp Ile Gln Met Thr 20 25 30 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 35 40 45 Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln 50 55 60 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe 65 70 75 80 Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr 85 90 95 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 100 105 110 Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly 115 120 125 Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile 130 135 140 Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val 145 150 155 160 Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys 165 170 175 Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu 180 185 190 Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu 195 200 205 Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr 210 215 220 His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu 225 230 235 240 Cys 121236PRTArtificial Sequencep97 fragment - linker - Trastuzumab FL light chain fusion protein 121Asp Ser Ser His Ala Phe Thr Leu Asp Glu Leu Arg Gly Gly Gly Gly 1 5 10 15 Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45 Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys 50 55

60 Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr 85 90 95 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 100 105 110 His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 115 120 125 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 122471PRTHomo sapiens 122Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 20 25 30 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn 35 40 45 Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly 50 55 60 Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr 65 70 75 80 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys 85 90 95 Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 225 230 235 240 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 370 375 380 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 123234PRTHomo sapiens 123Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp 35 40 45 Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60 Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr 100 105 110 Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 12415PRTArtificial SequenceLinker peptide 124Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys 1 5 10 15 1253519DNAArtificial SequenceTZM HC-MTf fusion protein polynucleotide 125atggagaccg acaccctgct gctctgggtg ctgctgctct gggtgcccgg ctccaccgga 60gaggtgcagc tggtggagag cggcggaggc ctcgtgcagc ccggcggatc tctgcggctg 120agctgcgccg ctagcggctt caacatcaag gacacctaca tccactgggt gcgccaggcc 180cccggcaagg gcctggagtg ggtggcccgg atctacccca ccaacggcta cacccgctac 240gccgacagcg tgaagggccg gttcaccatc agcgccgaca cctccaagaa caccgcctac 300ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcag ccggtggggc 360ggcgacggat tctacgccat ggactactgg ggacagggca ccctggtgac cgtgagcagc 420gcctctacca agggccccag cgtgttccct ctggccccca gcagcaagag caccagcggc 480ggaaccgccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt gaccgtgtcc 540tggaacagcg gcgctctgac cagcggagtg cacaccttcc ctgccgtgct gcagagcagc 600ggcctgtact ccctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc 660tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagaa ggtggagcct 720aagagctgcg acaagaccca cacctgccct ccctgccccg cccccgagct gctgggcgga 780cccagcgtgt tcctgttccc tcccaagccc aaggacaccc tgatgatcag ccgcaccccc 840gaggtgacct gcgtggtggt ggacgtgagc cacgaggacc ccgaggtgaa gttcaactgg 900tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc ctcgggagga gcagtacaac 960tccacctacc gcgtggtgag cgtgctgacc gtgctgcacc aggactggct gaacggcaag 1020gagtacaagt gcaaggtgag caacaaggcc ctgcccgctc ccatcgagaa gaccatcagc 1080aaggccaagg gccagccccg ggagcctcag gtgtacaccc tgccccccag ccgcgacgag 1140ctgaccaaga accaggtgag cctgacctgc ctggtgaagg gcttctaccc ctccgacatc 1200gccgtggagt gggagagcaa cggccagcct gagaacaact acaagaccac ccctcccgtg 1260ctggacagcg acggcagctt cttcctgtac agcaagctga ccgtggacaa gtcccggtgg 1320cagcagggca acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc 1380cagaagagcc tgagcctgag ccccggaaag ggtggcggag gatctggcgg aggcggatcc 1440ggcatggaag tgcgttggtg cgccacctct gaccccgagc agcacaagtg cggcaacatg 1500tccgaggcct tcagagaggc cggcatccag ccttctctgc tgtgtgtgcg gggcacctct 1560gccgaccatt gcgtgcagct gatcgccgcc caggaagccg acgctatcac actggatggc 1620ggcgctatct acgaggctgg caaagagcac ggcctgaagc ccgtcgtggg cgaggtgtac 1680gatcaggaag tgggcacctc ctactacgcc gtggctgtcg tgcggagatc ctcccacgtg 1740accatcgaca ccctgaaggg cgtgaagtcc tgccacaccg gcatcaacag aaccgtgggc 1800tggaacgtgc ccgtgggcta cctggtggaa tccggcagac tgtccgtgat gggctgcgac 1860gtgctgaagg ccgtgtccga ttacttcggc ggctcttgtg tgcctggcgc tggcgagaca 1920tcctactccg agtccctgtg cagactgtgc aggggcgact cttctggcga gggcgtgtgc 1980gacaagtccc ctctggaacg gtactacgac tactccggcg ccttcagatg cctggctgaa 2040ggtgctggcg acgtggcctt cgtgaagcac tccaccgtgc tggaaaacac cgacggcaag 2100accctgcctt cttggggcca ggcactgctg tcccaggact tcgagctgct gtgccgggat 2160ggctccagag ccgatgtgac agagtggcgg cagtgccacc tggccagagt gcctgcccat 2220gctgtggtcg tgcgcgccga tacagatggc ggcctgatct tccggctgct gaacgagggc 2280cagcggctgt tctctcacga gggctccagc ttccagatgt tctccagcga ggcctacggc 2340cagaaggacc tgctgttcaa ggactccacc tccgagctgg tgcctatcgc cacccagacc 2400tatgaggctt ggctgggcca cgagtacctg cacgctatga agggactgct gtgcgacccc 2460aaccggctgc ctccttatct gaggtggtgc gtgctgtcca cccccgagat ccagaaatgc 2520ggcgatatgg ccgtggcctt tcggcggcag agactgaagc ctgagatcca gtgcgtgtct 2580gccaagagcc ctcagcactg catggaacgg atccaggccg aacaggtgga cgccgtgaca 2640ctgtccggcg aggatatcta caccgccgga aagacctacg gcctggtgcc agctgctggc 2700gagcattacg cccctgagga ctcctccaac agctactacg tggtggcagt cgtgcgccgg 2760gactcctctc acgcctttac cctggatgag ctgcggggca agagaagctg tcacgccggc 2820tttggaagcc ctgccggatg ggatgtgcct gtgggcgctc tgatccagcg gggcttcatc 2880agacccaagg actgtgatgt gctgaccgcc gtgtctgagt tcttcaacgc ctcctgtgtg 2940cccgtgaaca accccaagaa ctacccctcc agcctgtgcg ccctgtgtgt gggagatgag 3000cagggccgga acaaatgcgt gggcaactcc caggaaagat attacggcta cagaggcgcc 3060ttccggtgtc tggtggaaaa cgccggggat gtggcttttg tgcggcacac caccgtgttc 3120gacaacacca atggccacaa ctccgagcct tgggccgctg agctgagatc cgaggattac 3180gaactgctgt gtcccaacgg cgccagggct gaggtgtccc agtttgccgc ctgtaacctg 3240gcccagatcc ctccccacgc tgtgatggtg cgacccgaca ccaacatctt caccgtgtac 3300ggcctgctgg acaaggccca ggatctgttc ggcgacgacc acaacaagaa cgggttcaag 3360atgttcgact ccagcaacta ccacggacag gatctgctgt ttaaagatgc caccgtgcgg 3420gccgtgccag tgggcgaaaa gaccacctac agaggatggc tgggactgga ctacgtggcc 3480gccctggaag gcatgtcctc ccagcagtgt tccggctag 3519126705DNAHomo sapiens 126atggagaccg acaccctgct gctctgggtg ctgctgctct gggtgcccgg ctccaccgga 60gacatccaga tgacccagag cccttccagc ctgagcgcca gcgtgggcga ccgggtgacc 120atcacctgcc gcgctagcca ggacgtgaac accgccgtgg cctggtacca gcagaagccc 180ggaaaggccc ccaagctgct gatctactct gctagcttcc tgtacagcgg cgtgcccagc 240cggttcagcg gatctcgcag cggcaccgac ttcaccctga ccatcagcag cctgcagcct 300gaggacttcg ccacctacta ctgccagcag cactacacca cgcctcccac cttcggacag 360ggcaccaagg tggagatcaa gcggaccgtg gccgccccca gcgtgttcat cttccctccc 420agcgacgagc agctgaagtc tggcaccgcc agcgtggtgt gcctgctgaa caacttctac 480ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 540gagagcgtga ccgagcagga ctccaaggac agcacctaca gcctgagcag caccctgacc 600ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccaggga 660ctgtctagcc ccgtgaccaa gagcttcaac cggggcgagt gctaa 7051271482DNAArtificial SequenceMTfp NH-TZM fusion protein polynucleotide 127atggagaccg acaccctgct gctctgggtg ctgctgctct gggtgcccgg ctccaccgga 60gactcctctc acgccttcac cctggacgag ctgcggtacg gtggcggagg atctggcgga 120ggcggatccg aggtgcagct ggtggagagc ggcggaggcc tcgtgcagcc cggcggatct 180ctgcggctga gctgcgccgc tagcggcttc aacatcaagg acacctacat ccactgggtg 240cgccaggccc ccggcaaggg cctggagtgg gtggcccgga tctaccccac caacggctac 300acccgctacg ccgacagcgt gaagggccgg ttcaccatca gcgccgacac ctccaagaac 360accgcctacc tgcagatgaa cagcctgcgc gccgaggaca ccgccgtgta ctactgcagc 420cggtggggcg gcgacggatt ctacgccatg gactactggg gacagggcac cctggtgacc 480gtgagcagcg cctctaccaa gggccccagc gtgttccctc tggcccccag cagcaagagc 540accagcggcg gaaccgccgc cctgggctgc ctggtgaagg actacttccc cgagcccgtg 600accgtgtcct ggaacagcgg cgctctgacc agcggagtgc acaccttccc tgccgtgctg 660cagagcagcg gcctgtactc cctgagcagc gtggtgaccg tgcccagcag cagcctgggc 720acccagacct acatctgcaa cgtgaaccac aagccctcca acaccaaggt ggacaagaag 780gtggagccta agagctgcga caagacccac acctgccctc cctgccccgc ccccgagctg 840ctgggcggac ccagcgtgtt cctgttccct cccaagccca aggacaccct gatgatcagc 900cgcacccccg aggtgacctg cgtggtggtg gacgtgagcc acgaggaccc cgaggtgaag 960ttcaactggt acgtggacgg cgtggaggtg cacaacgcca agaccaagcc tcgggaggag 1020cagtacaact ccacctaccg cgtggtgagc gtgctgaccg tgctgcacca ggactggctg 1080aacggcaagg agtacaagtg caaggtgagc aacaaggccc tgcccgctcc catcgagaag 1140accatcagca aggccaaggg ccagccccgg gagcctcagg tgtacaccct gccccccagc 1200cgcgacgagc tgaccaagaa ccaggtgagc ctgacctgcc tggtgaaggg cttctacccc 1260tccgacatcg ccgtggagtg ggagagcaac ggccagcctg agaacaacta caagaccacc 1320cctcccgtgc tggacagcga cggcagcttc ttcctgtaca gcaagctgac cgtggacaag 1380tcccggtggc agcagggcaa cgtgttcagc tgcagcgtga tgcacgaggc cctgcacaac 1440cactacaccc agaagagcct gagcctgagc cccggaaagt aa 1482128705DNAHomo sapiens 128atggagaccg acaccctgct gctctgggtg ctgctgctct gggtgcccgg ctccaccgga 60gacatccaga tgacccagag cccttccagc ctgagcgcca gcgtgggcga ccgggtgacc 120atcacctgcc gcgctagcca ggacgtgaac accgccgtgg cctggtacca gcagaagccc 180ggaaaggccc ccaagctgct gatctactct gctagcttcc tgtacagcgg cgtgcccagc 240cggttcagcg gatctcgcag cggcaccgac ttcaccctga ccatcagcag cctgcagcct 300gaggacttcg ccacctacta ctgccagcag cactacacca cgcctcccac cttcggacag 360ggcaccaagg tggagatcaa gcggaccgtg gccgccccca gcgtgttcat cttccctccc 420agcgacgagc agctgaagtc tggcaccgcc agcgtggtgt gcctgctgaa caacttctac 480ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 540gagagcgtga ccgagcagga ctccaaggac agcacctaca gcctgagcag caccctgacc 600ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccaggga 660ctgtctagcc ccgtgaccaa gagcttcaac cggggcgagt gctaa 7051291479PRTArtificial SequenceTZM HC-MTfp fusion protein polynucleotid 129Ala Thr Gly Gly Ala Gly Ala Cys Cys Gly Ala Cys Ala Cys Cys Cys 1 5 10 15 Thr Gly Cys Thr Gly Cys Thr Cys Thr Gly Gly Gly Thr Gly Cys Thr 20 25 30 Gly Cys Thr Gly Cys Thr Cys Thr Gly Gly Gly Thr Gly Cys Cys Cys 35 40 45 Gly Gly Cys Thr Cys Cys Ala Cys Cys Gly Gly Ala Gly Ala Gly Gly 50 55 60 Thr Gly Cys Ala Gly Cys Thr Gly Gly Thr Gly Gly Ala Gly Ala Gly 65 70 75 80 Cys Gly Gly Cys Gly Gly Ala Gly Gly Cys Cys Thr Cys Gly Thr Gly 85 90 95 Cys Ala Gly Cys Cys Cys Gly Gly Cys Gly Gly Ala Thr Cys Thr Cys 100 105 110 Thr Gly Cys Gly Gly Cys Thr Gly Ala Gly Cys Thr Gly Cys Gly Cys 115 120 125 Cys Gly Cys Thr Ala Gly Cys Gly Gly Cys Thr Thr Cys Ala Ala Cys 130 135 140 Ala Thr Cys Ala Ala Gly Gly Ala Cys Ala Cys Cys Thr Ala Cys Ala 145 150 155 160 Thr Cys Cys Ala Cys Thr Gly Gly Gly Thr Gly Cys Gly Cys Cys Ala 165 170 175 Gly Gly Cys Cys Cys Cys Cys Gly Gly Cys Ala Ala Gly Gly Gly Cys 180 185 190 Cys Thr Gly Gly Ala Gly Thr Gly Gly Gly Thr Gly Gly Cys Cys Cys 195 200 205 Gly Gly Ala Thr Cys Thr Ala Cys Cys Cys Cys Ala Cys Cys Ala Ala 210 215 220 Cys Gly Gly Cys Thr Ala Cys Ala Cys Cys Cys Gly Cys Thr Ala Cys 225 230 235 240 Gly Cys Cys Gly Ala Cys Ala Gly Cys Gly Thr Gly Ala Ala Gly Gly 245 250 255 Gly Cys Cys Gly Gly Thr Thr Cys Ala Cys Cys Ala Thr Cys Ala Gly 260 265 270 Cys Gly Cys Cys Gly Ala Cys Ala Cys Cys Thr Cys Cys Ala Ala Gly 275 280 285 Ala Ala Cys Ala Cys Cys Gly Cys Cys Thr Ala Cys Cys Thr Gly Cys 290 295 300 Ala Gly Ala Thr Gly Ala Ala Cys Ala Gly Cys Cys Thr Gly Cys Gly 305 310 315 320 Cys Gly Cys Cys Gly Ala Gly Gly Ala Cys Ala Cys Cys Gly Cys Cys 325 330 335 Gly Thr Gly Thr Ala Cys Thr Ala Cys Thr Gly Cys Ala Gly Cys Cys 340 345 350 Gly Gly Thr Gly Gly Gly Gly Cys Gly Gly Cys Gly Ala Cys Gly Gly 355 360 365 Ala Thr Thr Cys Thr Ala Cys Gly

Cys Cys Ala Thr Gly Gly Ala Cys 370 375 380 Thr Ala Cys Thr Gly Gly Gly Gly Ala Cys Ala Gly Gly Gly Cys Ala 385 390 395 400 Cys Cys Cys Thr Gly Gly Thr Gly Ala Cys Cys Gly Thr Gly Ala Gly 405 410 415 Cys Ala Gly Cys Gly Cys Cys Thr Cys Thr Ala Cys Cys Ala Ala Gly 420 425 430 Gly Gly Cys Cys Cys Cys Ala Gly Cys Gly Thr Gly Thr Thr Cys Cys 435 440 445 Cys Thr Cys Thr Gly Gly Cys Cys Cys Cys Cys Ala Gly Cys Ala Gly 450 455 460 Cys Ala Ala Gly Ala Gly Cys Ala Cys Cys Ala Gly Cys Gly Gly Cys 465 470 475 480 Gly Gly Ala Ala Cys Cys Gly Cys Cys Gly Cys Cys Cys Thr Gly Gly 485 490 495 Gly Cys Thr Gly Cys Cys Thr Gly Gly Thr Gly Ala Ala Gly Gly Ala 500 505 510 Cys Thr Ala Cys Thr Thr Cys Cys Cys Cys Gly Ala Gly Cys Cys Cys 515 520 525 Gly Thr Gly Ala Cys Cys Gly Thr Gly Thr Cys Cys Thr Gly Gly Ala 530 535 540 Ala Cys Ala Gly Cys Gly Gly Cys Gly Cys Thr Cys Thr Gly Ala Cys 545 550 555 560 Cys Ala Gly Cys Gly Gly Ala Gly Thr Gly Cys Ala Cys Ala Cys Cys 565 570 575 Thr Thr Cys Cys Cys Thr Gly Cys Cys Gly Thr Gly Cys Thr Gly Cys 580 585 590 Ala Gly Ala Gly Cys Ala Gly Cys Gly Gly Cys Cys Thr Gly Thr Ala 595 600 605 Cys Thr Cys Cys Cys Thr Gly Ala Gly Cys Ala Gly Cys Gly Thr Gly 610 615 620 Gly Thr Gly Ala Cys Cys Gly Thr Gly Cys Cys Cys Ala Gly Cys Ala 625 630 635 640 Gly Cys Ala Gly Cys Cys Thr Gly Gly Gly Cys Ala Cys Cys Cys Ala 645 650 655 Gly Ala Cys Cys Thr Ala Cys Ala Thr Cys Thr Gly Cys Ala Ala Cys 660 665 670 Gly Thr Gly Ala Ala Cys Cys Ala Cys Ala Ala Gly Cys Cys Cys Thr 675 680 685 Cys Cys Ala Ala Cys Ala Cys Cys Ala Ala Gly Gly Thr Gly Gly Ala 690 695 700 Cys Ala Ala Gly Ala Ala Gly Gly Thr Gly Gly Ala Gly Cys Cys Thr 705 710 715 720 Ala Ala Gly Ala Gly Cys Thr Gly Cys Gly Ala Cys Ala Ala Gly Ala 725 730 735 Cys Cys Cys Ala Cys Ala Cys Cys Thr Gly Cys Cys Cys Thr Cys Cys 740 745 750 Cys Thr Gly Cys Cys Cys Cys Gly Cys Cys Cys Cys Cys Gly Ala Gly 755 760 765 Cys Thr Gly Cys Thr Gly Gly Gly Cys Gly Gly Ala Cys Cys Cys Ala 770 775 780 Gly Cys Gly Thr Gly Thr Thr Cys Cys Thr Gly Thr Thr Cys Cys Cys 785 790 795 800 Thr Cys Cys Cys Ala Ala Gly Cys Cys Cys Ala Ala Gly Gly Ala Cys 805 810 815 Ala Cys Cys Cys Thr Gly Ala Thr Gly Ala Thr Cys Ala Gly Cys Cys 820 825 830 Gly Cys Ala Cys Cys Cys Cys Cys Gly Ala Gly Gly Thr Gly Ala Cys 835 840 845 Cys Thr Gly Cys Gly Thr Gly Gly Thr Gly Gly Thr Gly Gly Ala Cys 850 855 860 Gly Thr Gly Ala Gly Cys Cys Ala Cys Gly Ala Gly Gly Ala Cys Cys 865 870 875 880 Cys Cys Gly Ala Gly Gly Thr Gly Ala Ala Gly Thr Thr Cys Ala Ala 885 890 895 Cys Thr Gly Gly Thr Ala Cys Gly Thr Gly Gly Ala Cys Gly Gly Cys 900 905 910 Gly Thr Gly Gly Ala Gly Gly Thr Gly Cys Ala Cys Ala Ala Cys Gly 915 920 925 Cys Cys Ala Ala Gly Ala Cys Cys Ala Ala Gly Cys Cys Thr Cys Gly 930 935 940 Gly Gly Ala Gly Gly Ala Gly Cys Ala Gly Thr Ala Cys Ala Ala Cys 945 950 955 960 Thr Cys Cys Ala Cys Cys Thr Ala Cys Cys Gly Cys Gly Thr Gly Gly 965 970 975 Thr Gly Ala Gly Cys Gly Thr Gly Cys Thr Gly Ala Cys Cys Gly Thr 980 985 990 Gly Cys Thr Gly Cys Ala Cys Cys Ala Gly Gly Ala Cys Thr Gly Gly 995 1000 1005 Cys Thr Gly Ala Ala Cys Gly Gly Cys Ala Ala Gly Gly Ala Gly 1010 1015 1020 Thr Ala Cys Ala Ala Gly Thr Gly Cys Ala Ala Gly Gly Thr Gly 1025 1030 1035 Ala Gly Cys Ala Ala Cys Ala Ala Gly Gly Cys Cys Cys Thr Gly 1040 1045 1050 Cys Cys Cys Gly Cys Thr Cys Cys Cys Ala Thr Cys Gly Ala Gly 1055 1060 1065 Ala Ala Gly Ala Cys Cys Ala Thr Cys Ala Gly Cys Ala Ala Gly 1070 1075 1080 Gly Cys Cys Ala Ala Gly Gly Gly Cys Cys Ala Gly Cys Cys Cys 1085 1090 1095 Cys Gly Gly Gly Ala Gly Cys Cys Thr Cys Ala Gly Gly Thr Gly 1100 1105 1110 Thr Ala Cys Ala Cys Cys Cys Thr Gly Cys Cys Cys Cys Cys Cys 1115 1120 1125 Ala Gly Cys Cys Gly Cys Gly Ala Cys Gly Ala Gly Cys Thr Gly 1130 1135 1140 Ala Cys Cys Ala Ala Gly Ala Ala Cys Cys Ala Gly Gly Thr Gly 1145 1150 1155 Ala Gly Cys Cys Thr Gly Ala Cys Cys Thr Gly Cys Cys Thr Gly 1160 1165 1170 Gly Thr Gly Ala Ala Gly Gly Gly Cys Thr Thr Cys Thr Ala Cys 1175 1180 1185 Cys Cys Cys Thr Cys Cys Gly Ala Cys Ala Thr Cys Gly Cys Cys 1190 1195 1200 Gly Thr Gly Gly Ala Gly Thr Gly Gly Gly Ala Gly Ala Gly Cys 1205 1210 1215 Ala Ala Cys Gly Gly Cys Cys Ala Gly Cys Cys Thr Gly Ala Gly 1220 1225 1230 Ala Ala Cys Ala Ala Cys Thr Ala Cys Ala Ala Gly Ala Cys Cys 1235 1240 1245 Ala Cys Cys Cys Cys Thr Cys Cys Cys Gly Thr Gly Cys Thr Gly 1250 1255 1260 Gly Ala Cys Ala Gly Cys Gly Ala Cys Gly Gly Cys Ala Gly Cys 1265 1270 1275 Thr Thr Cys Thr Thr Cys Cys Thr Gly Thr Ala Cys Ala Gly Cys 1280 1285 1290 Ala Ala Gly Cys Thr Gly Ala Cys Cys Gly Thr Gly Gly Ala Cys 1295 1300 1305 Ala Ala Gly Thr Cys Cys Cys Gly Gly Thr Gly Gly Cys Ala Gly 1310 1315 1320 Cys Ala Gly Gly Gly Cys Ala Ala Cys Gly Thr Gly Thr Thr Cys 1325 1330 1335 Ala Gly Cys Thr Gly Cys Ala Gly Cys Gly Thr Gly Ala Thr Gly 1340 1345 1350 Cys Ala Cys Gly Ala Gly Gly Cys Cys Cys Thr Gly Cys Ala Cys 1355 1360 1365 Ala Ala Cys Cys Ala Cys Thr Ala Cys Ala Cys Cys Cys Ala Gly 1370 1375 1380 Ala Ala Gly Ala Gly Cys Cys Thr Gly Ala Gly Cys Cys Thr Gly 1385 1390 1395 Ala Gly Cys Cys Cys Cys Gly Gly Ala Ala Ala Gly Gly Gly Thr 1400 1405 1410 Gly Gly Cys Gly Gly Ala Gly Gly Ala Thr Cys Thr Gly Gly Cys 1415 1420 1425 Gly Gly Ala Gly Gly Cys Gly Gly Ala Thr Cys Cys Gly Ala Cys 1430 1435 1440 Thr Cys Cys Thr Cys Thr Cys Ala Cys Gly Cys Cys Thr Thr Cys 1445 1450 1455 Ala Cys Cys Cys Thr Gly Gly Ala Cys Gly Ala Gly Cys Thr Gly 1460 1465 1470 Cys Gly Gly Thr Ala Gly 1475 130705DNAHomo sapiens 130atggagaccg acaccctgct gctctgggtg ctgctgctct gggtgcccgg ctccaccgga 60gacatccaga tgacccagag cccttccagc ctgagcgcca gcgtgggcga ccgggtgacc 120atcacctgcc gcgctagcca ggacgtgaac accgccgtgg cctggtacca gcagaagccc 180ggaaaggccc ccaagctgct gatctactct gctagcttcc tgtacagcgg cgtgcccagc 240cggttcagcg gatctcgcag cggcaccgac ttcaccctga ccatcagcag cctgcagcct 300gaggacttcg ccacctacta ctgccagcag cactacacca cgcctcccac cttcggacag 360ggcaccaagg tggagatcaa gcggaccgtg gccgccccca gcgtgttcat cttccctccc 420agcgacgagc agctgaagtc tggcaccgcc agcgtggtgt gcctgctgaa caacttctac 480ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 540gagagcgtga ccgagcagga ctccaaggac agcacctaca gcctgagcag caccctgacc 600ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccaggga 660ctgtctagcc ccgtgaccaa gagcttcaac cggggcgagt gctaa 7051313534DNAArtificial SequenceTZM/MTf fusion protein polynucleotide 131atggagaccg acaccctgct gctctgggtg ctgctgctct gggtgcccgg ctccaccgga 60gaggtgcagc tggtggagag cggcggaggc ctcgtgcagc ccggcggatc tctgcggctg 120agctgcgccg ctagcggctt caacatcaag gacacctaca tccactgggt gcgccaggcc 180cccggcaagg gcctggagtg ggtggcccgg atctacccca ccaacggcta cacccgctac 240gccgacagcg tgaagggccg gttcaccatc agcgccgaca cctccaagaa caccgcctac 300ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcag ccggtggggc 360ggcgacggat tctacgccat ggactactgg ggacagggca ccctggtgac cgtgagcagc 420gcctctacca agggccccag cgtgttccct ctggccccca gcagcaagag caccagcggc 480ggaaccgccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt gaccgtgtcc 540tggaacagcg gcgctctgac cagcggagtg cacaccttcc ctgccgtgct gcagagcagc 600ggcctgtact ccctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc 660tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagaa ggtggagcct 720aagagctgcg acaagaccca cacctgccct ccctgccccg cccccgagct gctgggcgga 780cccagcgtgt tcctgttccc tcccaagccc aaggacaccc tgatgatcag ccgcaccccc 840gaggtgacct gcgtggtggt ggacgtgagc cacgaggacc ccgaggtgaa gttcaactgg 900tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc ctcgggagga gcagtacaac 960tccacctacc gcgtggtgag cgtgctgacc gtgctgcacc aggactggct gaacggcaag 1020gagtacaagt gcaaggtgag caacaaggcc ctgcccgctc ccatcgagaa gaccatcagc 1080aaggccaagg gccagccccg ggagcctcag gtgtacaccc tgccccccag ccgcgacgag 1140ctgaccaaga accaggtgag cctgacctgc ctggtgaagg gcttctaccc ctccgacatc 1200gccgtggagt gggagagcaa cggccagcct gagaacaact acaagaccac ccctcccgtg 1260ctggacagcg acggcagctt cttcctgtac agcaagctga ccgtggacaa gtcccggtgg 1320cagcagggca acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc 1380cagaagagcc tgagcctgag ccccggaaag ggaggtggcg gttctggtgg cggaggatct 1440ggcggaggcg gatccggcat ggaagtgcgt tggtgcgcca cctctgaccc cgagcagcac 1500aagtgcggca acatgtccga ggccttcaga gaggccggca tccagccttc tctgctgtgt 1560gtgcggggca cctctgccga ccattgcgtg cagctgatcg ccgcccagga agccgacgct 1620atcacactgg atggcggcgc tatctacgag gctggcaaag agcacggcct gaagcccgtc 1680gtgggcgagg tgtacgatca ggaagtgggc acctcctact acgccgtggc tgtcgtgcgg 1740agatcctccc acgtgaccat cgacaccctg aagggcgtga agtcctgcca caccggcatc 1800aacagaaccg tgggctggaa cgtgcccgtg ggctacctgg tggaatccgg cagactgtcc 1860gtgatgggct gcgacgtgct gaaggccgtg tccgattact tcggcggctc ttgtgtgcct 1920ggcgctggcg agacatccta ctccgagtcc ctgtgcagac tgtgcagggg cgactcttct 1980ggcgagggcg tgtgcgacaa gtcccctctg gaacggtact acgactactc cggcgccttc 2040agatgcctgg ctgaaggtgc tggcgacgtg gccttcgtga agcactccac cgtgctggaa 2100aacaccgacg gcaagaccct gccttcttgg ggccaggcac tgctgtccca ggacttcgag 2160ctgctgtgcc gggatggctc cagagccgat gtgacagagt ggcggcagtg ccacctggcc 2220agagtgcctg ctcatgctgt ggtcgtgcgc gccgatacag atggcggcct gatcttccgg 2280ctgctgaacg agggccagcg gctgttctct cacgagggct ccagcttcca gatgttctcc 2340agcgaggcct acggccagaa ggacctgctg ttcaaggact ccacctccga gctggtgcct 2400atcgccaccc agacctatga ggcttggctg ggccacgagt acctgcacgc tatgaaggga 2460ctgctgtgcg accccaaccg gctgcctcct tatctgaggt ggtgcgtgct gtccaccccc 2520gagatccaga aatgcggcga tatggccgtg gcctttcggc ggcagagact gaagcctgag 2580atccagtgcg tgtccgccaa gagccctcag cactgcatgg aacggatcca ggccgaacag 2640gtggacgccg tgacactgtc cggcgaggat atctacaccg ccggaaagac ctacggcctg 2700gtgccagctg ctggcgagca ttacgcccct gaggactcct ccaacagcta ctacgtggtg 2760gcagtcgtgc gccgggactc ctctcacgcc tttaccctgg atgagctgcg gggcaagaga 2820agctgtcacg ccggctttgg aagccctgcc ggatgggatg tgcctgtggg cgctctgatc 2880cagcggggct tcatcagacc caaggactgt gatgtgctga ccgccgtgtc tgagttcttc 2940aacgcctcct gtgtgcccgt gaacaacccc aagaactacc cctccagcct gtgcgccctg 3000tgtgtgggag atgagcaggg ccggaacaaa tgcgtgggca actcccagga aagatattac 3060ggctacagag gcgccttccg gtgtctggtg gaaaacgccg gggatgtggc ttttgtgcgg 3120cacaccaccg tgttcgacaa caccaatggc cacaactccg agccttgggc cgctgagctg 3180agatccgagg attacgaact gctgtgtccc aacggcgcca gggctgaggt gtcccagttt 3240gccgcctgta acctggccca gatccctccc cacgctgtga tggtgcgacc cgacaccaac 3300atcttcaccg tgtacggcct gctggacaag gcccaggatc tgttcggcga cgaccacaac 3360aagaacgggt tcaagatgtt cgactccagc aactaccacg gacaggatct gctgtttaaa 3420gatgccaccg tgcgggccgt gccagtgggc gaaaagacca cctacagagg atggctggga 3480ctggactacg tggccgccct ggaaggcatg tcctcccagc agtgttccgg ctag 3534132705DNAHomo sapiens 132atggagaccg acaccctgct gctctgggtg ctgctgctct gggtgcccgg ctccaccgga 60gacatccaga tgacccagag cccttccagc ctgagcgcca gcgtgggcga ccgggtgacc 120atcacctgcc gcgctagcca ggacgtgaac accgccgtgg cctggtacca gcagaagccc 180ggaaaggccc ccaagctgct gatctactct gctagcttcc tgtacagcgg cgtgcccagc 240cggttcagcg gatctcgcag cggcaccgac ttcaccctga ccatcagcag cctgcagcct 300gaggacttcg ccacctacta ctgccagcag cactacacca cgcctcccac cttcggacag 360ggcaccaagg tggagatcaa gcggaccgtg gccgccccca gcgtgttcat cttccctccc 420agcgacgagc agctgaagtc tggcaccgcc agcgtggtgt gcctgctgaa caacttctac 480ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 540gagagcgtga ccgagcagga ctccaaggac agcacctaca gcctgagcag caccctgacc 600ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccaggga 660ctgtctagcc ccgtgaccaa gagcttcaac cggggcgagt gctaa 7051331497DNAHomo sapiens 133atggagaccg acaccctgct gctctgggtg ctgctgctct gggtgcccgg ctccaccgga 60gaggtgcagc tggtggagag cggcggaggc ctcgtgcagc ccggcggatc tctgcggctg 120agctgcgccg ctagcggctt caacatcaag gacacctaca tccactgggt gcgccaggcc 180cccggcaagg gcctggagtg ggtggcccgg atctacccca ccaacggcta cacccgctac 240gccgacagcg tgaagggccg gttcaccatc agcgccgaca cctccaagaa caccgcctac 300ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcag ccggtggggc 360ggcgacggat tctacgccat ggactactgg ggacagggca ccctggtgac cgtgagcagc 420gcctctacca agggccccag cgtgttccct ctggccccca gcagcaagag caccagcggc 480ggaaccgccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt gaccgtgtcc 540tggaacagcg gcgctctgac cagcggagtg cacaccttcc ctgccgtgct gcagagcagc 600ggcctgtact ccctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc 660tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagaa ggtggagcct 720aagagctgcg acaagaccca cacctgccct ccctgccccg cccccgagct gctgggcgga 780cccagcgtgt tcctgttccc tcccaagccc aaggacaccc tgatgatcag ccgcaccccc 840gaggtgacct gcgtggtggt ggacgtgagc cacgaggacc ccgaggtgaa gttcaactgg 900tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc ctcgggagga gcagtacaac 960tccacctacc gcgtggtgag cgtgctgacc gtgctgcacc aggactggct gaacggcaag 1020gagtacaagt gcaaggtgag caacaaggcc ctgcccgctc ccatcgagaa gaccatcagc 1080aaggccaagg gccagccccg ggagcctcag gtgtacaccc tgccccccag ccgcgacgag 1140ctgaccaaga accaggtgag cctgacctgc ctggtgaagg gcttctaccc ctccgacatc 1200gccgtggagt gggagagcaa cggccagcct gagaacaact acaagaccac ccctcccgtg 1260ctggacagcg acggcagctt cttcctgtac agcaagctga ccgtggacaa gtcccggtgg 1320cagcagggca acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc 1380cagaagagcc tgagcctgag ccccggaaag gaggccgctg ctaaagaggc tgccgccaaa 1440gaagccgccg ctaaggactc ctctcacgcc ttcaccctgg acgagctgcg gtactaa 1497134789DNAArtificial SequenceLC-MTfp/Fc-MTfp fusion protein polynucleotid 134atggagaccg acaccctgct gctctgggtg ctgctgctct gggtgcccgg ctccaccgga 60gacatccaga tgacccagag cccttccagc ctgagcgcca gcgtgggcga ccgggtgacc 120atcacctgcc gcgctagcca ggacgtgaac accgccgtgg cctggtacca gcagaagccc 180ggaaaggccc ccaagctgct gatctactct gctagcttcc tgtacagcgg cgtgcccagc 240cggttcagcg gatctcgcag cggcaccgac ttcaccctga ccatcagcag cctgcagcct 300gaggacttcg ccacctacta ctgccagcag cactacacca cgcctcccac cttcggacag 360ggcaccaagg tagagatcaa gcggaccgtg gccgccccca gcgtgttcat cttccctccc 420agcgacgagc agctgaagtc tggcaccgcc agcgtggtgt gcctgctgaa caacttctac 480ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 540gagagcgtga ccgagcagga ctccaaggac agcacctaca gcctgagcag caccctgacc 600ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccaggga 660ctgtctagcc ccgtgaccaa gagcttcaac cggggcgagt gcgaggccgc tgctaaagag 720gctgccgcca aagaagccgc cgctaaggac tcctctcacg ccttcaccct ggacgagctg 780cggtactaa 7891351497DNAHomo sapiens 135atggagaccg acaccctgct gctctgggtg ctgctgctct gggtgcccgg ctccaccgga 60gaggtgcagc tggtggagag cggcggaggc ctcgtgcagc ccggcggatc tctgcggctg 120agctgcgccg

ctagcggctt caacatcaag gacacctaca tccactgggt gcgccaggcc 180cccggcaagg gcctggagtg ggtggcccgg atctacccca ccaacggcta cacccgctac 240gccgacagcg tgaagggccg gttcaccatc agcgccgaca cctccaagaa caccgcctac 300ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcag ccggtggggc 360ggcgacggat tctacgccat ggactactgg ggacagggca ccctggtgac cgtgagcagc 420gcctctacca agggccccag cgtgttccct ctggccccca gcagcaagag caccagcggc 480ggaaccgccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt gaccgtgtcc 540tggaacagcg gcgctctgac cagcggagtg cacaccttcc ctgccgtgct gcagagcagc 600ggcctgtact ccctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc 660tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagaa ggtggagcct 720aagagctgcg acaagaccca cacctgccct ccctgccccg cccccgagct gctgggcgga 780cccagcgtgt tcctgttccc tcccaagccc aaggacaccc tgatgatcag ccgcaccccc 840gaggtgacct gcgtggtggt ggacgtgagc cacgaggacc ccgaggtgaa gttcaactgg 900tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc ctcgggagga gcagtacaac 960tccacctacc gcgtggtgag cgtgctgacc gtgctgcacc aggactggct gaacggcaag 1020gagtacaagt gcaaggtgag caacaaggcc ctgcccgctc ccatcgagaa gaccatcagc 1080aaggccaagg gccagccccg ggagcctcag gtgtacaccc tgccccccag ccgcgacgag 1140ctgaccaaga accaggtgag cctgacctgc ctggtgaagg gcttctaccc ctccgacatc 1200gccgtggagt gggagagcaa cggccagcct gagaacaact acaagaccac ccctcccgtg 1260ctggacagcg acggcagctt cttcctgtac agcaagctga ccgtggacaa gtcccggtgg 1320cagcagggca acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc 1380cagaagagcc tgagcctgag ccccggaaag gaggccgctg ctaaagaggc tgccgccaaa 1440gaagccgccg ctaaggactc ctctcacgcc ttcaccctgg acgagctgcg gtactaa 1497136705DNAHomo sapiens 136atggagaccg acaccctgct gctctgggtg ctgctgctct gggtgcccgg ctccaccgga 60gacatccaga tgacccagag cccttccagc ctgagcgcca gcgtgggcga ccgggtgacc 120atcacctgcc gcgctagcca ggacgtgaac accgccgtgg cctggtacca gcagaagccc 180ggaaaggccc ccaagctgct gatctactct gctagcttcc tgtacagcgg cgtgcccagc 240cggttcagcg gatctcgcag cggcaccgac ttcaccctga ccatcagcag cctgcagcct 300gaggacttcg ccacctacta ctgccagcag cactacacca cgcctcccac cttcggacag 360ggcaccaagg tggagatcaa gcggaccgtg gccgccccca gcgtgttcat cttccctccc 420agcgacgagc agctgaagtc tggcaccgcc agcgtggtgt gcctgctgaa caacttctac 480ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 540gagagcgtga ccgagcagga ctccaaggac agcacctaca gcctgagcag caccctgacc 600ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccaggga 660ctgtctagcc ccgtgaccaa gagcttcaac cggggcgagt gctaa 7051371413DNAHomo sapiens 137atggagaccg acaccctgct gctctgggtg ctgctgctct gggtgcccgg ctccaccgga 60gaggtgcagc tggtggagag cggcggaggc ctcgtgcagc ccggcggatc tctgcggctg 120agctgcgccg ctagcggctt caacatcaag gacacctaca tccactgggt gcgccaggcc 180cccggcaagg gcctggagtg ggtggcccgg atctacccca ccaacggcta cacccgctac 240gccgacagcg tgaagggccg gttcaccatc agcgccgaca cctccaagaa caccgcctac 300ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcag ccggtggggc 360ggcgacggat tctacgccat ggactactgg ggacagggca ccctggtgac cgtgagcagc 420gcctctacca agggccccag cgtgttccct ctggccccca gcagcaagag caccagcggc 480ggaaccgccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt gaccgtgtcc 540tggaacagcg gcgctctgac cagcggagtg cacaccttcc ctgccgtgct gcagagcagc 600ggcctgtact ccctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc 660tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagaa ggtggagcct 720aagagctgcg acaagaccca cacctgccct ccctgccccg cccccgagct gctgggcgga 780cccagcgtgt tcctgttccc tcccaagccc aaggacaccc tgatgatcag ccgcaccccc 840gaggtgacct gcgtggtggt ggacgtgagc cacgaggacc ccgaggtgaa gttcaactgg 900tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc ctcgggagga gcagtacaac 960tccacctacc gcgtggtgag cgtgctgacc gtgctgcacc aggactggct gaacggcaag 1020gagtacaagt gcaaggtgag caacaaggcc ctgcccgctc ccatcgagaa gaccatcagc 1080aaggccaagg gccagccccg ggagcctcag gtgtacaccc tgccccccag ccgcgacgag 1140ctgaccaaga accaggtgag cctgacctgc ctggtgaagg gcttctaccc ctccgacatc 1200gccgtggagt gggagagcaa cggccagcct gagaacaact acaagaccac ccctcccgtg 1260ctggacagcg acggcagctt cttcctgtac agcaagctga ccgtggacaa gtcccggtgg 1320cagcagggca acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc 1380cagaagagcc tgagcctgag ccccggaaag taa 1413138789DNAArtificial SequenceLC-MTfp/Fc fusion protein polynucleotide 138atggagaccg acaccctgct gctctgggtg ctgctgctct gggtgcccgg ctccaccgga 60gacatccaga tgacccagag cccttccagc ctgagcgcca gcgtgggcga ccgggtgacc 120atcacctgcc gcgctagcca ggacgtgaac accgccgtgg cctggtacca gcagaagccc 180ggaaaggccc ccaagctgct gatctactct gctagcttcc tgtacagcgg cgtgcccagc 240cggttcagcg gatctcgcag cggcaccgac ttcaccctga ccatcagcag cctgcagcct 300gaggacttcg ccacctacta ctgccagcag cactacacca cgcctcccac cttcggacag 360ggcaccaagg tagagatcaa gcggaccgtg gccgccccca gcgtgttcat cttccctccc 420agcgacgagc agctgaagtc tggcaccgcc agcgtggtgt gcctgctgaa caacttctac 480ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 540gagagcgtga ccgagcagga ctccaaggac agcacctaca gcctgagcag caccctgacc 600ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccaggga 660ctgtctagcc ccgtgaccaa gagcttcaac cggggcgagt gcgaggccgc tgctaaagag 720gctgccgcca aagaagccgc cgctaaggac tcctctcacg ccttcaccct ggacgagctg 780cggtactaa 789

Claims

1. A p97 fusion protein, comprising a trastuzumab heavy chain or light chain sequence fused to a p97 sequence and an optional linker in between.

2. The p97 fusion protein of claim 1, comprising SEQ ID NO:84 (MTfp NH-TZM) or 82 (TZM HC-MTf), or a variant/fragment thereof.

3. The p97 fusion protein of claim 1, comprising a trastuzumab heavy chain sequence fused to the N-terminus of the p97 sequence.

4. The p97 fusion protein of claim 1, comprising a trastuzumab heavy chain sequence fused to the C-terminus of the p97 sequence.

5. The p97 fusion protein of claim 4, comprising a truncated trastuzumab heavy chain sequence fused to the C-terminus of the p97 sequence.

6. The p97 fusion protein of claim 5, where the truncated trastuzumab heavy chain sequence consists essentially of the heavy chain constant region or a fragment thereof and substantially or entirely lacks the heavy chain variable region.

7. The p97 fusion protein of claim 6, where the truncated trastuzumab heavy chain sequence consists essentially of the CH1 domain or a fragment thereof, the hinge region, the CH2 domain, and the CH3 domain.

8. The p97 fusion protein of claim 6, where the truncated trastuzumab heavy chain sequence consists essentially of the hinge region or a fragment thereof, the CH2 domain, and the CH3 domain.

9. The p97 fusion protein of any of the preceding claims, comprising (a) a heavy chain amino acid sequence set forth in SEQ ID NOs:37-46 or 96-109; (b) a heavy chain amino acid sequence at least 90% identical to a sequence set forth in SEQ ID NOs:37-46 or 96-109; (c) or a heavy chain amino acid sequence that differs from SEQ ID NOs:37-46 or 96-109 by addition, substitution, insertion, or deletion of about 1-50 amino acids.

10. The p97 fusion protein of claim 9, comprising an amino acid sequence set forth in SEQ ID NOs:37-46 or 96-109.

11. The p97 fusion protein of claim 1, comprising a trastuzumab light chain sequence fused to the N-terminus of the p97 sequence.

12. The p97 fusion protein of claim 1, comprising a trastuzumab light chain sequence fused to the C-terminus of the p97 sequence.

13. The p97 fusion protein of any of claims 1 or 11-12, comprising (a) a light amino acid sequence set forth in SEQ ID NOs:110-121; (b) a light chain amino acid sequence at least 90% identical to a sequence set forth in SEQ ID NOs: 110-121; (c) or a light chain amino acid sequence that differs from SEQ ID NOs: 110-121 by addition, substitution, insertion, or deletion of about 1-50 amino acids.

14. The p97 fusion protein of claim 13, comprising an amino acid sequence set forth in SEQ ID NOs:110-121, or a variant/fragment thereof.

15. The p97 fusion protein of any of the preceding claims, where the p97 sequence comprises SEQ ID NO:2 or 14, or a variant/fragment thereof.

16. The p97 fusion protein of any of the preceding claims, comprising a peptide linker between the trastuzumab heavy chain or light chain and the p97 sequence.

17. An isolated polynucleotide which encodes a p97 fusion protein of any of the preceding claims.

18. The isolated polynucleotide of claim 17, which is codon-optimized for expression in a host cell.

19. The isolated polynucleotide of claim 18, where the host cell is a mammalian cell, an insect cell, a yeast cell, or a bacterial cell.

20. A recombinant host cell, comprising an isolated polynucleotide of any of the preceding claims, where the isolated polynucleotide is operably linked to one or more regulatory elements.

21. The recombinant host cell of claim 20, further comprising an isolated polynucleotide that encodes a (non-fusion) trastuzumab light chain sequence, which is operably linked to one or more regulatory elements.

22. The recombinant host cell of claim 20, further comprising an isolated polynucleotide that encodes a (non-fusion) trastuzumab heavy chain sequence, which is operably linked to one or more regulatory elements.

23. The recombinant host cell of claim 20, further comprising an isolated polynucleotide that encodes a (non-fusion) trastuzumab light chain sequence, and an isolated polynucleotide that encodes a (non-fusion) trastuzumab heavy chain sequence, which are operably linked to one or more regulatory elements.

24. A vector, comprising an isolated polynucleotide, which encodes a p97 fusion protein of any of the preceding claims, which is operably linked to one or more regulatory elements.

25. The vector of claim 24, further comprising an isolated polynucleotide that encodes a (non-fusion) trastuzumab light chain sequence, which is operably linked to one or more regulatory elements.

26. The vector of claim 24, further comprising an isolated polynucleotide that encodes a (non-fusion) trastuzumab heavy chain sequence, which is operably linked to one or more regulatory elements.

27. The vector of claim 24, further comprising an isolated polynucleotide that encodes a (non-fusion) trastuzumab light chain sequence, and an isolated polynucleotide that encodes a (non-fusion) trastuzumab heavy chain sequence, which are operably linked to one or more regulatory elements.

28. A recombinant host cell, comprising a vector of any of the preceding claims.

29. A p97-antibody fusion protein that comprises two (non-fusion) trastuzumab light chain sequences, and one or two p97-trastuzumab heavy chain fusion proteins of any of the preceding claims, where the one or two p97-trastuzumab heavy chain fusion protein(s) comprise a trastuzumab heavy chain sequence fused to the N-terminus or C-terminus of a p97 sequence and an optional linker in between.

30. The p97-antibody fusion protein of claim 29, comprising two p97-trastuzumab heavy chain fusion proteins.

31. The p97-antibody fusion protein of claim 29, comprising one p97-trastuzumab heavy chain fusion protein and one (non-fusion) trastuzumab heavy chain sequence.

32. A p97-antibody fusion protein that comprises one or two trastuzumab light chain sequences, one trastuzumab heavy chain sequence, and one p97-trastuzumab heavy chain fusion protein of any of the preceding claims, where the p97-trastuzumab heavy chain fusion protein comprises a trastuzumab heavy chain fused to the C-terminus of a p97 sequence and an optional linker in between.

33. The p97-antibody fusion protein of claim 32, comprising only one light chain sequence, where the trastuzumab heavy chain is a truncated trastuzumab heavy chain.

34. A p97-antibody fusion protein that comprises one or two p97-trastuzumab light chain fusion proteins of any of the preceding claims, and two trastuzumab heavy chain sequences, where the one or two p97-trastuzumab light chain fusion protein(s) comprise a trastuzumab light chain sequence fused to the N-terminus a p97 sequence and an optional linker in between.

35. The p97-antibody fusion protein of claim 34, comprising two p97-trastuzumab light chain fusion proteins.

36. The p97-antibody fusion protein of claim 34, comprising one p97-trastuzumab light chain fusion protein and one (non-fusion) trastuzumab light chain sequence.

37. A p97-antibody fusion protein that comprises one or two p97-trastuzumab light chain fusion proteins of any of the preceding claims, and one or two p97-trastuzumab heavy chain fusion proteins of any of the preceding claims, where the one or two p97-trastuzumab light chain fusion protein(s) comprise a trastuzumab light chain sequence fused to the N-terminus a p97 sequence and an optional linker in between, and where the one or two p97-trastuzumab heavy chain fusion protein(s) comprise a trastuzumab heavy chain sequence fused to the N-terminus or C-terminus of a p97 sequence and an optional linker in between.

38. The p97-antibody fusion protein of claim 37, comprising two p97-trastuzumab light chain fusion proteins and two p97-trastuzumab heavy chain fusion proteins, where the two p97-trastuzumab heavy chain fusion protein comprise a trastuzumab heavy chain sequence fused to the N-terminus of a p97 sequence and an optional linker in between.

39. A p97-antibody fusion protein that comprises two sets of heavy and light chains, where at least one set is selected from one or more of: a) the heavy chain of SEQ ID NO:82 and the light chain of SEQ ID NO:83; b) the heavy chain of SEQ ID NO:84 and the light chain of SEQ ID NO:85; c) the heavy chain of SEQ ID NO:86 and the light chain of SEQ ID NO:87; d) the heavy chain of SEQ ID NO:88 and the light chain of SEQ ID NO:89; e) the heavy chain of SEQ ID NO:90 and the light chain of SEQ ID NO:91; f) the heavy chain of SEQ ID NO:92 and the light chain of SEQ ID NO:93; and g) the heavy chain of SEQ ID NO:94 and the light chain of SEQ ID NO:95; including fragments/variants thereof.

40. The p97-antibody fusion protein of claim 39, where the fusion protein is a homodimer that comprises two sets of a), two sets of b), two sets of c), two sets of d), two sets of e), two sets of f), or two sets of g).

41. The p97-antibody fusion protein of claim 39, where the fusion protein is a heterodimer that comprises a first set of heavy and light chains selected from a)-g), and a second set of heavy and light chains selected from any combination of (i) the p97-trastuzumab heavy and light chains of SEQ ID NOS:37-46, 82-109, or 110-121, and (ii) the trastuzumab (non-fusion) heavy and light chains of SEQ ID NOs: 29-35 or 122 (heavy chains) and 36 or 123 (light chains).

42. A recombinant host cell, comprising a p97-antibody fusion protein of any of the preceding claims.

43. The recombinant host cell of any of the preceding claims, where the host cell is a mammalian cell, an insect cell, a yeast cell, or a bacterial cell.

44. The recombinant host cell of claim 43, where the mammalian cell is a Chinese hamster ovary (CHO) cell or a HEK-293 cell.

45. A pharmaceutical composition, comprising a pharmaceutically-acceptable carrier and a p97-antibody fusion protein of any of the preceding claims.

46. A method for the treatment of a HER2-overexpressing cancer in a subject in need thereof, comprising administering to the subject a p97-antibody fusion protein or pharmaceutical composition of any of the preceding claims.

47. The method of claim 46, where the HER2-overexpressing cancer is at risk for metastasizing to the CNS of the subject.

48. The method of claim 46, where the HER2-overexpressing cancer has metastasized to the CNS of the subject.

49. The method of any of the preceding claims, where the HER2-overexpressing cancer is a breast cancer, ovarian cancer, gastric cancer, or uterine cancer.

50. The method of any of claims 46-48, where the HER2-overexpressing cancer is a HER2-overexpressing metastatic breast cancer.

51. The method of claim 50, where the HER2-overexpressing metastatic breast cancer is at risk for metastasizing to the CNS of the subject.

52. The method of claim 50, where the HER2-overexpressing breast cancer has metastasized to the CNS of the subject.

53. The method of any of claims 46-48, where the HER2-overexpressing cancer is a HER2-overexpressing metastatic gastric or gastroesophageal junction adenocarcinoma.

54. The method of claim 53, where the HER2-overexpressing metastatic gastric or gastroesophageal junction adenocarcinoma is at risk for metastasizing to the CNS of the subject.

55. The method of claim 53, where the HER2-overexpressing metastatic gastric or gastroesophageal junction adenocarcinoma has metastasized to the CNS of the subject.

56. The method of any of claims 46-48, where the HER2-overexpressing cancer is a HER2-overexpressing uterine serous carcinoma (USC).

57. The method of claim 56, where the HER2-overexpressing USC is at risk for metastasizing to the CNS of the subject.

58. The method of claim 56, where the HER2-overexpressing USC has metastasized to the CNS of the subject.

59. The method of any of claims 46-48, comprising administering the p97-antibody fusion protein or pharmaceutical composition as part of an adjuvant treatment for a HER2-overexpressing breast cancer.

60. The method of claim 59, where the adjuvant treatment comprises doxorubicin, cyclophosphamide, and either paclitaxel or docetaxel.

61. The method of claim 59, where the adjuvant treatment comprises docetaxel and carboplatin.

62. The method of claim 59, comprising administering the p97-antibody fusion protein or pharmaceutical composition as a single agent following multi-modality anthracycline based therapy.

63. The method of any of the preceding claims, where the subject is a female human.

64. The method of any of the preceding claims, comprising administering the p97-antibody fusion protein or pharmaceutical composition by intravenous (IV) infusion.

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