Patent application title: ANTI-AMYLOID BETA ANTIBODIES AND THEIR USE
Inventors:
Michael Bardroff (Munchen, DE)
Hoffmann-La Roche Inc. (Nutley, NJ, US)
Bernd Bohrmann (Freiburg, DE)
Morphosys Ag (Martinsried, DE)
Manfred Brockhaus (Bettingen, CH)
Walter Huber (Kaiseraugst, CH)
Titus Kretzschmar (Hurlach, DE)
Hansruedi Loetscher (Mohlin, CH)
Corinna Lohning (Stockdorf, DE)
Christer Nordstedt (Sodertalje, SE)
Christine Rothe (Dachau, DE)
Christine Rothe (Dachau, DE)
Assignees:
MORPHOSYS AG
Hoffmann-La Roche Inc.
IPC8 Class: AC07K1618FI
USPC Class:
4241391
Class name: Drug, bio-affecting and body treating compositions immunoglobulin, antiserum, antibody, or antibody fragment, except conjugate or complex of the same with nonimmunoglobulin material binds antigen or epitope whose amino acid sequence is disclosed in whole or in part (e.g., binds specifically-identified amino acid sequence, etc.)
Publication date: 2013-05-30
Patent application number: 20130136747
Abstract:
The present invention relates to antibody molecules capable of
specifically recognizing two regions of the β-A4 peptide, wherein
the first region comprises the amino acid sequence AEFRHDSGY as shown in
SEQ ID NO: 1 or a fragment thereof and wherein the second region
comprises the amino acid sequence VHHQKLVFFAEDVG as shown in SEQ ID NO: 2
or a fragment thereof. Furthermore, nucleic acid molecules encoding the
inventive antibody molecules and vectors and hosts comprising said
nucleic acid molecules are disclosed. In addition, the present invention
provides for compositions, preferably pharmaceutical or diagnostic
compositions, comprising the compounds of the invention as well as for
specific uses of the antibody molecules, nucleic acid molecules, vectors
or hosts of the invention.Claims:
1-16. (canceled)
17. A method for treating a disease associated with amyloidogenesis and/or amyloid-plaque formation in a subject in need thereof comprising administering to the subject an antibody comprising: (a) a variable VL-Region comprising complementary determining regions, L-CDR1, L-CDR2, L-CDR3, wherein: (1) L-CDR1 comprises SEQ ID NO: 143; (2) L-CDR2 comprises SEQ ID NO: 144; and (3) L-CDR3 comprises SEQ ID NO: 95; and (b) a variable VH-Region comprising complementary determining regions, H-CDR1, H-CDR2, H-CDR3, wherein: (1) H-CDR1 comprises SEQ ID NO: 146; (2) H-CDR2 comprises SEQ ID NOs: 192; and (3) H-CDR3 comprises SEQ ID NOs: 93 in an amount effective to treat the disease.
18. A method for detecting a disease associated with amyloidogenesis and/or amyloid-plaque formation in a subject comprising: (i) providing a brain tissue from the subject; (ii) contacting the brain tissue with a composition comprising an antibody comprising: (a) a variable VL-Region comprising complementary determining regions, L-CDR1, L-CDR2, L-CDR3, wherein: (1) L-CDR1 comprises SEQ ID NO: 143; (2) L-CDR2 comprises SEQ ID NO: 144; and (3) L-CDR3 comprises SEQ ID NO: 95; and (b) a variable VH-Region comprising complementary determining regions, H-CDR1, H-CDR2, H-CDR3, wherein: (1) H-CDR1 comprises SEQ ID NO: 146; (2) H-CDR2 comprises SEQ ID NOs: 192; and (3) H-CDR3 comprises SEQ ID NOs: 93; and (iii) detecting the binding of antibody to the brain tissue.
19. A method for disintegrating β-amyloid plaques comprising contacting said β-amyloid plaque with an antibody comprising: (a) a variable VL-Region comprising complementary determining regions, L-CDR1, L-CDR2, L-CDR3, wherein: (1) L-CDR1 comprises SEQ ID NO: 143; (2) L-CDR2 comprises SEQ ID NO: 144; and (3) L-CDR3 comprises SEQ ID NO: 95; and (b) variable VH-Region comprising complementary determining regions, H-CDR1, H-CDR2, H-CDR3, wherein: (1) H-CDR1 comprises SEQ ID NO: 146; (2) H-CDR2 comprises SEQ ID NOs: 192; and (3) H-CDR3 comprises SEQ ID NOs: 93.
20. A method for passively immunizing a subject in need thereof against β-amyloid plaque formation comprising administering to the subject an effective amount of an antibody comprising: (a) a variable VL-Region comprising complementary determining regions, L-CDR1, L-CDR2, L-CDR3, wherein: (1) L-CDR1 comprises SEQ ID NO: 143; (2) L-CDR2 comprises SEQ ID NO: 144; and (3) L-CDR3 comprises SEQ ID NO: 95; and (b) a variable VH-Region comprising complementary determining regions, H-CDR1, H-CDR2, H-CDR3, wherein: (1) H-CDR1 comprises SEQ ID NO: 146; (2) H-CDR2 comprises SEQ ID NOs: 192; and (3) H-CDR3 comprises SEQ ID NOs: 93.
21. The method according to claim 17, wherein said disease is selected from the group consisting of dementia, Alzheimer's disease, motor neuropathy, Down's syndrome, Creutzfeld Jacob disease, hereditary cerebral hemorrhage with amyloidosis Dutch type, Parkinson's disease, HIV-related dementia, amyotropic lateral sclerosis (ALS), and neuronal disorders related to aging.
22-28. (canceled)
29. The method according to claim 17, wherein the antibody is of the IgG1 subtype.
30. The method according to claim 17, wherein the variable VH-region comprises SEQ ID NO: 89; and the variable VL-region comprises SEQ ID NO: 91.
31. The method according to claim 30, wherein the antibody is of the IgG1 subtype.
32. The method according to claim 17, wherein the variable VH-region comprises SEQ ID NO: 425; and the variable VL-region comprises SEQ ID NO: 91.
33. The method according to claim 32, wherein the antibody is of the IgG1 subtype.
34. The method according to claim 17, wherein the antibody is administered as a part of a pharmaceutical composition comprising the antibody and a pharmaceutically acceptable diluent.
35. The method according to claim 31, wherein the antibody is administered as a part of a pharmaceutical composition comprising the antibody and a pharmaceutically acceptable diluent.
36. The method according to claim 33, wherein the antibody is administered as a part of a pharmaceutical composition comprising the antibody and a pharmaceutically acceptable diluent.
37. The method according to claim 18, wherein said disease is selected from the group consisting of dementia, Alzheimer's disease, motor neuropathy, Down's syndrome, Creutzfeld Jacob disease, hereditary cerebral hemorrhage with amyloidosis Dutch type, Parkinson's disease, HIV-related dementia, amyotropic lateral sclerosis (ALS), and neuronal disorders related to aging.
38. The method according to claim 18, wherein the brain tissue is a brain section.
39. The method according to claim 18, wherein the antibody is of the IgG1 subtype.
40. The method according to claim 18, wherein the variable VH-region comprises SEQ ID NO: 89; and the variable VL-region comprises SEQ ID NO: 91.
41. The method according to claim 40, wherein the antibody is of the IgG1 subtype.
42. The method according to claim 18, wherein the variable VH-region comprises SEQ ID NO: 425; and the variable VL-region comprises SEQ ID NO: 91.
43. The method according to claim 42, wherein the antibody is of the IgG1 subtype.
44. The method according to claim 19, wherein disintegrating of the β-amyloid plaque occurs in vivo.
45. The method according to claim 19, wherein the antibody is of the IgG1 subtype.
46. The method according to claim 19, wherein the variable VH-region comprises SEQ ID NO: 89; and the variable VL-region comprises SEQ ID NO: 91.
47. The method according to claim 46, wherein the antibody is of the IgG1 subtype.
48. The method according to claim 19, wherein the variable VH-region comprises SEQ ID NO: 425; and the variable VL-region comprises SEQ ID NO: 91.
49. The method according to claim 48, wherein the antibody is of the IgG1 subtype.
50. The method according to claim 20, wherein the antibody is of the IgG1 subtype.
51. The method according to claim 20, wherein the variable VH-region comprises SEQ ID NO: 89; and the variable VL-region comprises SEQ ID NO: 91.
52. The method according to claim 51, wherein the antibody is of the IgG1 subtype.
53. The method according to claim 20, wherein the variable VH-region comprises SEQ ID NO: 425; and the variable VL-region comprises SEQ ID NO: 91.
54. The method according to claim 53, wherein the antibody is of the IgG1 subtype.
55. The method according to claim 20, wherein the antibody is administered as a part of a pharmaceutical composition comprising the antibody and a pharmaceutically acceptable diluent.
56. The method according to claim 53, wherein the antibody is administered as a part of a pharmaceutical composition comprising the antibody and a pharmaceutically acceptable diluent.
57. The method according to claim 55, wherein the antibody is administered as a part of a pharmaceutical composition comprising the antibody and a pharmaceutically acceptable diluent.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of and claims benefit to U.S. patent application Ser. No. 13/472,781 filed May 16, 2012, which is a divisional of and claims benefit to U.S. patent application Ser. No. 12/462,119, filed on Jul. 28, 2009, now issued as U.S. Pat. No. 8,216,577. The '119 application is a divisional of and claims benefit to U.S. patent application Ser. No. 10/505,313, filed Aug. 20, 2004, now issued as U.S. Pat. No. 7,794,719. The '313 application is the national stage of PCT/EP2003/001759, filed Feb. 20, 2003, which claims priority to European Application No. 02003844.4, filed Feb. 20, 2002.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
[0002] This application contains references to amino acids and/or nucleic acid sequences that have been filed concurrently herewith as sequence listing text file "0342525_sequence.txt", file size of 217 KB, created on Oct. 10, 2012. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. §1.52(e)(5).
FIELD OF THE INVENTION
[0003] The present invention relates to antibody molecules capable of specifically recognizing two regions of the β-A4 peptide, wherein the first region comprises the amino acid sequence AEFRHDSGY as shown in SEQ ID NO: 1 or a fragment thereof and wherein the second region comprises the amino acid sequence VHHQKLVFFAEDVG as shown in SEQ ID NO: 2 or a fragment thereof. Furthermore, nucleic acid molecules encoding the inventive antibody molecules and vectors and hosts comprising said nucleic acid molecules are disclosed. In addition, the present invention provides for compositions, preferably pharmaceutical or diagnostic compositions, comprising the compounds of the invention as well as for specific uses of the antibody molecules, nucleic acid molecules, vectors or hosts of the invention.
BACKGROUND OF THE INVENTION
[0004] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including any manufacturers specifications, instructions, etc.) are hereby incorporated by reference.
[0005] About 70% of all cases of dementia are due to Alzheimer's disease which is associated with selective damage of brain regions and neural circuits critical for cognition. Alzheimer's disease is characterized by neurofibrillary tangles in particular in pyramidal neurons of the hippocampus and numerous amyloid plaques containing mostly a dense core of amyloid deposits and defused halos.
[0006] The extracellular neuritic plaques contain large amounts of a pre-dominantly fibrillar peptide termed "amyloid β", "A-beta", "Aβ4", "β-A4" or "Aβ"; see Selkoe (1994), Ann. Rev. Cell Biol. 10, 373-403, Koo (1999), PNAS Vol. 96, pp. 9989-9990, U.S. Pat. No. 4,666,829 or Glenner (1984), BBRC 12, 1131. This amyloid β is derived from "Alzheimer precursor protein/β-amyloid precursor protein" (APP). APPs are integral membrane glycoproteins (see Sisodia (1992), PNAS Vol. 89, pp. 6075) and are endoproteolytically cleaved within the Aβ sequence by a plasma membrane protease, α-secretase (see Sisodia (1992), loc. cit.). Furthermore, further secretase activity, in particular β-secretase and γ-secretase activity leads to the extracellular release of amyloid-β (Aβ) comprising either 39 amino acids (Aβ39), 40 amino acids (Aβ40), 42 amino acids (Aβ42) or 43 amino acids (Aβ43); see Sinha (1999), PNAS 96, 11094-1053; Price (1998), Science 282, 1078 to 1083; WO 00/72880 or Hardy (1997), TINS 20, 154.
[0007] It is of note that Aβ has several naturally occurring forms, whereby the human forms are referred to as the above mentioned Aβ39, Aβ40, Aβ41, Aβ42 and Aβ43. The most prominent form, Aβ42, has the amino acid sequence (starting from the N-terminus): DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO: 27). In Aβ41, Aβ40, Aβ39, the C-terminal amino acids A, IA and VIA are missing, respectively. In the Aβ43-form an additional threonine residue is comprised at the C-terminus of the above depicted sequence (SEQ ID NO: 27).
[0008] The time required to nucleate Aβ40 fibrils was shown to be significantly longer than that to nucleate Aβ42 fibrils; see Koo, loc. cit. and Harper (1997), Ann. Rev. Biochem. 66, 385-407. As reviewed in Wagner (1999), J. Clin. Invest. 104, 1239-1332, the Aβ42 is more frequently found associated with neuritic plaques and is considered to be more fibrillogenic in vitro. It was also suggested that Aβ42 serves as a "seed" in the nucleation-dependent polymerization of ordered non-crystalline Aβ peptides; Jarrett (1993), Cell 93, 1055-1058.
[0009] It has to be stressed that modified APP processing and/or the generation of extracellular plaques containing proteinaceous depositions are not only known from Alzheimer's pathology but also from subjects suffering from other neurological and/or neurodegenerative disorders. These disorders comprise, inter alia, Down's syndrome, Hereditary cerebral hemorrhage with amyloidosis Dutch type, Parkinson's disease, ALS (amyotrophic lateral sclerosis), Creutzfeld Jacob disease, HIV-related dementia and motor neuropathy.
[0010] In order to prevent, treat and/or ameliorate disorders and/or diseases related to the pathological deposition of amyloid plaques, means and methods have to be developed which either interfere with β-amyloid plaque formation, which are capable of preventing Aβ aggregation and/or are useful in de-polymerization of already formed amyloid deposits or amyloid-β aggregates.
[0011] Accordingly, and considering the severe defects of modified and/or pathological amyloid biology, means and methods for treating amyloid related disorders are highly desirable. In particular, efficient drugs which either interfere with pathological amyloid aggregation or which are capable of de-polymerization of aggregated Aβ are desired. Furthermore, diagnostic means are desirable to detect, inter alia, amyloid plaques.
[0012] Thus, the technical problem of the present invention is to comply with the needs described herein above.
BRIEF SUMMARY OF THE INVENTION
[0013] Accordingly, the present invention relates to an antibody molecule capable of specifically recognizing two regions of the β-A4/Aβ4 peptide, wherein the first region comprises the amino acid sequence AEFRHDSGY (SEQ ID NO: 1) or a fragment thereof and wherein the second region comprises the amino acid sequence VHHQKLVFFAEDVG (SEQ ID NO: 2) or a fragment thereof.
[0014] In context of the present invention, the term "antibody molecule" relates to full immunoglobulin molecules, preferably IgMs, IgDs, IgEs, IgAs or IgGs, more preferably IgG1, IgG2a, IgG2b, IgG3 or IgG4 as well as to parts of such immunoglobulin molecules, like Fab-fragments or VL-, VH- or CDR-regions. Furthermore, the term relates to modified and/or altered antibody molecules, like chimeric and humanized antibodies. The term also relates to modified or altered monoclonal or polyclonal antibodies as well as to recombinantly or synthetically generated/synthesized antibodies. The term also relates to intact antibodies as well as to antibody fragments/parts thereof, like, separated light and heavy chains, Fab, Fab/c, Fv, Fab', F(ab')2. The term "antibody molecule" also comprises antibody derivatives, the bifunctional antibodies and antibody constructs, like single chain Fvs (scFv), bispecific scFvs or antibody-fusion proteins. Further details on the term "antibody molecule" of the invention are provided herein below.
[0015] The term "specifically recognizing" means in accordance with this invention that the antibody molecule is capable of specifically interacting with and/or binding to at least two amino acids of each of the two regions of β-A4 as defined herein. Said term relates to the specificity of the antibody molecule, i.e. to its ability to discriminate between the specific regions of the β-A4 peptide as defined herein and another, not related region of the β-A4 peptide or another, not APP-related protein/peptide/(unrelated) tests-peptide. Accordingly, specificity can be determined experimentally by methods known in the art and methods as disclosed and described herein. Such methods comprise, but are not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-tests and peptide scans. Such methods also comprise the determination of KD-values as, inter alia, illustrated in the appended examples. The peptide scan (pepspot assay) is routinely employed to map linear epitopes in a polypeptide antigen. The primary sequence of the polypeptide is synthesized successively on activated cellulose with peptides overlapping one another. The recognition of certain peptides by the antibody to be tested for its ability to detect or recognize a specific antigen/epitope is scored by routine colour development (secondary antibody with horseradish peroxidase and 4-chloronaphthol and hydrogenperoxide), by a chemoluminescence reaction or similar means known in the art. In the case of, inter alia, chemoluminescence reactions, the reaction can be quantified. If the antibody reacts with a certain set of overlapping peptides one can deduce the minimum sequence of amino acids that are necessary for reaction; see illustrative Example 6 and appended Table 2.
[0016] The same assay can reveal two distant clusters of reactive peptides, which indicate the recognition of a discontinuous, i.e. conformational epitope in the antigenic polypeptide (Geysen (1986), Mol. Immunol. 23, 709-715).
[0017] In addition to the pepspot assay, standard ELISA assay can be carried out. As demonstrated in the appended examples small hexapeptides may be coupled to a protein and coated to an immunoplate and reacted with antibodies to be tested. The scoring may be carried out by standard colour development (e.g. secondary antibody with horseradish peroxidase and tetramethyl benzidine with hydrogenperoxide). The reaction in certain wells is scored by the optical density, for example at 450 nm. Typical background (=negative reaction) may be 0.1 OD, typical positive reaction may be 1 OD. This means the difference (ratio) positive/negative can be more than 10 fold. Further details are given in the appended examples. Additional, quantitative methods for determining the specificity and the ability of "specifically recognizing" the herein defined two regions of the β-A4 peptide are given herein below.
[0018] The term "two regions of the β-A4 peptide" relates to two regions as defined by their amino acid sequences shown in SEQ ID NOs: 1 and 2, relating to the N-terminal amino acids 2 to 10 and to the central amino acids 12 to 25 of β-A4 peptide. The term "β-A4 peptide" in context of this invention relates to the herein above described Aβ39, Aβ41, Aβ43, preferably to Aβ40 and Aβ42. Aβ42 is also depicted in appended SEQ ID NO: 27. It is of note that the term "two regions of the β-A4 peptide" also relates to an "epitope" and/or an "antigenic determinant" which comprises the herein defined two regions of the β-A4 peptide or parts thereof. In accordance with this invention, said two regions of the β-A4 peptide are separated (on the level of the amino acid sequence) in the primary structure of the β-A4 peptide by at least one amino acid, preferably by at least two amino acids, more preferably by at least three amino acids, more preferably by at least four amino acids, more preferably by at least five amino acids, more preferably at least six amino acids, more preferably at least nine amino acids and most preferably at least twelve amino acids. As shown herein and as documented in the appended examples, the inventive antibodies/antibody molecules detect/interact with and/or bind to two regions of the β-A4 peptide as defined herein, whereby said two regions are separated (on the primary structure level of the amino acid sequence) by at least one amino acid and wherein the sequence separating said two regions/"epitope" may comprise more then ten amino acids, preferably 14 amino acids, more preferably 15 amino acids or 16 amino acids. For example, MSR-3 Fab (as an inventive antibody molecule) recognizes detects/interacts with two regions on the β-A4 peptide, wherein said first region comprises amino acids 3 and 4 (EF) and said second regions comprises amino acids 18 to 23 (VFFAED, SEQ ID NO: 421). Accordingly, the separating sequence between the region/epitopes to be detected/recognized has a length of 13 amino acids on the primary amino acid sequence structure. Similarly, MSR #3.4H7 IgG1, an optimized and matured antibody molecules derived from MSR-3 and comprised in an IgG1-framework, detects/interacts with/binds to two epitopes/regions of β-A4 which comprise in the first region positions 1 to 4 (DAEF) and in the second region positions 19 to 24 (FFAEDV, SEQ ID NO: 423) of β-A4 as defined herein. Accordingly, MSR #3.4H7 IgG1 recognizes/detects/interacts with/binds to two epitopes/regions which are, on the primary amino acid sequence level, separated by 14 amino acids. As detailed in the appended examples, affinity maturation and conversion of monovalent inventive Fab fragments to full-length IgG1 antibodies may result in a certain broadening of the epitopes/regions detected in pepspot, ELISA assays and the like. Therefore, the antibody molecules of the invention are capable of simultaneously and independently recognizing two regions of the β-A4 peptide/Aβ4 wherein said regions comprise the amino acid sequence as shown in SEQ ID NO: 1 (or parts thereof) and the amino acid sequence as shown in SEQ ID NO: 2 (or (a) part(s) thereof). Due to the potential broadening of epitopes as detailed herein it is, however, also envisaged that amino acids in close proximity to the sequences of SEQ ID NO: 1 and 2 are detected/recognized, i.e. that additional amino acids are part of the two regions to be detected/recognized. Accordingly, it is also envisaged that, e.g. the first amino acid of Aβ (1-42) as defined herein, namely D (Aspartic acid) in part of one epitope to be detected/recognized or that amino acids located after the region of Aβ (1-42) as defined in SEQ ID NO: 2 are detected/recognized. Said additional amino acid may, e.g., be the amino acid on position 26 of SEQ ID NO: 27 (βA4/Aβ(1-42)), namely S (Serine).
[0019] The term may also relate to a conformational epitope, a structural epitope or a discountinuous epitope consisting of said two regions or parts thereof; see also Geysen (1986), loc. cit. In context of this invention, a conformational epitope is defined by two or more discrete amino acid sequences separated in the primary sequence which come together on the surface when the polypeptide folds to the native protein (Sela, (1969) Science 166, 1365 and Layer, (1990) Cell 61, 553-6). The antibody molecules of the present invention are envisaged to specifically bind to/interact with a conformational/structural epitope(s) composed of and/or comprising the two regions of β-A4 described herein or parts thereof as disclosed herein below. The "antibody molecules" of the present invention are thought to comprise a simultaneous and independent dual specificity to (a) an amino acid stretch comprising amino acids 2 to 10 (or (a) part(s) thereof) of β-A4 and (b) an amino acid stretch comprising amino acids 12 to 25 (or (a) part(s) thereof) of β-A4 (SEQ ID NO. 27). Fragments or parts of these stretches comprise at least two, more preferably at least three amino acids. Preferred fragments or parts are in the first region/stretch of SEQ ID NO: 27 the amino acid sequences AEFRHD (SEQ ID NO: 415), EF, EFR, FR, EFRHDSG (SEQ ID NO: 416), EFRHD (SEQ ID NO: 417) or HDSG (SEQ ID NO: 418), and in the second region/stretch of SEQ ID NO: 27 the amino acid sequences HHQKL (SEQ ID NO: 419), LV, LVFFAE (SEQ ID NO: 420), VFFAED (SEQ ID NO: 421), VFFA (SEQ ID NO: 422) or FFAEDV (SEQ ID NO: 423). As mentioned above, said fragments may also comprise additional amino acids or may be parts of the fragments defined herein. Specific examples are DAE, DAEF, FRH or RHDSG.
DETAILED DESCRIPTION OF THE INVENTION
[0020] A number of antibodies specifically recognizing Aβ peptides have been described in the art. These antibodies have mainly been obtained by immunizing animals with Aβ1-40 or Aβ1-42 or fragments thereof using standard technologies. According to published data monoclonal antibodies that were generated by immunization with the complete Aβ peptide (1-40 or 1-42) recognize exclusively an epitope close to the N-terminus of Aβ. Further, examples are the antibodies BAP-1 and BAP-2 (Brockhaus, unpublished) which were generated by immunization of mice with Aβ1-40 and which recognize the amino acids 4-6 in the context of larger Aβ peptides; see appended Example 7, Table 2 and Example 12, Table 7. Antibodies that recognize the middle part of Aβ derive from immunizations with smaller peptides. For example, the antibody 4G8 was generated by immunization with the Aβ peptide 1-24 and recognizes exclusively the sequence 17-24 (Kim, (1988) Neuroscience Research Communications 2, 121-130). Many other monoclonal antibodies have been generated by immunizing mice with Aβ-derived fragments, and antibodies recognizing the C-terminal end of Aβ1-40 and Aβ1-42 are widely used to distinguish and quantitate the corresponding Aβ peptides in biological fluids and tissues by ELISA, Western blot and immunohistochemistry analysis (Ida et al, (1996) J. Biol. Chem. 271, 22908-22914; Johnson-Wood et al., (1997), Proc. Natl. Acad. Sci. USA (1994), 1550-1555; Suzuki et al., (1994), Science 264, 1336-1340; Brockhaus (1998), Neuro Rep. 9, 1481-1486). BAP-17 is a mouse monoclonal antibody which has been generated by immunizing mice with Aβ fragment 35-40. It specifically recognizes the C-terminal end of Aβ1-40 (Brockhaus (1998) Neuroreport 9, 1481-1486).
[0021] It is believed that the immunization with T-cell dependent antigens (often poor immunogens) requires a proteolytic cleavage of the antigen in the endosomes of antigen presenting cells. The in vivo selection of high affinity antibodies after immunization is driven by the contact of helper T cells to antigen presenting cells. The antigen presenting cells only present short peptides and not polypeptides of large size. Accordingly, these cells have a complicated (but well known) machinery to endocytose antigen(s), degrade the antigen(s) in endosomes, combine selected peptides with suitable MHC class II molecules, and to export the peptide-MHC complex to the cell surface. This is where the antigen specific recognition by T cells occurs, with the aim to provide help to maturing B cells. The B cells which receive most T cell help have the best chance to develop into antibody secreting cells and to proliferate. This shows that antigen processing by proteolysis is an important step for the generation of an high affinity antibody response in vivo and may explain the dominance of the N-terminal Aβ epitope in prior art monoclonal and polyclonal antibodies derived by immunization.
[0022] In contrast, the selection of antibodies/antibody molecules of the present invention is driven by the physical adherence of Fab expressing phages to the antigen. There is no degradation of the antigen involved in this in vitro selection process. The phages which express the Fab with the highest affinity towards the antigen are selected and propagated. A synthetic library as employed in the appended examples to select for specific antibody molecules according to this invention is particularly suited for avoiding any bias for single, continuous epitopes that is often found in libraries derived from immunized B cells.
[0023] It is of note that the prior art has not described antibody molecules recognizing two, independent regions of Aβ4 which specifically recognizes (a) discontinuous/structural/conformational epitope(s) and/or which are capable of simultaneously and independently recognizing two regions/epitopes of Aβ4.
[0024] Vaccination of transgenic mice overexpressing mutant human APP.sub.V717F (PDAPP mice) with Aβ1-42 resulted in an almost complete prevention of amyloid deposition in the brain when treatment was initiated in young animals, i.e. before the onset of neuropathologies, whereas in older animals a reduction of already formed plaques was observed suggesting antibody-mediated clearance of plaques (Schenk et al., (1999), Nature 400, 173-177). The antibodies generated by this immunization procedure were reactive against the N-terminus of Aβ4 covering an epitope around amino acids 3-7 (Schenk et al., (1999), loc. cit.; WO 00/72880). Active immunization with Aβ1-42 also reduced behavioural impairment and memory loss in different transgenic models for Alzheimer's Disease (Janus et al., (2000) Nature 408, 979-982; Morgan et al., (2000) Nature 408, 982-985). Subsequent studies with peripherally administered antibodies, i.e. passive immunization, have confirmed that antibodies can enter the central nervous system, decorate plaques and induce clearance of preexisting amyloid plaques in APP transgenic mice (PDAPP mice) (Bard et al., (2000) Nat. Med. 6, 916-919; WO 00/72880). In these studies, the monoclonal antibodies with the most potent in vivo and ex vivo efficacy (triggering of phagocytosis in exogenous microglial cells) were those which recognized Aβ4 N-terminal epitopes 1-5 (mab 3D6, IgG2b) or 3-6 (mab 10D5, IgG1). Likewise, polyclonal antibodies isolated from mice, rabbits or monkeys after immunization with Aβ1-42 displayed a similar N-terminal epitope specificity and were also efficacious in triggering phagocytosis and in vivo plaque clearing. In contrast, C-terminal specific antibodies binding to Aβ1-40 or Aβ1-42 with high affinity did not induce phagocytosis in the ex vivo assay and were not efficacious in vivo (WO 00/72880). Monoclonal antibody m266 (WO 00/72880) was raised against Aβ13-28 (central domain of Aβ) and epitope mapping confirmed the antibody specificity to cover amino acids 16-24 in the Aβ sequence. This antibody does not bind well to aggregated Aβ and amyloid deposits and merely reacts with soluble (monomeric) Aβ, i.e. properties which are similar to another well-known and commercially available monoclonal antibody (4G8; Kim, (1988) Neuroscience Research Communications 2, 121-130; commercially available from Signet Laboratories Inc. Dedham, Mass. USA) which recognizes the same epitope.
[0025] In vivo, the m266 antibody was recently found to markedly reduce Aβ deposition in PDAPP mice after peripheral administration (DeMattos, (2001) Proc. Natl. Acad. Sci. USA 98, 8850-8855). However, and in contrast to N-terminal specific antibodies, m266 did not decorate amyloid plaques in vivo, and it was therefore hyothesized that the brain Aβ burden was reduced by an antibody-induced shift in equilibrium between CNS and plasma Aβ resulting in the accumulation of brain-derived Aβ in the periphery, firmly complexed to m266 (DeMattos, (2001) loc. cit.).
[0026] The antibodies/antibody molecules of the present invention, by simultaneously (for example in a structural/conformational epitope formed by the N-terminal and central region of βA4 as described herein) and independently (for example in pepspot assays as documented in the appended experimental part) binding to the N-terminal and central epitopes, combine the properties of an N-terminal-specific antibody and a central epitope-specific antibody in a single molecule. Antibodies with the dual epitope specificity, as described in the present invention, are considered to be more efficacious in vivo, in particular in medical and diagnostic settings for, e.g., reducing amyloid plaque burden or amyloidogenesis or for the detection of amyloid deposits and plaques. It is well known that in the process of Aβ4 aggregation and amyloid deposition conformational changes occur, and while the central epitope is easily accessible in soluble Aβ4 it appears to be hidden and less reactive in aggregated or fibrillar Aβ4. The fact that the central/middle epitope-specific antibody m266 is efficacious in vivo indicates that neutralization of soluble Aβ4 may also be a critical parameter. The antibodies/antibody molecules of the present invention, due to the dual epitope specificity, can bind to both fibrillar and soluble Aβ4 with similar efficacy, thus allowing interaction with amyloid plaques as well as neutralization of soluble Aβ4. The term "simultaneously and independently binding to the N-terminal and central/middle epitopes of β-A4" as employed herein in context of the inventive antibody molecules relates to the fact that the antibodies/antibody molecules described herein may detect and/or bind to both epitopes simultaneously, i.e. at the same time (for example on conformational/structural epitopes formed by the N-terminal epitope (or (a) part(s) thereof) and central epitopes (or (a) part(s) thereof) of βA4 as defined herein) and that the same antibody molecules, however, are also capable of detecting/binding to each of the defined epitopes in an independent fashion, as inter alia, demonstrated in the pepspot analysis shown in the examples.
[0027] Clearance of amyloid plaques in vivo in PDAPP mice after direct application of the antibodies to the brain is not dependent on the IgG subtype and may also involve a mechanism which is not Fc-mediated, i.e. no involvement of activated microglia in plaque clearance (Bacskai, (2001), Abstract Society for Neuroscience 31st Annual Meeting, Nov. 10-15, 2001, San Diego). This observation is in contrast to what has been postulated in an earlier study by Bard (2000), loc. cit.
[0028] In another study antibodies raised against A61-28 and Aβ1-16 peptides were found to be effective in disaggregating Aβ fibrils in vitro, whereas an antibody specific for A613-28 was much less active in this assay (Solomon, (1997) Proc. Natl. Acad. Sci. USA 94, 4109-4112). Prevention of Aβ aggregation by an anti-M1-28 antibody (AMY-33) has also been reported (Solomon, (1996) Proc. Natl. Acad. Sci. USA 93, 452-455). In the same study, antibody 6F/3D which has been raised against Aβ fragment 8-17 slightly interfered with Zn2+-induced Aβ aggregation but had no effect on the self aggregation induced by other aggregation-inducing agents.
[0029] The efficacy of the various antibodies in these in vitro assays correlates with the accessibility of their epitopes in Aβ4 aggregates. The N-terminus is exposed and N-terminal specific antibodies clearly induce de-polymerization, whereas the central region and the C-terminus are hidden and not easily accessible and thus antibodies against these epitope are much less effective.
[0030] Investigations with respect to epitope accessibilty for antibodies have shown that in aggregated Aβ the N-terminal epitope is exposed and reacts with the BAP-1 antibody, whereas the middle or central epitope indeed remains cryptic, i.e. no binding of the 4G8 antibody was observed. However, in monomeric Aβ both epitopes are overt and are equally recognized by both prior art antibodies.
[0031] In contrast, in the present invention, it was surprisingly found that the herein described antibody molecules recognize two discontinuous amino acid sequences, e.g. a conformational/structural epitope on the Aβ peptide. Two "discontinuous amino acid sequences" in accordance with this invention means that said two amino acid sequences forming the N-terminal and central/middle epitopes, respectively, are separated on β-A4 in its primary structure by at least two amino acids which are not part of either epitope.
[0032] The binding area of an antibody Fab (=paratope) occupies a molecular surface of approximately 30×30 Å in size (Layer, Cell 61 (1990), 553-556). This is enough to contact 15 to 22 amino acid residues which may be present on several surface loops. The discontinuous epitope recognized by the inventive antibody molecules resembles a conformation in which the N-terminal (residues 2 to 10 or parts thereof) and middle A6 peptide sequences (residues 12 to 25 or parts thereof) are in close proximity. Only within this conformation, the maximum number of antigen-antibody contacts and the lowest free energy state are obtained.
[0033] Based on energetic calculations it has been suggested that a smaller subset of 5-6 residues, which are not arranged in a linear sequence but are scattered over the epitope surface, contributes most of the binding energy while surrounding residues may merely constitute a complementary array (Laver (1990) loc. cit.).
[0034] The inventive antibodies/antibody molecules are capable of binding to aggregated Aβ and strongly react with amyloid plaques in the brain of AD patients (as documented in the appended examples). In addition, they are capable of de-polymerizing/disintegrating amyloid aggregates.
[0035] Without being bound by theory, the conformational/structural epitope (composed by the two regions of Aβ4 or (a) part(s) of said regions as described herein) is believed to be partially exposed in aggregated Aβ. However, it is known that major part of the middle/second epitope/region alone is not freely accessible in these Aβ aggregates (based on the poor reactivities of middle epitope-specific antibodies 4G8 and m266). On the other hand, and in view of the considerations mentioned above, it is likely that one or several residues of the middle region are components of the conformational epitope and, in conjunction with the residues from the N-terminal region, are accessible to the antibodies of the present invention, thereby significantly contributing to the binding energy of the antibody-Aβ4 interaction. The reactivity of the inventive antibody molecules with the conformational epitope in aggregated Aβ is therefore unique and clearly distinct from α-Aβ4 antibodies described in the prior art. Yet, as pointed out herein above, a further unique feature of the inventive antibodies/antibody molecules is their capacity to simultaneously and independently binding to/recognizing two separate epitopes on β-A4, as defined herein and in the appended examples.
[0036] In a preferred embodiment of the invention, the inventive antibody molecule is an antibody molecule wherein the least two regions of the β-A4 to be specifically recognized by said antibody form a conformational/structural epitope or a discontinuous epitope; see Geysen (1986), loc. cit.; Ghoshal (2001), J. Neurochem. 77, 1372-1385; Hochleitner (2000), J. Imm. 164, 4156-4161; Laver (1990), loc. cit. The term "discontinuous epitope" means in context of the invention non-linear epitopes that are assembled from residues from distant portions of the polypeptide chain. These residues come together on the surface when the polypeptide chain folds into a three-dimensional structure to constitute a conformational/structural epitope. The present invention provides for preferred, unexpected epitopes within β-A4, which result in the inventive generation of specific antibody molecules, capable of specifically interacting with these epitopes. These inventive antibodies/antibody molecules provide the basis for increased efficacy, and a reduced potential for side effects. As pointed out above, the inventive antibodies, however, were also capable of independently interacting with each of the defined two regions/epitopes of β-A4, for example in Pepspot assays as documented in the appended examples.
[0037] The present invention, accordingly, provides for unique tools which may be employed to de-polymerize aggregated Aβ-fibrils in vivo and in vitro and/or which are capable of stabilizing and/or neutralizing a conformational epitope of monomeric Aβ and thereby capable of preventing the pathological Aβ aggregation.
[0038] It is furthermore envisaged that the inventive antibodies bind to Aβ deposits at the rim of amyloid plaques in, inter alia, Alzheimer's brain and efficiently dissolve the pathological protofibrils and fibrils.
[0039] In a preferred embodiment, the antibody molecule of the invention recognizes at least two consecutive amino acids within the two regions of Aβ4 defined herein, more preferably said antibody molecule recognizes in the first region an amino acid sequence comprising the amino acids: AEFRHD (SEQ ID NO: 415), EF, EFR, FR, EFRHDSG (SEQ ID NO: 416), EFRHD (SEQ ID NO: 417) or HDSG (SEQ ID NO: 418), and in the second region an amino acid sequence comprising the amino acids: HHQKL (SEQ ID NO: 419), LV, LVFFAE (SEQ ID NO: 420), VFFAED (SEQ ID NO: 421), VFFA (SEQ ID NO: 422) or FFAEDV (SEQ ID NO: 423). Further fragments or broadened parts comprise: DAE, DAEF, FRH or RHDSG.
[0040] It is particularly preferred that the antibody molecule of the invention comprises a variable VH-region as encoded by a nucleic acid molecule as shown in SEQ ID NO: 3, 5 or 7 or a variable VH-region as shown in the amino acid sequences depicted in SEQ ID NOs: 4, 6 or 8. The sequences as shown in SEQ ID NOs: 3 and 4 depict the coding region and the amino acid sequence, respectively, of the VH-region of the inventive, parental antibody MSR-3 (MS-Roche 3), the sequences in SEQ ID NOs: 5 and 6 depict the coding region and the amino acid sequence, respectively, of the VH-region of the inventive, parental antibody MSR-7 (MS-Roche 7) and SEQ ID NOs: 7 and 8 depict the coding region and the amino acid sequence, respectively, of the VH-region of the inventive, parental antibody MSR-8 (MS-Roche 8). Accordingly, the invention also provides for antibody molecules which comprise a variable VL-region as encoded by a nucleic acid molecule as shown in a SEQ ID NO selected from the group consisting of SEQ ID NO: 9, 11 or 13 or a variable VL-region as shown in the amino acid sequences depicted in SEQ ID NOs: 10, 12 or 14. SEQ ID NOs: 9 and 10 correspond to the VL-region of MSR-3, SEQ ID NOs: 11 and 12 correspond to the VL-region of MSR-7 and SEQ ID NOs: 13 and 14 correspond to the VL-region of MSR-8. As illustrated in the appended examples, the parental antibodies MSR-3, -7 and -8, are employed to further generate optimized antibody molecules with even better properties and/or binding affinities. Some of the corresponding and possible strategies are exemplified and shown in the appended examples.
[0041] The optimization strategy as illustrated in the appended examples lead to a plurality of inventive, optimized antibodies. These optimized antibodies share with their parental antibodies the CDR-3 domain of the VH-region. Whereas the original framework region (as shown in appended FIG. 1) remains the same, in the matured/optimized antibody molecules, CDR1, CDR2 and/or VL CDR3-regions are changed. Illustrative, modified sequence motives for optimized antibody molecules are shown in appended table 1. Accordingly, within the scope of the present invention are also optimized antibody molecules which are derived from the herein disclosed MSR-3, -7 and -8 and which are capable of specifically reacting with/specifically recognizing the two regions of the β-A4 peptide as defined herein. In particular, CDR-regions, preferably CDR1s, more preferably CDR1s and CDR2s, most preferably CDR1s, CDR2s and CDR3s as defined herein may be employed to generate further inventive antibodies/antibody molecules, inter alia, by CDR-grafting methods known in the art; see Jones (1986), Nature 321, 522-515 or Riechmann (1988), Nature 332, 323-327. Most preferably the inventive antibodies/antibody molecules as well as antibody fragments or derivatives are derived from the parental antibodies as disclosed herein and share, as disclosed above, the CDR-3 domain of the VH-region with at least one of said parental antibodies. As illustrated below, it is also envisaged that cross-cloned antibodies are generated which are to be considered as optimized/maturated antibodies/antibody molecules of the present invention. Accordingly, preferred antibody molecules may also comprise or may also be derived from antibodies/antibody molecules which are characterized by VH-regions as shown in any of SEQ ID NOs: 32 to 45 or VL-regions as shown in SEQ ID NOs: 46 to 59 or which may comprise a CDR-3 region as defined in any of SEQ ID NOs: 60 to 87. In a particular preferred embodiment, the optimized antibody molecule of the present invention comprises VH-regions and VL-regions as depicted in SEQ ID NOs: 88/89 and 90/91, respectively, or parts thereof. Apart thereof may be (a) CDR-region(s), preferably (a) CDR3-region(s). A particularly preferred antibody molecule of the optimized type comprises a H-CDR3 as characterized in SEQ ID NOs: 92 or 93 and/or a L-CDR3 as characterized in SEQ ID NOs: 94 or 95.
[0042] It is preferred that the antibodies/antibody molecules of the invention are characterized by their specific reactivity with β-A4 and/or peptides derived from said β-A4. For example, optical densities in ELISA-tests, as illustrated in the appended examples, may be established and the ratio of optical densities may be employed to define the specific reactivity of the parental or the optimized antibodies. Accordingly, a preferred antibody of the invention is an antibody which reacts in an ELISA-test with β-A4 to arrive at an optical density measured at 450 nm that is 10 times higher than the optical density measured without β-A4, i.e. 10 times over background. Preferably the measurement of the optical density is performed a few minutes (e.g. 1, 2, 3, 4, 5, 6, or 7 minutes) after initiation of the color developing reaction in order to optimize signal to background ratio.
[0043] In a particular preferred embodiment, the inventive antibody molecule comprises at least one CDR3 of an VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NOs: 15, 17 or 19 or at least one CDR3 amino acid sequence of an VL-region as shown in SEQ ID NOs: 16, 18 or 20 and/or said antibody molecule comprises at least one CDR3 of an VH-region as encoded by a nucleic acid molecule as shown in SEQ ID NOs: 21, 23 or 25 or at least one CDR3 amino acid sequence of an VH-region as shown in SEQ ID NOs: 22, 24 or 26. Most preferred are antibodies comprising at least one CDR3 of an VH-region as defined herein. The CDR-3 domains mentioned herein above relate to the inventive, illustrative parental antibody molecules MSR-3, -7, or -8. However, as illustrated in the appended tables 1, 8 or 10, matured and/or optimized antibody molecules obtainable by the methods disclosed in the appended examples may comprise modified VH-, VL-, CDR1, CDR2 and CDR3 regions. Accordingly, the antibody molecule of the invention is preferably selected from the group consisting of MSR-3, -7 and -8 or an affinity-matured version of MSR-3, -7 or -8. Affinity-matured as well as cross-cloned versions of MSR-3, -7 and -8 comprise, inter alia, antibody molecules comprising CDR1, CDR2 and/or CDR3 regions as shown in table 1 or 8 or characterized in any of SEQ ID NOs: 15 to 20, 21 to 26, 60 to 74, 75 to 87, 92 and 93 or 94 and 95 as well as in SEQ ID NOs: 354 to 413. Most preferably, the antibody of the invention comprises at least one CDR, preferably a CDR1, more preferably a CDR2, most preferably a CDR3 as shown in the appended table 1, 8 or as documented in appended table 10.
[0044] It is of note that affinity-maturation techniques are known in the art, described in the appended examples and, inter alia, in Knappik (2000), J. Mol. Biol. 296, 55; Krebs (2000), J. Imm. Meth. 254, 67-84; WO 01/87337; WO 01/87338; U.S. Pat. No. 6,300,064; EP 96 92 92 78.8 and further references cited herein below.
[0045] In a more preferred embodiment of the invention, the antibody molecule is a full antibody (immunoglobulin, like an IgG1, an IgG2, an IgG2b, an IgG3, an IgG4, an IgA, an IgM, an IgD or an IgE), an F(ab)-, Fabc-, Fv-, Fab'-, F(ab')2fragment, a single-chain antibody, a chimeric antibody, a CDR-grafted antibody, a bivalent antibody-construct, an antibody-fusion protein, a cross-cloned antibody or a synthetic antibody. Also envisaged are genetic variants of immunoglobulin genes. Genetic variants of, e.g., immunoglobulin heavy G chain subclass 1 (IgG1) may comprise the G1m(17) or G1m(3) allotypic markers in the CH1 domain, or the G1m(1) or the G1m(non-1) allotypic marker in the CH3 domain. The antibody molecule of the invention also comprises modified or mutant antibodies, like mutant IgG with enhanced or attenuated Fc-receptor binding or complement activation. It is also envisaged that the antibodies of the invention are produced by conventional means, e.g. the production of specific monoclonal antibodies generated by immunization of mammals, preferably mice, with peptides comprising the two regions of βA4 as defined herein, e.g. the N-terminal and central region/epitope comprising (a) amino acids 2 to 10 (or (a) part(s) thereof) of β-A4 and (b) an amino acid stretch comprising amino acids 12 to 25 (or (a) part(s) thereof) of β-A4 (SEQ ID NO. 27). Accordingly, the person skilled in the art may generate monoclonal antibodies against such a peptide and may screen the obtained antibodies for the capacity to simultaneously and independently binding to/reacting with the N-terminal and central region/epitope of βA4 as defined herein. Corresponding screening methods are disclosed in the appended examples.
[0046] As illustrated in the appended examples, the inventive antibodies/antibody molecules can readily and preferably be recombinantly constructed and expressed. Preferably, the antibody molecule of the invention comprises at least one, more preferably at least two, preferably at least three, more preferably at least four, more preferably at least five and most preferably six CDRs of the herein defined MSR-3, MSR-7 or MSR-8 parental antibodies or of affinity-matured/optimized antibodies derived from said parental antibodies. It is of note that also more than six CDRs may be comprised in recombinantly produced antibodies of the invention. The person skilled in the art can readily employ the information given in the appended examples to deduce corresponding CDRs of the parental as well as the affinity optimized antibodies. Examples of optimized antibodies which have been obtained by maturation/optimization of the parental antibodies are, inter alia, shown in appended table 1. An maturated/optimized antibody molecule of the invention is, e.g. MSR 7.9H7 which is also characterized by sequences appended herein, which comprise SEQ ID NOs: 88 to 95 and which depict the VH-region of MSR 7.9H7 (SEQ ID NOs: 88 and 89), the VL-region of MSR 7.9H7 (SEQ ID NOs: 90 and 91), the H-CDR3 of MSR 7.9H7 (SEQ ID NOs: 92 and 93) as well as the L-CDR3 of MSR 7.9H7 (SEQ ID NOs: 94 and 95). Illustrative antibody molecule 7.9H7 is derived from parental antibody MSR7 and is a particular preferred inventive example of an optimized/matured antibody molecule of the present invention. This antibody molecule may be further modified in accordance with this invention, for example in form of cross-cloning, see herein below and appended examples.
[0047] As documented in the appended examples, the antibodies of the invention also comprise cross-cloned antibodies, i.e. antibodies comprising different antibody regions (e.g. CDR-regions) from one or more parental or affinity-optimized antibody(ies) as described herein. These cross-cloned antibodies may be antibodies in several, different frameworks, whereby the most preferred framework is an IgG-framework, even more preferred in an IgG1-, IgG2a or an IgG2b-framework. It is particularly preferred that said antibody framework is a mammalian, most preferably a human framework. The domains on the light and heavy chains have the same general structure and each domain comprises four framework regions, whose sequences are relatively conserved, joined by three hypervariable domains known as complementarity determining regions (CDR1-3).
[0048] As used herein, a "human framework region" relates to a framework region that is substantially identical (about 85% or more, usually 90-95% or more) to the framework region of a naturally occurring human immunoglobulin. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDR's. The CDR's are primarily responsible for binding to an epitope of an antigen. It is of note that not only cross-cloned antibodies described herein may be presented in a preferred (human) antibody framework, but also antibody molecules comprising CDRs from, inter alia, the parental antibodies MSR-3, -7 or -8 as described herein or of matured antibodies derived from said parental antibodies, may be introduced in an immunoglobulin framework. Preferred frameworks are IgG1, IgG2a and IgG2b. Most preferred are human frameworks and human IgG1 frameworks.
[0049] As shown in the appended examples, it is, inter alia possible, to transfer, by genetic engineering known in the art whole light chains from an optimized donor clone to an optimized recipient clone. Example for an optimized donor clone is, e.g. L-CDR1 (L1) and an example for an optimized recipient clone is H-CDR2 (H2). Epitope specificity may be conserved by combining clones which possess the same H-CDR-3 regions. Further details are given in illustrative Example 13.
[0050] Preferred cross-cloned antibody molecules of the invention are selected from the group consisting of MS-R #3.3H1×3.4L9, MS-R #3.4H1×3.4L9, MS-R #3.4H3×3.4L7, MS-R #3.4H3×3.4L9, MS-R #3.4H7×3.4L9, MS-R #3.4H7×3.4L7, MS-R #3.6H5×3.6L1, MS-R #3.6H5×3.6L2, MS-R #3.6.H8×3.6.L2, MS-R #7.2H2×7.2L1, MS-R #7.4H2×7.2L1, MS-R #7.4H2×7.12L2, MS-R #7.9H2×7.2L1(L1), MS-R #7.9H2×7.12L1, MS-R #7.9H2×7.12L2, MS-R #7.9H2×7.12L2(L1+2), MS-R #7.9H4×7.12.L2, MS-R #7.11H1×7.2L1, MS-R #7.11H1×7.11L1, MS-R #7.11H2×7.2L1(L1), MS-R #7.11H2×7.9L1 (L1), MS-R #7.11H2×7.12L1 or MS-R #8.1H1×8.2L1.
[0051] The generation of cross-cloned antibodies is also illustrated in the appended examples. The above mentioned preferred cross-cloned antibodies/antibody molecules are optimized/matured antibody molecules derived from parental antibodies disclosed herein, in particular from MSR-3 and MSR-7. in addition, further characterizing CDR-sequences and V-regions of the cross-cloned antibody molecules/antibodies are given in appended SEQ ID NOs: 32, 33, 46 and 47 (MSR 3.6H5×3.6.L2; VH-, VL-region); 34, 35, 48 and 49 (MSR 3.6H8×3.6.L2; VH-, VL-regions); 36, 37, 50 and 51 (MSR 7.4H2×7.2.L1; VH-, VL-regions); 38, 39, 52 and 53 (MSR 7.9H2×7.12.L2; VH-, VL-regions); 40, 41, 54 and 55 (MSR #7.9H4×7.12.L2; VH-, VL-regions); 42, 43, 56 and 57 (MSR #7.11H1×7.11.L1; VH-, VL-regions); and 44, 45, 58 and 59 (MSR #7.11H1×7.2.L1; VH-, VL-regions). Corresponding CDR3 regions of these particular preferred cross-cloned antibody molecules are depicted in SEQ ID NOs: 60 to 87. For further MSR antibody molecules, VH-, VL-, CDR-regions can be deduced from appended Tables 8 or 10 and from the appended sequence listing, in particular SEQ ID NOS: 32 to 95 for MS-R antibodies/antibody molecules #3.6H5 x 3.6L2, #3.6H8 x 3.6L2, #7.4H2 x 7.2L1, #7.9H2 x 7.12L2, #7.9H4 x 7.12L2, #7.11H1 x 7.11L1, #7.11H1 x 7.2L1 and #7.9H7 or SEQ ID NOS: 294 to 413 for MSR-R antibodies/antibody molecules MS-R #3.3H1×3.4L9, #3.4H1 x 3.4L9, #3.4H3 x 3.4L7, #3.4H3 x 3.4L9, #3.4H7 x 3.4L9, #3.4H7 x 3.4L7, #3.6H5 x 3.6L1, #7.2H2 x 7.2L1, #7.4H2 x 7.12L2, #7.9H2 x 7.2L1, #7.9H2 x 7.12L1, #7.11H2 x 7.2L1, #7.11H2 x 7.9L1, #7.11H2 x 7.12L1 or #8.1H1 x 8.2L1. Accordingly, besides VH-regions defined above, preferred antibody molecules of the invention may comprise VH-regions as defined in any one of SEQ ID NOs: 294 to 323. Similarly, SEQ ID NOs: 324 to 353 depict preferred VL-regions which, besides to VL-regions defined above which may be comprised in the inventive antibody molecules. Corresponding CDR-3 regions are defined above, as well as in additional sequences shown in SEQ ID NOs: 354 to 413.
[0052] Inventive antibody molecules can easily be produced in sufficient quantities, inter alia, by recombinant methods known in the art, see, e.g. Bentley, Hybridoma 17 (1998), 559-567; Racher, Appl. Microbiol. Biotechnol. 40 (1994), 851-856; Samuelsson, Eur. J. Immunol. 26 (1996), 3029-3034.
[0053] Theoretically, in soluble β-A4 (monomeric/oligomeric) both the N-terminal and the middle epitopes are accessible for antibody interaction and antibody molecules of the present invention may either bind to the N-terminal or middle epitope separately, but under these conditions maximum affinity will not be obtained. However, it is more likely that an optimal contact to the antibody paratope will be attained by simultaneous binding to both epitopes, i.e. similar to the interaction with aggregated β-A4. Thus, antibodies of the present invention are unique anti-Aβ antibodies in that they bind to aggregated β-A4 (via interaction with the N-terminal and middle epitope), and at the same time are also able to stabilize and neutralize the conformational epitope in soluble β-A4. These antibodies are distinct to prior art antibodies.
[0054] Most preferred are antibody molecules of the invention which have an affinity to Aβ or defined fragments thereof with a KD value lower than 2000 nM, preferably lower than 100 nM, more preferably lower than 10 nM, most preferably lower than 1 nM. The measurement of such affinity/affinities may be carried out by methods illustrated in the examples and known in the art. Such methods comprise, but are not limited to BIACORE®-assays (www.biacore.com; Malmquist (1999), Biochem. Soc. Trans 27, 335-340) and solid phase assays using labeled antibodies or labeled Aβ.
[0055] Preferably, the antibody molecule of the invention is capable of decorating/reacting with/binding to amyloid plaques in in vitro (post-mortem) brain sections from patients suffering from amyloid-related disorders, like Alzheimer's disease. Yet, it is also preferred that the inventive antibody/antibody molecules prevent Aβ-aggregation in vivo as well as in in vitro assays, as illustrated in the appended examples. Similarly, the antibody molecules of the present invention are preferred to de-polymerize Aβ-aggregate in vivo and/or in in vitro assays shown in the examples. This capacity of the inventive antibodies/antibody molecules is, inter alia, to be employed in medical settings, in particular in pharmaceutical compositions described herein below.
[0056] The invention also provides for a nucleic acid molecule encoding an inventive antibody molecule as defined herein.
[0057] Said nucleic acid molecule may be a naturally nucleic acid molecule as well as a recombinant nucleic acid molecule. The nucleic acid molecule of the invention may, therefore, be of natural origin, synthetic or semi-synthetic. It may comprise DNA, RNA as well as PNA and it may be a hybrid thereof.
[0058] It is evident to the person skilled in the art that regulatory sequences may be added to the nucleic acid molecule of the invention. For example, promoters, transcriptional enhancers and/or sequences which allow for induced expression of the polynucleotide of the invention may be employed. A suitable inducible system is for example tetracycline-regulated gene expression as described, e.g., by Gossen and Bujard (Proc. Natl. Acad. Sci. USA 89 (1992), 5547-5551) and Gossen et al. (Trends Biotech. 12 (1994), 58-62), or a dexamethasone-inducible gene expression system as described, e.g. by Crook (1989) EMBO J. 8, 513-519.
[0059] Furthermore, it is envisaged for further purposes that nucleic acid molecule may contain, for example, thioester bonds and/or nucleotide analogues. Said modifications may be useful for the stabilization of the nucleic acid molecule against endo- and/or exonucleases in the cell. Said nucleic acid molecules may be transcribed by an appropriate vector containing a chimeric gene which allows for the transcription of said nucleic acid molecule in the cell. In this respect, it is also to be understood that the polynucleotide of the invention can be used for "gene targeting" or "gene therapeutic" approaches. In another embodiment said nucleic acid molecules are labeled. Methods for the detection of nucleic acids are well known in the art, e.g., Southern and Northern blotting, PCR or primer extension. This embodiment may be useful for screening methods for verifying successful introduction of the inventive nucleic acid molecules during gene therapy approaches.
[0060] The nucleic acid molecule(s) of the invention may be a recombinantly produced chimeric nucleic acid molecule comprising any of the aforementioned nucleic acid molecules either alone or in combination. Preferably, the nucleic acid molecule of the invention is part of a vector.
[0061] The present invention therefore also relates to a vector comprising the nucleic acid molecule of the present invention.
[0062] The vector of the present invention may be, e.g., a plasmid, cosmid, virus, bacteriophage or another vector used e.g. conventionally in genetic engineering, and may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions.
[0063] Furthermore, the vector of the present invention may, in addition to the nucleic acid sequences of the invention, comprise expression control elements, allowing proper expression of the coding regions in suitable hosts. Such control elements are known to the artisan and may include a promoter, a splice cassette, translation initiation codon, translation and insertion site for introducing an insert into the vector. Preferably, the nucleic acid molecule of the invention is operatively linked to said expression control sequences allowing expression in eukaryotic or prokaryotic cells.
[0064] Control elements ensuring expression in eukaryotic and prokaryotic cells are well known to those skilled in the art. As mentioned herein above, they usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally-associated or heterologous promoter regions. Possible regulatory elements permitting expression in for example mammalian host cells comprise the CMV-HSV thymidine kinase promoter, SV40, RSV-promoter (Rous Sarcoma Virus), human elongation factor 1α-promoter, the glucocorticoid-inducible MMTV-promoter (Moloney Mouse Tumor Virus), metallothionein- or tetracyclin-inducible promoters, or enhancers, like CMV enhancer or SV40-enhancer. For expression in neural cells, it is envisaged that neurofilament-, PGDF-, NSE-, PrP-, or thy-1-promoters can be employed. Said promoters are known in the art and, inter alia, described in Charron (1995), J. Biol. Chem. 270, 25739-25745. For the expression in prokaryotic cells, a multitude of promoters including, for example, the tac-lac-promoter or the trp promoter, has been described. Besides elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide. In this context, suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc/CMV, pcDNA1, pcDNA3 (In-vitrogene), pSPORT1 (GIBCO BRL), pX (Pagano (1992) Science 255, 1144-1147), yeast two-hybrid vectors, such as pEG202 and dpJG4-5 (Gyuris (1995) Cell 75, 791-803), or prokaryotic expression vectors, such as lambda gt11 or pGEX (Amersham-Pharmacia). Beside the nucleic acid molecules of the present invention, the vector may further comprise nucleic acid sequences encoding for secretion signals. Such sequences are well known to the person skilled in the art. Furthermore, depending on the expression system used leader sequences capable of directing the peptides of the invention to a cellular compartment may be added to the coding sequence of the nucleic acid molecules of the invention and are well known in the art. The leader sequence(s) is (are) assembled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein, or a protein thereof, into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusionprotein including an C- or N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and, as desired, the collection and purification of the antibody molecules or fragments thereof of the invention may follow. The invention also relates, accordingly, to hosts/host cells which comprise a vector as defined herein. Such hosts may be useful for in processes for obtaining antibodies/antibody molecules of the invention as well as in medical/pharmaceutical settings. Said host cells may also comprise transduced or transfected neuronal cells, like neuronal stem cells, preferably adult neuronal stem cells. Such host cells may be useful in transplantation therapies.
[0065] Furthermore, the vector of the present invention may also be an expression, a gene transfer or gene targeting vector. Gene therapy, which is based on introducing therapeutic genes into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer. Transgenic mice expressing a neutralizing antibody directed against nerve growth factor have been generated using the "neuroantibody" technique; Capsoni, Proc. Natl. Acad. Sci. USA 97 (2000), 6826-6831 and Biocca, Embo J. 9 (1990), 101-108. Suitable vectors, methods or gene-delivering systems for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813, Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Onodua, Blood 91 (1998), 30-36; Verzeletti, Hum. Gene Ther. 9 (1998), 2243-2251; Verma, Nature 389 (1997), 239-242; Anderson, Nature 392 (Supp. 1998), 25-30; Wang, Gene Therapy 4 (1997), 393-400; Wang, Nature Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957; U.S. Pat. No. 5,580,859; U.S. Pat. No. 5,589,466; U.S. Pat. No. 4,394,448 or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640, and references cited therein. In particular, said vectors and/or gene delivery systems are also described in gene therapy approaches in neurological tissue/cells (see, inter alia Blomer, J. Virology 71 (1997) 6641-6649) or in the hypothalamus (see, inter alia, Geddes, Front Neuroendocrinol. (1999), 296-316 or Geddes, Nat. Med. 3 (1997), 1402-1404). Further suitable gene therapy constructs for use in neurological cells/tissues are known in the art, for example in Meier (1999), J. Neuropathol. Exp. Neurol. 58, 1099-1110. The nucleic acid molecules and vectors of the invention may be designed for direct introduction or for introduction via liposomes, viral vectors (e.g. adenoviral, retroviral), electroporation, ballistic (e.g. gene gun) or other delivery systems into the cell. Additionally, a baculoviral system can be used as eukaryotic expression system for the nucleic acid molecules of the invention. The introduction and gene therapeutic approach should, preferably, lead to the expression of a functional antibody molecule of the invention, whereby said expressed antibody molecule is particularly useful in the treatment, amelioration and/or prevention of neurological disorders related to abnormal amyloid synthesis, assembly and/or aggregation, like, Alzheimer's disease and the like.
[0066] Accordingly, the nucleic acid molecule of the present invention and/or the above described vectors/hosts of the present invention may be particularly useful as pharmaceutical compositions. Said pharmaceutical compositions may be employed in gene therapy approaches. In this context, it is envisaged that the nucleic acid molecules and/or vectors of the present invention may be employed to modulate, alter and/or modify the (cellular) expression and/or concentration of the antibody molecules of the invention or of (a) fragment(s) thereof.
[0067] For gene therapy applications, nucleic acids encoding the peptide(s) of the invention or fragments thereof may be cloned into a gene delivering system, such as a virus and the virus used for infection and conferring disease ameliorating or curing effects in the infected cells or organism.
[0068] The present invention also relates to a host cell transfected or transformed with the vector of the invention or a non-human host carrying the vector of the present invention, i.e. to a host cell or host which is genetically modified with a nucleic acid molecule according to the invention or with a vector comprising such a nucleic acid molecule. The term "genetically modified" means that the host cell or host comprises in addition to its natural genome a nucleic acid molecule or vector according to the invention which was introduced into the cell or host or into one of its predecessors/parents. The nucleic acid molecule or vector may be present in the genetically modified host cell or host either as an independent molecule outside the genome, preferably as a molecule which is capable of replication, or it may be stably integrated into the genome of the host cell or host.
[0069] The host cell of the present invention may be any prokaryotic or eukaryotic cell. Suitable prokaryotic cells are those generally used for cloning like E. coli or Bacillus subtilis. Furthermore, eukaryotic cells comprise, for example, fungal or animal cells. Examples for suitable fungal cells are yeast cells, preferably those of the genus Saccharomyces and most preferably those of the species Saccharomyces cerevisiae. Suitable animal cells are, for instance, insect cells, vertebrate cells, preferably mammalian cells, such as e.g. HEK293, NSO, CHO, MDCK, U2-OSHela, NIH3T3, MOLT-4, Jurkat, PC-12, PC-3, IMR, NT2N, Sk-n-sh, CaSki, C33A. These host cells, e.g. CHO-cells, may provide post-translational modifications to the antibody molecules of the invention, including leader peptide removal, folding and assembly of H (heavy) and L (light) chains, glycosylation of the molecule at correct sides and secretion of the functional molecule. Further suitable cell lines known in the art are obtainable from cell line depositories, like the American Type Culture Collection (ATCC). In accordance with the present invention, it is furthermore envisaged that primary cells/cell cultures may function as host cells. Said cells are in particular derived from insects (like insects of the species Drosophila or Blatta) or mammals (like human, swine, mouse or rat). Said host cells may also comprise cells from and/or derived from cell lines like neuroblastoma cell lines. The above mentioned primary cells are well known in the art and comprise, inter alia, primary astrocytes, (mixed) spinal cultures or hippocampal cultures.
[0070] In a more preferred embodiment the host cell which is transformed with the vector of the invention is a neuronal cell, a neuronal stem cell (e.g. an adult neuronal stem cell), a brain cell or a cell (line) derived therefrom. However, also a CHO-cell comprising the nucleic acid molecule of the present invention may be particularly useful as host. Such cells may provide for correct secondary modifications on the expressed molecules, i.e. the antibody molecules of the present invention. These modifications comprise, inter alia, glycosylations and phosphorylations.
[0071] Hosts may be non-human mammals, most preferably mice, rats, sheep, calves, dogs, monkeys or apes. Said mammals may be indispensable for developing a cure, preferably a cure for neurological and/or neurodegenerative disorders mentioned herein. Furthermore, the hosts of the present invention may be particularly useful in producing the antibody molecules (or fragments thereof) of the invention. It is envisaged that said antibody molecules (or fragments thereof) be isolated from said host. It is, inter alia, envisaged that the nucleic acid molecules and or vectors described herein are incorporated in sequences for transgenic expression. The introduction of the inventive nucleic acid molecules as transgenes into non-human hosts and their subsequent expression may be employed for the production of the inventive antibodies. For example, the expression of such (a) transgene(s) in the milk of the transgenic animal provide for means of obtaining the inventive antibody molecules in quantitative amounts; see inter alia, U.S. Pat. No. 5,741,957, U.S. Pat. No. 5,304,489 or U.S. Pat. No. 5,849,992. Useful transgenes in this respect comprise the nucleic acid molecules of the invention, for example, coding sequences for the light and heavy chains of the antibody molecules described herein, operatively linked to promotor and/or enhancer structures from a mammary gland specific gene, like casein or beta-lactoglobulin.
[0072] The invention also provides for a method for the preparation of an antibody molecule of the invention comprising culturing the host cell described herein above under conditions that allow synthesis of said antibody molecule and recovering said antibody molecule from said culture.
[0073] The invention also relates to a composition comprising an antibody molecule of the invention or produced by the method described herein above, a nucleic acid molecule encoding the antibody molecule of the invention, a vector comprising said nucleic acid molecule or a host-cell as defined herein above and optionally, further molecules, either alone or in combination, like e.g. molecules which are capable of interfering with the formation of amyloid plaques or which are capable of depolymerizing already formed amyloid-plaques. The term "composition" as employed herein comprises at least one compound of the invention. Preferably, such a composition is a pharmaceutical or a diagnostic composition.
[0074] The composition may be in solid or liquid form and may be, inter alia, in a form of (a) powder(s), (a) tablet(s), (a) solution(s) or (an) aerosol(s). Said composition may comprise on or more antibodies/antibody molecules of the invention or nucleic acid molecules, vector or hosts of the invention. It is also envisaged that said composition comprises at least two, preferably three, more preferably four, most preferably five antibody molecules of the invention or nucleic acid molecule(s) encoding said antibody molecule(s). Said composition may also comprise optimized, inventive antibodies/antibody molecules obtainable by the methods described herein below and in the appended examples.
[0075] It is preferred that said pharmaceutical composition, optionally comprises a pharmaceutically acceptable carrier and/or diluent. The herein disclosed pharmaceutical composition may be particularly useful for the treatment of neurological and/or neurodegenerative disorders. Said disorders comprise, but are not limited to Alzheimer's disease, amyothrophic lateral sclerosis (ALS), hereditary cerebral hemorrhage with amyloidosis Dutch type, Down's syndrome, HIV-dementia, Parkinson's disease and neuronal disorders related to aging. The pharmaceutical composition of the invention is, inter alia, envisaged as potent inhibitors of amyloid plaque formation or as a potent stimulator for the de-polymerization of amyloid plaques. Therefore, the present invention provides for pharmaceutical compositions comprising the compounds of the invention to be used for the treatment of diseases/disorders associated with pathological APP proteolysis and/or amyloid plaque formation.
[0076] Examples of suitable pharmaceutical carriers, excipients and/or diluents are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose.
[0077] Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. It is particularly preferred that said administration is carried out by injection and/or delivery, e.g., to a site in a brain artery or directly into brain tissue. The compositions of the invention may also be administered directly to the target site, e.g., by biolistic delivery to an external or internal target site, like the brain. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Proteinaceous pharmaceutically active matter may be present in amounts between 1 ng and 10 mg/kg body weight per dose; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. If the regimen is a continuous infusion, it should also be in the range of 1 μg to 10 mg units per kilogram of body weight per minute.
[0078] Progress can be monitored by periodic assessment. The compositions of the invention may be administered locally or systemically. It is of note that peripherally administered antibodies can enter the central nervous system, see, inter alia, Bard (2000), Nature Med. 6, 916-919. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Furthermore, the pharmaceutical composition of the invention may comprise further agents depending on the intended use of the pharmaceutical composition. Said agents may be drugs acting on the central nervous system, like, neuroprotective factors, cholinesterase inhibitors, agonists of M1 muscarinic receptor, hormones, antioxidants, inhibitors of inflammation etc. It is particularly preferred that said pharmaceutical composition comprises further agents like, e.g. neurotransmitters and/or substitution molecules for neurotransmitters, vitamin E, or alpha-lipoic acid.
[0079] The pharmaceutical compositions, as well as the methods of the invention or the uses of the invention described infra can be used for the treatment of all kinds of diseases hitherto unknown or being related to or dependent on pathological APP aggregation or pathological APP processing. They may be particularly useful for the treatment of Alzheimer's disease and other diseases where extracellular deposits of amyloid-β, appear to play a role. They may be desirably employed in humans, although animal treatment is also encompassed by the methods, uses and compositions described herein.
[0080] In a preferred embodiment of the invention, the composition of the present invention as disclosed herein above is a diagnostic composition further comprising, optionally, suitable means for detection. The diagnostic composition comprises at least one of the aforementioned compounds of the invention.
[0081] Said diagnostic composition may comprise the compounds of the invention, in particular and preferably the antibody molecules of the present invention, in soluble form/liquid phase but it is also envisaged that said compounds are bound to/attached to and/or linked to a solid support.
[0082] Solid supports may be used in combination with the diagnostic composition as defined herein or the compounds of the present invention may be directly bound to said solid supports. Such supports are well known in the art and comprise, inter alia, commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, membranes, sheets, duracytes, wells and walls of reaction trays, plastic tubes etc. The compound(s) of the invention, in particular the antibodies of the present invention, may be bound to many different carriers. Examples of well-known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. The nature of the carrier can be either soluble or insoluble for the purposes of the invention. Appropriate labels and methods for labeling have been identified above and are furthermore mentioned herein below. Suitable methods for fixing/immobilizing said compound(s) of the invention are well known and include, but are not limited to ionic, hydrophobic, covalent interactions and the like.
[0083] It is particularly preferred that the diagnostic composition of the invention is employed for the detection and/or quantification of APP and/or APP-processing products, like amyloid-β or for the detection and/or quantification of pathological and/or (genetically) modified APP-cleavage sides.
[0084] As illustrated in the appended examples, the compounds of the present invention, in particular the inventive antibody molecules are particularly useful as diagnostic reagents in the detection of genuine human amyloid plaques in brain sections of Alzheimer's Disease patients by indirect immunofluorescence.
[0085] It is preferred that said compounds of the present invention to be employed in a diagnostic composition are detectably labeled. A variety of techniques are available for labeling biomolecules, are well known to the person skilled in the art and are considered to be within the scope of the present invention. Such techniques are, e.g., described in Tijssen, "Practice and theory of enzyme immuno assays", Burden, R H and von Knippenburg (Eds), Volume 15 (1985), "Basic methods in molecular biology"; Davis L G, Dibmer M D; Battey Elsevier (1990), Mayer et al., (Eds) "Immunochemical methods in cell and molecular biology" Academic Press, London (1987), or in the series "Methods in Enzymology", Academic Press, Inc.
[0086] There are many different labels and methods of labeling known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include enzymes, radioisotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds, and bioluminescent compounds.
[0087] Commonly used labels comprise, inter alia, fluorochromes (like fluorescein, rhodamine, Texas Red, etc.), enzymes (like horse radish peroxidase, β-galactosidase, alkaline phosphatase), radioactive isotopes (like 32P or 125I), biotin, digoxygenin, colloidal metals, chemi- or bioluminescent compounds (like dioxetanes, luminol or acridiniums). Labeling procedures, like covalent coupling of enzymes or biotinyl groups, iodinations, phosphorylations, biotinylations, etc. are well known in the art.
[0088] Detection methods comprise, but are not limited to, autoradiography, fluorescence microscopy, direct and indirect enzymatic reactions, etc. Commonly used detection assays comprise radioisotopic or non-radioisotopic methods. These comprise, inter alia, Westernblotting, overlay-assays, RIA (Radioimmuno Assay) and IRMA (Immune Radioimmunometric Assay), EIA (Enzyme Immuno Assay), ELISA (Enzyme Linked Immuno Sorbent Assay), FIA (Fluorescent Immuno Assay), and CLIA (Chemioluminescent Immune Assay).
[0089] Furthermore, the present invention provides for the use of an antibody molecule of invention, or an antibody molecule produced by the method of the invention, of a nucleic acid molecule, vector of or a host of the invention for the preparation of a pharmaceutical or a diagnostic composition for the prevention, treatment and/or diagnosis of a disease associated with amyloidogenesis and/or amyloid-plaque formation. It is further preferred that the compounds described herein, in particular the antibody molecules of the invention, be employed in the prevention and/or treatment of neuropathologies associated with modified or abnormal APP-processing and/or amyloidogenesis. The antibody molecules, e.g in format of (engineered) immunoglobulins, like antibodies in a IgG framework, in particular in an IgG1-framework, or in the format of chimeric antibodies, bispecific antibodies, single chain Fvs (scFvs) or bispecific scFvs and the like are to employed in the preparation of the pharmaceutical compositions provided herein. Yet, the antibody molecules are also useful in diagnostic settings as documented in the appended examples, since the antibody molecules of the invention specifically interact with/detect Aβ4 and/or amyloid deposits/plaques.
[0090] Therefore an inventive use of the compounds of the present invention is the use for the preparation of a pharmaceutical composition for a neurological disorder which calls for amelioration, for example by disintegration of β-amyloid plaques, by amyloid (plaque) clearance or by passive immunization against β-amyloid plaque formation. As illustrated in the appended examples, the inventive antibody molecules are particularly useful in preventing Aβ aggregation and in de-polymerization of already formed amyloid aggregates. Accordingly, the inventive antibodies are to be employed in the reduction of pathological amyloid deposits/plaques, in the clearance of amyloid plaques/plaque precursors as well as in neuronal protection. It is in particular envisaged that the antibody molecules of the invention be employed in the in vivo prevention of amyloid plaques as well as in in vivo clearance of pre-existing amyloid plaques/deposits. Furthermore, the antibody molecules of the invention may be employed in passive immunization approaches against Aβ4. Clearance of Aβ4/Aβ4 deposits may, inter alia, be achieved by the medical use of antibodies of the present invention which comprise an Fc-part. Said Fc-part of an antibody may be particularly useful in Fc-receptor mediated immune responses, e.g. the attraction of macrophages (phagocytic cells and/or microglia) and/or helper cells. For the mediation of Fc-part-related immunoresponses, the antibody molecule of the invention is preferably in an (human) IgG1framework. As discussed herein, the preferred subject to be treated with the inventive antibody molecules, the nucleic acid molecules encoding the same or parts thereof, the vectors of the invention or the host cells of this invention is a human subject. Other frameworks, like IgG2a- or IgG2b-frameworks for the inventive antibody molecules are also envisaged. Immunoglobulin frameworks in IgG2a and IgG2b format are particular envisaged in mouse settings, for example in scientific uses of the inventive antibody molecules, e.g. in tests on transgenic mice expressing (human) wildtype or mutated APP, APP-fragments and/or Aβ4.
[0091] The above recited diseases associated with amyloidogenesis and/or amyloid-plaque formation comprise, but are not limited to dementia, Alzheimer's disease, motor neuropathy, Parkinson's disease, ALS (amyotrophic lateral sclerosis), scrapie, HIV-related dementia as well as Creutzfeld-Jakob disease, hereditary cerebral hemorrhage, with amyloidis Dutch type, Down's syndrome and neuronal disorders related to aging. The antibody molecules of the invention and the compositions provided herein may also be useful in the amelioration and or prevention of inflammatory processes relating to amyloidogenesis and/or amyloid plaque formation.
[0092] Accordingly, the present invention also provides for a method for treating, preventing and/or delaying neurological and/or neurodegenerative disorders comprising the step of administering to a subject suffering from said neurological and/or neurodegenerative disorder and/or to a subject susceptible to said neurological and/or neurodegenerative disorder an effective amount of a antibody molecule of the invention, a nucleic acid molecule of invention and/or a composition as defined herein above.
[0093] In yet another embodiment, the present invention provides for a kit comprising at least one antibody molecule, at least one nucleic acid molecule, at least one vector or at least one host cell of the invention. Advantageously, the kit of the present invention further comprises, optionally (a) buffer(s), storage solutions and/or remaining reagents or materials required for the conduct of medical, scientific or diagnostic assays and purposes. Furthermore, parts of the kit of the invention can be packaged individually in vials or bottles or in combination in containers or multicontainer units.
[0094] The kit of the present invention may be advantageously used, inter alia, for carrying out the method of the invention and could be employed in a variety of applications referred herein, e.g., as diagnostic kits, as research tools or medical tools. Additionally, the kit of the invention may contain means for detection suitable for scientific, medical and/or diagnostic purposes. The manufacture of the kits follows preferably standard procedures which are known to the person skilled in the art.
[0095] The invention also provides for a method for the optimization of an antibody molecule as defined herein above comprising the steps of
(a) constructing a library of diversified Fab antibody fragments derived from an antibody comprising at least one CDR3 of an VH-region as encoded by a nucleic acid molecule as shown in SEQ ID NOs: 21, 23 or 25 or at least one CDR3 amino acid sequence of an VH-region as shown in SEQ ID NOs: 22, 24 or 26; (b) testing the resulting Fab optimization library by panning against Aβ/Aβ4; (c) identifying optimized clones; and (d) expressing of selected, optimized clones.
[0096] Optimization of the antibodies/antibody molecules of the invention is also documented in the appended examples and may comprise the selection for, e.g. higher affinity for one or both regions/epitopes of β-A4 as defined herein or selection for improved expression and the like. In one embodiment, said selection for to higher affinity for one or both regions/epitopes of β-A4 comprises the selection for high affinity to (a) an amino acid stretch comprising amino acids 2 to 10 (or (a) part(s) thereof) of β-A4 and/or (b) an amino acid stretch comprising amino acids 12 to 25 (or (a) part(s) thereof) of β-A4 (SEQ ID NO. 27).
[0097] The person skilled in the art can readily carry out the inventive method employing the teachings of the present invention. Optimization protocols for antibodies are known in the art. These optimization protocols comprise, inter alia, CDR walking mutagenesis as disclosed and illustrated herein and described in Yang (1995), J. Mol. Biol. 25, 392-403; Schier (1996), J. Mol. Biol. 263, 551-567; Barbas (1996), Trends. Biotech 14, 230-34 or Wu (1998), PNAS 95, 6037-6042; Schier (1996), Human Antibodies Hybridomas 7, 97; Moore (1997), J. Mol. Biol. 272, 336.
[0098] "Panning"-techniques are also known in the art, see, e.g. Kay (1993), Gene 128, 59-65. Furthermore, publications like Borrebaeck (1995), "Antibody Engineering", Oxford University, 229-266; McCafferty (1996), "Antibody Engineering", Oxford University Press; Kay (1996), A Laboratory Manual, Academic Press provide for optimization protocols which may be modified in accordance with this invention.
[0099] The optimization method may further comprise a step (ca), whereby the optimized clones are further optimized by cassette mutagenesis, as illustrated in the appended examples.
[0100] The method for the optimization of an antibody molecule described herein is further illustrated in the appended examples as affinity maturation of parental antibodies/antibody molecules capable of specifically recognizing two regions of the beta-A4 peptide/Abeta4/Aβ4/Aβ4/βA4.
[0101] Preferably, said Aβ4/Aβ4 (also designated as βA4 in context of this invention) in step (b) of the method described herein above is aggregated Aβ4/Aβ4. Said panning may be carried out (as described in the appended examples) with increased stringency of binding. Stringency may be increased, inter alia, by reducing the Aβ4/Aβ4 concentration or by elevating the (assay) temperature. The testing of the optimized library by panning is known to the skilled artisan and described in Kay (1993), loc. cit. Preferably, the identification in step (c) is carried out by ranking according to the lowest KD-values.
[0102] Most preferably said identification in step (c) is carried out by koff-ranking. Koff-ranking is known to the skilled artisan and described in Schier (1996), loc. cit.; Schier (1996), J. Mol. Biol. 255, 28-43 or Duenas (1996), Mol. Immunol. 33, 279-286. Furthermore, koff-ranking is illustrated in the appended examples. The off-rate constant may be measured as described in the appended examples.
[0103] As mentioned herein above, the identified clones may, for further evaluation, be expressed. The expression may be carried out by known methods, inter alia, illustrated in the appended examples. The expression may, inter alia, lead to expressed Fab-fragments, scFvs, bispecific immunoglobulins, bispecific antibody molecules, Fab- and/or Fv fusion proteins, or full antibodies, like IgGs, in particular IgG1.
[0104] Optimized antibodies, in particular optimized Fabs or optimized IgGs, preferably IgG1 s, may be tested by methods as illustrated in the appended examples. Such methods comprise, but are not limited to, the testing of binding affinities, the determination of KD values, pepspot analysis, ELISA-assays, RIA-assays, CLIA-assays, (immuno-) histological studies (for example staining of amyloid plaques), de-polymerization assays or antibody-dependent β-A4 phagocytoses.
[0105] In a further embodiment of the present invention, a method is provided wherein optimized antibodies are generated by cross-cloning. This method is also illustrated in the appended examples and comprises the step of combining independently optimized CDR-regions, for example, by combining independently optimized H-CDR2 and L-CDR2 from matured clones with H-CDR3, preferably the same H-CDR3.
[0106] In a preferred embodiment, the invention relates to a method for the preparation of a pharmaceutical composition comprising the steps of
(a) optimization of an antibody according to the method described herein and illustrated in the appended examples; and (b) formulating the optimized antibody/antibody molecule with an physiologically acceptable carrier, as described herein above.
[0107] Accordingly, the invention also provides for a pharmaceutical composition prepared by the method disclosed herein and comprising further optimized antibody molecules capable of specifically recognizing two regions of the beta-A4 petide/Abeta4/Aβ/A4β/βA4, as described herein above.
Exemplified Sequences as Recited Herein:
TABLE-US-00001
[0108] SEQ ID NO: 1 AEFRHDSGY First region of β-A4 peptide, "N-terminal region/epitope" SEQ ID NO: 2 VHHQKLVFFAEDVG Second region of β-A4 peptide, "Central/middle region/epitope" SEQ ID NO: 3 VH-region of MS-Roche#3 (nucleic acid sequence) (SEQ ID NO: 3) CAGGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGC GTCTGAGCTGCGCGGCCTCCGGATTTACCTTTAGCAGCTATGCGATGAGCTGGGTGC GCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCGCGATTAGCGGTAGCGGCGGC AGCACCTATTATGCGGATAGCGTGAAAGGCCGTTTTACCATTTCACGTGATAATTCGA AAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTA TTGCGCGCGTCTTACTCATTATGCTCGTTATTATCGTTATTTTGATGTTTGGGGCCAAG GCACCCTGGTGACGGTTAGCTCAGC SEQ ID NO: 4 VH-region of MS-Roche#3 (amino acid sequence) (SEQ ID NO: 4) QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGST YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLTHYARYYRYFDVWGQGTL VTVSS SEQ ID NO: 5 VH-region of MS-Roche#7 (nucleic acid sequence) (SEQ ID NO: 5) CAGGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGC GTCTGAGCTGCGCGGCCTCCGGATTTACCTTTAGCAGCTATGCGATGAGCTGGGTGC GCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCGCGATTAGCGGTAGCGGCGGC AGCACCTATTATGCGGATAGCGTGAAAGGCCGTTTACCATTTCACGTGATAATTCGAA AAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTAT TGCGCGCGTGGTAAGGGTAATACTCATAAGCCTTATGGTTATGTTCGTTATTTTGATGT TTGGGGCCAAGGCACCCTGGTGACGGTTAGCTCAGC SEQ ID NO: 6 VH-region of MS-Roche#7 (amino acid sequence) (SEQ ID NO: 6) QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGST YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWG QGTLVTVSS SEQ ID NO: 7 VH-region of MS-Roche#8 (nucleic acid sequence) (SEQ ID NO: 7) CAGGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGC GTCTGAGCTGCGCGGCCTCCGGATTTACCTTTAGCAGCTATGCGATGAGCTGGGTGC GCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCGCGATTAGCGGTAGCGGCGGC AGCACCTATTATGCGGATAGCGTGAAAGGCCGTTTTACCATTTCACGTGATAATTCGA AAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTA TTGCGCGCGTCTTCTTTCTCGTGGTTATAATGGTTATTATCATAAGTTTGATGTTTGGG GCCAAGGCACCCTGGTGACGGTTAGCTCAGC SEQ ID NO: 8 VH-region of MS-Roche#8 (amino acid sequence) (SEQ ID NO: 8) QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGST YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLLSRGYNGYYHKFDVWGQG TLVTVSS SEQ ID NO: 9 VL-region of MS-Roche#3 (nucleic acid sequence) (SEQ ID NO: 9) GATATCGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGTCTCCGGGCGAACGTGC GACCCTGAGCTGCAGAGCGAGCCAGAGCGTGAGCAGCAGCTATCTGGCGTGGTACC AGCAGAAACCAGGTCAAGCACCGCGTCTATTAATTTATGGCGCGAGCAGCCGTGCAA CTGGGGTCCCGGCGCGTTTTAGCGGCTCTGGATCCGGCACGGATTTTACCCTGACCA TTAGCAGCCTGGAACCTGAAGACTTTGCGGTTTATTATTGCCAGCAGGTTTATAATCCT CCTGTTACCTTTGGCCAGGGTACGAAAGTTGAAATTAAACGTACG SEQ ID NO: 10 VL-region of MS-Roche #3 (amino acid sequence) (SEQ ID NO: 10) DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAVVYQQKPGQAPRLLIYGASSRATGVPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQVYNPPVTFGQGTKVEIKRT SEQ ID NO: 11 VL-region of MS-Roche#7 (nucleic acid sequence) (SEQ ID NO. 11) GATATCGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGTCTCCGGGCGAACGTGC GACCCTGAGCTGCAGAGCGAGCCAGAGCGTGAGCAGCAGCTATCTGGCGTGGTACC AGCAGAAACCAGGTCAAGCACCGCGTCTATTAATTTATGGCGCGAGCAGCCGTGCAA CTGGGGTCCCGGCGCGTTTTAGCGGCTCTGGATCCGGCACGGATTTTACCCTGACCA TTAGCAGCCTGGAACCTGAAGACTTTGCGACTTATTATTGCTTTCAGCTTTATTCTGAT CCTTTTACCTTTGGCCAGGGTACGAAAGTTGAAATTAAACGTACG SEQ ID NO: 12 VL-region of MS-Roche#7 (amino acid sequence) (SEQ ID NO: 12) DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAVVYQQKPGQAPRLLIYGASSRATGVPA RFSGSGSGTDFTLTISSLEPEDFATYYCFQLYSDPFTFGQGTKVEIKRT SEQ ID NO: 13 VL-region of MS-Roche#8 (nucleic acid sequence) (SEQ ID NO: 13) GATATCGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGTCTCCGGGCGAACGTGC GACCCTGAGCTGCAGAGCGAGCCAGAGCGTGAGCAGCAGCTATCTGGCGTGGTACC AGCAGAAACCAGGTCAAGCACCGCGTCTATTAATTTATGGCGCGAGCAGCCGTGCAA CTGGGGTCCCGGCGCGTTTTAGCGGCTCTGGATCCGGCACGGATTTTACCCTGACCA TTAGCAGCCTGGAACCTGAAGACTTTGCGACTTATTATTGCCAGCAGCTTTCTTCTTTT CCTCCTACCTTTGGCCAGGGTACGAAAGTTGAAATTAAACGTACG SEQ ID NO: 14 VL-region of MS-Roche#8 (amino acid sequence) (SEQ ID NO: 14) DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAVVYQQKPGQAPRLLIYGASSRATGVPA RFSGSGSGTDFTLTISSLEPEDFATYYCQQLSSFPPTFGQGTKVEIKRT SEQ ID NO: 15 CDR3 of VL-region of MSR-3 (nucleic acid sequence) (SEQ ID NO: 15) CAGCAGGTTTATAATCCTCCTGTT SEQ ID NO: 16 CDR3 of VL-region of MSR-3 (amino acid sequence) (SEQ ID NO: 16) QQVYNPPV SEQ ID NO: 17 CDR3 of VL-region of MSR-7 (nucleic acid sequence) (SEQ ID NO: 17) TTTCAGCTTTATTCTGATCCTTTT SEQ ID NO: 18 CDR3 of VL-region of MSR-7 (amino acid sequence) (SEQ ID NO. 18) FQLYSDPF SEQ ID NO: 19 CDR3 of VL-region of MSR-8 (nucleic acid sequence) (SEQ ID NO. 19) CAG CAG CTT TCT TCT TTT CCT CCT SEQ ID NO: 20 CDR3 of VL-region of MSR-8 (amino acid sequence) (SEQ ID NO: 20) QQLSSFPP SEQ ID NO: 21 CDR of VH-region of MSR-3 (nucleic acid sequence) (SEQ ID NO: 21) CTT ACT CAT TAT GCT CGT TAT TAT CGT TAT TTT GAT GTT SEQ ID NO: 22 CDR of VH-region of MSR-3 (amino acid sequence) (SEQ ID NO: 22) LTHYARYYRYFDV SEQ ID NO: 23 CDR of VH-region of MSR-7 (nucleic acid sequence) (SEQ ID NO: 23) GGT AAG GGT AAT ACT CAT AAG CCT TAT GGT TAT GTT CGT TAT TTT GAT GTT SEQ ID NO: 24 CDR of VH-region of MSR-7 (amino acid sequence) (SEQ ID NO: 24) GKGNTHKPYGYVRYFDV SEQ ID NO: 25 CDR of VH-region of MSR-8 (nucleic acid sequence) (SEQ ID NO. 25) CTT CTT TCT CGT GGT TAT AAT GGT TAT TAT CAT AAG TTT GAT GTT SEQ ID NO: 26 CDR of VH-region of MSR-8 (amino acid sequence) (SEQ ID NO: 26) LLSRGYNGYYHKFDV SEQ ID NO: 27 Aβ4 (amino acids 1 to 42) (SEQ ID NO: 27) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA SEQ ID NO: 28 primer (SEQ ID NO: 28) 5'-GTGGTGGTTCCGATATC-3' SEQ ID NO: 29 primer (SEQ ID NO: 29) 5'-AGCGTCACACTCGGTGCGGCTTTCGGCTGGCCAAGAACGGTTA-3' SEQ ID NO: 30 primer (SEQ ID NO: 30) 5'-CAGGAAACAGCTATGAC-3' SEQ ID NO: 31 primer (SEQ ID NO: 31) 5'-TACCGTTGCTCTTCACCCC-3' SEQ ID NO: 32 VH of MS-Roche#3.6H5 × 3.6L2; DNA; artificial sequence (SEQ ID NO: 32) CAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAG CTGCGCGGCCTCCGGATTTACCTTTAGCAGCTATGCGATGAGCTGGGTGCGCCAAGC CCCTGGGAAGGGTCTCGAGTGGGTGAGCGCTATTTCTGAGTCTGGTAAGACTAAGTA TTATGCTGATTCTGTTAAGGGTCGTTTTACCATTTCACGTGATAATTCGAAAAACACCC TGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGC GTCTTACTCATTATGCTCGTTATTATCGTTATTTTGATGTTTGGGGCCAAGGCACCCTG GTGACGGTTAGCTCA SEQ ID NO. 33: prot VH region of MS-Roche#3.6H5 × 3.6L2; protein/1;
artificial sequence (SEQ ID NO: 33) QLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISESGKTKYYA DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLTHYARYYRYFDVWGQGTLVTVS S SEQ ID NO: 34 VH region of MS-Roche#3.6H8 × 3.6L2; DNA; artificial sequence (SEQ ID NO: 34) CAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAG CTGCGCGGCCTCCGGATTTACCTTTAGCAGCTATGCGATGAGCTGGGTGCGCCAAGC CCCTGGGAAGGGTCTCGAGTGGGTGAGCGCTATTTCTGAGTATTCTAAGTTTAAGTAT TATGCTGATTCTGTTAAGGGTCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCT GTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCG TCTTACTCATTATGCTCGTTATTATCGTTATTTTGATGTTTGGGGCCAAGGCACCCTGG TGACGGTTAGCTCA SEQ ID NO: 35 prot VH region of MS-Roche#3.6H8 × 3.6L2; protein/1; artificial sequence (SEQ ID NO: 35) QLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISEYSKFKYYA DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLTHYARYYRYFDVWGQGTLVTVS S SEQ ID NO: 36 VH region of MS-Roche#7.4H2 × 7.2L1; DNA; artificial sequence (SEQ ID NO: 36) CAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAG CTGCGCGGCCTCCGGATTTACCTTTAGCAGCTATGCGATGAGCTGGGTGCGCCAAGC CCCTGGGAAGGGTCTCGAGTGGGTGAGCGCTATTAATTATAATGGTGCTCGTATTTAT TATGCTGATTCTGTTAAGGGTCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCT GTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCG TGGTAAGGGTAATACTCATAAGCCTTATGGTTATGTTCGTTATTTTGATGTTTGGGGCC AAGGCACCCTGGTGACGGTTAGCTCA SEQ ID NO: 37 prot VH region of MS-Roche#7.4H2 × 7.2L1; protein/1; artificial sequence (SEQ ID NO: 37) QLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINYNGARIYYA DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQGT LVTVSS SEQ ID NO: 38 VH region of MS-Roche#7.9H2 × 7.12 L2; DNA; artificial sequence (SEQ ID NO: 38) CAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAG CTGCGCGGCCTCCGGATTTACCTTTAGCAGCTATGCGATGAGCTGGGTGCGCCAAGC CCCTGGGAAGGGTCTCGAGTGGGTGAGCGCTATTAATGCTGATGGTAATCGTAAGTA TTATGCTGATTCTGTTAAGGGTCGTTTTACCATTTCACGTGATAATTCGAAAAACACCC TGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGC GTGGTAAGGGTAATACTCATAAGCCTTATGGTTATGTTCGTTATTTTGATGTTTGGGGC CAAGGCACCCTGGTGACGGTTAGCTCA SEQ ID NO: 39 prot VH region of MS-Roche#7.9H2 × 7.12 L2; protein/1; artificial sequence (SEQ ID NO: 39) QLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINADGNRKYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQG TLVTVSS SEQ ID NO: 40 VH region of MS-Roche#7.9H4 × 7.12L2; DNA; artificial sequence (SEQ ID NO: 40) CAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAG CTGCGCGGCCTCCGGATTTACCTTTAGCAGCTATGCGATGAGCTGGGTGCGCCAAGC CCCTGGGAAGGGTCTCGAGTGGGTGAGCGCTATTAATGCTGTTGGTATGAAGAAGTT TTATGCTGATTCTGTTAAGGGTCGTTTTACCATTTCACGTGATAATTCGAAAAACACCC TGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGC GTGGTAAGGGTAATACTCATAAGCCTTATGGTTATGTTCGTTATTTTGATGTTTGGGGC CAAGGCACCCTGGTGACGGTTAGCTCA SEQ ID NO: 41 prot VH region of MS-Roche#7.9H4 × 7.12L2; protein/1; artificial sequence (SEQ ID NO: 41) QLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINAVGMKKFY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQG TLVTVSS SEQ ID NO: 42 VH region of MS-Roche#7.11H1 × 7.11L1; DNA; artificial sequence (SEQ ID NO: 42) CAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAG CTGCGCGGCCTCCGGATTTACCTTTAGCAGCTATGCGATGAGCTGGGTGCGCCAAGC CCCTGGGAAGGGTCTCGAGTGGGTGAGCGGTATTAATGCTGCTGGTTTTCGTACTTAT TATGCTGATTCTGTTAAGGGTCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCT GTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCG TGGTAAGGGTAATACTCATAAGCCTTATGGTTATGTTCGTTATTTTGATGTTTGGGGCC AAGGCACCCTGGTGACGGTTAGCTCA SEQ ID NO. 43 prot VH region of MS-Roche#7.11H1 × 7.11L1; protein/1; artificial sequence (SEQ ID NO: 43) QLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEVVVSGINAAGFRTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQG TLVTVSS SEQ ID NO: 44 VH region of MS-Roche#7.11H1 × 7.2L1; DNA; artificial sequence (SEQ ID NO: 44) CAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAG CTGCGCGGCCTCCGGATTTACCTTTAGCAGCTATGCGATGAGCTGGGTGCGCCAAGC CCCTGGGAAGGGTCTCGAGTGGGTGAGCGGTATTAATGCTGCTGGTTTTCGTACTTAT TATGCTGATTCTGTTAAGGGTCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCT GTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCG TGGTAAGGGTAATACTCATAAGCCTTATGGTTATGTTCGTTATTTTGATGTTTGGGGCC AAGGCACCCTGGTGACGGTTAGCTCA SEQ ID NO: 45 prot VH region of MS-Roche#7.11H1 × 7.2L1; protein/1; artificial sequence (SEQ ID NO: 45) QLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGINAAGFRTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQG TLVTVSS SEQ ID NO: 46 VL region of MS-Roche#3.6H5 × 3.6L2; DNA; artificial sequence (SEQ ID NO: 46) GATATCGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGTCTCCGGGCGAACGTGC GACCCTGAGCTGCAGAGCGAGCCAGTTTCTTTCTCGTTATTATCTGGCGTGGTACCAG CAGAAACCAGGTCAAGCACCGCGTCTATTAATTTATGGCGCGAGCAGCCGTGCAACT GGGGTCCCGGCGCGTTTTAGCGGCTCTGGATCCGGCACGGATTTTACCCTGACCATT AGCAGCCTGGAACCTGAAGACTTTGCGGTTTATTATTGCCAGCAGACTTATAATTATCC TCCTACCTTTGGCCAGGGTACGAAAGTTGAAATTAAACGTACG SEQ ID NO: 47 prot VL region of MS-Roche#3.6H5 × 3.6L2; protein/1; artificial sequence (SEQ ID NO: 47) DIVLTQSPATLSLSPGERATLSCRASQFLSRYYLAWYQQKPGQAPRLLIYGASSRATGVPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQTYNYPPTFGQGTKVEIKRT SEQ ID NO: 48 VL region of MS-Roche#3.6H8 × 3.6L2; DNA; artificial sequence (SEQ ID NO: 48) GATATCGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGTCTCCGGGCGAACGTGC GACCCTGAGCTGCAGAGCGAGCCAGTTTCTTTCTCGTTATTATCTGGCGTGGTACCAG CAGAAACCAGGTCAAGCACCGCGTCTATTAATTTATGGCGCGAGCAGCCGTGCAACT GGGGTCCCGGCGCGTTTTAGCGGCTCTGGATCCGGCACGGATTTTACCCTGACCATT AGCAGCCTGGAACCTGAAGACTTTGCGGTTTATTATTGCCAGCAGACTTATAATTATCC TCCTACCTTTGGCCAGGGTACGAAAGTTGAAATTAAACGTACG SEQ ID NO: 49 prot VL region of MS-Roche#3.6H8 × 3.6L2; protein/1; artificial sequence (SEQ ID NO: 49) DIVLTQSPATLSLSPGERATLSCRASQFLSRYYLAWYQQKPGQAPRLLIYGASSRATGVPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQTYNYPPTFGQGTKVEIKRT SEQ ID NO: 50 VL region of MS-Roche#7.4H2 × 7.2L1; DNA; artificial sequence (SEQ ID NO: 50) GATATCGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGTCTCCGGGCGAACGTGC GACCCTGAGCTGCAGAGCGAGCCAGTATGTTGATCGTACTTATCTGGCGTGGTACCA GCAGAAACCAGGTCAAGCACCGCGTCTATTAATTTATGGCGCGAGCAGCCGTGCAAC TGGGGTCCCGGCGCGTTTTAGCGGCTCTGGATCCGGCACGGATTTTACCCTGACCAT TAGCAGCCTGGAACCTGAAGACTTTGCGACTTATTATTGCCAGCAGATTTATTCTTTTC CTCATACCTTTGGCCAGGGTACGAAAGTTGAAATTAAACGTACG SEQ ID NO: 51 prot VL region of MS-Roche#7.4H2 × 7.2L1; protein/1; artificial sequence (SEQ ID NO: 51) DIVLTQSPATLSLSPGERATLSCRASQYVDRTYLAWYQQKPGQAPRLLIYGASSRATGVP ARFSGSGSGTDFTLTISSLEPEDFATYYCQQIYSFPHTFGQGTKVEIKRT SEQ ID NO: 52 VL region of MS-Roche#7.9H2 × 7.12 L2; DNA; artificial sequence (SEQ ID NO: 52) GATATCGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGTCTCCGGGCGAACGTGC GACCCTGAGCTGCAGAGCGAGCCAGCGTTTTTTTTATAAGTATCTGGCGTGGTACCAG CAGAAACCAGGTCAAGCACCGCGTCTATTAATTTCTGGTTCTTCTAACCGTGCAACTG GGGTCCCGGCGCGTTTTAGCGGCTCTGGATCCGGCACGGATTTTACCCTGACCATTA GCAGCCTGGAACCTGAAGACTTTGCGGTTTATTATTGCCTTCAGCTTTATAATATTCCT
AATACCTTTGGCCAGGGTACGAAAGTTGAAATTAAACGTACG SEQ ID NO: 53 prot VL region of MS-Roche#7.9H2 × 7.12 L2; protein/1; artificial sequence (SEQ ID NO: 53) DIVLTQSPATLSLSPGERATLSCRASQRFFYKYLAWYQQKPGQAPRLLISGSSNRATGVP ARFSGSGSGTDFTLTISSLEPEDFAVYYCLQLYNIPNTFGQGTKVEIKRT SEQ ID NO: 54 VL region of MS-Roche#7.9H4 × 7.12L2; DNA; artificial sequence (SEQ ID NO: 54) GATATCGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGTCTCCGGGCGAACGTGC GACCCTGAGCTGCAGAGCGAGCCAGCGTTTTTTTTATAAGTATCTGGCGTGGTACCAG CAGAAACCAGGTCAAGCACCGCGTCTATTAATTTCTGGTTCTTCTAACCGTGCAACTG GGGTCCCGGCGCGTTTTAGCGGCTCTGGATCCGGCACGGATTTTACCCTGACCATTA GCAGCCTGGAACCTGAAGACTTTGCGGTTTATTATTGCCTTCAGCTTTATAATATTCCT AATACCTTTGGCCAGGGTACGAAAGTTGAAATTAAACGTACG SEQ ID NO: 55 prot VL region of MS-Roche#7.9H4 × 7.12L2; protein/1; artificial sequence (SEQ ID NO: 55) DIVLTQSPATLSLSPGERATLSCRASQRFFYKYLAWYQQKPGQAPRLLISGSSNRATGVP ARFSGSGSGTDFTLTISSLEPEDFAVYYCLQLYNIPNTFGQGTKVEIKRT SEQ ID NO: 56 VL region of MS-Roche#7.11H1 × 7.11L1; DNA; artificial sequence (SEQ ID NO: 56) GATATCGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGTCTCCGGGCGAACGTGC GACCCTGAGCTGCAGAGCGAGCCAGCGTATTCTTCGTATTTATCTGGCGTGGTACCA GCAGAAACCAGGTCAAGCACCGCGTCTATTAATTTATGGCGCGAGCAGCCGTGCAAC TGGGGTCCCGGCGCGTTTTAGCGGCTCTGGATCCGGCACGGATTTTACCCTGACCAT TAGCAGCCTGGAACCTGAAGACTTTGCGACTTATTATTGCCAGCAGGTTTATTCTCCTC CTCATACCTTTGGCCAGGGTACGAAAGTTGAAATTAAACGTACG (SEQ ID NO: 56) SEQ ID NO: 57 prot VL region of MS-Roche#7.11H1 × 7.11L1; protein/1; artificial sequence (SEQ ID NO: 57) DIVLTQSPATLSLSPGERATLSCRASQRILRIYLAWYQQKPGQAPRLLIYGASSRATGVPAR FSGSGSGTDFTLTISSLEPEDFATYYCQQVYSPPHTFGQGTKVEIKRT SEQ ID NO: 58 VL region of MS-Roche#7.11H1 × 7.2L1; DNA; artificial sequence (SEQ ID NO: 58) GATATCGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGTCTCCGGGCGAACGTGC GACCCTGAGCTGCAGAGCGAGCCAGTATGTTGATCGTACTTATCTGGCGTGGTACCA GCAGAAACCAGGTCAAGCACCGCGTCTATTAATTTATGGCGCGAGCAGCCGTGCAAC TGGGGTCCCGGCGCGTTTTAGCGGCTCTGGATCCGGCACGGATTTTACCCTGACCAT TAGCAGCCTGGAACCTGAAGACTTTGCGACTTATTATTGCCAGCAGATTTATTCTTTTC CTCATACCTTTGGCCAGGGTACGAAAGTTGAAATTAAACGTACG SEQ ID NO: 59 prot VL region of MS-Roche#7.11H1 × 7.2L1; protein/1; artificial sequence (SEQ ID NO: 59) DIVLTQSPATLSLSPGERATLSCRASQYVDRTYLAWYQQKPGQAPRLLIYGASSRATGVP ARFSGSGSGTDFTLTISSLEPEDFATYYCQQIYSFPHTFGQGTKVEIKRT SEQ ID NO: 60 HCDR3 region of MS-Roche#3.6H5 × 3.6L2; DNA; artificial sequence (SEQ ID NO: 60) CTTACTCATTATGCTCGTTATTATCGTTATTTTGATGTT SEQ ID NO: 61 prot HCDR3 region of MS-Roche#3.6H5 × 3.6L2; protein/1; artificial sequence (SEQ ID NO: 61) LTHYARYYRYFDV SEQ ID NO: 62 HCDR3 region of MS-Roche#3.6H8 × 3.6L2; DNA; artificial sequence (SEQ ID NO: 62) CTTACTCATTATGCTCGTTATTATCGTTATTTTGATGTT SEQ ID NO: 63 prot HCDR3 region of MS-Roche#3.6H8 × 3.6L2; protein/1; artificial sequence (SEQ ID NO: 63) LTHYARYYRYFDV SEQ ID NO: 64 HCDR3 region of MS-Roche#7.4H2 × 7.2L1; DNA; artificial sequence (SEQ ID NO: 64) GGTAAGGGTAATACTCATAAGCCTTATGGTTATGTTCGTTATTTTGATGTT SEQ ID NO: 65 prot HCDR3 region of MS-Roche#7.4H2 × 7.2L1; protein/1; artificial sequence (SEQ ID NO: 65) GKGNTHKPYGYVRYFDV SEQ ID NO: 66 HCDR3 region of MS-Roche#7.9H2 × 7.12 L2; DNA; artificial sequence (SEQ ID NO: 66) GGTAAGGGTAATACTCATAAGCCTTATGGTTATGTTCGTTATTTTGATGTT SEQ ID NO: 67 prot HCDR3 region of#MS-Roche 7.9H2 × 7.12 L2; protein/1; artificial sequence (SEQ ID NO: 67) GKGNTHKPYGYVRYFDV SEQ ID NO: 68 HCDR3 region of MS-Roche#7.9H4 × 7.12L2; DNA; artificial sequence (SEQ ID NO: 68) GGTAAGGGTAATACTCATAAGCCTTATGGTTATGTTCGTTATTTTGATGTT SEQ ID NO: 69 prot HCDR3 region of MS-Roche#7.9H4 × 7.12L2; protein/1; artificial sequence (SEQ ID NO: 69) GKGNTHKPYGYVRYFDV SEQ ID NO: 70 HCDR3 region of MS-Roche#7.11H1 × 7.11L1; DNA; artificial sequence (SEQ ID NO: 70) GGTAAGGGTAATACTCATAAGCCTTATGGTTATGTTCGTTATTTTGATGTT SEQ ID NO: 71 prot HCDR3 region of MS-Roche#7.11H1 × 7.11L1; protein/1; artificial sequence (SEQ ID NO: 71) GKGNTHKPYGYVRYFDV SEQ ID NO: 72 HCDR3 region of MS-Roche#7.11H1 × 7.2L1; DNA; artificial sequence (SEQ ID NO: 72) GGTAAGGGTAATACTCATAAGCCTTATGGTTATGTTCGTTATTTTGATGTT SEQ ID NO: 73 prot HCDR3 region of MS-Roche#7.11H1 × 7.2L1; protein/1; artificial sequence (SEQ ID NO: 73) GKGNTHKPYGYVRYFDV SEQ ID NO: 74 LCDR3 region of MS-Roche#3.6H5 × 3.6L2; DNA; artificial sequence (SEQ ID NO: 74) CAGCAGACTTATAATTATCCTCCT SEQ ID NO: 75 prot LCDR3 region of MS-Roche#3.6H5 × 3.6L2; protein/1; artificial sequence (SEQ ID NO: 75) QQTYNYPP SEQ ID NO: 76 LCDR3 region of MS-Roche#3.6H8 × 3.6L2; DNA; artificial sequence (SEQ ID NO: 76) CAGCAGACTTATAATTATCCTCCT SEQ ID NO: 77 prot LCDR3 region of MS-Roche#3.6H8 × 3.6L2; protein/1; artificial sequence (SEQ ID NO: 77) QQTYNYPP SEQ ID NO: 78 LCDR3 region of MS-Roche#7.4H2 × 7.2L1; DNA; artificial sequence (SEQ ID NO: 78) CAGCAGATTTATTCTTTTCCTCAT SEQ ID NO: 79 prot LCDR3 region of MS-Roche#7.4H2 × 7.2L1; protein/1; artificial sequence (SEQ ID NO: 79) QQIYSFPH SEQ ID NO: 80 LCDR3 region of MS-Roche#7.9H2 × 7.12 L2; DNA; artificial sequence (SEQ ID NO: 80) CTTCAGCTTTATAATATTCCTAAT SEQ ID NO: 81 prot LCDR3 region of MS-Roche#7.9H2 × 7.12 L2; protein/1; artificial sequence (SEQ ID NO: 81) LQLYNIPN SEQ ID NO: 82 LCDR3 region of MS-Roche#7.9H4 × 7.12L2; DNA; artificial sequence (SEQ ID NO: 82) CTTCAGCTTTATAATATTCCTAAT SEQ ID NO: 83 prot LCDR3 region of MS-Roche#7.9H4 × 7.12L2; protein/1; artificial sequence (SEQ ID NO: 83) LQLYNIPN SEQ ID NO: 84 LCDR3 region of MS-Roche#7.11H1 × 7.11L1; DNA; artificial sequence (SEQ ID NO: 84) CAGCAGGTTTATTCTCCTCCTCAT SEQ ID NO: 85 prot LCDR3 region of MS-Roche#7.11H1 × 7.11L1; protein/1; artificial sequence (SEQ ID NO: 85) QQVYSPPH SEQ ID NO: 86 LCDR3 region of MS-Roche#7.11H1 × 7.2L1; DNA; artificial sequence (SEQ ID NO: 86) CAGCAGATTTATTCTTTTCCTCAT SEQ ID NO: 87 prot LCDR3 region of MS-Roche#7.11H1 × 7.2L1; protein/1; artificial sequence (SEQ ID NO: 87) QQIYSFPH SEQ ID NO: 88 VH region of MS-Roche#7.9H7; DNA; artificial sequence (SEQ ID NO: 88) Caggtgcaattggtggaaagcggcggcggcctggtgcaaccgggcggcagcctgcgtctgagctgcgcggcctc- cggattt acctttagcagctatgcgatgagctgggtgcgccaagcccctgggaagggtctcgagtgggtgagcgctattaa- tgcttctggta ctcgtacttattatgctgattctgttaagggtcgttttaccatttcacgtgataattcgaaaaacaccctgtat- ctgcaaatgaacagc ctgcgtgcggaagatacggccgtgtattattgcgcgcgtggtaagggtaatactcataagccttatggttatgt- tcgttattttgatgtt tggggccaaggcaccctggtgacggttagctca SEQ ID NO: 89 prot VH region of MS-Roche#7.9H7; protein/1; artificial
sequence (SEQ ID NO: 89) QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINASGTRT YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWG QGTLVTVSS SEQ ID NO: 90 VL region of MS-Roche#7.9H7; DNA; artificial sequence (SEQ ID NO: 90) Gatatcgtgctgacccagagcccggcgaccctgagcctgtctccgggcgaacgtgcgaccctgagctgcagagc- gagcca gagcgtgagcagcagctatctggcgtggtaccagcagaaaccaggtcaagcaccgcgtctattaatttatggcg- cgagcagc cgtgcaactggggtcccggcgcgttttagcggctctggatccggcacggattttaccctgaccattagcagcct- ggaacctgaa gactttgcgacttattattgccttcagatttataatatgcctattacctttggccagggtacgaaagttgaaat- taaacgtacg SEQ ID NO: 91 prot VL region of MS-Roche#7.9H7; protein/1; artificial sequence (SEQ ID NO: 91) DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGVP ARFSGSGSGTDFTLTISSLEPEDFATYYCLQIYNMPITFGQGTKVEIKRT SEQ ID NO: 92 HCDR3 region of MS-Roche#7.9H7; DNA; artificial sequence (SEQ ID NO: 92) Ggtaagggtaatactcataagccttatggttatgttcgttattttgatgtt SEQ ID NO: 93 prot HCDR3 region of MS-Roche#7.9H7; protein/1; artificial sequence (SEQ ID NO: 93) GKGNTHKPYGYVRYFDV SEQ ID NO: 94 LCDR3 region of MS-Roche#7.9H7; DNA; artificial sequence (SEQ ID NO: 94) Cttcagatttataatatgcctatt SEQ ID NO: 95 prot LCDR3 region of MS-Roche#7.9H7; protein/1; artificial sequence (SEQ ID NO: 95) LQIYNMPI
[0109] Further illustrative sequences are depicted in the appended sequence listing and are also shown in the appended tables, in particular tables 1, 8 and 10.
BRIEF DESCRIPTION OF THE DRAWINGS
[0110] The Figures show:
[0111] FIG. 1 Sequence summary of HuCAL®-Fab1 Library
[0112] The numbering is according to VBASE except the gap in VLλ position 9. In VBASE the gap is set at position 10 (Chothia et al., 1992). In the sequence summary all CDR3 residues which were kept constant are indicated. Corresponding sequences employed for the HuCAL-Fab1 library can be found in the appended sequence listing.
[0113] A: amino acid sequence
[0114] B: DNA sequence
[0115] FIG. 2 Fab display vector pMORPH®18_Fab
[0116] Vector map and DNA sequence including restriction sites
[0117] FIG. 3 Fab expression vector pMORPH®x9_Fab
[0118] Vector map and DNA sequence including restriction sites
[0119] FIG. 4 Sequences of the parental Fab fragments MS-Roche-3, MS-Roche-7 and MS-Roche 8
[0120] A: amino acid sequence
[0121] B: DNA sequence
[0122] FIG. 5: Indirect immunofluorescence of amyloid-plaques from a cryostat section of human temporal cortex. The plaques were labeled with MS-R #3.2 Fab (upper panels) and MS-R #7.4 Fab (lower panels) at 20 μg/ml (left panels) and 5 μg/ml (right panels) under stringent blocking conditions. Bound MS-R Fab was revealed by goat anti-human-Cy3.
[0123] FIG. 6: Indirect immunofluorescence of amyloid-plaques from a cryostat section of human temporal cortex. The plaques were labeled with MS-R #3.3 IgG1 (upper panels) and MS-R #7.12 IgG1(lower panels) at 0.05 μg/ml (left panels) and 0.01 μg/ml (right panels) under stringent blocking conditions. Bound MS-R IG1 antibody was revealed by goat anti-human (H+L)-Cy3.
[0124] FIG. 7: Indirect immunofluorescence of amyloid-plaques from a cryostat section of human temporal cortex using antibodies after final affinity maturation. The plaques were labeled with MS-R #7.9.H7 IgG1(MAB 31, top panel), MS-R #7.11.H1×7.2.L1 IgG1(MAB 11, middle panel) and MS-R #3.4.H7, bottom panel). Antibodies were used at 0.05 μg/ml (left panels) and 0.01 μg/ml (right panels) under stringent blocking conditions. Bound MS-R IgG1 antibody was revealed by goat anti-human (H+L)-Cy3.
[0125] Scale: 8.5 mm=150 μm.
[0126] FIG. 8: Polymerization Assay. Anti-Aβ antibodies prevent incorporation of biotinylated Aβ into preformed Aβ aggregates.
[0127] FIGS. 9A-D: De-polymerization Assay. Anti-Aβ antibodies induce release of biotinylated Aβ from aggregated Aβ.
[0128] FIG. 10: In vivo decoration of amyloid plaques in an APP/PS2 double transgenic mouse after intravenous injection of 1 mg MS-Roche IgG #7.9.H2 x 7.12.L2. After three days the mouse was perfused with phosphate-buffered saline and sacrificed. The presence of human IgG bound to amyloid plaques was revealed by confocal microscopy after labelling cryostat sections from the frontal cortex with a goat anti-human IgG-Cy3 conjugate (panel B). The same section was counterstained with an anti-Abeta mouse monoclonal antibody (BAP-2-Alexa488 conjugate, panel A) to visualize the position of amyloid plaques. Individual red (panel B) and green (panel A) channels, merged image (panel D) and colocalized (pancel C) signals are shown.
Scale: 1 cm=50 μm
[0129] FIGS. 11A-D: In vivo decoration of amyloid plaques in an APP/PS2 double transgenic mouse after intravenous injection of 1 mg MS-Roche IgG #7.9.H4 x 7.12.L2. Experimental conditions and staining procedure were identical to those described in the legend of FIG. 10.
[0130] Scale: 1.6 cm=50 μm
[0131] FIGS. 12A-D: In vivo decoration of amyloid plaques in an APP/PS2 double transgenic mouse after intravenous injection of 1 mg MS-Roche IgG #7.11.H1 x 7.2.L1 (MAB 11). Experimental conditions and staining procedure were identical to those described in the legend of FIG. 10.
[0132] Scale: 1.4 cm=70 μm
[0133] FIG. 13: In vivo decoration of amyloid plaques in an APP/PS2 double transgenic mouse after intravenous injection of 2 mg MS-Roche IgG #7.9.H7 (MAB 31) at day 0, 3, and 6. After nine days the mouse was perfused with phosphate-buffered saline and sacrificed. The presence of human IgG bound to amyloid plaques was revealed by confocal microscopy after labelling cryostat sections from the frontal cortex with a goat anti-human IgG-Cy3 conjugate (panel B). The same section was counterstained with an anti-Abeta mouse monoclonal antibody (BAP-2-Alexa488 conjugate, panel A) to visualize the position of amyloid plaques. Individual red (panel B) and green (panel A) channels, merged image (panel D) and colocalized (panel C) signals and are shown.
[0134] Scale: 1.6 cm=80 μm (panels A, B, C); 1.0 cm=50 μm (panel D)
[0135] FIGS. 14A-D: In vivo decoration of amyloid plaques in an APP/PS2 double transgenic mouse after intravenous injection of 2 mg MS-Roche IgG #7.11.H1 x 7.2.L1 (MAB 11) at day 0, 3 and 6. Experimental conditions and staining procedure were identical to those described in the legend of FIG. 13.
[0136] Scale: 1.6 cm=80 μm
[0137] FIG. 15: Binding analysis of anti-Aβ antibodies to cell surface APP. Antibody binding to human APP-transfected HEK293 cells and non-transfected control cells was analyzed by flow cytometry.
[0138] The examples illustrate the invention.
EXAMPLES
Example 1
Construction and Screening of a Human Combinatorial Antibody Library (HuCAL®-Fab 1)
Cloning of HuCAL®-Fab 1
[0139] HuCAL®-Fab 1 is a fully synthetic, modular human antibody library in the Fab antibody fragment format. HuCAL®-Fab 1 was assembled starting from an antibody library in the single-chain format (HuCAL®-scFv; Knappik, (2000), J. Mol. Biol. 296, 57-86).
Vλ Positions 1 and 2.
[0140] The original HuCAL® master genes were constructed with their authentic N-termini: VLλ1: QS (CAGAGC), VLλ2: QS (CAGAGC), and VLλ3: SY (AGCTAT). Sequences containing these amino acids are shown in WO 97/08320. During HuCAL® library construction, the first two amino acids were changed to DI to facilitate library cloning (EcoRI site). All HuCAL® libraries contain VLλ genes with the EcoRV site GATATC (DI) at the 5'-end. All HuCAL® kappa genes (master genes and all genes in the library) contain DI at the 5'-end (FIGS. 1A and B).
VH Position 1.
[0141] The original HuCAL® master genes were constructed with their authentic N-termini: VH1A, VH1B, VH2, VH4, and VH6 with Q(=CAG) as the first amino acid and VH3 and VH5 with E (=GAA) as the first amino acid. Sequences containing these amino acids are shown in WO 97/08320. During cloning of the HuCAL®-Fab1 library, amino acid at position 1 of VH was changed to Q (CAG) in all VH genes (FIGS. 1A and B).
Design of the CDR Libraries
Vκ1/Vκ3 Position 85.
[0142] Because of the cassette mutagenesis procedure used to introduce the CDR3 library (Knappik, (2000), loc. cit.), position 85 of Vκ1 and Vκ3 can be either T or V. Thus, during HuCAL®-scFv1 library construction, position 85 of Vκ1 and Vκ3 was varied as follows: Vκ1 original, 85T (codon ACC); Vκ1 library, 85T or 85V (TRIM codons ACT or GTT); Vκ3 original, 85V (codon GTG); Vκ3 library, 85T or 85V (TRIM codons ACT or GTT); the same applies to HuCAL®-Fab1.
CDR3 Design.
[0143] All CDR3 residues, which were kept constant, are indicated in FIGS. 1A and B.
CDR3 Length.
[0144] The designed CDR3 length distribution is as follows. Residues, which were varied are shown in brackets (x) in FIG. 1. V kappa CDR3, 8 amino acid residues (position 89 to 96) (occasionally 7-10 residues), with Q89, S90, and D92 fixed; and VH CDR3, 5 to 28 amino acid residues (position 95 to 102) (occasionally 4-28), with D101 fixed. HuCAL®-Fab 1 was cloned into a phagemid expression vector pMORPH° 18_Fab1 (FIG. 2). This vector comprises the Fd fragment with a phoA signal sequence fused at the C-terminus to a truncated gene III protein of filamentous phage, and further comprises the light chain VL-CL with an ompA signal sequence. Both chains are under the control of the lac operon. The constant domains Cλ Cκ and CH1 are synthetic genes fully compatible with the modular system of HuCAL® (Knappik, (2000), loc. cit.). The whole VH-chain (MunI/StyI-fragment) was replaced by a 1205 bp dummy fragment containing the β-lactamase transcription unit (bla), thereby facilitating subsequent steps for vector fragment preparation and allowing for selection of complete VH removal.
[0145] After VH-replacement, VLA, was removed by EcoRI/DraIII and VLκ by EcoRI/BsIWI and replaced with bacterial alkaline phosphatase (bap) gene fragment (1420 bp).
[0146] As the variability of the light chains is lower than that of the heavy chains, cloning was started with the light chain libraries. The VL.sub.λ, and VL.sub.κ light chain libraries diversified in L-CDR3, which were generated for the HuCAL®-scFv library (Knappik, (2000), loc. cit.) were also used for cloning of HuCAL®-Fab1. In case of λ they consisted of the λ1-, λ2- and λ3-HuCAL®-framework and had a total variability of 5.7×106. VL.sub.λ fragments were amplified by 15 PCR cycles (Pwo-polymerase) with primers 5'-GTGGTGGTTCCGATATC-3' (SEQ ID NO: 28) and 5'-AGCGTCACACTCGGTGCGGCTTTCGGCTGGCCAAGAACGGTTA-3' (SEQ ID NO: 29). PCR-products were digested with EcoRV/DraIII and gel-purified. In case of the VL.sub.λ-library, the bap-dummy was removed by EcoRV/DraIII from the library vector. 2 μg of gelpurified vector were ligated with a 3-fold molar excess of VL-chains for 16 h at 16° C., and the ligation mixtures were electroporated in 800 μl E. coli TOP10F cells (Invitrogen), yielding altogether 4.1×108 independent colonies. The transformants were amplified about 2000-fold in 2×YT/1% glucose/34 μg/ml chloramphenicol/100 μg/ml ampicillin, harvested and stored in 20% (w/v) glycerol at -80° C.
[0147] The κ libraries comprise the κ1-, κ2-, κ3- and κ4-HuCAL® master genes with a total variability of 5.7×106. VL.sub.κ-chains were obtained by restriction digest with EcoRV/BsiWI and gel-purified. In case of the VL.sub.κ-library, the bap-dummy was removed by EcoRV/BsIWI from the library vector. 2 μg of gel-purified vector were mixed with a 5-fold molar excess of VL.sub.κ-chains. Ligation and transformation into E. coli TOP10F cells (Invitrogen) was performed as described for VL.sub.λ-chains, yielding altogether 1.6×108 independent colonies. DNA of the two light chain libraries was prepared and the bla-dummy was removed by MunIIStyI, thereby generating the two vectors for insertion of the VH sub-libraries. The VH libraries of HuCAL®-scFv were used for the generation of HuCAL®-Fab1. The VH libraries of HuCAL®-scFv consist of the master genes VH1A/B-6 diversified with two VH-CDR3 trinucleotide library cassettes differing in CDR3 length separately, and each VH-library combined with the VL.sub.κ- and with the VL-library. For the generation of the HuCAL®-Fab1 DNA from these VH-libraries was prepared preserving the original variability. The DNA was digested with MunIIStyI and gel-purified. A 5-fold molar excess of the VH-chains was ligated with 3 μg of the VL.sub.λ-library vector and with 3 μg of the VL.sub.κ-library vector for 4 h at 22° C. The ligation mixtures were electroporated for each vector in 1200 μl E. coli TOP10F cells (Invitrogen), yielding altogether 2.1×1010 independent colonies. The transformants were amplified about 4000-fold in 2×YT/1% glucose/34 μg/ml chloramphenicol/10 μg/ml tetracycline, harvested and stored in 20% (w/v) glycerol at -80° C. As quality control the light chain and heavy chain of single clones was sequenced with 5''-CAGGAAACAGCTATGAC-3' (SEQ ID NO: 30) and 5'-TACCGTTGCTCTTCACCCC-3' (SEQ ID NO: 31), respectively.
Phagemid Rescue, Phage Amplification and Purification
[0148] HuCAL®-Fab 1 was amplified in 2×TY medium containing 34 μg/ml chloramphenicol, 10 μg/ml tetracycline and 1% glucose (2×TY-CG). After helper phage infection (VCSM13) at 37° C. at an OD600 of about 0.5, centrifugation and resuspension in 2×TY/34 μg/ml chloramphenicol/50 μg/ml kanamycin cells were grown overnight at 30° C. Phage were PEG-precipitated from the supernatant (Ausubel, (1998), Current protocols in molecular biology. John Wiley & Sons, Inc., New York, USA), resuspended in PBS/20% glycerol and stored at -80° C. Phage amplification between two panning rounds was conducted as follows: mid-log phase TG1-cells were infected with eluted phage and plated onto LB-agar supplemented with 1% of glucose and 34 μg/ml of chloramphenicol. After overnight incubation at 30° C. colonies were scraped off, adjusted to an OD600 of 0.5 and helper phage added as described above.
Example 2
Solid Phase Panning
[0149] Wells of MaxiSorp® microtiterplates F96 (Nunc) were coated with 100 μl 2.5 μM human Aβ (1-40) peptide (Bachem) dissolved in TBS containing NaN3 (0.05% v/v) and the sealed plate was incubated for 3 days at 37° C. where the peptide is prone to aggregate on the plate. After blocking with 5% non-fat dried milk in TBS, 1-5×1012 HuCAC-Fab phage purified as above were added for 1 h at 20° C. After several washing steps, bound phages were eluted by pH-elution with 500 mM NaCl, 100 mM glycin pH 2.2 and subsequent neutralisation with 1M TRIS-CI pH 7. Three rounds of panning were performed with phage amplification conducted between each round as described above, the washing stringency was increased from round to round.
Example 3
Subcloning of Selected Fab Fragments for Expression
[0150] The Fab-encoding inserts of the selected HuCAL®-Fab fragments were subcloned into the expression vector pMORPH° x7_FS to facilitate rapid expression of soluble Fab. The DNA preparation of the selected HuCAC-Fab clones was digested with XbaI/EcoRI, thus cutting out the Fab encoding insert (ompA-VL and phoA-Fd). Subcloning of the purified inserts into the XbaI/EcoRI cut vector pMORPH° x7, previously carrying a scFv insert, leads to a Fab expression vector designated pMORPH° x9_Fab1 (FIG. 3). Fabs expressed in this vector carry two C-terminal tags (FLAG and Strep) for detection and purification.
Example 4
Identification of Aβ-Binding Fab Fragments by ELISA
[0151] Wells of Maxisorp® microtiterplates F384 (Nunc) were coated with 20 μl 2.5 μM human Aβ (1-40) peptide (Bachem) dissolved in TBS containing NaN3 (0.05% v/v) and the sealed plate was incubated for 3 days at 37° C., where the peptide is prone to aggregate on the plate. Expression of individual Fab was induced with 1 mM IPTG for 16 h at 22° C. Soluble Fab was extracted from E. coli by BEL lysis (boric acid, NaCl, EDTA and lysozyme containing buffer pH 8) and used in an ELISA. The Fab fragment was detected with an alkaline phosphatase-conjugated goat anti-Fab antibody (Dianova/Jackson Immuno Research). After excitation at 340 nm the emission at 535 nm was read out after addition of AttoPhos fluorescence substrate (Roche Diagnostics).
Example 5
Optimization of Antibody Fragments
[0152] In order to optimize the binding affinity of the selected Aβ binding antibody fragments, some of the Fab fragments, MS-Roche-3 (MSR-3), MS-Roche-7 (MSR-7) and MS-Roche-8 (MSR-8) (FIG. 4), were used to construct a library of Fab antibody fragments by replacing the parental VL κ3 chain by the pool of all kappa chains κ1-3 diversified in CDR3 from the HuCAL® library (Knappik et al., 2000).
[0153] The Fab fragments MS-Roche-3, 7 and 8 were cloned via XbaI/EcoRI from pMORPH® x9_FS into pMORPH° 18, a phagemid-based vector for phage display of Fab fragments, to generate pMORPH° 18_Fab1 (FIG. 2). A kappa chain pool was cloned into pMORPH° 18_Fab1 via XbaI/SphI restriction sites.
[0154] The resulting Fab optimization library was screened by panning against aggregated human Aβ (1-40) peptide coated to a solid support as described in example 2. Optimized clones were identified by koff-ranking in a Biacore assay as described in Example 8. The optimized clones MS-Roche-3.2, 3.3, 3.4, 3.6, 7.2, 7.3, 7.4, 7.9, 7.11, 7.12, 8.1, 8.2, were further characterized and showed improved affinity and biological activity compared to the starting fragment MS-Roche-3, MS-Roche-7 and MS-Roche-8 (FIG. 4). The CDRs listed refer to the HuCAL® consensus-based antibody gene VH3 kappa3. The Fab fragment MS-Roche-7.12 was obtained by cloning the HCDR3 of parental clone MS-R 7 into a HuCAL®-Fab library, carrying diversity in all 6 CDR regions using a design procedure identical with that for CDR3 cassettes described in Knappik et al., 2000. The library cassettes were designed strongly biased for the known natural distribution of amino acids and following the concept of canonical CDR conformations established by Allazikani (Allazikani et al., 1997). However in contrast to the HuCAL® master genes, the clone MS-Roche 7.12 contains amino acid S at position 49 of the VL chain (see appended table 1).
[0155] The optimized Fabs after the first affinity maturation round showed improved characteristics over the starting MS-Roche-3, MS-Roche-7 and MS-Roche-8 clones (FIG. 4). The binding affinities of the maturated Fabs to Aβ1-40 and Aβ1-42 were significantly increased yielding KD values in the range of 22-240 nM in comparison to 850-1714 nM of the parental clones (Table 3). Immunohistochemistry analysis of amyloid plaques in human AD brain sections also showed a significantly increased staining profile of the maturated clones, i.e. better signal to background ratios were obtained and positive plaque staining was detected at relatively low concentrations of the maturated Fabs (FIG. 5).
[0156] For further optimization, the VH CDR2 regions and the VL CDR1 regions of a set of antibody fragments derived from L-CDR3 optimized MS-Roche-3, -7 and -8 (table 1; FIG. 4) were optimized by cassette mutagenesis using trinucleotide-directed mutagenesis (Virnekas et al., 1994). Therefore, a trinucleotide-based HCDR2 cassette and a trinucleotide-based LCDR1 cassette were constructed using a design procedure identical with that for CDR3 cassettes described in Knappik et al., 2000. The library cassettes were designed strongly biased for the known natural distribution of amino acids and following the concept of canonical CDR conformations established by Allazikani (Allazikani et al., 1997). The protocol used for the optimization of the initial selected antibody fragments would mimic the process of affinity maturation by somatic hypermutation observed during the natural immune response.
[0157] The resulting libraries were screened separately as described above leading to optimized clones either in the H-CDR2 or in the L-CDR1 region. All clones were identified as above by an improved koff towards Aβ1-40-fibers after a koff-ranking in the Biacore and showed improved affinity either to Aβ1-40 or Aβ-42 or both when compared to the corresponding parent clone (Table 3). Table 1 contains the sequence characteristics of the parental as well as sequences of the optimized clones. The CDRs listed refer to the HuCAL® consensus-based antibody gene VH3 kappa3.
[0158] For example, the affinity of the MS-Roche-7 parental Fab towards Ab1-40 was improved over 35-fold from 1100 nM to 31 nM after L-CDR3 optimization (MS-Roche-7.9) and further improved to 5 nM after H-CDR2 optimization (MS-Roche-7.9H2) as illustrated in Table 3. The H-CDR2 and L-CDR1 optimization procedure not only increased the affinity but also resulted for some of the clones in a significantly improved staining of amyloid plaques in AD brain section, as particularly seen with MS-Roche 7.9H2 and 7.9H3.
TABLE-US-00002 TABLE 1 pos. pos. pos. Binder name L-CDR1 49 L-CDR2 85 L-CDR3 H-CDR1 47 H-CDR2 H-CDR3 MS-Roche #3 RASQSVSSSYLA Y GASSRAT V QQVYNPPV GFTFSSYAMS W AISGSGGSTYYADS LTHYARYYRYFDV VKG MS-Roche #3.1 RASQSVSSSYLA Y GASSRAT T QQVYSVPP GFTFSSYAMS W AISGSGGSTYYADS LTHYARYYRYFDV VKG MS-Roche #3.2 RASQSVSSSYLA Y GASSRAT V QQIYSYPP GFTFSSYAMS W AISGSGGSTYYADS LTHYARYYRYFDV VKG MS-Roche #3.3 RASQSVSSSYLA Y GASSRAT V HQMSSYPP GFTFSSYAMS W AISGSGGSTYYADS LTHYARYYRYFDV VKG MS-Roche #3.4 RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISGSGGSTYYADS LTHYARYYRYFDV VKG MS-Roche #3.5 RASQSVSSSYLA Y GASSRAT T QQIYDYPP GFTFSSYAMS W AISGSGGSTYYADS LTHYARYYRYFDV VKG MS-Roche #3.6 RASQSVSSSYLA Y GASSRAT V QQTYNYPP GFTFSSYAMS W AISGSGGSTYYADS LTHYARYYRYFDV VKG MS-Roche #3.2.H1 RASQSVSSSYLA Y GASSRAT V QQIYSYPP GFTFSSYAMS W AISEHGLNIYYADS LTHYARYYRYFDV VKG MS-Roche #3.2.H2 RASQSVSSSYLA Y GASSRAT V QQIYSYPP GFTFSSYAMS W AISQRGQFTYYADS LTHYARYYRYFDV VKG MS-Roche #3.3.H1 RASQSVSSSYLA Y GASSRAT V HQMSSYPP GFTFSSYAMS W VISEKSRFIYYADS LTHYARYYRYFDV VKG MS-Roche #3.3.H2 RASQSVSSSYLA Y GASSRAT V HQMSSYPP GFTFSSYAMS W VISQESQYKYYADS LTHYARYYRYFDV VKG MS-Roche #3.3.H3 RASQSVSSSYLA Y GASSRAT V HQMSSYPP GFTFSSYAMS W AISQNGFHIYYADS LTHYARYYRYFDV VKG MS-Roche #3.4.H1 RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISETSIRKYYADS LTHYARYYRYFDV VKG MS-Roche #3.4.H2 RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W VIDMVGHTYYADSV LTHYARYYRYFDV KG MS-Roche #3.4.H3 RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W VISQTGRKIYYADS LTHYARYYRYFDV VKG MS-Roche #3.4.H4 RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISETGMHIYYADS LTHYARYYRYFDV VKG MS-Roche #3.4.H5 RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W VISQVGAHIYYADS LTHYARYYRYFDV VKG MS-Roche #3.4.H6 RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISESGWSTYYADS LTHYARYYRYFDV VKG MS-Roche #3.4.H7 RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W VISETGKNIYYADS LTHYARYYRYFDV VKG MS-Roche #3.4.H8 RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISEHGRFKYYADS LTHYARYYRYFDV VKG MS-Roche #3.4.H9 RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISESSKNKYYADS LTHYARYYRYFDV VKG MS-Roche #3.4.H10 RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISESGRGKYYADS LTHYARYYRYFDV VKG MS-Roche #3.4.H11 RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISEFGKNIYYADS LTHYARYYRYFDV VKG MS-Roche #3.4.H12 RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W VISQTGQNIYYADS LTHYARYYRYFDV VKG MS-Roche #3.4.H13 RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISEQGRNIYYADS LTHYARYYRYFDV VKG MS-Roche #3.4.H14 RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISESGQYKYYADS LTHYARYYRYFDV VKG MS-Roche #3.4.H16 RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISESGVNIYYADS LTHYARYYRYFDV VKG MS-Roche #3.4.H17 RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISEFGQFIYYADS LTHYARYYRYFDV VKG MS-Roche #3.4.H18 RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISQQSNFIYYADS LTHYARYYRYFDV VKG MS-Roche #3.4.L7 RASQRLGRLYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISGSGGSTYYADS LTHYARYYRYFDV VKG MS-Roche #3.4.L8 RASQWITKSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISGSGGSTYYADS LTHYARYYRYFDV VKG MS-Roche #3.4.L9 RASRRIHVYYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISGSGGSTYYADS LTHYARYYRYFDV VKG MS-Roche #3.4.L11 RASQLVGRAYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISGSGGSTYYADS LTHYARYYRYFDV VKG MS-Roche #3.6.H1 RASQSVSSSYLA Y GASSRAT V QQTYNYPP GFTFSSYAMS W VISESGQYKYYADS LTHYARYYRYFDV VKG MS-Roche #3.6.H2 RASQSVSSSYLA Y GASSRAT V QQTYNYPP GFTFSSYAMS W VISERGINTYYADS LTHYARYYRYFDV VKG MS-Roche #3.6.H3 RASQSVSSSYLA Y GASSRAT V QQTYNYPP GFTFSSYAMS W VISETGKFIYYADS LTHYARYYRYFDV VKG MS-Roche #3.6.H4 RASQSVSSSYLA Y GASSRAT V QQTYNYPP GFTFSSYAMS W AISERGRHIYYADS LTHYARYYRYFDV VKG MS-Roche #3.6.H5 RASQSVSSSYLA Y GASSRAT V QQTYNYPP GFTFSSYAMS W AISESGKTKYYADS LTHYARYYRYFDV VKG MS-Roche #3.6.H6 RASQSVSSSYLA Y GASSRAT V QQTYNYPP GFTFSSYAMS W AISEHGTNIYYADS LTHYARYYRYFDV VKG MS-Roche #3.6.H8 RASQSVSSSYLA Y GASSRAT V QQTYNYPP GFTFSSYAMS W AISEYSKFKYYADS LTHYARYYRYFDV VKG MS-Roche #3.6.L1 RASQFIQRFYLA Y GASSRAT V QQTYNYPP GFTFSSYAMS W AISGSGGSTYYADS LTHYARYYRYFDV VKG MS-Roche #3.6.L2 RASQFLSRYYLA Y GASSRAT V QQTYNYPP GFTFSSYAMS W AISGSGGSTYYADS LTHYARYYRYFDV VKG MS-Roche #7 RASQSVSSSYLA Y GASSRAT T FQLYSDPF GFTFSSYAMS W AISGSGGSTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.1 RASQSVSSSYLA Y GASSRAT V HQLYSSPY GFTFSSYAMS W AISGSGGSTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.2 RASQSVSSSYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS W AISGSGGSTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.3 RASQSVSSSYLA Y GASSRAT V HQVYSHPF GFTFSSYAMS W AISGSGGSTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.4 RASQSVSSSYLA Y GASSRAT V QQIYNFPH GFTFSSYAMS W AISGSGGSTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.5 RASQSVSSSYLA Y GASSRAT T HQVYSSPF GFTFSSYAMS W AISGSGGSTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.6 RASQSVSSSYLA Y GASSRAT V HQLYSPPY GFTFSSYAMS W AISGSGGSTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.7 RASQSVSSSYLA Y GASSRAT T HQVYSAPF GFTFSSYAMS W AISGSGGSTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.8 RASQSVSSSYLA Y GASSRAT V HQVYSFPI GFTFSSYAMS W AISGSGGSTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.9 RASQSVSSSYLA Y GASSRAT T LQIYNMPI GFTFSSYAMS W AISGSGGSTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.10 RASQSVSSSYLA Y GASSRAT T QQVYNPPH GFTFSSYAMS W AISGSGGSTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.11 RASQSVSSSYLA Y GASSRAT T QQVYSPPH GFTFSSYAMS W AISGSGGSTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.12 RASQYVSSPYLA S GSSNRAT V LQLYNIPN GFTFSSYGMS W NISGSGSSTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.13 RASQSVSSSYLA Y GASSRAT V HQVYSPPF GFTFSSYAMS W AISGSGGSTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.2.H1 RASQSVSSSYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS W AINANGLKKYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.2.H2 RASQSVSSSYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS W AINGTGMKKYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.2.H3 RASQSVSSSYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS W AINANGYKTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.2.H4 RASQSVSSSYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS W AINSKGSRIYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.2.H5 RASQSVSSSYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS W AINATGRSKYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.2.H6 RASQSVSSSYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS W
AINARGNRTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.2.H7 RASQSVSSSYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS W AINSRGSDTHYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.2.H8 RASQSVSSSYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS W AINASGHKTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.2.L1 RASQYVDRTYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS W AISGSGGSTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.2.L2 RASQYISFRYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS W AISGSGGSTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.2.L4 RASQFIRRSYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS W AISGSGGSTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.3.H1 RASQSVSSSYLA Y GASSRAT V HQVYSHPF GFTFSSYAMS W AISAISNKTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.3.L1 RASQYLHYGYLA Y GASSRAT V HQVYSHPF GFTFSSYAMS W AISGSGGSTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.4.H1 RASQSVSSSYLA Y GASSRAT V QQIYNFPH GFTFSSYAMS W AINATGYRTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.4.H2 RASQSVSSSYLA Y GASSRAT V QQIYNFPH GFTFSSYAMS W AINYNGARIYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.9.H1 RASQSVSSSYLA Y GASSRAT T LQIYNMPI GFTFSSYAMS W AINANGQRKFYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.9.H2 RASQSVSSSYLA Y GASSRAT T LQIYNMPI GFTFSSYAMS W AINADGNRKYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.9.H3 RASQSVSSSYLA Y GASSRAT T LQIYNMPI GFTFSSYAMS W AINYQGNRKYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.9.H4 RASQSVSSSYLA Y GASSRAT T LQIYNMPI GFTFSSYAMS W AINAVGMKKFYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.9.H5 RASQSVSSSYLA Y GASSRAT T LQIYNMPI GFTFSSYAMS W AINHAGNKKYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.9.L1 RASQRLSPRYLA Y GASSRAT T LQIYNMPI GFTFSSYAMS W AISGSGGSTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.9.L2 RASQYLHKRYLA Y GASSRAT T LQIYNMPI GFTFSSYAMS W AISGSGGSTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.9.H6 RASQSVSSSYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS W AINARGNRTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.9.H7 RASQSVSSSYLA Y GASSRAT T LQIYNMPI GFTFSSYAMS W AINASGTRTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.9.H8 RASQSVSSSYLA Y GASSRAT T LQIYNMPI GFTFSSYAMS W AINASGSKIYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.9.H9 RASQSVSSSYLA Y GASSRAT T LQIYNMPI GFTFSSYAMS W AINGKGNKKYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.11.H1 RASQSVSSSYLA Y GASSRAT T QQVYSPPH GFTFSSYAMS W GINAAGFRTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.11.H2 RASQSVSSSYLA Y GASSRAT T QQVYSPPH GFTFSSYAMS W AINANGYKKYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.11.H3 RASQSVSSSYLA Y GASSRAT T QQVYSPPH GFTFSSYAMS W GINANGNRTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.11.H4 RASQSVSSSYLA Y GASSRAT T QQVYSPPH GFTFSSYAMS W AINANGYKTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.11.H5 RASQSVSSSYLA Y GASSRAT T QQVYSPPH GFTFSSYAMS W AINAHGQRTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.11.L1 RASQRILRIYLA Y GASSRAT T QQVYSPPH GFTFSSYAMS W AISGSGGSTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.12.H1 RASQYVFRRYLA S GSSNRAT V LQLYNIPN GFTFSSYGMS W NINGNGNRKYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.12.L1 RASQYVFRRYLA S GSSNRAT V LQLYNIPN GFTFSSYGMS W NISGSGSSTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.12.L2 RASQRFFYKYLA S GSSNRAT V LQLYNIPN GFTFSSYGMS W NISGSGSSTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.12.L3 RASQFVRRGFLA S GSSNRAT V LQLYNIPN GFTFSSYGMS W NISGSGSSTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.12.L4 RASQRLKRSYLA S GSSNRAT V LQLYNIPN GFTFSSYGMS W NISGSGSSTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.12.L5 RASQRLKRSYLA S GSSNRAT V LQLYNIPN GFTFSSYGMS W NISGSGSSTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.12.L6 RASQYLWYRYLA S GSSNRAT V LQLYNIPN GFTFSSYGMS W NISGSGSSTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #7.12.L7 RASQWIRKTYLA S GSSNRAT V LQLYNIPN GFTFSSYGMS W NISGSGSSTYYADS GKGNTHKPYGYVRYF VKG DV MS-Roche #8 RASQSVSSSYLA Y GASSRAT T QQLSSFPP GFTFSSYAMS W AISGSGGSTYYADS LLSRGYNGYYHKFDV VKG DV MS-Roche #8.1 RASQSVSSSYLA Y GASSRAT T QQLSNYPP GFTFSSYAMS W AISGSGGSTYYADS LLSRGYNGYYHKFDV VKG DV MS-Roche #8.2 RASQSVSSSYLA Y GASSRAT T QQLSSYPP GFTFSSYAMS W AISGSGGSTYYADS LLSRGYNGYYHKFDV VKG DV MS-Roche #8.1.H1 RASQSVSSSYLA Y GASSRAT T QQLSNYPP GFTFSSYAMS W AISRSGSNIYYADS LLSRGYNGYYHKFDV VKG DV MS-Roche #8.2.H1 RASQSVSSSYLA Y GASSRAT T QQLSSYPP GFTFSSYAMS W AISITGRRKYYADS LLSRGYNGYYHKFDV VKG DV MS-Roche #8.2.H2 RASQSVSSSYLA Y GASSRAT T QQLSSYPP GFTFSSYAMS W AISRTGSKTYYADS LLSRGYNGYYHKFDV VKG DV MS-Roche #8.2.H4 RASQSVSSSYLA Y GASSRAT T QQLSSYPP GFTFSSYAMS W ATSVKGKTYYADSV LLSRGYNGYYHKFDV KG DV MS-Roche #8.2.L1 RASQRVSGRYLA Y GASSRAT T QQLSSYPP GFTFSSYAMS W AISGSGGSTYYADS LLSRGYNGYYHKFDV VKG DV Sequences belonging to VH3 and Vκ3 HuCAL consensus sequences see FIG. 1 A
Example 6
Construction of HuCAL® Immunoglobulin Expression Vectors
Heavy Chain Cloning.
[0159] The multiple cloning site of pcDNA3.1+ (invitrogen) was removed (NheI/ApaI), and a stuffer compatible with the restriction sites used for HuCAL® design was inserted for the ligation of the leader sequences (NheI/EcoRI), VH-domains (MunI/), and the immunoglobulin constant regions (BlpI/ApaI). The leader sequence (EMBL 83133) was equipped with a Kozak sequence (Kozak, 1987). The constant regions of human IgG (PIR A02146), IgG4 (EMBL K01316), and serum IgA1 (EMBL J00220) were dissected into overlapping oligonucleotides with length of about 70 bases. Silent mutations were introduced to remove restriction sites non-compatible with the HuCAL® design. The oligonucleotides were spliced by overlap extension-PCR.
[0160] During sub-cloning from Fab into IgG, the VH DNA sequence of the Fab is cut out via Mfe I/Blp I and ligated into the IgG vector opened via EcoR I/Blp I. EcoR I (g/aattc) and Mfe I (c/aattg) share compatible cohesive ends (aatt) and the DNA sequence of the original Mfe I site in the Fab changes from: c/aattg to: g/aattg after ligation into the IgG expression vector, thereby destroying both Mfe I and EcoR I site, and thus also leading to an amino acid change from Q (codon: caa) to E (codon: gaa). The VH DNA sequence of the IgG of antibody molecule 7.9H7 after subcloning is shown in SEQ ID No.: 424, and the corresponding amino acid sequence is shown in SEQ ID No: 425.
Light Chain Cloning.
[0161] The multiple cloning site of pcDNA3.1/Zeo+ (Invitrogen) was replaced by two different stuffers. The K-stuffer provided restriction sites for insertion of a κ-leader (NheI/EcoRV), HuCAL®-scFv Vλ-domains (EcoRV/BsiWI), and the κ-chain constant region (BsiWI/ApaI). The corresponding restriction sites in the λ-stuffer were NheI/EcoRV (λ-leader), EcoRV/HpaI (Vλ-domains), and HpaI/ApaI (λ-chain constant region). The κ-leader (EMBL Z00022) as well as the λ-leader (EMBL J00241) were both equipped with Kozak sequences. The constant regions of the human κ-(EMBL L00241) and λ-chain (EMBL M18645) were assembled by overlap extension-PCR as described above.
Generation of IgG-Expressing CHO-Cells.
[0162] CHO-K1 cells were co-transfected with an equimolar mixture of IgG heavy and light chain expression vectors. Double-resistant transfectants were selected with 600 μg/ml G418 and 300 μg/ml Zeocin (Invitrogen) followed by limiting dilution. The supernatant of single clones was assessed for IgG expression by capture-ELISA. Positive clones were expanded in RPMI-1640 medium supplemented with 10% ultra-low IgG-FCS (Life Technologies). After adjusting the pH of the supernatant to 8.0 and sterile filtration, the solution was subjected to standard protein A column chromatography (Poros 20 A, PE Biosystems).
Example 7
Pepspot Analysis with Decapeptides
[0163] The following aminoacid sequence encompassing Aβ (1-42) was divided into 43 overlapping decapeptides with a frameshift of 1 aminoacid.
[0164] ISEVKM1DAEF RHDSGYEVHH QKLVFFAEDV GSNKGAIIGL MVGGVVI42ATV IV (SEQ ID NO: 414). Accordingly, DAEF RHDSGYEVHH QKLVFFAEDV GSNKGAIIGL MVGGVVIA (SEQ ID NO: 27) as enclosed represents amino acids 1 to 42 of Aβ4/β-A4 peptide. The 43 decapeptides were synthesized with N-terminal acetylation and C-terminal covalent attachment to a cellulose sheet ("pepspot") by a commercial supplier (Jerini BioTools, Berlin). The cellulose sheet is incubated for 2 hours on a rocking platform with monoclonal antibody (2 μg/ml) in blocking buffer (50 mM Tris HCl, 140 mM NaCl, 5 mM NaEDTA, 0.05% NP40 (Fluka), 0.25% gelatine (Sigma), 1% bovine serum albumine fraction V (Sigma), pH 7.4). The sheet is washed 3 times 3 minutes on a rocking platform with TBS (10 mM Tris.HCl, 150 mM NaCl, pH 7.5). It is then wetted with cathode buffer (25 mM Tris base, 40 mM 6-Aminohexane acid, 0.01% SDS, 20% methanol) and transferred to a semi-dry blotting stack with the peptide side facing a PVDF membrane (Biorad) of equal size. The semi-dry blotting stack consists out of freshly wetted filter papers (Whatman No. 3) slightly larger than the peptide sheet:
3 papers wetted with Cathode buffer the peptide sheet a sheet of PVDF membrane wetted with methanol 3 papers wetted with Anode buffer 1 (30 mM Tris base, 20% methanol) 3 papers wetted with Anode buffer 2 (0.3 mM Tris base, 20% methanol)
[0165] The transfer is conducted at a current density between Cathode and Anode of 0.8 mA/cm2 for 40 minutes which is sufficient to elute most of the antibody from the cellulose sheet and deposit it on the PVDF membrane. The PVDF membrane is then exchanged for a 2nd PVDF membrane and transferred for another 40 minutes to ensure complete elution from the cellulose sheet.
[0166] The PVDF membrane is immersed in blocking buffer for 10 minutes. Then HRP-labeled anti-human Ig H+L (Pierce) is added at 1:1000 dilution and the membrane is incubated on a rocking platform for 1 hour. It is washed 3×10 minutes with TBST (TBS with 0.005% Tween20). Color is developed by immersing the membrane into a solution made of 3 mg 4-chloronaphthol dissolved in 9 ml methanol with 41 ml PBS (20 mM Na-phosphate, 150 mM NaCl, pH 7.2) an 10 μl 30% hydrogen peroxide (Merck). After the development of blue-black spots the membrane is washed extensively with water and dried.
[0167] The assignment of antibody-reactive pepspots is made by visual inspection through a transparent spot matrix. The epitopes of the antibody in question is defined as the minimal aminoacid sequence in reactive peptides. For comparison mouse monoclonal antibodies (BAP-2, BAP-1, BAP-17 BAP-21, BAP-24, and 4G8) are analyzed in the same way, except using HRP-labeled anti-mouse Ig instead of anti-human 1 g.
[0168] It is of note that affinity maturation and conversion of the monovalent Fab fragments into full-length IgG1 antibodies results usually in some broadening of the epitope recognition sequence as indicated by pepspot and ELISA analyses. This may be related to the recruitment of more contact points in the antibody-antigen interaction area as a consequence of the affinity maturation or to a stronger binding to the minimal epitope such that also weak interactions with adjacent amino acid can be detected. The latter may be the case when Aβ-derived peptides are probed with full-length IgG antibodies. As illustrated in Table 2 for the pepspot analysis, the recognition sequences of the N-terminal and middle epitopes are extended by up to three amino acids when parent Fabs and corresponding fully maturated IgG antibodies are compared. However, it has to be kept in mind that the decapeptides are modified for covalent attachment at the C-terminal amino acid and this amino acid may therefore not easily be accessible to the full-length antibody due to steric hindrance. If this is the case the last C-terminal amino acid does not significantly contribute to the epitope recognition sequence and a potential reduction of the minimal recognition sequence by one amino acid at the C-terminal end has to be considered in the pepspot analysis as used in the present invention.
TABLE-US-00003 TABLE 2 Pepspot analysis of binding Fabs and full-length IgG antibodies to decapeptides on a cellulose sheet. The numbers refer to the essential amino acids from the Aβ1-40 sequence which have to be present in the decapeptide for optimal binding of antibody. A weak peptide reactivity, and hence a weak contribution to the epitope, is indicated by brackets. antibody position position MSR-3 Fab 3-4 18-23 MSR-7 Fab 3-5 19-24 MSR-8 Fab 4-5 18-21 MSR-9 Fab (1)3-9 18-24 MSR-10 Fab (4-10) 19-20 MSR-11 Fab 3-7 (18-20) MSR-26 Fab 3-5 (16)-19-23 MSR-27 Fab (3)6-9 13-18(20) MSR-29 Fab 14-16(20) MSR-37 Fab (4-6) (19-24) MSR-41 Fab 3-7 (17-21) MSR-42 Fab (4-9) (18-24) MSR 3.4.H7 IgG1 1-3 19-26 MSR 7.9.H2 IgG1 1-4 19-24 MSR 7.9.H7 IgG1 4-6 19-26 MSR 7.2.H2 × 7.2.L1 IgG1 (1-4) 5-9 18-26 MSR 7.11.H1 × 7.2.L1 IgG1 4-6 19-26 BAP-2 4-6 4G8 19-20(23) BAP-21 32-34 BAP-24 38-40 BAP-1 4-6 BAP-17 38-40
Example 8
Determination of KD Values for MS-R Fab and MS-R IgG1 Antibody Binding to Aβ1-40 and A(31-42 Fibers In Vitro by Surface Plasmon Resonance (SPR)
[0169] Binding of anti-Aβ antibodies (Fabs and IgG1) to fibrillar Aβ was measured online by surface plasmon resonance (SPR), and the affinities of the molecular interactions were determined as described by Johnson, Anal. Biochem. 1991, 198, 268-277, and Richalet-Secordel, Anal. Biochem. 1997, 249, 165-173. Biacore2000 and Biacore3000 instruments were used for these measurements. Aβ1-40 and Aβ1-42 fibers were generated in vitro by incubation of synthetic peptides at a concentration of 200 μg/ml in 10 mM Na-acetat buffer (pH 4.0) for three days at 37° C. Electron microscopic analysis confirmed a fibrillar structure for both peptides, Aβ1-40 showing predominantly shorter (<1 micron) and Aβ1-42 predominantly longer (>1 micron) fibers. These fibers are assumed to represent aggregated Aβ peptides in human AD brain more closely than ill-defined mixtures of amorphous aggregates and unstructured precipitates. The fibers were diluted 1:10 and directly coupled to a "Pioneer Sensor Chip F1" as described in the Instruction Manual of the manufacturer (BIAapplication Handbook, version AB, Biacore AB, Uppsala, 1998). In initial experiments it was found that selected MS-Roche Fabs differed substantially in their reaction kinetics and therefore the mode of data analysis had to be chosen accordingly. For binders with slow kinetics KD values were calculated by curve fitting of the time-dependent sensor responses, i.e. from the ratio of koff/kon. Binders with fast kinetics were analyzed by fitting the concentration-dependent sensor responses at equilibrium (adsorption-isotherms). KD values were calculated from the Biacore sensograms based on the total Fab concentration as determined by a protein assay. For the clones derived from the 1st and 2nd affinity maturation cycle the content of active Fab in each preparation was determined in the Biacore according to a method described by Christensen, Analytical Biochemistry (1997) 249, 153-164. Briefly, time-dependent protein binding to Aβ1-40 fibers immobilized on the Biacore chip was measured during the association phase under mass-limited conditions at different flow rates of the analyte solution. The conditions of mass limitation were realized by immobilizing high amounts of Aβ fibers (2300 response units) on the chip surface of a measuring channel and by working at relatively low analyte concentrations, i.e. 160 nM (based on the total Fab protein concentration).
[0170] A summary of the KD values of selected MS-Roche clones identified in the primary screen of the HuCAL library and their corresponding maturated derivatives after the 1st and 2nd affinity maturation cycle is shown in Table 3. In the 1st affinity maturation cycle the heavy chain CDR3 (VH-CDR3) was kept constant and optimization was focussed on diversification of the light chain CDR3 (VL-CDR3). In the 2nd affinity cycle diversification of VL-CDR1 and VH-CDR2 was performed. Some of the binders from the 1st maturation cycle were converted to full-length human IgG1 antibodies according to the technology developed by MorphoSys as described in Example 6 and KD values determined in the Biacore as described above. The KD values for full-length IgG1 binding to Aβ1-40 and Aβ1-42 fibers are shown in Table 4.
[0171] Matured derivatives from both the L-CDR1 as well as H-CDR2 library after the 2nd maturation cycle were identified and allowed combination of light and heavy chains. The cross-cloning strategy is described in Example 13. Either whole light chains, LCDR1 or L-CDR1+2 were exchanged. KD values of selected cross-cloned Fabs are shown in Table 8. Some of the Fabs from the 1st and 2nd maturation cycles and from the cross-cloned binders were converted to full-length human IgG1 antibodies according to the technology developed by MorphoSys as described in Example 6. KD values of IgG binding to Aβ1-40 and Aβ1-42 fibers were determined in the Biacore. Briefly, a kinetic model for the stepwise formation of a bivalent complex was used, and KD values were calculated by Scatchard type analysis of equilibrium binding. Due to the very slow association process at low antibody concentration (several hours to reach equilibrium) equilibrium binding data were obtained by extrapolation of the association curves to long time intervals. The on- and off rates for the formation of the monovalent and bivalent complex were determined via the curve fit procedure and used for the extrapolation. Based on these Req values a Scatchard analysis was performed and KD values for the formation of the monovalent and the bivalent complex were determined. The data are summarized in Table 5. From the curvilinear Scatchard plot a higher (bivalent) and lower (monovalent) affinity interaction was derived for the MS-R IgGs derived from the 2nd affinity maturation cycle and cross-clones. These two affinities represent the lower and upper KD values of the range indicated in Table 5.
TABLE-US-00004 TABLE 3 KD Aβ1-40 KD Aβ1-42 KD Aβ1-40 KD Aβ1-42 KD Aβ1-40 KD Aβ1-42 Secreted clones from MS-R # nM nM MS-R # nM nM MS-R # nM nM primary screen 3 930 1300 7 1100 1714 8 850 1000 1st affinity maturation 3.2 52 240 7.2 22 58 8.1 24 42 3.3 38 104 7.3 23 88 8.2 24 64 3.4 32 103 7.4 28 103 3.6 40 68 7.9 31 93 7.11 22 74 7.12 28 60 2nd affinity maturation 3.2H1 4.4 3.3 7.2H1 9.3 10.2 8.1H1 13.6 9.2 3.2H2 5.2 1.1 7.2H2 8.2 8.2 8.2H1 1.6a 2.1a 3.3H1 17.1 19.4 7.2H3 45.4 5.3 8.2H3 n.d. 3.1 3.3H2 10.6 22.8 7.2H4 5.9 5.0 8.2H4 12.1 11.9 3.3H3 1.4 3.3 7.2H5 8.0 10.1 8.2L1 4.8 3.7 3.4H1 13.5 14.0 7.2H6 1.0 n.d. 3.4H3 6.7 8.4 7.2H7 15.5 8.1 3.4H4 33.0 43.0 7.2H8 1.5 2.1 3.4H5 26.5 36.0 7.2L1 13.3 12.7 3.4H6 49.0 60.0 7.2L2 5.6 4.0 3.4H7 19.2 31.7 7.2L4 1.1 1.1 3.4H8 10.7 26.5 7.3H1 8.0 11.2 3.4H9 21.7 18.6 7.3L1 4.5 6.0 3.4H10 8.1 10.1 7.4H1 8.0 6.6 3.4H11 19.5 8.3 7.4H2 9.9 6.2 3.4H12 25.5 27.0 7.9H1 4.9 5.4 3.4H13 32.3 18.8 7.9H2 5.0 5.7 3.4H14 13.3 16.8 7.9H3 4.2 2.8 3.4H16 25.5 15.6 7.9H4 4.8 4.2 3.4H17 2.0 4.3 7.9H5 1.7 1.8 3.4H18 17.1 10.0 7.9H6 1.2 1.2 3.4L7 9.3 9.3 7.9H7 1.0 0.9 3.4L8 6.2 13.0 7.9H8 0.8 0.7 3.4L9 16.3 9.1 7.9H9 0.9 0.9 3.4L11 5.3 2.6 7.9L1 1.0 1.1 3.6H1 18.9 23.1 7.9L2 1.0 0.5 3.6H2 19.8 54.0 7.11H1 12.7 6.7 3.6H3 5.4 7.5 7.11H2 0.3 0.3 3.6H4 13.0 7.8 7.11H3 6.6 4.4 3.6H5 8.2 6.0 7.11H4 1.0 1.7 3.6H6 36.0 11.8 7.11H5 3.4 1.7 3.6H8 2.5 2.5 7.11L1 1.1 1.2 3.6L1 15.6 11.1 7.12H1 0.6 0.8 3.6L2 13.7 13.1 7.12L1 n.d. 3.8 7.12L2 4.0 5.4 7.12L3 0.8 0.9 7.12L4 2.0 0.6 7.12L5 0.8 0.6 7.12L6 n.d. n.d. 7.12L7 n.d. n.d. Table 3: KD values for MS-R Fab binding to Aβ1-40 and Aβ1-42 fibers as determined in the Biacore. For the clones derived from the 1st and 2nd affinity maturation cycle the values are corrected for the content of active Fab present in each sample as described in the text. avalues were calculated from the concentration-dependent sensor responses at equilibrium; n.d., not determined.
TABLE-US-00005 TABLE 4 Table 4: KD values for MS-R IgG1 binding to Aβ1-40 and Aβ1-42 fibers as determined in the Biacore. The IgGs were derived from MS-R Fabs selected after the 1st affinity maturation cycle. The values are corrected for the content of active MS-R IgGs present in each sample as described in the text. KD Aβ1-40 KD Aβ1-42 MS-R # nM nM 3.3 IgG1 3.7 6.6 7.11 IgG1 2.3 5.7 7.12 IgG1 3.1 13.7 8.1 IgG1 6.6 12.3
TABLE-US-00006 TABLE 5 KD values for MS-R IgG1 binding to Aβ1-40 and Aβ1-42 fibers as determined in the Biacore. The IgGs were derived from MS-R Fabs selected after the 1st and 2nd affinity maturation cycle and from crosscloned Fabs. The values are corrected for the content of active MS-R IgGs present in each sample as described in the text. The two KD values given for MS-R IgGs derived from the 2nd affinity maturation step and cross-cloned binders represent higher and lower affinity interaction as calculated from the curvilinear Scatchard plots. With a number of additional MS-R IgGs (for example MS-R IgG 7.9.H2 × 7.12.L2 and MS-R IgG 7.9.H4 × 7.12.L2), complex curvilinear Scatchard blots were obtained and determination of KD-values was therefore not possible. KD Aβ1-40 KD Aβ1-42 Selected clones from MS-R IgG1 nM nM 1st affinity maturation 3.3 3.7 6.6 7.11 2.3 5.7 7.12 3.1 13.7 8.1 6.6 12.3 2nd affinity maturation 3.4.H7 0.10-0.30 0.10-0.30 7.2.H4 0.09-0.30 0.10-0.66 7.9.H2 0.12-0.42 0.11-0.38 7.9.H3 0.10-0.50 0.10-0.40 7.9.H7 0.25-0.69 0.24-0.70 7.12.L1 1.20-3.50 0.74-2.90 8.2.H2 0.16-1.00 0.12-0.92 cross-cloned Fabs 3.6.H5 × 3.6.L2 0.20-1.03 0.20-0.95 3.6.H8 × 3.6.L2 0.22-0.95 0.22-0.82 7.4.H2 × 7.2.L1 0.12-0.63 0.12-0.56 7.11.H1 × 7.2.L1 0.14-0.66 0.15-0.67 7.11.H1 × 7.11.L1 0.11-0.70 0.13-0.70
Example 9
Staining of Genuine Human Amyloid Plaques in Brain Sections of an Alzheimer's Disease Patient by Indirect Immunofluorescence
[0172] Selected MS-Roche Fabs and full-length IgG1 were tested for binding to β-amyloid plaques by immunohistochemistry analysis. Cryostat sections of unfixed tissue from human temporal cortex (obtained postmortem from a patient that was positively diagnosed for Alzheimer's disease) were labeled by indirect immunofluorescence using MS-Roche Fabs or full-length human IgG1 antibodies at various concentrations. Fabs and IgG1 antibodies were revealed by goat anti-human affinity-purified F(ab')2 fragment conjugated to Cy3 and goat anti-human (H+L) conjugated to Cy3, respectively. Both secondary reagents were obtained from Jackson Immuno Research. Controls included an unrelated Fab and the secondary antibodies alone, which all gave negative results. Typical examples of plaque stainings with selected MS-Roche Fabs and MS-Roche IgG1 antibodies are shown in FIGS. 5 to 7.
Example 10
Polymerization Assay: Prevention of Aβ Aggregation
[0173] Synthetic Aβ when incubated in aqueous buffer over several days spontaneously aggregates and forms fibrillar structures which are similar to those seen in amyloid deposits in the brains of Alzheimer's Disease patients. We have developed an in vitro assay to measure incorporation of biotinylated Aβ into preformed Aβ aggregates in order to analyze the Aβ-neutralizing potential of anti-Aβ antibodies and other Aβ-binding proteins such as albumin (Bohrmann et al., 1999, J. Biol. Chem. 274, 15990-15995). The effect of small molecules on Aβ aggregation can also be analyzed in this assay.
Experimental Procedure:
[0174] NUNC Maxisorb microtiter plates (MTP) are coated with a 1:1 mixture of Aβ1-40 and Aβ1-42 (2 μM each, 100 μl per well) at 37° C. for three days. Under these conditions highly aggregated, fibrillar Aβ is adsorbed and immobilized on the surface of the well. The coating solution is then removed and the plates are dried at room temperature for 2-4 hours. (The dried plates can be stored at -20° C.). Residual binding sites are blocked by adding 300 μl/well phosphate-buffered saline containing 0.05% Tween 20 (T-PBS) and 1% bovine serum albumin (BSA). After 1-2 hours incubation at room temperature the plates are washed 1× with 300 μl T-PBS. A solution of 20 nM biotinylated Aβ1-40 in 20 mM Tris-HCl, 150 mM NaCl pH 7.2 (TBS) containing 0.05% NaN3 and serially diluted antibody is added (100 μl/well) and the plate incubated at 37° C. overnight. After washing 3× with 300 μl T-PBS a streptavidin-POD conjugate (Roche Molecular Biochemicals), diluted 1:1000 in T-PBS containing 1% BSA, is added (100 μl/well) and incubated at room temperature for 2 hours. The wells are washed 3× with T-PBS and 100 μl/well of a freshly prepared tetramethyl-benzidine (TMB) solution are added. [Preparation of the TMB solution: 10 ml 30 mM citric acid pH 4.1 (adjusted with KOH)+0.5 ml TMB (12 mg TMB in 1 ml acetone+9 ml methanol)+0.01 ml 35% H2O2]. The reaction is stopped by adding 100 μl/well 1 N H2SO4 and absorbance is read at 450 nm in a microtiter plate reader.
Result:
[0175] FIG. 8 shows that MS-Roche IgG1 antibodies prevented incorporation of biotinylated Aβ1-40 into preformed Aβ1-40/Aβ1-42 aggregates. The Aβ-neutralizing capacity of these full-length human IgGs was similar to that of the mouse monoclonal antibody BAP-1 which had been generated by a standard immunization procedure and specifically recognizes amino acid residues 4-6 of the Aβ peptide when analyzed by the Pepspot technique as described in example 7. Mouse monoclonal antibody BAP-2 which also reacts exclusively with amino acids 4-6 (Brockhaus, unpublished) was significantly less active in this assay. An even lower activity was found with the Aβ1-40 C-terminal specific antibody BAP-17 (Brockhaus, Neuroreport 9 (1998), 1481-1486) and the monoclonal antibody 4G8 which recognizes an epitope between position 17 and 24 in the Aβ sequence (Kim, 1988, Neuroscience Research Communication Vol. 2, 121-130). BSA at a concentration of up to 10 μg/ml did not affect incorporation of biotinylated Aβ and served as a negative control. However, at higher concentrations, i.e. >100 μg/ml, BSA has been reported to inhibit binding of biotinylated Aβ into preformed Aβ fibers (Bohrmann, (1999) J Biol Chem 274 (23), 15990-5) indicating that the interaction of BSA with Aβ is not of high affinity.
Example 11
De-Polymerization Assay: Release of Biotinylated Aβ from Aggregated Aβ
[0176] In a similar experimental setup we have tested the potential of MS-Roche IgG antibodies to induce depolymerization of aggregated Aβ. Biotinylated Aβ1-40 was first incorporated into preformed Aβ1-40/Aβ1-42 fibers before treatment with various anti-Aβ antibodies. Liberation of biotinylated Aβ was measured using the same assay as described in the polymerization assay.
Experimental Procedure:
[0177] NUNC Maxisorb microtiter plates (MTP) are coated with a 1:1 mixture of Aβ1-40 and Aβ1-42 as described in the polymerization assay. For incorporation of biotinylated Aβ the coated plates are incubated with 200 μl/well 20 nM biotinylated Aβ1-40 in TBS containing 0.05 NaN3 at 37° C. overnight. After washing the plate with 3×300 μl/well T-PBS, antibodies serially diluted in TBS containing 0.05% NaN3 were added and incubated at 37° C. for 3 hours. The plate was washed and analyzed for the presence of biotinylated Aβ1-40 as described above.
Result:
[0178] FIGS. 9A to D shows that the inventive antibodies induced de-polymerization of aggregated Aβ as measured by the release of incorporated biotinylated Aβ1-40. The MS-R antibodies and the mouse monoclonal antibody BAP-1 were similarly active whereas the BAP-2, BAP-17 and 4G8 antibodies were clearly less efficient in liberating biotinylated Aβ from the bulk of immobilized Aβ aggregates. BAP-1 can clearly be differentiated from the MS-R antibodies by its reactivity with cell surface full-length APP (see FIG. 15), and antibodies like BAP-1 with such properties are not useful for therapeutic applications as potential autoimmunological reactions may be induced. It is interesting to note that BAP-2, despite its specificity for amino acid residue 4-6 which is exposed in aggregated Aβ has a clearly lower activity in this assay indicating that not all N-terminus specific antibodies a priori are equally efficient in releasing Aβ from preformed aggregates. The MS-Roche IgGs are clearly superior to BAP-2 with respect to the depolymerizing activity. The relatively low efficiency of BAP-17 (C-terminus-specific) and 4G8 (amino acid residues 16-24-specific) in this assay is due to the cryptic nature of these two epitopes in aggregated Aβ. As already noted in the polymerization assay, BSA at the concentrations used here had no effect on aggregated Aβ.
[0179] The MS-R antibodies derived from the 2nd affinity maturation cycle and from the cross-cloned binders show in general a higher efficacy in the de-polymerization assay (comparison of FIG. 9A with FIGS. 9B and C), which is consistent with the increased binding affinity of these antibodies (see tables 3-5). The monoclonal antibodies AMY-33 and 6F/3D have been reported to prevent Aβ aggregation in vitro under certain experimental conditions (Solomon, (1996) Proc. Natl. Acad. Sci. USA 93, 452-455; AMY-33 and 6F/3D antibodies were obtained from Zymed Laboratories Inc., San Francisco (Order No. 13-0100) and Dako Diagnostics AG, Zug, Switzerland (Order No. M087201), respectively). As demonstrated in FIG. 9D both of these antibodies were completely inactive in the de-polymerization assay.
Example 12
Epitope Analysis by ELISA on Peptide Conjugates
[0180] The following heptapeptides (single letter code) were obtained by solid-phase synthesis and purified by liquid chromatography using the techniques known in the art.
TABLE-US-00007 AEFRHDC EFRHDSC FRHDSGC RHDSGYC HDSGYEC DSGYEVC SGYEVHC YEVHHQC EVHHQKC VHHQKLC HHQKLVC HQKLVFC QKLVFFC KLVFFAC LVFFAEC VFFAEDC FFAEDVC FAEDVGC AEDVGSC EDVGSNC DVGSNKC VGSNKGC GSNKGAC CSNKGAI CNKGAII CKGAIIG CGLMVGG CMVGGVV CGGVVIA
[0181] The peptides were dissolved in DMSO to arrive at 10 mM concentration.
[0182] Bovine Albumin (essentially fatty acid free BSA, Sigma Lot 112F-9390) was dissolved to 10 mg/ml in 0.1M sodium bicarbonate and activated by addition per ml of 50 μl of a 26 mg/ml solution of N-succinmidyl-maleinimido propionate (NSMP, Pierce) in DMSO. After 15 minutes reaction at room temperature the activated BSA was purified by gel filtration (NAP-10, Pharmacia) in PBS with 0.1% sodium azide as solvent. 50 μl of NSMP activated BSA (6.7 mg/ml) was diluted with 50 μl of PBS, 0.1% sodium azide and 10 μl of peptide solution (1 mM in DMSO) was added. As negative control activated BSA was mock-treated without peptide addition. After 4 hrs at room temperature the reaction was stopped by addition of 10 μl of 10 mM Cystein. An aliquot of the conjugate reaction mixture was diluted 1:100 with 0.1M sodium bicarbonate buffer and immediately filled into the wells (100 μl) of ELISA plates (Nunc Immuno-Plate). After standing 16 hrs at 4° C. 100 μl blocking buffer (as above) was added to each well and incubated for another 30 minutes. The plates were washed with 2×300 μl/well TBST (as above) and filled with 100 μl antibody at 10 μg/ml or 2 μg/ml in blocking buffer. The plates were kept 16 hours at 4° C. and washed with 2×300 μl TBST. 100 μl/well HRP-conjugated anti-human Ig H+L (Pierce, dilution 1:1000 with blocking buffer) was added and incubated for 1 hour at ambient temperature. The plates were washed with 3×300 ul/well TBST. Colour development was started by addition of 100 μl tetra-methyl benzidine/hydrogen peroxide reagent. The reaction was stopped after 5 minutes by addition of 100 μl/well 1M sulfuric acid and the optical density is measured by an opticalreader (Microplate Reader 3550, BioRad) at 450 nm. For comparison mouse monoclonal antibodies were analysed in the same way, except using as revealing agent HRP-labelled anti-mouse Ig instead of anti-human Ig.
[0183] Employing specific of the above described heptapeptides derived from Aβ, specific ELISA-tests as described herein above were carried out. Preferably, inventive antibodies comprise antibodies which show, as measured by of optical densities, a signal to background ratio above "10" when their reactivity with an A-beta derived peptide (AEFRHD, SEQ ID NO: 415; amino acid 2 to 7 of A-beta) is compared to an non-related protein/peptide like BSA. Most preferably, the ratio of optical densities is above "5" for a corresponding reaction with at least one of the following three Aβ derived peptides: (VFFAED, SEQ ID NO: 421; amino acid 18 to 23 of Aβ) or (FFAEDV, SEQ ID NO: 423; amino acid 19 to 24 of Aβ) or (LVFFAE, SEQ ID NO: 420; amino acid 17 to 22 of Aβ).
[0184] Corresponding results for the inventive parental and/or maturated antibodies are shown in the following two tables:
TABLE-US-00008 TABLE 6 Reactivity of MS-R Fabs with BSA-conjugated Abeta heptapeptides 2-7 (AEFRHD, SEQ ID NO: 415), 17-22 (LVFFAE, SEQ ID NO: 420), 18-23 (VFFAED, SEQ ID NO: 421) and 19-24 (FFAEDV, SEQ ID NO: 423). The ratios of the ELISA read-out (optical density) obtained with peptide-conjugated and non-conjugated BSA are given. The signal intensities obtained with the 17-22, 18-23 and 19-24 peptides in relation to the 2-7 peptide are also indicated. Peptide2-7 Peptide 17-22 Peptide 18-23 Peptide 19-24 Peptide-ratio Peptide-ratio Peptide-ration MS-R # 2-7/BSA 17-22/BSA 18-23/BSA 19-24/BSA 17-22/2-7 18-23/2-7 19-24/2-7 7 24 4 7 4 0.17 0.29 0.17 8 28 10 29 25 0.36 1.04 0.89 7.2 34 12 16 9 0.35 0.47 0.26 7.3 34 11 15 9 0.32 0.44 0.26 7.4 36 10 13 6 0.28 0.36 0.17 7.9 28 9 13 8 0.32 0.46 0.29 7.11 37 11 15 9 0.30 0.41 0.24 7.12 38 6 8 7 0.16 0.21 0.18 8.1 30 1 11 8 0.03 0.37 0.27 8.2 32 4 28 23 0.13 0.88 0.72 3.2H2 26 12 23 20 0.46 0.88 0.77 3.3H1 23 4 12 8 0.17 0.52 0.35 3.3H3 31 2 5 2 0.06 0.16 0.06 3.4H1 27 2 8 2 0.07 0.30 0.07 3.4H2 16 11 1 1 0.69 0.06 0.06 3.4H3 22 9 17 11 0.41 0.77 0.50 3.4H5 28 5 13 4 0.18 0.46 0.14 3.4H7 24 2 6 5 0.08 0.25 0.21 3.4H17 28 5 12 11 0.18 0.43 0.39 3.4L11 31 6 20 5 0.19 0.65 0.16 3.6H6 25 1 4 7 0.04 0.16 0.28 3.6H1 23 3 13 5 0.13 0.57 0.22 3.6H2 19 2 8 3 0.11 0.42 0.16 7.2H1 38 8 11 9 0.21 0.29 0.24 7.2H2 16 10 10 10 0.63 0.63 0.63 7.2H3 33 17 20 18 0.52 0.61 0.55 7.2H4 23 12 13 12 0.52 0.57 0.52 7.2H5 30 13 18 15 0.43 0.60 0.50 7.2L1 24 14 16 11 0.57 0.68 0.45 7.4H1 31 16 20 16 0.52 0.65 0.51 7.4H2 36 17 20 16 0.47 0.56 0.46 7.9H1 32 7 12 6 0.23 0.36 0.19 7.9H2 35 3 6 8 0.08 0.16 0.23 7.9H3 35 11 20 9 0.31 0.57 0.27 7.9H4 30 10 15 7 0.32 0.49 0.22 7.11H1 31 8 9 8 0.25 0.29 0.25 7.11H2 34 10 12 14 0.29 0.36 0.41 7.12L1 16 10 12 10 0.60 0.70 0.59 8.1H1 29 22 25 25 0.77 0.88 0.86 8.2H1 22 7 23 20 0.34 1.05 0.94 8.2L1 26 15 32 31 0.60 1.26 1.22
TABLE-US-00009 TABLE 7 Reactivity of MS-R IgGs and mouse monoclonal antibodies BAP-1, BAP-2, 4G8, 6E10 Amy-33 and 6F/3D with BSA-conjugated Aβ heptapeptides 2-7 (AEFRHD, SEQ ID NO: 415), 17-22 (LVFFAE, SEQ ID NO: 420), 18-23 (VFFAED, SEQ ID NO: 421) and 19-24 (FFAEDV, SEQ ID NO: 423). The ratios of the ELISA read-out (optical density) obtained with peptide-conjugated and non-conjugated BSA are given. The signal intensities obtained with the 17-22, 18-23 and 19-24 peptides in relation to the 2-7 peptide are also indicated. AEFRHD LVFFAE VFFAED FFAEDV (SEQ ID (SEQ ID No. (SEQ ID No. (SEQ ID Peptide- Peptide- No. 415) 420) 421) No. 423) ratio ratio Peptide-ratio MS-R IgG # 2-7/BSA 17-22/BSA 18-23/BSA 19-24/BSA 17-22/2-7 18-23/2-7 19-24/2-7 3.3 17 11 16 11 0.65 0.94 0.65 7.12 19 11 13 11 0.58 0.68 0.58 8.1 16 7 16 14 0.44 1.00 0.88 3.4H7 22 3 16 15 0.14 0.73 0.68 7.9H2 13 5 8 6 0.38 0.62 0.46 7.9H3 13 6 8 6 0.46 0.62 0.46 7.9.H7 30 5 16 10 0.17 0.53 0.33 7.11H2 10 6 7 6 0.60 0.70 0.60 8.2.H2 18 10 15 14 0.56 0.83 0.78 3.6.H5 × 3.6.L2 11 7 9 8 0.64 0.82 0.73 7.11.H2 × 7.9.L1 14 8 10 9 0.57 0.71 0.64 (L1) 8.2.H2 × 8.2.L1 13 20 25 25 1.54 1.92 1.92 Mouse mab BAP-1 21 1 1 1 0.05 0.05 0.05 BAP-2 21 1 1 1 0.05 0.05 0.05 4G8 1 23 20 1 23 20 1 6E10 18 1 1 1 0.06 0.06 0.06 6F/3D* 1 1 1 1 1 1 1 Amy 33 16 2 1 3 0.13 0.06 0.19 *this antibody is specific for sequence 8-17 and does not recognize N-terminal or middle epitope sequences.
Example 13
Combination of Optimized H-CDR2 and L-CDR1 by Cross-Cloning
[0185] The modular design of the HuCAL library allows exchange of complementarity determining regions (CDRs) of two different Fab encoding genes in a simple cloning step. For a further improvement of affinity the independently optimized H-CDR2 and L-CDR1 from matured clones with the same H-CDR3 were combined, because there was a high probability that this combination would lead to a further gain of affinity (Yang et al., 1995, J. Mol. Biol. 254, 392-403; Schier et al., 1996b, J. Mol. Biol. 263, 551-567; Chen et al., 1999, J. Mol. Biol. 293, 865-881). Whole light chains, or fragments thereof, were transferred from an L-CDR1 optimized donor clone to a H-CDR2 optimized recipient clone. Donor and recipient clones were only combined, if both carried identical H-CDR3 sequences. All donor and recipient clones carried the VH3-Vκ3 framework.
[0186] This was accomplished by transferring whole light chains from the L-CDR1-optimized donor clone to the H-CDR2-optimized recipient clone. Epitope specificity was conserved by only combining clones with the same H-CDR3. By light chain exchange a H-CDR2-optimized clone obtained only an optimized L-CDR1, if the exchange occurred between clones with the same L-CDR3. If the L-CDR3 of the clones to be combined was different, the H-CDR2-optimized clone acquired in addition to the optimized L-CDR1 another L-CDR3 (L-CDR2 remained the HuCAL consensus sequence (Knappik et al., 2000)) and when derivatives of MS-Roche #7.12 were used as donors of the light chain L-CDR1, 2 and 3 were exchanged in the H-CDR2-optimized acceptor clone. Three different cloning strategies were employed:
[0187] 1) Using restriction endonucleases XbaI and SphI the whole antibody light chain fragment was excised from plasmid 1 (e.g. pMx9_Fab_MS-Roche#7.11.H1_FS) and the thereby obtained vector backbone was then ligated to the light chain fragment of plasmid 2 (e.g. pMx9_Fab_MS-Roche#7.2.L1_FS) generated by XbaI and SphI digest. Thereby a new plasmid (nomenclature: pMx9_Fab_MS-Roche#7.11.H1×7.2.1--1_FS) was created encoding L-CDR1,2,3 of parental clone #7.2.L1 and H-CDR1,2,3 of parental clone #7.11.H1.
[0188] 2) Using restriction endonucleases XbaI and Acc65I an L-CDR1 coding fragment was excised from plasmid 1 (e.g. pMx9_Fab_MS-Roche#7.11.H2_FS) and the thereby obtained vector backbone was then ligated to the L-CDR1 fragment of plasmid 2 (e.g. pMx9_Fab_MS-Roche#7.12.L1_FS) generated by XbaI and Acc65I. Thereby a new plasmid (nomenclature: pMx9_Fab_MS-Roche#7.11.H2×7.12.L1(L-CDR1)_FS) was created encoding L-CDR1 of parental clone #7.12.L1 while L-CDR2,3 and H-CDR1,2,3 are derived from parental clone #7.11.H2.
[0189] 3) Using restriction endonucleases XbaI and BamHI an L-CDR1 and L-CDR2 coding fragment was excised from plasmid 1 (e.g. pMx9_Fab_MS-Roche#7.11.H2_FS) and the thereby obtained vector backbone was then ligated to the L-CDR1 and L-CDR2 fragment of plasmid 2 (e.g. pMx9_Fab_MS-Roche#7.12.L1_FS) generated by XbaI and BamHI digest. Thereby a new plasmid (nomenclature: pMx9_Fab_MS-Roche#7.11.H2×7.12.L1(L-CDR1+2)_FS) was created encoding L-CDR1 and L-CDR2 of parental clone #7.12.L1 while L-CDR3 and H-CDR1,2,3 are derived from parental clone #7.11.H2.
[0190] Illustrative examples for the different cloning strategies as well as for sequences donor and recipient clones are given in table 8.
[0191] After large scale expression and purification their affinities were determined on Aβ (1-40) fibers. Furthermore, KD values for selected cross-cloned MS-R Fab/antibodies are given in appended Table 9.
TABLE-US-00010 TABLE 8 Binder pos. pos. pos. name L-CDR1 49 L-CDR2 85 L-CDR3 H-CDR1 47 H-CDR2 H-CDR3 cloning strategy 1) ##STR00001## ##STR00002## MS-Roche RASQSVSSSYLA Y GASSRAT T QQVYSPPH GFTFSSYAMS W GINAAGFRTYYADSVKG GKGNTHKPYGYVRYF #7.11.H1 DV MS-Roche RASQYVDRTYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS W AISGSGGSTYYADSVKG GKGNTHKPYGYVRYF #7.2.L1 DV MS-Roche RASQYVDRTYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS W GINAAGFRTYYADSVKG GKGNTHKPYGYVRYF #7.11.H1 × DV 7.2.L1 cloning strategy 2) ##STR00003## ##STR00004## MS-Roche RASQSVSSSYLA Y GASSRAT T QQVYSPPH GFTFSSYAMS W AINANGYKKYYADSVKG GKGNTHKPYGYVRYF #7.11.H2 DV MS-Roche RASQYVFRRYLA S GSSNRAT V LQLYNIPN GFTFSSYGMS W NISGSGSSTYYADSVKG GKGNTHKPYGYVRYF #7.12.L1 DV MS Roche RASQYVFRRYLA Y GASSRAT T QQVYSPPH GFTFSSYAMS W AINANGYKKYYADSVKG GKGNTHKPYGYVRYF #7.11.H2 × DV 7.12.L1 (LCDR1) cloning strategy 3) ##STR00005## ##STR00006## MS-Roche RASQSVSSSYLA Y GASSRAT T QQVYSPPH GFTFSSYAMS W AINANGYKKYYADSVKG GKGNTHKPYGYVRYF #7.11.H2 DV MS-Roche RASQYVFRRYLA S GSSNRAT V LQLYNIPN GFTFSSYGMS W NISGSGSSTYYADSVKG GKGNTHKPYGYVRYF #7.12.L1 DV MS Roche RASQYVFRRYLA S GSSNRAT T QQVYSPPH GFTFSSYAMS W AINANGYKKYYADSVKG GKGNTHKPYGYVRYF #7.11.H2 × DV 7.12.L1 (LCDR1 + 2) MS-Roche RASQSVSSSYLA Y GASSRAT V QQTYNYPP GFTFSSYAMS W AISESGKTKYYADSVKG LTHYARYYRYFDV #3.6H5 MS-Roche RASQFLSRYYLA Y GASSRAT V QQTYNYPP GFTFSSYAMS W AISGSGGSTYYADSVKG LTHYARYYRYFDV #3.6L2 MS-Roche RASQFLSRYYLA Y GASSRAT V QQTYNYPP GFTFSSYAMS W AISESGKTKYYADSVKG LTHYARYYRYFDV #3.6H5 × 3.6L2 MS-Roche RASQSVSSSYLA Y GASSRAT V QQTYNYPP GFTFSSYAMS W AISEYSKFKYYADSVKG LTHYARYYRYFDV #3.6H8 MS-Roche RASQFLSRYYLA Y GASSRAT V QQTYNYPP GFTFSSYAMS W AISGSGGSTYYADSVKG LTHYARYYRYFDV #3.6L2 MS-Roche RASQFLSRYYLA Y GASSRAT V QQTYNYPP GFTFSSYAMS W AISEYSKFKYYADSVKG LTHYARYYRYFDV #3.6H8 × 3.6L2 MS-Roche RASQSVSSSYLA Y GASSRAT V QQIYNFPH GFTFSSYAMS W AINYNGARIYYADSVKG GKGNTHKPYGYVRYF #7.4.H2 DV MS-Roche RASQYVDRTYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS W AISGSGGSTYYADSVKG GKGNTHKPYGYVRYF #7.2.L1 DV MS-Roche RASQYVDRTYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS W AINYNGARIYYADSVKG GKGNTHKPYGYVRYF #7.4.H2 × DV 7.2.L1 MS-Roche RASQSVSSSYLA Y GASSRAT T LQIYNMPI GFTFSSYAMS W AINADGNRKYYADSVKG GKGNTHKPYGYVRYF #7.9H2 DV MS-Roche RASQRFFYKYLA S GSSNRAT V LQLYNIPN GFTFSSYGMS W NISGSGSSTYYADSVKG GKGNTHKPYGYVRYF #7.12L2 DV MS-Roche RASQRFFYKYLA S GSSNRAT V LQLYNIPN GFTFSSYAMS W AINADGNRKYYADSVKG GKGNTHKPYGYVRYF #7.9H2 × DV 7.12L2 MS-Roche RASQSVSSSYLA Y GASSRAT T LQIYNMPI GFTFSSYAMS W AINAVGMKKFYADSVKG GKGNTHKPYGYVRYF #7.9H4 DV MS-Roche RASQRFFYKYLA S GSSNRAT V LQLYNIPN GFTFSSYGMS W NISGSGSSTYYADSVKG GKGNTHKPYGYVRYF #7.12.L2 DV MS-Roche RASQRFFYKYLA S GSSNRAT V LQLYNIPN GFTFSSYAMS W AINAVGMKKFYADSVKG GKGNTHKPYGYVRYF #7.9H4 × DV 7.12L2 MS-Roche RASQSVSSSYLA Y GASSRAT T QQVYSPPH GFTFSSYAMS W GINAAGFRTYYADSVKG GKGNTHKPYGYVRYF #7.11H1 DV MS-Roche RASQRILRIYLA Y GASSRAT T QQVYSPPH GFTFSSYAMS W AISGSGGSTYYADSVKG GKGNTHKPYGYVRYF #7.11L1 DV MS-Roche RASQRILRIYLA Y GASSRAT T QQVYSPPH GFTFSSYAMS W GINAAGFRTYYADSVKG GKGNTHKPYGYVRYF #7.11H1 × DV 7.11L1 MS-Roche RASQSVSSSYLA Y GASSRAT T QQVYSPPH GFTFSSYAMS W GINAAGFRTYYADSVKG GKGNTHKPYGYVRYF #7.11H1 DV MS-Roche RASQYVDRTYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS W AISGSGGSTYYADSVKG GKGNTHKPYGYVRYF #7.2L1 DV MS-Roche RASQYVDRTYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS W GINAAGFRTYYADSVKG GKGNTHKPYGYVRYF #7.11H1 × DV 7.2L1 MS-Roche RASQSVSSSYLA Y GASSRAT V HQMSSYPP GFTFSSYAMS W VISEKSRFIYYADSVKG LTHYARYYRYFDV #3.3H1 MS-Roche RASRRIHVYYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISGSGGSTYYADSVKG LTHYARYYRYFDV #3.4L9 MS-Roche RASRRIHVYYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W VISEKSRFIYYADSVKG LTHYARYYRYFDV #3.3H1 × 3.4L9 MS-Roche RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISETSIRKYYADSVKG LTHYARYYRYFDV #3.4H1 MS-Roche RASRRIHVYYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISGSGGSTYYADSVKG LTHYARYYRYFDV #3.4L9 MS-Roche RASRRIHVYYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISETSIRKYYADSVKG LTHYARYYRYFDV #3.4H1 × 3.4L9 MS-Roche RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W VISQTGRKIYYADSVKG LTHYARYYRYFDV #3.4H3 MS-Roche RASQRLGRLYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISGSGGSTYYADSVKG LTHYARYYRYFDV #3.4L7 MS-Roche RASQRLGRLYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W VISQTGRKIYYADSVKG LTHYARYYRYFDV #3.4H3 × 3.4L7 MS-Roche RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W VISQTGRKIYYADSVKG LTHYARYYRYFDV #3.4H3 MS-Roche RASRRIHVYYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISGSGGSTYYADSVKG LTHYARYYRYFDV #3.4L9 MS-Roche RASRRIHVYYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W VISQTGRKIYYADSVKG LTHYARYYRYFDV #3.4H3 × 3.4L9 MS-Roche RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W VISETGKNIYYADSVKG LTHYARYYRYFDV #3.4H7 MS-Roche RASRRIHVYYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISGSGGSTYYADSVKG LTHYARYYRYFDV #3.4L9 MS-Roche RASRRIHVYYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W VISETGKNIYYADSVKG LTHYARYYRYFDV #3.4H7 × 3.4L9 MS-Roche RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W VISETGKNIYYADSVKG LTHYARYYRYFDV #3.4H7 MS-Roche RASQRLGRLYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISGSGGSTYYADSVKG LTHYARYYRYFDV #3.4L7 MS-Roche RASQRLGRLYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W VISETGKNIYYADSVKG LTHYARYYRYFDV #3.4H7 × 3.4L7 MS-Roche RASQSVSSSYLA Y GASSRAT V QQTYNYPP GFTFSSYAMS W AISESGKTKYYADSVKG LTHYARYYRYFDV #3.6H5 MS-Roche RASQFIQRFYLA Y GASSRAT V QQTYNYPP GFTFSSYAMS W AISGSGGSTYYADSVKG LTHYARYYRYFDV #3.6L1 MS-Roche RASQFIQRFYLA Y GASSRAT V QQTYNYPP GFTFSSYAMS W AISESGKTKYYADSVKG LTHYARYYRYFDV #3.6H5 × 3.6L1 MS-Roche RASQSVSSSYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS W AINGTGMKKYYADSVKG GKGNTHKPYGYVRYF #7.2H2 DV MS-Roche RASQYVDRTYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS W AISGSGGSTYYADSVKG GKGNTHKPYGYVRYF #7.2L1 DV MS-Roche RASQYVDRTYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS W AINGTGMKKYYADSVKG GKGNTHKPYGYVRYF #7.2H2 × DV 7.2L1 MS-Roche RASQSVSSSYLA Y GASSRAT V QQIYNFPH GFTFSSYAMS W AINYNGARIYYADSVKG GKGNTHKPYGYVRYF #7.4H2 DV MS-Roche RASQRFFYKYLA S GSSNRAT V LQLYNIPN GFTFSSYGMS W NISGSGSSTYYADSVKG GKGNTHKPYGYVRYF #7.12L2 DV MS-Roche RASQRFFYKYLA S GSSNRAT V LQLYNIPN GFTFSSYAMS W AINYNGARIYYADSVKG GKGNTHKPYGYVRYF #7.4H2 × DV 7.12L2 MS-Roche RASQSVSSSYLA Y GASSRAT T LQIYNMPI GFTFSSYAMS W AINADGNRKYYADSVKG GKGNTHKPYGYVRYF #7.9H2 DV MS-Roche RASQYVDRTYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS W AISGSGGSTYYADSVKG GKGNTHKPYGYVRYF #7.2L1 DV MS-Roche RASQYVDRTYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS W AINADGNRKYYADSVKG GKGNTHKPYGYVRYF #7.9H2 × DV 7.2L1 MS-Roche RASQSVSSSYLA Y GASSRAT T QQVYSPPH GFTFSSYAMS W AINANGYKKYYADSVKG GKGNTHKPYGYVRYF #7.11H2 DV MS-Roche RASQYVDRTYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS W AISGSGGSTYYADSVKG GKGNTHKPYGYVRYF #7.2L1 DV MS-Roche RASQYVDRTYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS W AINANGYKKYYADSVKG GKGNTHKPYGYVRYF #7.11H2 × DV 7.2L1 MS-Roche RASQSVSSSYLA Y GASSRAT T LQIYNMPI GFTFSSYAMS W AINADGNRKYYADSVKG GKGNTHKPYGYVRYF #7.9H2 DV MS-Roche RASQYVFRRYLA S GSSNRAT V LQLYNIPN GFTFSSYGMS W NISGSGSSTYYADSVKG GKGNTHKPYGYVRYF #7.12L1 DV MS-Roche RASQYVFRRYLA S GSSNRAT V LQLYNIPN GFTFSSYAMS W AINADGNRKYYADSVKG GKGNTHKPYGYVRYF #7.9H2 × DV 7.12L1 MS-Roche RASQSVSSSYLA Y GASSRAT T QQVYSPPH GFTFSSYAMS W AINANGYKKYYADSVKG GKGNTHKPYGYVRYF #7.11H2 DV MS-Roche RASQRLSPRYLA Y GASSRAT T LQIYNMPI GFTFSSYAMS W AISGSGGSTYYADSVKG GKGNTHKPYGYVRYF #7.9L1 DV MS-Roche RASQRLSPRYLA Y GASSRAT T LQIYNMPI GFTFSSYAMS W AINANGYKKYYADSVKG GKGNTHKPYGYVRYF #7.11H2 × DV 7.9L1 MS-Roche RASQSVSSSYLA Y GASSRAT T QQLSNYPP GFTFSSYAMS W AISRSGSNIYYADSVKG LLSRGYNGYYHKFDV #8.1H1 MS-Roche RASQRVSGRYLA Y GASSRAT T QQLSSYPP GFTFSSYAMS W AISGSGGSTYYADSVKG LLSRGYNGYYHKFDV
#8.2L1 MS-Roche RASQRVSGRYLA Y GASSRAT T QQLSSYPP GFTFSSYAMS W AISRSGSNIYYADSVKG LLSRGYNGYYHKFDV #8.1H1 × 8.2L1 MS-Roche RASQSVSSSYLA Y GASSRAT T QQVYSPPH GFTFSSYAMS W AINANGYKKYYADSVKG GKGNTHKPYGYVRYF #7.11H2 DV MS-Roche RASQYVFRRYLA S GSSNRAT V LQLYNIPN GFTFSSYGMS W NISGSGSSTYYADSVKG GKGNTHKPYGYVRYF #7.12L1 DV MS-Roche RASQYVFRRYLA S GSSNRAT V LQLYNIPN GFTFSSYAMS W AINANGYKKYYADSVKG GKGNTHKPYGYVRYF #7.11H2 × DV 7.12L1 Arrows indicate the location of restriction enzyme sites used to digest corresponding plasmids
TABLE-US-00011 TABLE 9 KD values for crosscloned MS-R Fab binding to Aβ1-40 and Aβ1-42 fibers as determined in the Biacore. The preparation of crosscloned Fabs is described in example 13. The KD values were determined by kinetic curve fittings and corrected for the content of active Fab present in each sample as described in the text. Some of the Fabs were additionally purified by size exclusion chromatography or preparative ultracentrifugation to remove aggregated material. (L1), the H-CDR2-matured acceptor clone received only L-CDR1 from the L-CDR1 improved donor clone; (L1 + 2), the H-CDR2-matured acceptor clone received L-CDR1 + 2 from the L-CDR1 improved donor clone. KD Aβ1-40 KD Aβ1-42 MS-R # nM nM 3.3H1 × 3.4L9 2.16 2.97 3.4H1 × 3.4L9 0.25 0.5 3.4H3 × 3.4L7 0.92 0.92 3.4H3 × 3.4L9 1.05 0.93 3.4H7 × 3.4L9 2.66 3.51 3.4H7 × 3.4L7 1.19 1.23 3.6H5 × 3.6L1 1.25 1.04 3.6H5 × 3.6L2 1.26 0.84 7.2H2 × 7.2L1 1.29 1.43 7.4H2 × 7.2L1 1.4 1.4 7.4H2 × 7.12L2 1.4 1.8 7.9H2 × 7.2L1(L1) 1.4 1.4 7.9H2 × 7.12L1 1.2 1.1 7.9H2 × 7.12L2(L1 + 2) 0.4 0.4 7.11H1 × 7.2L1 1.75 1.39 7.11H1 × 7.11L1 0.41 0.47 7.11H2 × 7.2L1(L1) 1 0.6 7.11H2 × 7.9L1 (L1) 0.1 1 8.1H1 × 8.2L1 1.3 1.6
Example 14
In Vivo Amyloid Plaque Decoration in a Mouse Model of Alzheimer's Disease as Revealed by Confocal Laser Scanning Microscopy and Colocalization Analysis
[0192] Selected MS-R IgG1 antibodies were tested in APP/PS2 double transgenic mice (Reference: Richards et al., Soc. Neurosci. Abstr., Vol. 27, Program No. 5467, 2001) for amyloid plaque decoration in vivo. The antibodies (1 mg/mouse) were administered i.v. and after 3 days the brains were perfused with saline and prepared for cryosection. In another study the mice were exposed to higher concentrations of the antibodies, i.e. 2 mg injected i.v. at day 0, 3, and 6, and sacrificed at day nine. The presence of the antibodies bound to amyloid plaques was assessed on unfixed cryostat sections by double-labeled indirect immunofluorescence using goat anti-human IgG (H+L) conjugated to either Cy3 (#109-165-003, Jackson Immuno Research) followed by BAP-2-Alexa488 immunoconjugate. Imaging was done by confocal laser microscopy and image processing for quantitative detection of colocalizations by IMARIS and COLOCALIZATION software (Bitplane, Switzerland). Typical examples are shown in FIGS. 10-14. All of the MS-R antibodies tested were found positive in immunodecoration of amyloid plaques in vivo, although some variability was noted.
Example 15
Investigation of Binding of Different Monoclonal Antibodies to Amyloid Precursor Protein (APP) on the Surface of HEK293 Cells
[0193] APP is widely expressed in the central nervous system. Binding of antibody to cell surface APP may lead to complement activation and cell destruction in healthy brain areas. Therefore, it is mandatory for therapeutic A-beta antibodies to be devoid of reactivity towards APP. High affinity antibodies against the N-terminal domain of A-beta (e.g. BAP-1, BAP-2) recognize the respective epitope also in the framework of APP. In contrast, the antibodies against the middle epitope (e.g. 4G8), and the antibodies of the invention are surprisingly unable to recognize to cell surface APP. Thus, antibodies of the invention which decorate A-beta, but not APP in vivo, are superior to non-selective antibodies.
[0194] The method of flow cytometry is well known in the art. Relative units of fluorescence (FL1-H) measured by flow cytometry indicate cell surface binding of the respective antibody. A fluorescence shift on APP transfected HEK293 compared to untransfected HEK293 cells indicates the unwanted reaction with cell surface APP. As an example, antibodies BAP-1 and BAP-2 against the N-terminal domain show a significant shift of FL-1 signal in HEK293/APP (thick line) compared to untransfected HEK293 cells (dotted line). The 4G8 antibody (specific for the middle A-beta epitope) and all antibodies of the invention (specific for N-terminal and middle A-beta epitopes) show no significant shift in fluorescence. Differences in basal fluorescence between HE293/APP ad HEK293 cells are due to different cell size. A FACScan instrument was used in combination with the Cellquest Pro Software package (both Becton Dickinson).
Example 16
List of Identified SEQ ID NOs Relating to Inventive Antibody Molecules
[0195] The appended table 10 relates to sequences as defined herein for some specific inventive antibody molecules.
TABLE-US-00012 TABLE 10 Identification of SEQ ID NOs for parental antibodies as well as optimized, matured and/or cross-cloned antibody molecules HCDR3 HCDR3 LCDR3 LCDR3 Molecule # VH prot VL prot VH DNA VL DNA prot DNA prot DNA 3 4 10 3 9 22 21 16 15 7 6 12 5 11 24 23 18 17 8 8 14 7 13 26 25 20 19 3.6H5 × 3.6L2 33 47 32 46 61 60 75 74 3.6H8 × 3.6L2 35 49 34 48 63 62 77 76 7.4H2 × 7.2L1 37 51 36 50 65 64 79 78 7.9H2 × 7.12L2 39 53 38 52 67 66 81 80 7.9H4 × 7.12L2 41 55 40 54 69 68 83 82 7.11H1 × 7.11L1 43 57 42 56 71 70 85 84 7.11H1 × 7.2L1 45 59 44 58 73 72 87 86 7.9H7 89 91 88 90 93 92 95 94 3.3H1 × 3.4L9 295 325 294 324 355 354 385 384 3.4H1 × 3.4L9 297 327 296 326 357 356 387 386 3.4H3 × 3.4L7 299 329 298 328 359 358 389 388 3.4H3 × 3.4L9 301 331 300 330 361 360 391 390 3.4H7 × 3.4L9 303 333 302 332 363 362 393 392 3.4H7 × 3.4L7 305 335 304 334 365 364 395 394 3.6H5 × 3.6L1 307 337 306 336 367 366 397 396 7.2H2 × 7.2L1 309 339 308 338 369 368 399 398 7.4H2 × 7.12L2 311 341 310 340 371 370 401 400 7.9H2 × 7.2L1 313 343 312 342 373 372 403 402 7.9H2 × 7.12L1 315 345 314 344 375 374 405 404 7.11H2 × 7.2L1 317 347 316 346 377 376 407 406 7.11H2 × 7.9L1 319 349 318 348 379 378 409 408 7.11H2 × 7.12L1 321 351 320 350 381 380 411 410 8.1H1 × 8.2L1 323 353 322 352 383 382 413 412
Sequence CWU
1
1
42519PRTartificial sequencesynthetic construct; first region of beta-A4
peptide 1Ala Glu Phe Arg His Asp Ser Gly Tyr 1 5
214PRTartificial sequencesynthetic construct; second region of
beta-A4 peptide 2Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val
Gly 1 5 10
3368DNAartificial sequencesynthetic construct; VH-region of MS-Roche#3
3caggtgcaat tggtggaaag cggcggcggc ctggtgcaac cgggcggcag cctgcgtctg
60agctgcgcgg cctccggatt tacctttagc agctatgcga tgagctgggt gcgccaagcc
120cctgggaagg gtctcgagtg ggtgagcgcg attagcggta gcggcggcag cacctattat
180gcggatagcg tgaaaggccg ttttaccatt tcacgtgata attcgaaaaa caccctgtat
240ctgcaaatga acagcctgcg tgcggaagat acggccgtgt attattgcgc gcgtcttact
300cattatgctc gttattatcg ttattttgat gtttggggcc aaggcaccct ggtgacggtt
360agctcagc
3684122PRTartificial sequencesynthetic construct; VH-region of MS-Roche#3
4Gln 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 Thr Phe Ser Ser Tyr 20
25 30 Ala Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp
Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Leu Thr His Tyr Ala Arg Tyr Tyr
Arg Tyr Phe Asp Val Trp 100 105
110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 5379DNAartificial sequencesynthetic construct;
VH-region of MS-Roche#7 5caggtgcaat tggtggaaag cggcggcggc ctggtgcaac
cgggcggcag cctgcgtctg 60agctgcgcgg cctccggatt tacctttagc agctatgcga
tgagctgggt gcgccaagcc 120cctgggaagg gtctcgagtg ggtgagcgcg attagcggta
gcggcggcag cacctattat 180gcggatagcg tgaaaggccg tttaccattt cacgtgataa
ttcgaaaaac accctgtatc 240tgcaaatgaa cagcctgcgt gcggaagata cggccgtgta
ttattgcgcg cgtggtaagg 300gtaatactca taagccttat ggttatgttc gttattttga
tgtttggggc caaggcaccc 360tggtgacggt tagctcagc
3796126PRTartificial sequencesynthetic construct;
VH-region of MS-Roche#7 6Gln 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 Thr Phe Ser Ser Tyr
20 25 30 Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Ala Ile Ser Gly Ser Gly Gly
Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr 65 70 75
80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Gly Lys
Gly Asn Thr His Lys Pro Tyr Gly Tyr Val Arg Tyr 100
105 110 Phe Asp Val Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 115 120 125
7374DNAartificial sequencesynthetic construct; VH-region of MS-Roche#8
7caggtgcaat tggtggaaag cggcggcggc ctggtgcaac cgggcggcag cctgcgtctg
60agctgcgcgg cctccggatt tacctttagc agctatgcga tgagctgggt gcgccaagcc
120cctgggaagg gtctcgagtg ggtgagcgcg attagcggta gcggcggcag cacctattat
180gcggatagcg tgaaaggccg ttttaccatt tcacgtgata attcgaaaaa caccctgtat
240ctgcaaatga acagcctgcg tgcggaagat acggccgtgt attattgcgc gcgtcttctt
300tctcgtggtt ataatggtta ttatcataag tttgatgttt ggggccaagg caccctggtg
360acggttagct cagc
3748124PRTartificial sequencesynthetic construct; VH-region of MS-Roche#8
8Gln 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 Thr Phe Ser Ser Tyr 20
25 30 Ala Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp
Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Leu Leu Ser Arg Gly Tyr Asn Gly
Tyr Tyr His Lys Phe Asp 100 105
110 Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 9330DNAartificial sequencesynthetic
construct; VL-region of MS-Roche#3 9gatatcgtgc tgacccagag cccggcgacc
ctgagcctgt ctccgggcga acgtgcgacc 60ctgagctgca gagcgagcca gagcgtgagc
agcagctatc tggcgtggta ccagcagaaa 120ccaggtcaag caccgcgtct attaatttat
ggcgcgagca gccgtgcaac tggggtcccg 180gcgcgtttta gcggctctgg atccggcacg
gattttaccc tgaccattag cagcctggaa 240cctgaagact ttgcggttta ttattgccag
caggtttata atcctcctgt tacctttggc 300cagggtacga aagttgaaat taaacgtacg
33010110PRTartificial sequencesynthetic
construct; VL-region of MS-Roche#3 10Asp Ile Val Leu Thr Gln Ser Pro Ala
Thr Leu Ser Leu Ser Pro Gly 1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser
Ser Ser 20 25 30
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45 Ile Tyr Gly Ala
Ser Ser Arg Ala Thr Gly Val Pro Ala Arg Phe Ser 50
55 60 Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Glu 65 70
75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Val
Tyr Asn Pro Pro 85 90
95 Val Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr
100 105 110 11330DNAartificial
sequencesynthetic construct; VL-region of MS-Roche#7 11gatatcgtgc
tgacccagag cccggcgacc ctgagcctgt ctccgggcga acgtgcgacc 60ctgagctgca
gagcgagcca gagcgtgagc agcagctatc tggcgtggta ccagcagaaa 120ccaggtcaag
caccgcgtct attaatttat ggcgcgagca gccgtgcaac tggggtcccg 180gcgcgtttta
gcggctctgg atccggcacg gattttaccc tgaccattag cagcctggaa 240cctgaagact
ttgcgactta ttattgcttt cagctttatt ctgatccttt tacctttggc 300cagggtacga
aagttgaaat taaacgtacg
33012110PRTartificial sequencesynthetic construct; VL-region of
MS-Roche#7 12Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro
Gly 1 5 10 15 Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser
20 25 30 Tyr Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35
40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr
Gly Val Pro Ala Arg Phe Ser 50 55
60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Glu 65 70 75
80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Leu Tyr Ser Asp Pro
85 90 95 Phe Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 100
105 110 13330DNAartificial sequencesynthetic construct;
VL-region of MS-Roche#8 13gatatcgtgc tgacccagag cccggcgacc ctgagcctgt
ctccgggcga acgtgcgacc 60ctgagctgca gagcgagcca gagcgtgagc agcagctatc
tggcgtggta ccagcagaaa 120ccaggtcaag caccgcgtct attaatttat ggcgcgagca
gccgtgcaac tggggtcccg 180gcgcgtttta gcggctctgg atccggcacg gattttaccc
tgaccattag cagcctggaa 240cctgaagact ttgcgactta ttattgccag cagctttctt
cttttcctcc tacctttggc 300cagggtacga aagttgaaat taaacgtacg
33014110PRTartificial sequencesynthetic construct;
VL-region of MS-Roche#8 14Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser
Leu Ser Pro Gly 1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser
20 25 30 Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35
40 45 Ile Tyr Gly Ala Ser Ser Arg Ala
Thr Gly Val Pro Ala Arg Phe Ser 50 55
60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Glu 65 70 75
80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Ser Ser Phe Pro
85 90 95 Pro Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 100
105 110 1524DNAartificial sequencesynthetic construct;
CDR3 of VL-region of MS-Roche#3 15cagcaggttt ataatcctcc tgtt
24168PRTartificial sequencesynthetic
construct; CDR3 of VL-region of MS-Roche#3 16Gln Gln Val Tyr Asn Pro
Pro Val 1 5 1724DNAartificial
sequencesynthetic construct; CDR3 of VL-region of MS-Roche#7
17tttcagcttt attctgatcc tttt
24188PRTartificial sequencesynthetic construct; CDR3 of VL-region of
MS-Roche#7 18Phe Gln Leu Tyr Ser Asp Pro Phe 1 5
1924DNAartificial sequencesynthetic construct; CDR3 of VL-region of
MS-Roche#8 19cagcagcttt cttcttttcc tcct
24208PRTartificial sequencesynthetic construct; CDR3 of
VL-region of MS-Roche#8 20Gln Gln Leu Ser Ser Phe Pro Pro 1
5 2139DNAartificial sequencesynthetic construct; CDR3
of VH-region of MS-Roche#3 21cttactcatt atgctcgtta ttatcgttat
tttgatgtt 392213PRTartificial
sequencesynthetic construct; CDR3 of VH-region of MS-Roche#3 22Leu
Thr His Tyr Ala Arg Tyr Tyr Arg Tyr Phe Asp Val 1 5
10 2351DNAartificial sequencesynthetic construct;
CDR3 of VH-region of MS-Roche#7 23ggtaagggta atactcataa gccttatggt
tatgttcgtt attttgatgt t 512417PRTartificial
sequencesynthetic construct; CDR3 of VH-region of MS-Roche#7 24Gly
Lys Gly Asn Thr His Lys Pro Tyr Gly Tyr Val Arg Tyr Phe Asp 1
5 10 15 Val 2545DNAartificial
sequencesynthetic construct; CDR3 of VH-region of MS-Roche#8
25cttctttctc gtggttataa tggttattat cataagtttg atgtt
452615PRTartificial sequencesynthetic construct; CDR3 of VH-region of
MS-Roche#8 26Leu Leu Ser Arg Gly Tyr Asn Gly Tyr Tyr His Lys Phe Asp Val
1 5 10 15
2742PRTartificial sequencesynthetic construct; beta-A4 peptide 27Asp Ala
Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5
10 15 Leu Val Phe Phe Ala Glu Asp
Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25
30 Gly Leu Met Val Gly Gly Val Val Ile Ala
35 40 2817DNAartificial sequencesynthetic
construct; VL-primer for 28gtggtggttc cgatatc
172943DNAartificial sequencesynthetic construct;
VL-primer back 29agcgtcacac tcggtgcggc tttcggctgg ccaagaacgg tta
433017DNAartificial sequencesynthetic construct; control
primer for 30caggaaacag ctatgac
173119DNAartificial sequencesynthetic construct; control primer
back 31taccgttgct cttcacccc
1932360DNAartificial sequencesynthetic construct; VH MS-Roche#3.6H5 x
3.6L2 32caattggtgg aaagcggcgg cggcctggtg caaccgggcg gcagcctgcg tctgagctgc
60gcggcctccg gatttacctt tagcagctat gcgatgagct gggtgcgcca agcccctggg
120aagggtctcg agtgggtgag cgctatttct gagtctggta agactaagta ttatgctgat
180tctgttaagg gtcgttttac catttcacgt gataattcga aaaacaccct gtatctgcaa
240atgaacagcc tgcgtgcgga agatacggcc gtgtattatt gcgcgcgtct tactcattat
300gctcgttatt atcgttattt tgatgtttgg ggccaaggca ccctggtgac ggttagctca
36033120PRTartificial sequencesynthetic construct; VH MS-Roche#3.6H5 x
3.6L2 33Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 1
5 10 15 Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met 20
25 30 Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val Ser Ala 35 40
45 Ile Ser Glu Ser Gly Lys Thr Lys Tyr Tyr Ala Asp Ser
Val Lys Gly 50 55 60
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65
70 75 80 Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85
90 95 Leu Thr His Tyr Ala Arg Tyr Tyr Arg
Tyr Phe Asp Val Trp Gly Gln 100 105
110 Gly Thr Leu Val Thr Val Ser Ser 115
120 34360DNAartificial sequencesynthetic construct; VH MS-Roche#3.6H8
x 3.6L2 34caattggtgg aaagcggcgg cggcctggtg caaccgggcg gcagcctgcg
tctgagctgc 60gcggcctccg gatttacctt tagcagctat gcgatgagct gggtgcgcca
agcccctggg 120aagggtctcg agtgggtgag cgctatttct gagtattcta agtttaagta
ttatgctgat 180tctgttaagg gtcgttttac catttcacgt gataattcga aaaacaccct
gtatctgcaa 240atgaacagcc tgcgtgcgga agatacggcc gtgtattatt gcgcgcgtct
tactcattat 300gctcgttatt atcgttattt tgatgtttgg ggccaaggca ccctggtgac
ggttagctca 36035120PRTartificial sequencesynthetic construct; VH
MS-Roche#3.6H8 x 3.6L2 35Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly Ser Leu 1 5 10
15 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met
20 25 30 Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala 35
40 45 Ile Ser Glu Tyr Ser Lys Phe Lys
Tyr Tyr Ala Asp Ser Val Lys Gly 50 55
60 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr Leu Gln 65 70 75
80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
85 90 95 Leu Thr His Tyr
Ala Arg Tyr Tyr Arg Tyr Phe Asp Val Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser Ser
115 120 36372DNAartificial sequencesynthetic
construct; VH MS-Roche#7.4H2 x 7.2L1 36caattggtgg aaagcggcgg cggcctggtg
caaccgggcg gcagcctgcg tctgagctgc 60gcggcctccg gatttacctt tagcagctat
gcgatgagct gggtgcgcca agcccctggg 120aagggtctcg agtgggtgag cgctattaat
tataatggtg ctcgtattta ttatgctgat 180tctgttaagg gtcgttttac catttcacgt
gataattcga aaaacaccct gtatctgcaa 240atgaacagcc tgcgtgcgga agatacggcc
gtgtattatt gcgcgcgtgg taagggtaat 300actcataagc cttatggtta tgttcgttat
tttgatgttt ggggccaagg caccctggtg 360acggttagct ca
37237124PRTartificial sequencesynthetic
construct; VH MS-Roche#7.4H2 x 7.2L1 37Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly Ser Leu 1 5 10
15 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr Ala Met 20 25 30
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala
35 40 45 Ile Asn Tyr Asn
Gly Ala Arg Ile Tyr Tyr Ala Asp Ser Val Lys Gly 50
55 60 Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr Leu Gln 65 70
75 80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Ala Arg 85 90
95 Gly Lys Gly Asn Thr His Lys Pro Tyr Gly Tyr Val Arg Tyr Phe Asp
100 105 110 Val Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
38372DNAartificial sequencesynthetic construct; VH
MS-Roche#7.9H2 x 7.12L2 38caattggtgg aaagcggcgg cggcctggtg caaccgggcg
gcagcctgcg tctgagctgc 60gcggcctccg gatttacctt tagcagctat gcgatgagct
gggtgcgcca agcccctggg 120aagggtctcg agtgggtgag cgctattaat gctgatggta
atcgtaagta ttatgctgat 180tctgttaagg gtcgttttac catttcacgt gataattcga
aaaacaccct gtatctgcaa 240atgaacagcc tgcgtgcgga agatacggcc gtgtattatt
gcgcgcgtgg taagggtaat 300actcataagc cttatggtta tgttcgttat tttgatgttt
ggggccaagg caccctggtg 360acggttagct ca
37239124PRTartificial sequencesynthetic construct;
VH MS-Roche#7.9H2 x 7.12L2 39Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly Ser Leu 1 5 10
15 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met
20 25 30 Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala 35
40 45 Ile Asn Ala Asp Gly Asn Arg
Lys Tyr Tyr Ala Asp Ser Val Lys Gly 50 55
60 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr Leu Gln 65 70 75
80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
85 90 95 Gly Lys Gly
Asn Thr His Lys Pro Tyr Gly Tyr Val Arg Tyr Phe Asp 100
105 110 Val Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 115 120
40372DNAartificial sequencesynthetic construct; VH MS-Roche#7.9H4 x
7.12L2 40caattggtgg aaagcggcgg cggcctggtg caaccgggcg gcagcctgcg
tctgagctgc 60gcggcctccg gatttacctt tagcagctat gcgatgagct gggtgcgcca
agcccctggg 120aagggtctcg agtgggtgag cgctattaat gctgttggta tgaagaagtt
ttatgctgat 180tctgttaagg gtcgttttac catttcacgt gataattcga aaaacaccct
gtatctgcaa 240atgaacagcc tgcgtgcgga agatacggcc gtgtattatt gcgcgcgtgg
taagggtaat 300actcataagc cttatggtta tgttcgttat tttgatgttt ggggccaagg
caccctggtg 360acggttagct ca
37241124PRTartificial sequencesynthetic construct; VH
MS-Roche#7.9H4 x 7.12L2 41Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly Ser Leu 1 5 10
15 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met
20 25 30 Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala 35
40 45 Ile Asn Ala Val Gly Met Lys Lys
Phe Tyr Ala Asp Ser Val Lys Gly 50 55
60 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr Leu Gln 65 70 75
80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
85 90 95 Gly Lys Gly Asn
Thr His Lys Pro Tyr Gly Tyr Val Arg Tyr Phe Asp 100
105 110 Val Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120
42372DNAartificial sequencesynthetic construct; VH MS-Roche#7.11H1 x
7.11L1 42caattggtgg aaagcggcgg cggcctggtg caaccgggcg gcagcctgcg
tctgagctgc 60gcggcctccg gatttacctt tagcagctat gcgatgagct gggtgcgcca
agcccctggg 120aagggtctcg agtgggtgag cggtattaat gctgctggtt ttcgtactta
ttatgctgat 180tctgttaagg gtcgttttac catttcacgt gataattcga aaaacaccct
gtatctgcaa 240atgaacagcc tgcgtgcgga agatacggcc gtgtattatt gcgcgcgtgg
taagggtaat 300actcataagc cttatggtta tgttcgttat tttgatgttt ggggccaagg
caccctggtg 360acggttagct ca
37243124PRTartificial sequencesynthetic construct; VH
MS-Roche#7.11H1 x 7.11L1 43Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly Ser Leu 1 5 10
15 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala
Met 20 25 30 Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly 35
40 45 Ile Asn Ala Ala Gly Phe
Arg Thr Tyr Tyr Ala Asp Ser Val Lys Gly 50 55
60 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr Leu Gln 65 70 75
80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
85 90 95 Gly Lys
Gly Asn Thr His Lys Pro Tyr Gly Tyr Val Arg Tyr Phe Asp 100
105 110 Val Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 115 120
44372DNAartificial sequencesynthetic construct; VH MS-Roche#7.11H1 x
7.2L1 44caattggtgg aaagcggcgg cggcctggtg caaccgggcg gcagcctgcg tctgagctgc
60gcggcctccg gatttacctt tagcagctat gcgatgagct gggtgcgcca agcccctggg
120aagggtctcg agtgggtgag cggtattaat gctgctggtt ttcgtactta ttatgctgat
180tctgttaagg gtcgttttac catttcacgt gataattcga aaaacaccct gtatctgcaa
240atgaacagcc tgcgtgcgga agatacggcc gtgtattatt gcgcgcgtgg taagggtaat
300actcataagc cttatggtta tgttcgttat tttgatgttt ggggccaagg caccctggtg
360acggttagct ca
37245124PRTartificial sequencesynthetic construct; VH MS-Roche#7.11H1 x
7.2L1 45Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 1
5 10 15 Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met 20
25 30 Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val Ser Gly 35 40
45 Ile Asn Ala Ala Gly Phe Arg Thr Tyr Tyr Ala Asp Ser
Val Lys Gly 50 55 60
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65
70 75 80 Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85
90 95 Gly Lys Gly Asn Thr His Lys Pro Tyr
Gly Tyr Val Arg Tyr Phe Asp 100 105
110 Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 46330DNAartificial
sequencesynthetic construct; VL MS-Roche#3.6H5 x 3.6L2 46gatatcgtgc
tgacccagag cccggcgacc ctgagcctgt ctccgggcga acgtgcgacc 60ctgagctgca
gagcgagcca gtttctttct cgttattatc tggcgtggta ccagcagaaa 120ccaggtcaag
caccgcgtct attaatttat ggcgcgagca gccgtgcaac tggggtcccg 180gcgcgtttta
gcggctctgg atccggcacg gattttaccc tgaccattag cagcctggaa 240cctgaagact
ttgcggttta ttattgccag cagacttata attatcctcc tacctttggc 300cagggtacga
aagttgaaat taaacgtacg
33047110PRTartificial sequencesynthetic construct; VL MS-Roche#3.6H5 x
3.6L2 47Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1
5 10 15 Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Phe Leu Ser Arg Tyr 20
25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu 35 40
45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Val Pro Ala
Arg Phe Ser 50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu 65
70 75 80 Pro Glu Asp Phe
Ala Val Tyr Tyr Cys Gln Gln Thr Tyr Asn Tyr Pro 85
90 95 Pro Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg Thr 100 105
110 48330DNAartificial sequencesynthetic construct; VL MS-Roche#3.6H8 x
3.6L2 48gatatcgtgc tgacccagag cccggcgacc ctgagcctgt ctccgggcga acgtgcgacc
60ctgagctgca gagcgagcca gtttctttct cgttattatc tggcgtggta ccagcagaaa
120ccaggtcaag caccgcgtct attaatttat ggcgcgagca gccgtgcaac tggggtcccg
180gcgcgtttta gcggctctgg atccggcacg gattttaccc tgaccattag cagcctggaa
240cctgaagact ttgcggttta ttattgccag cagacttata attatcctcc tacctttggc
300cagggtacga aagttgaaat taaacgtacg
33049110PRTartificial sequencesynthetic construct; VL MS-Roche#3.6H8 x
3.6L2 49Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1
5 10 15 Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Phe Leu Ser Arg Tyr 20
25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu 35 40
45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Val Pro Ala
Arg Phe Ser 50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu 65
70 75 80 Pro Glu Asp Phe
Ala Val Tyr Tyr Cys Gln Gln Thr Tyr Asn Tyr Pro 85
90 95 Pro Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg Thr 100 105
110 50330DNAartificial sequencesynthetic construct; VL MS-Roche#7.4H2 x
7.2L1 50gatatcgtgc tgacccagag cccggcgacc ctgagcctgt ctccgggcga acgtgcgacc
60ctgagctgca gagcgagcca gtatgttgat cgtacttatc tggcgtggta ccagcagaaa
120ccaggtcaag caccgcgtct attaatttat ggcgcgagca gccgtgcaac tggggtcccg
180gcgcgtttta gcggctctgg atccggcacg gattttaccc tgaccattag cagcctggaa
240cctgaagact ttgcgactta ttattgccag cagatttatt cttttcctca tacctttggc
300cagggtacga aagttgaaat taaacgtacg
33051110PRTartificial sequencesynthetic construct; VL MS-Roche#7.4H2 x
7.2L1 51Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1
5 10 15 Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Tyr Val Asp Arg Thr 20
25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu 35 40
45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Val Pro Ala
Arg Phe Ser 50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu 65
70 75 80 Pro Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Ile Tyr Ser Phe Pro 85
90 95 His Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg Thr 100 105
110 52330DNAartificial sequencesynthetic construct; VL MS-Roche#7.9H2 x
7.12L2 52gatatcgtgc tgacccagag cccggcgacc ctgagcctgt ctccgggcga
acgtgcgacc 60ctgagctgca gagcgagcca gcgttttttt tataagtatc tggcgtggta
ccagcagaaa 120ccaggtcaag caccgcgtct attaatttct ggttcttcta accgtgcaac
tggggtcccg 180gcgcgtttta gcggctctgg atccggcacg gattttaccc tgaccattag
cagcctggaa 240cctgaagact ttgcggttta ttattgcctt cagctttata atattcctaa
tacctttggc 300cagggtacga aagttgaaat taaacgtacg
33053110PRTartificial sequencesynthetic construct; VL
MS-Roche#7.9H2 x 7.12L2 53Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser
Leu Ser Pro Gly 1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Arg Phe Phe Tyr Lys
20 25 30 Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35
40 45 Ile Ser Gly Ser Ser Asn Arg Ala
Thr Gly Val Pro Ala Arg Phe Ser 50 55
60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Glu 65 70 75
80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Leu Gln Leu Tyr Asn Ile Pro
85 90 95 Asn Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 100
105 110 54330DNAartificial sequencesynthetic construct;
VL MS-Roche#7.9H4 x 7.12L2 54gatatcgtgc tgacccagag cccggcgacc ctgagcctgt
ctccgggcga acgtgcgacc 60ctgagctgca gagcgagcca gcgttttttt tataagtatc
tggcgtggta ccagcagaaa 120ccaggtcaag caccgcgtct attaatttct ggttcttcta
accgtgcaac tggggtcccg 180gcgcgtttta gcggctctgg atccggcacg gattttaccc
tgaccattag cagcctggaa 240cctgaagact ttgcggttta ttattgcctt cagctttata
atattcctaa tacctttggc 300cagggtacga aagttgaaat taaacgtacg
33055110PRTartificial sequencesynthetic construct;
VL MS-Roche#7.9H4 x 7.12L2 55Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu
Ser Leu Ser Pro Gly 1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Arg Phe Phe Tyr Lys
20 25 30 Tyr Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35
40 45 Ile Ser Gly Ser Ser Asn Arg
Ala Thr Gly Val Pro Ala Arg Phe Ser 50 55
60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Glu 65 70 75
80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Leu Gln Leu Tyr Asn Ile Pro
85 90 95 Asn Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 100
105 110 56330DNAartificial sequencesynthetic construct;
VL MS-Roche#7.11H1 x 7.11L1 56gatatcgtgc tgacccagag cccggcgacc
ctgagcctgt ctccgggcga acgtgcgacc 60ctgagctgca gagcgagcca gcgtattctt
cgtatttatc tggcgtggta ccagcagaaa 120ccaggtcaag caccgcgtct attaatttat
ggcgcgagca gccgtgcaac tggggtcccg 180gcgcgtttta gcggctctgg atccggcacg
gattttaccc tgaccattag cagcctggaa 240cctgaagact ttgcgactta ttattgccag
caggtttatt ctcctcctca tacctttggc 300cagggtacga aagttgaaat taaacgtacg
33057110PRTartificial sequencesynthetic
construct; VL MS-Roche#7.11H1 x 7.11L1 57Asp Ile Val Leu Thr Gln Ser
Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5
10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Arg Ile Leu Arg Ile 20 25
30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu 35 40 45 Ile
Tyr Gly Ala Ser Ser Arg Ala Thr Gly Val Pro Ala Arg Phe Ser 50
55 60 Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu 65 70
75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Val Tyr Ser Pro Pro 85 90
95 His Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr
100 105 110 58330DNAartificial
sequencesynthetic construct; VL MS-Roche#7.11H1 x 7.2L1 58gatatcgtgc
tgacccagag cccggcgacc ctgagcctgt ctccgggcga acgtgcgacc 60ctgagctgca
gagcgagcca gtatgttgat cgtacttatc tggcgtggta ccagcagaaa 120ccaggtcaag
caccgcgtct attaatttat ggcgcgagca gccgtgcaac tggggtcccg 180gcgcgtttta
gcggctctgg atccggcacg gattttaccc tgaccattag cagcctggaa 240cctgaagact
ttgcgactta ttattgccag cagatttatt cttttcctca tacctttggc 300cagggtacga
aagttgaaat taaacgtacg
33059110PRTartificial sequencesynthetic construct; VL MS-Roche#7.11H1 x
7.2L1 59Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1
5 10 15 Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Tyr Val Asp Arg Thr 20
25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu 35 40
45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Val Pro Ala
Arg Phe Ser 50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu 65
70 75 80 Pro Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Ile Tyr Ser Phe Pro 85
90 95 His Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg Thr 100 105
110 6039DNAartificial sequenceHCDR3 MS-Roche#3.6H5 x 3.6L2 60cttactcatt
atgctcgtta ttatcgttat tttgatgtt
396113PRTartificial sequenceHCDR3 MS-Roche#3.6H5 x 3.6L2 61Leu Thr His
Tyr Ala Arg Tyr Tyr Arg Tyr Phe Asp Val 1 5
10 6239DNAartificial sequenceHCDR3 MS-Roche#3.6H8 x 3.6L2
62cttactcatt atgctcgtta ttatcgttat tttgatgtt
396313PRTartificial sequenceHCDR3 MS-Roche#3.6H8 x 3.6L2 63Leu Thr His
Tyr Ala Arg Tyr Tyr Arg Tyr Phe Asp Val 1 5
10 6451DNAartificial sequenceHCDR3 MS-Roche#7.4H2x7.2L1
64ggtaagggta atactcataa gccttatggt tatgttcgtt attttgatgt t
516517PRTartificial sequenceHCDR3 MS-Roche#7.4H2x7.2L1 65Gly Lys Gly Asn
Thr His Lys Pro Tyr Gly Tyr Val Arg Tyr Phe Asp 1 5
10 15 Val 6651DNAartificial sequenceHCDR3
MS-Roche#7.9H2x7.12L2 66ggtaagggta atactcataa gccttatggt tatgttcgtt
attttgatgt t 516717PRTartificial sequenceHCDR3
MS-Roche#7.9H2x7.12L2 67Gly Lys Gly Asn Thr His Lys Pro Tyr Gly Tyr Val
Arg Tyr Phe Asp 1 5 10
15 Val 6851DNAartificial sequenceHCDR3 MS-Roche#7.9H4x7.12L2
68ggtaagggta atactcataa gccttatggt tatgttcgtt attttgatgt t
516917PRTartificial sequenceHCDR3 MS-Roche#7.9H4x7.12L2 69Gly Lys Gly Asn
Thr His Lys Pro Tyr Gly Tyr Val Arg Tyr Phe Asp 1 5
10 15 Val 7051DNAartificial sequenceHCDR3
MS-Roche#7.11H1x7.11L1 70ggtaagggta atactcataa gccttatggt tatgttcgtt
attttgatgt t 517117PRTartificial sequenceHCDR3
MS-Roche#7.11H1x7.11L1 71Gly Lys Gly Asn Thr His Lys Pro Tyr Gly Tyr Val
Arg Tyr Phe Asp 1 5 10
15 Val 7251DNAartificial sequenceHCDR3 MS-Roche#7.11H1x7.2L1
72ggtaagggta atactcataa gccttatggt tatgttcgtt attttgatgt t
517317PRTartificial sequenceHCDR3 MS-Roche#7.11H1x7.2L1 73Gly Lys Gly Asn
Thr His Lys Pro Tyr Gly Tyr Val Arg Tyr Phe Asp 1 5
10 15 Val 7424DNAartificial sequenceLCDR3
MS-Roche#3.6H5 x 3.6L2 74cagcagactt ataattatcc tcct
24758PRTartificial sequenceLCDR3 MS-Roche#3.6H5 x
3.6L2 75Gln Gln Thr Tyr Asn Tyr Pro Pro 1 5
7624DNAartificial sequenceLCDR3 MS-Roche#3.6H8 x 3.6L2 76cagcagactt
ataattatcc tcct
24778PRTartificial sequenceLCDR3 MS-Roche#3.6H8 x 3.6L2 77Gln Gln Thr Tyr
Asn Tyr Pro Pro 1 5 7824DNAartificial
sequenceLCDR3 MS-Roche#7.4H2x7.2L1 78cagcagattt attcttttcc tcat
24798PRTartificial sequenceLCDR3
MS-Roche#7.4H2x7.2L1 79Gln Gln Ile Tyr Ser Phe Pro His 1 5
8024DNAartificial sequenceLCDR3 MS-Roche#7.9H2x7.12L2
80cttcagcttt ataatattcc taat
24818PRTartificial sequenceLCDR3 MS-Roche#7.9H2x7.12L2 81Leu Gln Leu Tyr
Asn Ile Pro Asn 1 5 8224DNAartificial
sequenceLCDR3 MS-Roche#7.9H4x7.12L2 82cttcagcttt ataatattcc taat
24838PRTartificial sequenceLCDR3
MS-Roche#7.9H4x7.12L2 83Leu Gln Leu Tyr Asn Ile Pro Asn 1 5
8424DNAartificial sequenceLCDR3 MS-Roche#7.11H1x7.11L1
84cagcaggttt attctcctcc tcat
24858PRTartificial sequenceLCDR3 MS-Roche#7.11H1x7.11L1 85Gln Gln Val Tyr
Ser Pro Pro His 1 5 8624DNAartificial
sequenceLCDR3 MS-Roche#7.11H1x7.2L1 86cagcagattt attcttttcc tcat
24878PRTartificial sequenceLCDR3
MS-Roche#7.11H1x7.2L1 87Gln Gln Ile Tyr Ser Phe Pro His 1 5
88378DNAartificial sequencesynthetic construct; VH
MS-Roche#7.9H7 88caggtgcaat tggtggaaag cggcggcggc ctggtgcaac cgggcggcag
cctgcgtctg 60agctgcgcgg cctccggatt tacctttagc agctatgcga tgagctgggt
gcgccaagcc 120cctgggaagg gtctcgagtg ggtgagcgct attaatgctt ctggtactcg
tacttattat 180gctgattctg ttaagggtcg ttttaccatt tcacgtgata attcgaaaaa
caccctgtat 240ctgcaaatga acagcctgcg tgcggaagat acggccgtgt attattgcgc
gcgtggtaag 300ggtaatactc ataagcctta tggttatgtt cgttattttg atgtttgggg
ccaaggcacc 360ctggtgacgg ttagctca
37889126PRTartificial sequencesynthetic construct; VH
MS-Roche#7.9H7 89Gln 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 Thr Phe Ser Ser Tyr
20 25 30 Ala Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Ala Ile Asn Ala Ser Gly Thr Arg
Thr Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr 65 70 75
80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Gly Lys
Gly Asn Thr His Lys Pro Tyr Gly Tyr Val Arg Tyr 100
105 110 Phe Asp Val Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 115 120 125
90330DNAartificial sequencesynthetic construct; VL MS-Roche#7.9H7
90gatatcgtgc tgacccagag cccggcgacc ctgagcctgt ctccgggcga acgtgcgacc
60ctgagctgca gagcgagcca gagcgtgagc agcagctatc tggcgtggta ccagcagaaa
120ccaggtcaag caccgcgtct attaatttat ggcgcgagca gccgtgcaac tggggtcccg
180gcgcgtttta gcggctctgg atccggcacg gattttaccc tgaccattag cagcctggaa
240cctgaagact ttgcgactta ttattgcctt cagatttata atatgcctat tacctttggc
300cagggtacga aagttgaaat taaacgtacg
33091110PRTartificial sequencesynthetic construct; VL MS-Roche#7.9H7
91Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1
5 10 15 Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20
25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu Leu 35 40
45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Val Pro Ala Arg
Phe Ser 50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu 65
70 75 80 Pro Glu Asp Phe Ala
Thr Tyr Tyr Cys Leu Gln Ile Tyr Asn Met Pro 85
90 95 Ile Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg Thr 100 105 110
9251DNAartificial sequencesynthetic construct; HCDR3 MS-Roche#7.9H7
92ggtaagggta atactcataa gccttatggt tatgttcgtt attttgatgt t
519317PRTartificial sequencesynthetic construct; HCDR3 MS-Roche#7.9H7
93Gly Lys Gly Asn Thr His Lys Pro Tyr Gly Tyr Val Arg Tyr Phe Asp 1
5 10 15 Val
9424DNAartificial sequencesynthetic construct; LCDR3 MS-Roche#7.9H7
94cttcagattt ataatatgcc tatt
24958PRTartificial sequencesynthetic construct; LCDR3 MS-Roche#7.9H7
95Leu Gln Ile Tyr Asn Met Pro Ile 1 5
9612PRTartificial sequencesynthetic construct; LCDR1 of MS-Roche#3 96Arg
Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala 1 5
10 977PRTartificial sequencesynthetic construct; LCDR2 of
MS-Roche#3 97Gly Ala Ser Ser Arg Ala Thr 1 5
988PRTartificial sequencesynthetic construct; LCDR3 of MS-Roche#3 98Gln
Gln Val Tyr Asn Pro Pro Val 1 5
9910PRTartificial sequencesynthetic construct; HCDR1 of MS-Roche#3 99Gly
Phe Thr Phe Ser Ser Tyr Ala Met Ser 1 5
10 10017PRTartificial sequencesynthetic construct; HCDR2 of MS-Roche#3
100Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1
5 10 15 Gly
10113PRTartificial sequencesynthetic construct; HCDR3 of MS-Roche#3
101Leu Thr His Tyr Ala Arg Tyr Tyr Arg Tyr Phe Asp Val 1 5
10 1028PRTartificial sequencesynthetic
construct; LCDR3 of MS-Roche#3.1 102Gln Gln Val Tyr Ser Val Pro Pro 1
5 1038PRTartificial sequencesynthetic construct;
LCDR3 of MS-Roche#3.2 103Gln Gln Ile Tyr Ser Tyr Pro Pro 1
5 1048PRTartificial sequencesynthetic construct; LCDR3 of
MS-Roche#3.3 104His Gln Met Ser Ser Tyr Pro Pro 1 5
1058PRTartificial sequencesynthetic construct; LCDR3 of
MS-Roche#3.4 105Gln Gln Thr Tyr Asp Tyr Pro Pro 1 5
1068PRTartificial sequencesynthetic construct; LCDR3 of
MS-Roche#3.5 106Gln Gln Ile Tyr Asp Tyr Pro Pro 1 5
1078PRTartificial sequencesynthetic construct; LCDR3 of
MS-Roche#3.6 107Gln Gln Thr Tyr Asn Tyr Pro Pro 1 5
10817PRTartificial sequencesynthetic construct; HCDR2 of
MS-Roche#3.2H1 108Ala Ile Ser Glu His Gly Leu Asn Ile Tyr Tyr Ala Asp Ser
Val Lys 1 5 10 15
Gly 10917PRTartificial sequencesynthetic construct; HCDR2 of
MS-Roche#3.2H2 109Ala Ile Ser Gln Arg Gly Gln Phe Thr Tyr Tyr Ala Asp Ser
Val Lys 1 5 10 15
Gly 11017PRTartificial sequencesynthetic construct; HCDR2 of
MS-Roche#3.3H1 110Val Ile Ser Glu Lys Ser Arg Phe Ile Tyr Tyr Ala Asp Ser
Val Lys 1 5 10 15
Gly 11117PRTartificial sequencesynthetic construct; HCDR2 of
MS-Roche#3.3H2 111Val Ile Ser Gln Glu Ser Gln Tyr Lys Tyr Tyr Ala Asp Ser
Val Lys 1 5 10 15
Gly 11217PRTartificial sequencesynthetic construct; HCDR2 of
MS-Roche#3.3H3 112Ala Ile Ser Gln Asn Gly Phe His Ile Tyr Tyr Ala Asp Ser
Val Lys 1 5 10 15
Gly 11317PRTartificial sequencesynthetic construct; HCDR2 of
MS-Roche#3.4H1 113Ala Ile Ser Glu Thr Ser Ile Arg Lys Tyr Tyr Ala Asp Ser
Val Lys 1 5 10 15
Gly 11416PRTartificial sequencesynthetic construct; HCDR2 of
MS-Roche#3.4H2 114Val Ile Asp Met Val Gly His Thr Tyr Tyr Ala Asp Ser Val
Lys Gly 1 5 10 15
11517PRTartificial sequencesynthetic construct; HCDR2 of MS-Roche#3.4H3
115Val Ile Ser Gln Thr Gly Arg Lys Ile Tyr Tyr Ala Asp Ser Val Lys 1
5 10 15 Gly
11617PRTartificial sequencesynthetic construct; HCDR2 of MS-Roche#3.4H4
116Ala Ile Ser Glu Thr Gly Met His Ile Tyr Tyr Ala Asp Ser Val Lys 1
5 10 15 Gly
11717PRTartificial sequencesynthetic construct; HCDR2 of MS-Roche#3.4H5
117Val Ile Ser Gln Val Gly Ala His Ile Tyr Tyr Ala Asp Ser Val Lys 1
5 10 15 Gly
11817PRTartificial sequencesynthetic construct; HCDR2 of MS-Roche#3.4H6
118Ala Ile Ser Glu Ser Gly Trp Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1
5 10 15 Gly
11917PRTartificial sequencesynthetic construct; HCDR2 of MS-Roche#3.4H7
119Val Ile Ser Glu Thr Gly Lys Asn Ile Tyr Tyr Ala Asp Ser Val Lys 1
5 10 15 Gly
12017PRTartificial sequencesynthetic construct; HCDR2 of MS-Roche#3.4H8
120Ala Ile Ser Glu His Gly Arg Phe Lys Tyr Tyr Ala Asp Ser Val Lys 1
5 10 15 Gly
12117PRTartificial sequencesynthetic construct; HCDR2 of MS-Roche#3.4H9
121Ala Ile Ser Glu Ser Ser Lys Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1
5 10 15 Gly
12217PRTartificial sequencesynthetic construct; HCDR2 of MS-Roche#3.4H10
122Ala Ile Ser Glu Ser Gly Arg Gly Lys Tyr Tyr Ala Asp Ser Val Lys 1
5 10 15 Gly
12317PRTartificial sequencesynthetic construct; HCDR2 of MS-Roche#3.4H11
123Ala Ile Ser Glu Phe Gly Lys Asn Ile Tyr Tyr Ala Asp Ser Val Lys 1
5 10 15 Gly
12417PRTartificial sequencesynthetic construct; HCDR2 of MS-Roche#3.4H12
124Val Ile Ser Gln Thr Gly Gln Asn Ile Tyr Tyr Ala Asp Ser Val Lys 1
5 10 15 Gly
12517PRTartificial sequencesynthetic construct; HCDR2 of MS-Roche#3.4H13
125Ala Ile Ser Glu Gln Gly Arg Asn Ile Tyr Tyr Ala Asp Ser Val Lys 1
5 10 15 Gly
12617PRTartificial sequencesynthetic construct; HCDR2 of MS-Roche#3.4H14
126Ala Ile Ser Glu Ser Gly Gln Tyr Lys Tyr Tyr Ala Asp Ser Val Lys 1
5 10 15 Gly
12717PRTartificial sequencesynthetic construct; HCDR2 of MS-Roche#3.4H16
127Ala Ile Ser Glu Ser Gly Val Asn Ile Tyr Tyr Ala Asp Ser Val Lys 1
5 10 15 Gly
12817PRTartificial sequencesynthetic construct; HCDR2 of MS-Roche#3.4H17
128Ala Ile Ser Glu Phe Gly Gln Phe Ile Tyr Tyr Ala Asp Ser Val Lys 1
5 10 15 Gly
12917PRTartificial sequencesynthetic construct; HCDR2 of MS-Roche#3.4H18
129Ala Ile Ser Gln Gln Ser Asn Phe Ile Tyr Tyr Ala Asp Ser Val Lys 1
5 10 15 Gly
13012PRTartificial sequencesynthetic construct; LCDR1 of MS-Roche#3.4L7
130Arg Ala Ser Gln Arg Leu Gly Arg Leu Tyr Leu Ala 1 5
10 13112PRTartificial sequencesynthetic construct;
LCDR1 of MS-Roche#3.4L8 131Arg Ala Ser Gln Trp Ile Thr Lys Ser Tyr Leu
Ala 1 5 10 13212PRTartificial
sequencesynthetic construct; LCDR1 of MS-Roche#3.4L9 132Arg Ala Ser Arg
Arg Ile His Val Tyr Tyr Leu Ala 1 5 10
13312PRTartificial sequencesynthetic construct; LCDR1 of
MS-Roche#3.4L11 133Arg Ala Ser Gln Leu Val Gly Arg Ala Tyr Leu Ala 1
5 10 13417PRTartificial
sequencesynthetic construct; HCDR2 of MS-Roche#3.6H1 134Val Ile Ser Glu
Ser Gly Gln Tyr Lys Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly 13517PRTartificial
sequencesynthetic construct; HCDR2 of MS-Roche#3.6H2 135Val Ile Ser Glu
Arg Gly Ile Asn Thr Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly 13617PRTartificial
sequencesynthetic construct; HCDR2 of MS-Roche#3.6H3 136Val Ile Ser Glu
Thr Gly Lys Phe Ile Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly 13717PRTartificial
sequencesynthetic construct; HCDR2 of MS-Roche#3.6H4 137Ala Ile Ser Glu
Arg Gly Arg His Ile Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly 13817PRTartificial
sequencesynthetic construct; HCDR2 of MS-Roche#3.6H5 138Ala Ile Ser Glu
Ser Gly Lys Thr Lys Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly 13917PRTartificial
sequencesynthetic construct; HCDR2 of MS-Roche#3.6H6 139Ala Ile Ser Glu
His Gly Thr Asn Ile Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly 14017PRTartificial
sequencesynthetic construct; HCDR2 of MS-Roche#3.6H8 140Ala Ile Ser Glu
Tyr Ser Lys Phe Lys Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly 14112PRTartificial
sequencesynthetic construct; LCDR1 of MS-Roche#3.6L1 141Arg Ala Ser Gln
Phe Ile Gln Arg Phe Tyr Leu Ala 1 5 10
14212PRTartificial sequencesynthetic construct; LCDR1 of
MS-Roche#3.6L2 142Arg Ala Ser Gln Phe Leu Ser Arg Tyr Tyr Leu Ala 1
5 10 14312PRTartificial
sequencesynthetic construct; LCDR1 of MS-Roche#7 143Arg Ala Ser Gln Ser
Val Ser Ser Ser Tyr Leu Ala 1 5 10
1447PRTartificial sequencesynthetic construct; LCDR2 of MS-Roche#7
144Gly Ala Ser Ser Arg Ala Thr 1 5
1458PRTartificial sequencesynthetic construct; LCDR3 of MS-Roche#7 145Phe
Gln Leu Tyr Ser Asp Pro Phe 1 5
14610PRTartificial sequencesynthetic construct; HCDR1 of MS-Roche#7
146Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser 1 5
10 14717PRTartificial sequencesynthetic construct; HCDR2 of
MS-Roche#7 147Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
Lys 1 5 10 15 Gly
14817PRTartificial sequencesynthetic construct; HCDR3 of MS-Roche#7
148Gly Lys Gly Asn Thr His Lys Pro Tyr Gly Tyr Val Arg Tyr Phe Asp 1
5 10 15 Val
1498PRTartificial sequencesynthetic construct; LCDR3 of MS-Roche#7.1
149His Gln Leu Tyr Ser Ser Pro Tyr 1 5
1508PRTartificial sequencesynthetic construct; LCDR3of MS-Roche#7.2
150Gln Gln Ile Tyr Ser Phe Pro His 1 5
1518PRTartificial sequencesynthetic construct; LCDR3 of MS-Roche#7.3
151His Gln Val Tyr Ser His Pro Phe 1 5
1528PRTartificial sequencesynthetic construct; LCDR3 of MS-Roche#7.4
152Gln Gln Ile Tyr Asn Phe Pro His 1 5
1538PRTartificial sequencesynthetic construct; LCDR3 of MS-Roche#7.5
153His Gln Val Tyr Ser Ser Pro Phe 1 5
1548PRTartificial sequencesynthetic construct; LCDR3 of MS-Roche#7.6
154His Gln Leu Tyr Ser Pro Pro Tyr 1 5
1558PRTartificial sequencesynthetic construct; LCDR3 of MS-Roche#7.7
155His Gln Val Tyr Ser Ala Pro Phe 1 5
1568PRTartificial sequencesynthetic construct; LCDR3 of MS-Roche#7.8
156His Gln Val Tyr Ser Phe Pro Ile 1 5
1578PRTartificial sequencesynthetic construct; LCDR3 of MS-Roche#7.9
157Leu Gln Ile Tyr Asn Met Pro Ile 1 5
1588PRTartificial sequencesynthetic construct; LCDR3 of MS-Roche#7.10
158Gln Gln Val Tyr Asn Pro Pro His 1 5
1598PRTartificial sequencesynthetic construct; LCDR3 of MS-Roche#7.11
159Gln Gln Val Tyr Ser Pro Pro His 1 5
16012PRTartificial sequencesynthetic construct; LCDR1 of MS-Roche#7.12
160Arg Ala Ser Gln Tyr Val Ser Ser Pro Tyr Leu Ala 1 5
10 1617PRTartificial sequencesynthetic construct;
LCDR2 of MS-Roche#7.12 161Gly Ser Ser Asn Arg Ala Thr 1 5
1628PRTartificial sequencesynthetic construct; LCDR3 of
MS-Roche#7.12 162Leu Gln Leu Tyr Asn Ile Pro Asn 1 5
16310PRTartificial sequencesynthetic construct; HCDR1 of
MS-Roche#7.12 163Gly Phe Thr Phe Ser Ser Tyr Gly Met Ser 1
5 10 16417PRTartificial sequencesynthetic construct;
HCDR2 of MS-Roche#7.12 164Asn Ile Ser Gly Ser Gly Ser Ser Thr Tyr Tyr Ala
Asp Ser Val Lys 1 5 10
15 Gly 16517PRTartificial sequencesynthetic construct; HCDR3 of
MS-Roche#7.12 165Gly Lys Gly Asn Thr His Lys Pro Tyr Gly Tyr Val Arg Tyr
Phe Asp 1 5 10 15
Val 1668PRTartificial sequencesynthetic construct; LCDR3 of MS-Roche#7.13
166His Gln Val Tyr Ser Pro Pro Phe 1 5
16717PRTartificial sequencesynthetic construct; HCDR2 of MS-Roche#7.2H1
167Ala Ile Asn Ala Asn Gly Leu Lys Lys Tyr Tyr Ala Asp Ser Val Lys 1
5 10 15 Gly
16817PRTartificial sequencesynthetic construct; HCDR2 of MS-Roche#7.2H2
168Ala Ile Asn Gly Thr Gly Met Lys Lys Tyr Tyr Ala Asp Ser Val Lys 1
5 10 15 Gly
16917PRTartificial sequencesynthetic construct; HCDR2 of MS-Roche#7.2H3
169Ala Ile Asn Ala Asn Gly Tyr Lys Thr Tyr Tyr Ala Asp Ser Val Lys 1
5 10 15 Gly
17017PRTartificial sequencesynthetic construct; HCDR2 of MS-Roche#7.2H4
170Ala Ile Asn Ser Lys Gly Ser Arg Ile Tyr Tyr Ala Asp Ser Val Lys 1
5 10 15 Gly
17117PRTartificial sequencesynthetic construct; HCDR2 of MS-Roche#7.2H5
171Ala Ile Asn Ala Thr Gly Arg Ser Lys Tyr Tyr Ala Asp Ser Val Lys 1
5 10 15 Gly
17217PRTartificial sequencesynthetic construct; HCDR2 of MS-Roche#7.2H6
172Ala Ile Asn Ala Arg Gly Asn Arg Thr Tyr Tyr Ala Asp Ser Val Lys 1
5 10 15 Gly
17317PRTartificial sequencesynthetic construct; HCDR2 of MS-Roche#7.2H7
173Ala Ile Asn Ser Arg Gly Ser Asp Thr His Tyr Ala Asp Ser Val Lys 1
5 10 15 Gly
17417PRTartificial sequencesynthetic construct; HCDR2 of MS-Roche#7.2H8
174Ala Ile Asn Ala Ser Gly His Lys Thr Tyr Tyr Ala Asp Ser Val Lys 1
5 10 15 Gly
17512PRTartificial sequencesynthetic construct; LCDR1 of MS-Roche#7.2L1
175Arg Ala Ser Gln Tyr Val Asp Arg Thr Tyr Leu Ala 1 5
10 17612PRTartificial sequencesynthetic construct;
LCDR1 of MS-Roche#7.2L2 176Arg Ala Ser Gln Tyr Ile Ser Phe Arg Tyr Leu
Ala 1 5 10 17712PRTartificial
sequencesynthetic construct; LCDR1 of MS-Roche#7.2L4 177Arg Ala Ser Gln
Phe Ile Arg Arg Ser Tyr Leu Ala 1 5 10
1788PRTartificial sequencesynthetic construct; LCDR3 of
MS-Roche#7.3H1 178His Gln Val Tyr Ser His Pro Phe 1 5
17917PRTartificial sequencesynthetic construct; HCDR2 of
MS-Roche#7.3H1 179Ala Ile Ser Ala Ile Ser Asn Lys Thr Tyr Tyr Ala Asp Ser
Val Lys 1 5 10 15
Gly 18012PRTartificial sequencesynthetic construct; LCDR1 of
MS-Roche#7.3L1 180Arg Ala Ser Gln Tyr Leu His Tyr Gly Tyr Leu Ala 1
5 10 18117PRTartificial
sequencesynthetic construct; HCDR2 of MS-Roche#7.4H1 181Ala Ile Asn Ala
Thr Gly Tyr Arg Thr Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly 18217PRTartificial
sequencesynthetic construct; HCDR2 of MS-Roche#7.4H2 182Ala Ile Asn Tyr
Asn Gly Ala Arg Ile Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly 1838PRTartificial
sequencesynthetic construct; LCDR3 of MS-Roche#7.9H1 183Leu Gln Ile Tyr
Asn Met Pro Ile 1 5 18417PRTartificial
sequencesynthetic construct; HCDR2 of MS-Roche#7.9H1 184Ala Ile Asn Ala
Asn Gly Gln Arg Lys Phe Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly 18517PRTartificial
sequencesynthetic construct; HCDR2 of MS-Roche#7.9H2 185Ala Ile Asn Ala
Asp Gly Asn Arg Lys Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly 18617PRTartificial
sequencesynthetic construct; HCDR2 of MS-Roche#7.9H3 186Ala Ile Asn Tyr
Gln Gly Asn Arg Lys Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly 18717PRTartificial
sequencesynthetic construct; HCDR2 of MS-Roche#7.9H4 187Ala Ile Asn Ala
Val Gly Met Lys Lys Phe Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly 18817PRTartificial
sequencesynthetic construct; HCDR2 of MS-Roche#7.9H5 188Ala Ile Asn His
Ala Gly Asn Lys Lys Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly 18912PRTartificial
sequencesynthetic construct; LCDR1 of MS-Roche#7.9L1 189Arg Ala Ser Gln
Arg Leu Ser Pro Arg Tyr Leu Ala 1 5 10
19012PRTartificial sequencesynthetic construct; LCDR1 of
MS-Roche#7.9L2 190Arg Ala Ser Gln Tyr Leu His Lys Arg Tyr Leu Ala 1
5 10 19117PRTartificial
sequencesynthetic construct; HCDR2 of MS-Roche#7.9H6 191Ala Ile Asn Ala
Ser Gly Arg Leu Thr Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly 19217PRTartificial
sequencesynthetic construct; HCDR2 of MS-Roche#7.9H7 192Ala Ile Asn Ala
Ser Gly Thr Arg Thr Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly 19317PRTartificial
sequencesynthetic construct; HCDR2 of MS-Roche#7.9H8 193Ala Ile Asn Ala
Ser Gly Ser Lys Ile Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly 19417PRTartificial
sequencesynthetic construct; HCDR2 of MS-Roche#7.9H9 194Ala Ile Asn Gly
Lys Gly Asn Lys Lys Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly 19517PRTartificial
sequencesynthetic construct; HCDR2 of MS-Roche#7.11H1 195Gly Ile Asn Ala
Ala Gly Phe Arg Thr Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly 19617PRTartificial
sequencesynthetic construct; HCDR2 of MS-Roche#7.11H2 196Ala Ile Asn Ala
Asn Gly Tyr Lys Lys Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly 19717PRTartificial
sequencesynthetic construct; HCDR2 of MS-Roche#7.11H3 197Gly Ile Asn Ala
Asn Gly Asn Arg Thr Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly 19817PRTartificial
sequencesynthetic construct; HCDR2 of MS-Roche#7.11H4 198Ala Ile Asn Ala
Asn Gly Tyr Lys Thr Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly 19917PRTartificial
sequencesynthetic construct; HCDR2 of MS-Roche#7.11H5 199Ala Ile Asn Ala
His Gly Gln Arg Thr Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly 20012PRTartificial
sequencesynthetic construct; LCDR1 of MS-Roche#7.11L1 200Arg Ala Ser Gln
Arg Ile Leu Arg Ile Tyr Leu Ala 1 5 10
20112PRTartificial sequencesynthetic construct; LCDR1 of
MS-Roche#7.12H1 201Arg Ala Ser Gln Tyr Val Phe Arg Arg Tyr Leu Ala 1
5 10 2028PRTartificial
sequencesynthetic construct; LCDR3 of MS-Roche#7.12H1 202Leu Gln Leu Tyr
Asn Ile Pro Asn 1 5 20310PRTartificial
sequencesynthetic construct; HCDR1 of MS-Roche#7.12H1 203Gly Phe Thr Phe
Ser Ser Tyr Gly Met Ser 1 5 10
20417PRTartificial sequencesynthetic construct; HCDR2 of MS-Roche#7.12H1
204Asn Ile Asn Gly Asn Gly Asn Arg Lys Tyr Tyr Ala Asp Ser Val Lys 1
5 10 15 Gly
20517PRTartificial sequencesynthetic construct; HCDR2 of MS-Roche#7.12L1
205Asn Ile Ser Gly Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1
5 10 15 Gly
20612PRTartificial sequencesynthetic construct; LCDR1 of MS-Roche#7.12L2
206Arg Ala Ser Gln Arg Phe Phe Tyr Lys Tyr Leu Ala 1 5
10 20712PRTartificial sequencesynthetic construct;
LCDR1 of MS-Roche#7.12L3 207Arg Ala Ser Gln Phe Val Arg Arg Gly Phe Leu
Ala 1 5 10 20812PRTartificial
sequencesynthetic construct; LCDR1 of MS-Roche#7.12L4 208Arg Ala Ser Gln
Arg Leu Lys Arg Ser Tyr Leu Ala 1 5 10
20912PRTartificial sequencesynthetic construct; LCDR1 of
MS-Roche#7.12L6 209Arg Ala Ser Gln Tyr Leu Trp Tyr Arg Tyr Leu Ala 1
5 10 21012PRTartificial
sequencesynthetic construct; LCDR1 of MS-Roche#7.12L7 210Arg Ala Ser Gln
Trp Ile Arg Lys Thr Tyr Leu Ala 1 5 10
21112PRTartificial sequencesynthetic construct; LCDR1 of MS-Roche#8
211Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala 1 5
10 2127PRTartificial sequencesynthetic construct;
LCDR2 of MS-Roche#8 212Gly Ala Ser Ser Arg Ala Thr 1 5
2138PRTartificial sequencesynthetic construct; LCDR3 of MS-Roche#8
213Gln Gln Leu Ser Ser Phe Pro Pro 1 5
21410PRTartificial sequencesynthetic construct; HCDR1 of MS-Roche#8
214Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser 1 5
10 21517PRTartificial sequencesynthetic construct; HCDR2 of
MS-Roche#8 215Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
Lys 1 5 10 15 Gly
21615PRTartificial sequencesynthetic construct; HCDR3 of MS-Roche#8
216Leu Leu Ser Arg Gly Tyr Asn Gly Tyr Tyr His Lys Phe Asp Val 1
5 10 15 2178PRTartificial
sequencesynthetic construct; LCDR3 of MS-Roche#8.1 217Gln Gln Leu Ser Asn
Tyr Pro Pro 1 5 2188PRTartificial
sequencesynthetic construct; LCDR3 of MS-Roche#8.2 218Gln Gln Leu Ser Ser
Tyr Pro Pro 1 5 21917PRTartificial
sequencesynthetic construct; HCDR2 of MS-Roche#8.1H1 219Ala Ile Ser Arg
Ser Gly Ser Asn Ile Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly 2208PRTartificial
sequencesynthetic construct; LCDR3 of MS-Roche#8.2H1 220Gln Gln Leu Ser
Ser Tyr Pro Pro 1 5 22117PRTartificial
sequencesynthetic construct; HCDR2 of MS-Roche#8.2H1 221Ala Ile Ser Ile
Thr Gly Arg Arg Lys Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly 22217PRTartificial
sequencesynthetic construct; HCDR2 of MS-Roche#8.2H2 222Ala Ile Ser Arg
Thr Gly Ser Lys Thr Tyr Tyr Ala Asp Ser Val Lys 1 5
10 15 Gly 22316PRTartificial
sequencesynthetic construct; HCDR2 of MS-Roche#8.2H4 223Ala Thr Ser Val
Lys Gly Lys Thr Tyr Tyr Ala Asp Ser Val Lys Gly 1 5
10 15 22412PRTartificial
sequencesynthetic construct; LCDR1 of MS-Roche#8.2L1 224Arg Ala Ser Gln
Arg Val Ser Gly Arg Tyr Leu Ala 1 5 10
225109PRTartificial sequencesynthetic construct; VL kappa1 225Asp
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 Gly Ile Ser Ser Tyr 20
25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65
70 75 80 Glu Asp Phe Ala Xaa
Tyr Tyr Cys Xaa Gln Xaa Xaa Xaa Xaa Xaa Xaa 85
90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg Thr 100 105
226114PRTartificial sequencesynthetic construct; VL kappa2 226Asp Ile Val
Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5
10 15 Glu Pro Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser Leu Leu His Ser 20 25
30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro
Gly Gln Ser 35 40 45
Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50
55 60 Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70
75 80 Ser Arg Val Glu Ala Glu Asp Val Gly
Val Tyr Tyr Cys Xaa Gln Xaa 85 90
95 Xaa Xaa Xaa Xaa Xaa Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys 100 105 110
Arg Thr 227110PRTartificial sequencesynthetic construct; VL kappa3
227Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1
5 10 15 Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20
25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu Leu 35 40
45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Val Pro Ala Arg
Phe Ser 50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu 65
70 75 80 Pro Glu Asp Phe Ala
Xaa Tyr Tyr Cys Xaa Gln Xaa Xaa Xaa Xaa Xaa 85
90 95 Xaa Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg Thr 100 105 110
228115PRTartificial sequencesynthetic construct; VL kappa4 228Asp Ile Val
Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5
10 15 Glu Arg Ala Thr Ile Asn Cys Arg
Ser Ser Gln Ser Val Leu Tyr Ser 20 25
30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln 35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50
55 60 Pro Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70
75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val
Ala Val Tyr Tyr Cys Xaa Gln 85 90
95 Xaa Xaa Xaa Xaa Xaa Xaa Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile 100 105 110
Lys Arg Thr 115 229111PRTartificial sequencesynthetic construct;
VL lambda1 229Asp Ile Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly
Gln 1 5 10 15 Arg
Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn
20 25 30 Tyr Val Ser Trp Tyr
Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35
40 45 Ile Tyr Asp Asn Asn Gln Arg Pro Ser
Gly Val Pro Asp Arg Phe Ser 50 55
60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr
Gly Leu Gln 65 70 75
80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Xaa Asp Xaa Xaa Xaa
85 90 95 Xaa Xaa Xaa Val
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100
105 110 230112PRTartificial sequencesynthetic
construct; VL lambda2 230Asp Ile Ala Leu Thr Gln Pro Ala Ser Val Ser Gly
Ser Pro Gly Gln 1 5 10
15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr
20 25 30 Asn Tyr Val
Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35
40 45 Met Ile Tyr Asp Val Ser Asn Arg
Pro Ser Gly Val Ser Asn Arg Phe 50 55
60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile
Ser Gly Leu 65 70 75
80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Xaa Asp Xaa Xaa
85 90 95 Xaa Xaa Xaa Xaa
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100
105 110 231109PRTartificial
sequencesynthetic construct; VL lambda3 231Asp Ile Glu Leu Thr Gln Pro
Pro Ser Val Ser Val Ala Pro Gly Gln 1 5
10 15 Thr Ala Arg Ile Ser Cys Ser Gly Asp Ala Leu
Gly Asp Lys Tyr Ala 20 25
30 Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile
Tyr 35 40 45 Asp
Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50
55 60 Asn Ser Gly Asn Thr Ala
Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu 65 70
75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Xaa Asp
Xaa Xaa Xaa Xaa Xaa 85 90
95 Xaa Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105 232127PRTartificial
sequencesynthetic construct; VH1A 232Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ser 1 5 10
15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser
Ser Tyr 20 25 30
Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45 Gly Gly Ile Ile
Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Ile Thr Ala
Asp Glu Ser Thr Ser Thr Ala Tyr 65 70
75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90
95 Ala Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
100 105 110 Xaa Xaa Asp
Xaa Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 125 233127PRTartificial sequencesynthetic
construct; VH1B 233Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 Tyr Met His Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly
Thr Asn Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser
Thr Ala Tyr 65 70 75
80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100
105 110 Xaa Xaa Asp Xaa Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser 115 120
125 234128PRTartificial sequencesynthetic construct; VH2 234Gln
Val Gln Leu Lys Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln 1
5 10 15 Thr Leu Thr Leu Thr Cys
Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20
25 30 Gly Val Gly Val Gly Trp Ile Arg Gln Pro
Pro Gly Lys Ala Leu Glu 35 40
45 Trp Leu Ala Leu Ile Asp Trp Asp Asp Asp Lys Tyr Tyr Ser
Thr Ser 50 55 60
Leu Lys Thr Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65
70 75 80 Val Leu Thr Met Thr
Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85
90 95 Cys Ala Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 100 105
110 Xaa Xaa Xaa Asp Xaa Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 120 125
235127PRTartificial sequencesynthetic construct; VH3 235Gln 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 Thr Phe Ser Ser Tyr 20 25
30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 100 105 110 Xaa
Xaa Asp Xaa Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 125 236126PRTartificial
sequencesynthetic construct; VH4 236Gln Val Gln Leu Gln Glu Ser Gly Pro
Gly Leu Val Lys Pro Ser Glu 1 5 10
15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser
Ser Tyr 20 25 30
Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45 Gly Tyr Ile Tyr
Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50
55 60 Ser Arg Val Thr Ile Ser Val Asp
Thr Ser Lys Asn Gln Phe Ser Leu 65 70
75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val
Tyr Tyr Cys Ala 85 90
95 Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
100 105 110 Xaa Asp Xaa
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 125 237127PRTartificial sequencesynthetic
construct; VH5 237Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Glu 1 5 10 15
Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr
20 25 30 Trp Ile Gly Trp Val
Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35
40 45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp
Thr Arg Tyr Ser Pro Ser Phe 50 55
60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser
Thr Ala Tyr 65 70 75
80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
85 90 95 Ala Arg Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100
105 110 Xaa Xaa Asp Xaa Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser 115 120
125 238130PRTartificial sequencesynthetic construct; VH6 238Gln
Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1
5 10 15 Thr Leu Ser Leu Thr Cys
Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20
25 30 Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser
Pro Gly Arg Gly Leu Glu 35 40
45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp
Tyr Ala 50 55 60
Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn 65
70 75 80 Gln Phe Ser Leu Gln
Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85
90 95 Tyr Tyr Cys Ala Arg Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 100 105
110 Xaa Xaa Xaa Xaa Xaa Asp Xaa Trp Gly Gln Gly Thr Leu Val Thr
Val 115 120 125 Ser
Ser 130 239327DNAartificial sequencesynthetic construct; VL kappa1
239gatatccaga tgacccagag cccgtctagc ctgagcgcga gcgtgggtga tcgtgtgacc
60attacctgca gagcgagcca gggcattagc agctatctgg cgtggtacca gcagaaacca
120ggtaaagcac cgaaactatt aatttatgca gccagcagct tgcaaagcgg ggtcccgtcc
180cgttttagcg gctctggatc cggcactgat tttaccctga ccattagcag cctgcaacct
240gaagactttg cgnnntatta ttgcnnncag nnnnnnnnnn nnnnnnnnac ctttggccag
300ggtacgaaag ttgaaattaa acgtacg
327240328DNAartificial sequencesynthetic construct; VL kappa2
240gatatcgtga tgacccagag cccactgagc ctgccagtga ctccgggcga gcctgcgagc
60attagctgca gaagcagcca aagcctgctg catagcaacg gctataacta tctggattgg
120taccttcaaa aaccaggtca aagcccgcag ctattaattt atctgggcag caaccgtgcc
180agtggggtcc cggatcgttt tagcggctct ggatccggca ccgattttac cctgaaaatt
240agccgtgtgg aagctgaaga cgtgggcgtg tattattgcn cagnnnnnna cctttggcca
300gggtacgaaa gttgaaatta aacgtacg
328241330DNAartificial sequencesynthetic construct; VL kappa3
241gatatcgtgc tgacccagag cccggcgacc ctgagcctgt ctccgggcga acgtgcgacc
60ctgagctgca gagcgagcca gagcgtgagc agcagctatc tggcgtggta ccagcagaaa
120ccaggtcaag caccgcgtct attaatttat ggcgcgagca gccgtgcaac tggggtcccg
180gcgcgtttta gcggctctgg atccggcacg gattttaccc tgaccattag cagcctggaa
240cctgaagact ttgcgnnnta ttattgcnnn cagnnnnnnn nnnnnnnnnn nacctttggc
300cagggtacga aagttgaaat taaacgtacg
330242345DNAartificial sequencesynthetic construct; VL kappa4
242gatatcgtga tgacccagag cccggatagc ctggcggtga gcctgggcga acgtgcgacc
60attaactgca gaagcagcca gagcgtgctg tatagcagca acaacaaaaa ctatctggcg
120tggtaccagc agaaaccagg tcagccgccg aaactattaa tttattgggc atccacccgt
180gaaagcgggg tcccggatcg ttttagcggc tctggatccg gcactgattt taccctgacc
240atttcgtccc tgcaagctga agacgtggcg gtgtattatt gcnnncagnn nnnnnnnnnn
300nnnnnnacct ttggccaggg tacgaaagtt gaaattaaac gtacg
345243322DNAartificial sequencesynthetic construct; VL lambda1
243gatatcgtgc tgacccagcc gccttcagtg agtggcgcac caggtcagcg tgtgaccatc
60tcgtgtagcg gcagcagcag caacattggc agcaactatg tgagctggta ccagcagttg
120cccgggacgg cgccgaaact gctgatttat gataacaacc agcgtccctc aggcgtgccg
180gatcgtttta gcggatccaa aagcggcacc agcgcgagcc ttgcgattac gggcctgcaa
240agcgaagacg aagcggatta ttattgccag tctngatnnn nnngtgtttg gcggcggcac
300gaagttaacc gttcttggcc ag
322244336DNAartificial sequencesynthetic construct; VL lambda2
244gatatcgcac tgacccagcc agcttcagtg agcggctcac caggtcagag cattaccatc
60tcgtgtacgg gtactagcag cgatgtgggc ggctataact atgtgagctg gtaccagcag
120catcccggga aggcgccgaa actgatgatt tatgatgtga gcaaccgtcc ctcaggcgtg
180agcaaccgtt ttagcggatc caaaagcggc aacaccgcga gcctgaccat tagcggcctg
240caagcggaag acgaagcgga ttattattgc cagnnngatn nnnnnnnnnn nnnnnnngtg
300tttggcggcg gcacgaagtt aaccgttctt ggccag
336245327DNAartificial sequencesynthetic construct; VL lambda3
245gatatcgaac tgacccagcc gccttcagtg agcgttgcac caggtcagac cgcgcgtatc
60tcgtgtagcg gcgatgcgct gggcgataaa tacgcgagct ggtaccagca gaaacccggg
120caggcgccag ttctggtgat ttatgatgat tctgaccgtc cctcaggcat cccggaacgc
180tttagcggat ccaacagcgg caacaccgcg accctgacca ttagcggcac tcaggcggaa
240gacgaagcgg attattattg ccagnnngat nnnnnnnnnn nnnnnnnngt gtttggcggc
300ggcacgaagt taaccgttct tggccag
327246382DNAartificial sequencesynthetic construct; VH1A 246caggtgcaat
tggttcagtc tggcgcggaa gtgaaaaaac cgggcagcag cgtgaaagtg 60agctgcaaag
cctccggagg cacttttagc agctatgcga ttagctgggt gcgccaagcc 120cctgggcagg
gtctcgagtg gatgggcggc attattccga tttttggcac ggcgaactac 180gcgcagaagt
ttcagggccg ggtgaccatt accgcggatg aaagcaccag caccgcgtat 240atggaactga
gcagcctgcg tagcgaagat acggccgtgt attattgcgc gcgtnnnnnn 300nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn ngatnnntgg ggccaaggca 360ccctggtgac
ggttagctca gc
382247383DNAartificial sequencesynthetic construct; VH1B 247caggtgcaat
tggttcagag cggcgcggaa gtgaaaaaac cgggcgcgag cgtgaaagtg 60agctgcaaag
cctccggata tacctttacc agctattata tgcactgggt ccgccaagcc 120cctgggcagg
gtctcgagtg gatgggctgg attaacccga atagcggcgg cacgaactac 180gcgcagaagt
ttcagggccg ggtgaccatg acccgtgata ccagcattag caccgcgtat 240atggaactga
gcagcctgcg tagcgaagat acggccgtgt attattgcgc gcgtnnnnnn 300nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nngatnnntg gggccaaggc 360accctggtga
cggttagctc agc
383248386DNAartificial sequencesynthetic construct; VH2 248caggtgcaat
tgaaagaaag cggcccggcc ctggtgaaac cgacccaaac cctgaccctg 60acctgtacct
tttccggatt tagcctgtcc acgtctggcg ttggcgtggg ctggattcgc 120cagccgcctg
ggaaagccct cgagtggctg gctctgattg attgggatga tgataagtat 180tatagcacca
gcctgaaaac gcgtctgacc attagcaaag atacttcgaa aaatcaggtg 240gtgctgacta
tgaccaacat ggacccggtg gatacggcca cctattattg cgcgcgtnnn 300nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnngatnn ntggggccaa 360ggcaccctgg
tgacggttag ctcagc
386249349DNAartificial sequencesynthetic construct; VH3 249caggtgcaat
tggtggaaag cggcggcggc ctggtgcaac cgggcggcag cctgcgtctg 60agctgcgcgg
cctccggatt tacctttagc agctatgcga tgagctgggt gcgccaagcc 120cctgggaagg
gtctcgagtg ggtgagcgcg attagcggta gcggcggcag cacctattat 180gcggatagcg
tgaaaggccg ttttaccatt tcacgtgata attcgaaaaa caccctgtat 240ctgcaaatga
acagcctgcg tgcggaagat acggccgtgt attattgcgc gcgtnnnnnn 300nnnnnnnnnn
gatntggggc caaggcaccc tggtgacggt tagctcagc
349250346DNAartificial sequencesynthetic construct; VH4 250caggtgcaat
tgcaagaaag tggtccgggc ctggtgaaac cgagcgaaac cctgagcctg 60acctgcaccg
tttccggagg cagcattagc agctattatt ggagctggat tcgccagccg 120cctgggaagg
gtctcgagtg gattggctat atttattata gcggcagcac caactataat 180ccgagcctga
aaagccgggt gaccattagc gttgatactt cgaaaaacca gtttagcctg 240aaactgagca
gcgtgacggc ggcggatacg gccgtgtatt attgcgcgcg tnnnnnnnnn 300nnnnnnngat
ntggggccaa ggcaccctgg tgacggttag ctcagc
346251349DNAartificial sequencesynthetic construct; VH5 251caggtgcaat
tggttcagag cggcgcggaa gtgaaaaaac cgggcgaaag cctgaaaatt 60agctgcaaag
gttccggata ttcctttacg agctattgga ttggctgggt gcgccagatg 120cctgggaagg
gtctcgagtg gatgggcatt atttatccgg gcgatagcga tacccgttat 180tctccgagct
ttcagggcca ggtgaccatt agcgcggata aaagcattag caccgcgtat 240cttcaatgga
gcagcctgaa agcgagcgat acggccatgt attattgcgc gcgtnnnnnn 300nnnnnnnnnn
gatntggggc caaggcaccc tggtgacggt tagctcagc
349252392DNAartificial sequencesynthetic construct; VH6 252caggtgcaat
tgcaacagtc tggtccgggc ctggtgaaac cgagccaaac cctgagcctg 60acctgtgcga
tttccggaga tagcgtgagc agcaacagcg cggcgtggaa ctggattcgc 120cagtctcctg
ggcgtggcct cgagtggctg ggccgtacct attatcgtag caaatggtat 180aacgattatg
cggtgagcgt gaaaagccgg attaccatca acccggatac ttcgaaaaac 240cagtttagcc
tgcaactgaa cagcgtgacc ccggaagata cggccgtgta ttattgcgcg 300cgtnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn ngatnnntgg 360ggccaaggca
ccctggtgac ggttagctca gc
3922534151DNAartificial sequencesynthetic construct; pMORPH 18 Fab_5'
253tctagataac gagggcaaaa aatgaaaaag acagctatcg cgattgcagt ggcactggct
60ggtttcgcta ccgtagcgca ggccgatatc gtgctgaccc agagcccggc gaccctgagc
120ctgtctccgg gcgaacgtgc gaccctgagc tgcagagcga gccagagcgt gagcagcagc
180tatctggcgt ggtaccagca gaaaccaggt caagcaccgc gtctattaat ttatggcgcg
240agcagccgtg caactggggt cccggcgcgt tttagcggct ctggatccgg cacggatttt
300accctgacca ttagcagcct ggaacctgaa gactttgcgg tgtattattg ccagcagcat
360tataccaccc cgccgacctt tggccagggt acgaaagttg aaattaaacg tacggtggct
420gctccgagcg tgtttatttt tccgccgagc gatgaacaac tgaaaagcgg cacggcgagc
480gtggtgtgcc tgctgaacaa cttttatccg cgtgaagcga aagttcagtg gaaagtagac
540aacgcgctgc aaagcggcaa cagccaggaa agcgtgaccg aacaggatag caaagatagc
600acctattctc tgagcagcac cctgaccctg agcaaagcgg attatgaaaa acataaagtg
660tatgcgtgcg aagtgaccca tcaaggtctg agcagcccgg tgactaaatc ttttaatcgt
720ggcgaggcct gataagcatg cgtaggagaa aataaaatga aacaaagcac tattgcactg
780gcactcttac cgttgctctt cacccctgtt accaaagccg aagtgcaatt ggtggaaagc
840ggcggcggcc tggtgcaacc gggcggcagc ctgcgtctga gctgcgcggc ctccggattt
900acctttagca gctatgcgat gagctgggtg cgccaagccc ctgggaaggg tctcgagtgg
960gtgagcgcga ttagcggtag cggcggcagc acctattatg cggatagcgt gaaaggccgt
1020tttaccattt cacgtgataa ttcgaaaaac accctgtatc tgcaaatgaa cagcctgcgt
1080gcggaagata cggccgtgta ttattgcgcg cgttggggcg gcgatggctt ttatgcgatg
1140gattattggg gccaaggcac cctggtgacg gttagctcag cgtcgaccaa aggtccaagc
1200gtgtttccgc tggctccgag cagcaaaagc accagcggcg gcacggctgc cctgggctgc
1260ctggttaaag attatttccc ggaaccagtc accgtgagct ggaacagcgg ggcgctgacc
1320agcggcgtgc atacctttcc ggcggtgctg caaagcagcg gcctgtatag cctgagcagc
1380gttgtgaccg tgccgagcag cagcttaggc actcagacct atatttgcaa cgtgaaccat
1440aaaccgagca acaccaaagt ggataaaaaa gtggaaccga aaagcgaatt cgggggaggg
1500agcgggagcg gtgattttga ttatgaaaag atggcaaacg ctaataaggg ggctatgacc
1560gaaaatgccg atgaaaacgc gctacagtct gacgctaaag gcaaacttga ttctgtcgct
1620actgattacg gtgctgctat cgatggtttc attggtgacg tttccggcct tgctaatggt
1680aatggtgcta ctggtgattt tgctggctct aattcccaaa tggctcaagt cggtgacggt
1740gataattcac ctttaatgaa taatttccgt caatatttac cttccctccc tcaatcggtt
1800gaatgtcgcc cttttgtctt tggcgctggt aaaccatatg aattttctat tgattgtgac
1860aaaataaact tattccgtgg tgtctttgcg tttcttttat atgttgccac ctttatgtat
1920gtattttcta cgtttgctaa catactgcgt aataaggagt cttgataagc ttgacctgtg
1980aagtgaaaaa tggcgcagat tgtgcgacat tttttttgtc tgccgtttaa tgaaattgta
2040aacgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
2100caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
2160agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
2220gggcgaaaaa ccgtctatca gggcgatggc ccactacgag aaccatcacc ctaatcaagt
2280tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
2340agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
2400gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
2460gcgcttaatg cgccgctaca gggcgcgtgc tagccatgtg agcaaaaggc cagcaaaagg
2520ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
2580agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
2640accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
2700ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
2760gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
2820ccgttcagtc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
2880gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
2940taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
3000tatttggtat ctgcgctctg ctgtagccag ttaccttcgg aaaaagagtt ggtagctctt
3060gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
3120cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
3180agtggaacga aaactcacgt taagggattt tggtcagatc tagcaccagg cgtttaaggg
3240caccaataac tgccttaaaa aaattacgcc ccgccctgcc actcatcgca gtactgttgt
3300aattcattaa gcattctgcc gacatggaag ccatcacaaa cggcatgatg aacctgaatc
3360gccagcggca tcagcacctt gtcgccttgc gtataatatt tgcccatagt gaaaacgggg
3420gcgaagaagt tgtccatatt ggctacgttt aaatcaaaac tggtgaaact cacccaggga
3480ttggctgaga cgaaaaacat attctcaata aaccctttag ggaaataggc caggttttca
3540ccgtaacacg ccacatcttg cgaatatatg tgtagaaact gccggaaatc gtcgtggtat
3600tcactccaga gcgatgaaaa cgtttcagtt tgctcatgga aaacggtgta acaagggtga
3660acactatccc atatcaccag ctcaccgtct ttcattgcca tacggaactc cgggtgagca
3720ttcatcaggc gggcaagaat gtgaataaag gccggataaa acttgtgctt atttttcttt
3780acggtcttta aaaaggccgt aatatccagc tgaacggtct ggttataggt acattgagca
3840actgactgaa atgcctcaaa atgttcttta cgatgccatt gggatatatc aacggtggta
3900tatccagtga tttttttctc cattttagct tccttagctc ctgaaaatct cgataactca
3960aaaaatacgc ccggtagtga tcttatttca ttatggtgaa agttggaacc tcacccgacg
4020tctaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
4080cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
4140gattacgaat t
4151254638PRTartificial sequencesynthetic construct; pMORPH18_Fab protein
254Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala 1
5 10 15 Thr Val Ala Gln
Ala Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu 20
25 30 Ser Leu Ser Pro Gly Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln 35 40
45 Ser Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln 50 55 60
Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Val 65
70 75 80 Pro Ala Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 85
90 95 Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala
Val 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 Ala
Met Lys Gln Ser 225 230 235
240 Thr Ile Ala Leu Ala Leu Leu Pro Leu Leu Phe Thr Pro Val Thr Lys
245 250 255 Ala Gln Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 260
265 270 Gly Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ser 275 280
285 Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp 290 295 300
Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser 305
310 315 320 Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 325
330 335 Tyr Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr 340 345
350 Cys Ala Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr
Trp Gly 355 360 365
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 370
375 380 Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 385 390
395 400 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val 405 410
415 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala 420 425 430 Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 435
440 445 Pro Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His 450 455
460 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Glu 465 470 475
480 Phe Gly Gly Gly Ser Gly Ser Gly Asp Phe Asp Tyr Glu Lys Met Ala
485 490 495 Asn Ala
Asn Lys Gly Ala Met Thr Glu Asn Ala Asp Glu Asn Ala Leu 500
505 510 Gln Ser Asp Ala Lys Gly Lys
Leu Asp Ser Val Ala Thr Asp Tyr Gly 515 520
525 Ala Ala Ile Asp Gly Phe Ile Gly Asp Val Ser Gly
Leu Ala Asn Gly 530 535 540
Asn Gly Ala Thr Gly Asp Phe Ala Gly Ser Asn Ser Gln Met Ala Gln 545
550 555 560 Val Gly Asp
Gly Asp Asn Ser Pro Leu Met Asn Asn Phe Arg Gln Tyr 565
570 575 Leu Pro Ser Leu Pro Gln Ser Val
Glu Cys Arg Pro Phe Val Phe Gly 580 585
590 Ala Gly Lys Pro Tyr Glu Phe Ser Ile Asp Cys Asp Lys
Ile Asn Leu 595 600 605
Phe Arg Gly Val Phe Ala Phe Leu Leu Tyr Val Ala Thr Phe Met Tyr 610
615 620 Val Phe Ser Thr
Phe Ala Asn Ile Leu Arg Asn Lys Glu Ser 625 630
635 2555020DNAartificial sequencesynthetic construct;
pMORPH x9 255atcgtgctga cccagccgcc ttcagtgagt ggcgcaccag gtcagcgtgt
gaccatctcg 60tgtagcggca gcagcagcaa cattggcagc aactatgtga gctggtacca
gcagttgccc 120gggacggcgc cgaaactgct gatttatgat aacaaccagc gtccctcagg
cgtgccggat 180cgttttagcg gatccaaaag cggcaccagc gcgagccttg cgattacggg
cctgcaaagc 240gaagacgaag cggattatta ttgccagagc tatgacatgc ctcaggctgt
gtttggcggc 300ggcacgaagt ttaaccgttc ttggccagcc gaaagccgca ccgagtgtga
cgctgtttcc 360gccgagcagc gaagaattgc aggcgaacaa agcgaccctg gtgtgcctga
ttagcgactt 420ttatccggga gccgtgacag tggcctggaa ggcagatagc agccccgtca
aggcgggagt 480ggagaccacc acaccctcca aacaaagcaa caacaagtac gcggccagca
gctatctgag 540cctgacgcct gagcagtgga agtcccacag aagctacagc tgccaggtca
cgcatgaggg 600gagcaccgtg gaaaaaaccg ttgcgccgac tgaggcctga taagcatgcg
taggagaaaa 660taaaatgaaa caaagcacta ttgcactggc actcttaccg ttgctcttca
cccctgttac 720caaagcccag gtgcaattga aagaaagcgg cccggccctg gtgaaaccga
cccaaaccct 780gaccctgacc tgtacctttt ccggatttag cctgtccacg tctggcgttg
gcgtgggctg 840gattcgccag ccgcctggga aagccctcga gtggctggct ctgattgatt
gggatgatga 900taagtattat agcaccagcc tgaaaacgcg tctgaccatt agcaaagata
cttcgaaaaa 960tcaggtggtg ctgactatga ccaacatgga cccggtggat acggccacct
attattgcgc 1020gcgttctcct cgttatcgtg gtgcttttga ttattggggc caaggcaccc
tggtgacggt 1080tagctcagcg tcgaccaaag gtccaagcgt gtttccgctg gctccgagca
gcaaaagcac 1140cagcggcggc acggctgccc tgggctgcct ggttaaagat tatttcccgg
aaccagtcac 1200cgtgagctgg aacagcgggg cgctgaccag cggcgtgcat acctttccgg
cggtgctgca 1260aagcagcggc ctgtatagcc tgagcagcgt tgtgaccgtg ccgagcagca
gcttaggcac 1320tcagacctat atttgcaacg tgaaccataa accgagcaac accaaagtgg
ataaaaaagt 1380ggaaccgaaa agcgaattcg actataaaga tgacgatgac aaaggcgcgc
cgtggagcca 1440cccgcagttt gaaaaatgat aagcttgacc tgtgaagtga aaaatggcgc
agattgtgcg 1500acattttttt tgtctgccgt ttaattaaag gggggggggg gccggcctgg
gggggggtgt 1560acatgaaatt gtaaacgtta atattttgtt aaaattcgcg ttaaattttt
gttaaatcag 1620ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
aagaatagac 1680cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
agaacgtgga 1740ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
gagaaccatc 1800accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
accctaaagg 1860gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
aggaagggaa 1920gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
tgcgcgtaac 1980caccacaccc gccgcgctta atgcgccgct acagggcgcg tgctagacta
gtgtttaaac 2040cggaccgggg gggggcttaa gtgggctgca aaacaaaacg gcctcctgtc
aggaagccgc 2100ttttatcggg tagcctcact gcccgctttc cagtcgggaa acctgtcgtg
ccagctgcat 2160cagtgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggagcca
gggtggtttt 2220tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
cctgagagag 2280ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
tgatggtggt 2340cagcggcggg atataacatg agctgtcctc ggtatcgtcg tatcccacta
ccgagatgtc 2400cgcaccaacg cgcagcccgg actcggtaat ggcacgcatt gcgcccagcg
ccatctgatc 2460gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
tggtttgttg 2520aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
tttgattgcg 2580agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
ttaatgggcc 2640agctaacagc gcgatttgct ggtggcccaa tgcgaccaga tgctccacgc
ccagtcgcgt 2700accgtcctca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
catcaagaaa 2760taacgccgga acattagtgc aggcagcttc cacagcaata gcatcctggt
catccagcgg 2820atagttaata atcagcccac tgacacgttg cgcgagaaga ttgtgcaccg
ccgctttaca 2880ggcttcgacg ccgcttcgtt ctaccatcga cacgaccacg ctggcaccca
gttgatcggc 2940gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
tggaggtggc 3000aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
taggaatgta 3060attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
cgtggctggc 3120ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
cgacatcgta 3180taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
gctatcatgc 3240cataccgcga aaggttttgc gccattcgat gctagccatg tgagcaaaag
gccagcaaaa 3300ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
gcccccctga 3360cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
gactataaag 3420ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
ccctgccgct 3480taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
atagctcacg 3540ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
tgcacgaacc 3600ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
ccaacccggt 3660aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
gagcgaggta 3720tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
ctagaagaac 3780agtatttggt atctgcgctc tgctgtagcc agttaccttc ggaaaaagag
ttggtagctc 3840ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
agcagcagat 3900tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
ggtctgacgc 3960tcagtggaac gaaaactcac gttaagggat tttggtcaga tctagcacca
ggcgtttaag 4020ggcaccaata actgccttaa aaaaattacg ccccgccctg ccactcatcg
cagtactgtt 4080gtaattcatt aagcattctg ccgacatgga agccatcaca aacggcatga
tgaacctgaa 4140tcgccagcgg catcagcacc ttgtcgcctt gcgtataata tttgcccata
gtgaaaacgg 4200gggcgaagaa gttgtccata ttggctacgt ttaaatcaaa actggtgaaa
ctcacccagg 4260gattggctga gacgaaaaac atattctcaa taaacccttt agggaaatag
gccaggtttt 4320caccgtaaca cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa
tcgtcgtggt 4380attcactcca gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg
taacaagggt 4440gaacactatc ccatatcacc agctcaccgt ctttcattgc catacggaac
tccgggtgag 4500cattcatcag gcgggcaaga atgtgaataa aggccggata aaacttgtgc
ttatttttct 4560ttacggtctt taaaaaggcc gtaatatcca gctgaacggt ctggttatag
gtacattgag 4620caactgactg aaatgcctca aaatgttctt tacgatgcca ttgggatata
tcaacggtgg 4680tatatccagt gatttttttc tccattttag cttccttagc tcctgaaaat
ctcgataact 4740caaaaaatac gcccggtagt gatcttattt cattatggtg aaagttggaa
cctcacccga 4800cgtctaatgt gagttagctc actcattagg caccccaggc tttacacttt
atgcttccgg 4860ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
agctatgacc 4920atgattacga atttctagat aacgagggca aaaaatgaaa aagacagcta
tcgcgattgc 4980agtggcactg gctggtttcg ctaccgtagc gcaggccgat
50202567PRTartificial sequencesynthetic construct 256Ala Glu
Phe Arg His Asp Cys 1 5 2577PRTartificial
sequencesynthetic construct 257Glu Phe Arg His Asp Ser Cys 1
5 2587PRTartificial sequencesynthetic construct 258Phe Arg His
Asp Ser Gly Cys 1 5 2597PRTartificial
sequencesynthetic construct 259Arg His Asp Ser Gly Tyr Cys 1
5 2607PRTartificial sequencesynthetic construct 260His Asp Ser
Gly Tyr Glu Cys 1 5 2617PRTartificial
sequencesynthetic construct 261Asp Ser Gly Tyr Glu Val Cys 1
5 2627PRTartificial sequencesynthetic construct 262Ser Gly Tyr
Glu Val His Cys 1 5 2637PRTartificial
sequencesynthetic construct 263Tyr Glu Val His His Gln Cys 1
5 2647PRTartificial sequencesynthetic construct 264Glu Val His
His Gln Lys Cys 1 5 2657PRTartificial
sequencesynthetic construct 265Val His His Gln Lys Leu Cys 1
5 2667PRTartificial sequencesynthetic construct 266His His Gln
Lys Leu Val Cys 1 5 2677PRTartificial
sequencesynthetic construct 267His Gln Lys Leu Val Phe Cys 1
5 2687PRTartificial sequencesynthetic construct 268Gln Lys Leu
Val Phe Phe Cys 1 5 2697PRTartificial
sequencesynthetic construct 269Lys Leu Val Phe Phe Ala Cys 1
5 2707PRTartificial sequencesynthetic construct 270Leu Val Phe
Phe Ala Glu Cys 1 5 2717PRTartificial
sequencesynthetic construct 271Val Phe Phe Ala Glu Asp Cys 1
5 2727PRTartificial sequencesynthetic construct 272Phe Phe Ala
Glu Asp Val Cys 1 5 2737PRTartificial
sequencesynthetic construct 273Phe Ala Glu Asp Val Gly Cys 1
5 2747PRTartificial sequencesynthetic construct 274Ala Glu Asp
Val Gly Ser Cys 1 5 2757PRTartificial
sequencesynthetic construct 275Glu Asp Val Gly Ser Asn Cys 1
5 2767PRTartificial sequencesynthetic construct 276Asp Val Gly
Ser Asn Lys Cys 1 5 2777PRTartificial
sequencesynthetic construct 277Val Gly Ser Asn Lys Gly Cys 1
5 2787PRTartificial sequencesynthetic construct 278Gly Ser Asn
Lys Gly Ala Cys 1 5 2797PRTartificial
sequencesynthetic construct 279Cys Ser Asn Lys Gly Ala Ile 1
5 2807PRTartificial sequencesynthetic construct 280Cys Asn Lys
Gly Ala Ile Ile 1 5 2817PRTartificial
sequencesynthetic construct 281Cys Lys Gly Ala Ile Ile Gly 1
5 2827PRTartificial sequencesynthetic construct 282Cys Gly Leu
Met Val Gly Gly 1 5 2837PRTartificial
sequencesynthetic construct 283Cys Met Val Gly Gly Val Val 1
5 2847PRTartificial sequencesynthetic construct 284Cys Gly Gly
Val Val Ile Ala 1 5 2856PRTartificial
sequencesynthetic construct; peptide 1 A beta 285Ala Glu Phe Arg His Asp
1 5 2867PRTartificial sequencesynthetic construct;
peptide 2 A beta 286Glu Phe Arg His Asp Ser Gly 1 5
2875PRTartificial sequencesynthetic construct; peptide 3 A beta 287Glu
Phe Arg His Asp 1 5 2884PRTartificial sequencesynthetic
construct; peptide 4 A beta 288His Asp Ser Gly 1
2895PRTartificial sequencesynthetic construct; peptide 5 A beta 289His
His Gln Lys Leu 1 5 2906PRTartificial sequencesynthetic
construct; peptide 6 A beta 290Leu Val Phe Phe Ala Glu 1 5
2916PRTartificial sequencesynthetic construct; peptide 7 A beta
291Val Phe Phe Ala Glu Asp 1 5 2924PRTartificial
sequencesynthetic construct; peptide 8 A beta 292Val Phe Phe Ala 1
2936PRTartificial sequencesynthetic construct; peptide 9 A beta
293Phe Phe Ala Glu Asp Val 1 5 294360DNAartificial
sequencesynthetic construct 294caattggtgg aaagcggcgg cggcctggtg
caaccgggcg gcagcctgcg tctgagctgc 60gcggcctccg gatttacctt tagcagctat
gcgatgagct gggtgcgcca agcccctggg 120aagggtctcg agtgggtgag cgttatttct
gagaagtctc gttttattta ttatgctgat 180tctgttaagg gtcgttttac catttcacgt
gataattcga aaaacaccct gtatctgcaa 240atgaacagcc tgcgtgcgga agatacggcc
gtgtattatt gcgcgcgtct tactcattat 300gctcgttatt atcgttattt tgatgtttgg
ggccaaggca ccctggtgac ggttagctca 360295120PRTartificial
sequencesynthetic construct 295Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly Ser Leu 1 5 10
15 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala
Met 20 25 30 Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Val 35
40 45 Ile Ser Glu Lys Ser Arg
Phe Ile Tyr Tyr Ala Asp Ser Val Lys Gly 50 55
60 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr Leu Gln 65 70 75
80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
85 90 95 Leu Thr
His Tyr Ala Arg Tyr Tyr Arg Tyr Phe Asp Val Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser
Ser 115 120 296360DNAartificial sequencesynthetic
construct 296caattggtgg aaagcggcgg cggcctggtg caaccgggcg gcagcctgcg
tctgagctgc 60gcggcctccg gatttacctt tagcagctat gcgatgagct gggtgcgcca
agcccctggg 120aagggtctcg agtgggtgag cgctatttct gagacttcta ttcgtaagta
ttatgctgat 180tctgttaagg gtcgttttac catttcacgt gataattcga aaaacaccct
gtatctgcaa 240atgaacagcc tgcgtgcgga agatacggcc gtgtattatt gcgcgcgtct
tactcattat 300gctcgttatt atcgttattt tgatgtttgg ggccaaggca ccctggtgac
ggttagctca 360297120PRTartificial sequencesynthetic construct 297Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 1
5 10 15 Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met 20
25 30 Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val Ser Ala 35 40
45 Ile Ser Glu Thr Ser Ile Arg Lys Tyr Tyr Ala Asp Ser Val
Lys Gly 50 55 60
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65
70 75 80 Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85
90 95 Leu Thr His Tyr Ala Arg Tyr Tyr Arg Tyr
Phe Asp Val Trp Gly Gln 100 105
110 Gly Thr Leu Val Thr Val Ser Ser 115
120 298360DNAartificial sequencesynthetic construct 298caattggtgg
aaagcggcgg cggcctggtg caaccgggcg gcagcctgcg tctgagctgc 60gcggcctccg
gatttacctt tagcagctat gcgatgagct gggtgcgcca agcccctggg 120aagggtctcg
agtgggtgag cgttatttct cagactggtc gtaagattta ttatgctgat 180tctgttaagg
gtcgttttac catttcacgt gataattcga aaaacaccct gtatctgcaa 240atgaacagcc
tgcgtgcgga agatacggcc gtgtattatt gcgcgcgtct tactcattat 300gctcgttatt
atcgttattt tgatgtttgg ggccaaggca ccctggtgac ggttagctca
360299120PRTartificial sequencesynthetic construct 299Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 1 5
10 15 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr Ala Met 20 25
30 Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser
Val 35 40 45 Ile
Ser Gln Thr Gly Arg Lys Ile Tyr Tyr Ala Asp Ser Val Lys Gly 50
55 60 Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65 70
75 80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala Arg 85 90
95 Leu Thr His Tyr Ala Arg Tyr Tyr Arg Tyr Phe Asp Val Trp Gly Gln
100 105 110 Gly Thr
Leu Val Thr Val Ser Ser 115 120
300360DNAartificial sequencesynthetic construct 300caattggtgg aaagcggcgg
cggcctggtg caaccgggcg gcagcctgcg tctgagctgc 60gcggcctccg gatttacctt
tagcagctat gcgatgagct gggtgcgcca agcccctggg 120aagggtctcg agtgggtgag
cgttatttct cagactggtc gtaagattta ttatgctgat 180tctgttaagg gtcgttttac
catttcacgt gataattcga aaaacaccct gtatctgcaa 240atgaacagcc tgcgtgcgga
agatacggcc gtgtattatt gcgcgcgtct tactcattat 300gctcgttatt atcgttattt
tgatgtttgg ggccaaggca ccctggtgac ggttagctca 360301120PRTartificial
sequencesynthetic construct 301Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly Ser Leu 1 5 10
15 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala
Met 20 25 30 Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Val 35
40 45 Ile Ser Gln Thr Gly Arg
Lys Ile Tyr Tyr Ala Asp Ser Val Lys Gly 50 55
60 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr Leu Gln 65 70 75
80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
85 90 95 Leu Thr
His Tyr Ala Arg Tyr Tyr Arg Tyr Phe Asp Val Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser
Ser 115 120 302360DNAartificial sequencesynthetic
construct 302caattggtgg aaagcggcgg cggcctggtg caaccgggcg gcagcctgcg
tctgagctgc 60gcggcctccg gatttacctt tagcagctat gcgatgagct gggtgcgcca
agcccctggg 120aagggtctcg agtgggtgag cgttatttct gagactggta agaatattta
ttatgctgat 180tctgttaagg gtcgttttac catttcacgt gataattcga aaaacaccct
gtatctgcaa 240atgaacagcc tgcgtgcgga agatacggcc gtgtattatt gcgcgcgtct
tactcattat 300gctcgttatt atcgttattt tgatgtttgg ggccaaggca ccctggtgac
ggttagctca 360303120PRTartificial sequencesynthetic construct 303Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 1
5 10 15 Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met 20
25 30 Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val Ser Val 35 40
45 Ile Ser Glu Thr Gly Lys Asn Ile Tyr Tyr Ala Asp Ser Val
Lys Gly 50 55 60
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65
70 75 80 Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85
90 95 Leu Thr His Tyr Ala Arg Tyr Tyr Arg Tyr
Phe Asp Val Trp Gly Gln 100 105
110 Gly Thr Leu Val Thr Val Ser Ser 115
120 304360DNAartificial sequencesynthetic construct 304caattggtgg
aaagcggcgg cggcctggtg caaccgggcg gcagcctgcg tctgagctgc 60gcggcctccg
gatttacctt tagcagctat gcgatgagct gggtgcgcca agcccctggg 120aagggtctcg
agtgggtgag cgttatttct gagactggta agaatattta ttatgctgat 180tctgttaagg
gtcgttttac catttcacgt gataattcga aaaacaccct gtatctgcaa 240atgaacagcc
tgcgtgcgga agatacggcc gtgtattatt gcgcgcgtct tactcattat 300gctcgttatt
atcgttattt tgatgtttgg ggccaaggca ccctggtgac ggttagctca
360305120PRTartificial sequencesynthetic construct 305Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 1 5
10 15 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr Ala Met 20 25
30 Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser
Val 35 40 45 Ile
Ser Glu Thr Gly Lys Asn Ile Tyr Tyr Ala Asp Ser Val Lys Gly 50
55 60 Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65 70
75 80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala Arg 85 90
95 Leu Thr His Tyr Ala Arg Tyr Tyr Arg Tyr Phe Asp Val Trp Gly Gln
100 105 110 Gly Thr
Leu Val Thr Val Ser Ser 115 120
306360DNAartificial sequencesynthetic construct 306caattggtgg aaagcggcgg
cggcctggtg caaccgggcg gcagcctgcg tctgagctgc 60gcggcctccg gatttacctt
tagcagctat gcgatgagct gggtgcgcca agcccctggg 120aagggtctcg agtgggtgag
cgctatttct gagtctggta agactaagta ttatgctgat 180tctgttaagg gtcgttttac
catttcacgt gataattcga aaaacaccct gtatctgcaa 240atgaacagcc tgcgtgcgga
agatacggcc gtgtattatt gcgcgcgtct tactcattat 300gctcgttatt atcgttattt
tgatgtttgg ggccaaggca ccctggtgac ggttagctca 360307120PRTartificial
sequencesynthetic construct 307Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly Ser Leu 1 5 10
15 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala
Met 20 25 30 Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala 35
40 45 Ile Ser Glu Ser Gly Lys
Thr Lys Tyr Tyr Ala Asp Ser Val Lys Gly 50 55
60 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr Leu Gln 65 70 75
80 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
85 90 95 Leu Thr
His Tyr Ala Arg Tyr Tyr Arg Tyr Phe Asp Val Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser
Ser 115 120 308372DNAartificial sequencesynthetic
construct 308caattggtgg aaagcggcgg cggcctggtg caaccgggcg gcagcctgcg
tctgagctgc 60gcggcctccg gatttacctt tagcagctat gcgatgagct gggtgcgcca
agcccctggg 120aagggtctcg agtgggtgag cgctattaat ggtactggta tgaagaagta
ttatgctgat 180tctgttaagg gtcgttttac catttcacgt gataattcga aaaacaccct
gtatctgcaa 240atgaacagcc tgcgtgcgga agatacggcc gtgtattatt gcgcgcgtgg
taagggtaat 300actcataagc cttatggtta tgttcgttat tttgatgttt ggggccaagg
caccctggtg 360acggttagct ca
372309124PRTartificial sequencesynthetic construct 309Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 1 5
10 15 Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr Ala Met 20 25
30 Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val Ser Ala 35 40 45
Ile Asn Gly Thr Gly Met Lys Lys Tyr Tyr Ala Asp Ser Val Lys Gly
50 55 60 Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65
70 75 80 Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85
90 95 Gly Lys Gly Asn Thr His Lys Pro Tyr Gly Tyr
Val Arg Tyr Phe Asp 100 105
110 Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 310372DNAartificial sequencesynthetic
construct 310caattggtgg aaagcggcgg cggcctggtg caaccgggcg gcagcctgcg
tctgagctgc 60gcggcctccg gatttacctt tagcagctat gcgatgagct gggtgcgcca
agcccctggg 120aagggtctcg agtgggtgag cgctattaat tataatggtg ctcgtattta
ttatgctgat 180tctgttaagg gtcgttttac catttcacgt gataattcga aaaacaccct
gtatctgcaa 240atgaacagcc tgcgtgcgga agatacggcc gtgtattatt gcgcgcgtgg
taagggtaat 300actcataagc cttatggtta tgttcgttat tttgatgttt ggggccaagg
caccctggtg 360acggttagct ca
372311124PRTartificial sequencesynthetic construct 311Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 1 5
10 15 Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr Ala Met 20 25
30 Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val Ser Ala 35 40 45
Ile Asn Tyr Asn Gly Ala Arg Ile Tyr Tyr Ala Asp Ser Val Lys Gly
50 55 60 Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65
70 75 80 Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85
90 95 Gly Lys Gly Asn Thr His Lys Pro Tyr Gly Tyr
Val Arg Tyr Phe Asp 100 105
110 Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 312372DNAartificial sequencesynthetic
construct 312caattggtgg aaagcggcgg cggcctggtg caaccgggcg gcagcctgcg
tctgagctgc 60gcggcctccg gatttacctt tagcagctat gcgatgagct gggtgcgcca
agcccctggg 120aagggtctcg agtgggtgag cgctattaat gctgatggta atcgtaagta
ttatgctgat 180tctgttaagg gtcgttttac catttcacgt gataattcga aaaacaccct
gtatctgcaa 240atgaacagcc tgcgtgcgga agatacggcc gtgtattatt gcgcgcgtgg
taagggtaat 300actcataagc cttatggtta tgttcgttat tttgatgttt ggggccaagg
caccctggtg 360acggttagct ca
372313124PRTartificial sequencesynthetic construct 313Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 1 5
10 15 Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr Ala Met 20 25
30 Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val Ser Ala 35 40 45
Ile Asn Ala Asp Gly Asn Arg Lys Tyr Tyr Ala Asp Ser Val Lys Gly
50 55 60 Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65
70 75 80 Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85
90 95 Gly Lys Gly Asn Thr His Lys Pro Tyr Gly Tyr
Val Arg Tyr Phe Asp 100 105
110 Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 314372DNAartificial sequencesynthetic
construct 314caattggtgg aaagcggcgg cggcctggtg caaccgggcg gcagcctgcg
tctgagctgc 60gcggcctccg gatttacctt tagcagctat gcgatgagct gggtgcgcca
agcccctggg 120aagggtctcg agtgggtgag cgctattaat gctgatggta atcgtaagta
ttatgctgat 180tctgttaagg gtcgttttac catttcacgt gataattcga aaaacaccct
gtatctgcaa 240atgaacagcc tgcgtgcgga agatacggcc gtgtattatt gcgcgcgtgg
taagggtaat 300actcataagc cttatggtta tgttcgttat tttgatgttt ggggccaagg
caccctggtg 360acggttagct ca
372315124PRTartificial sequencesynthetic construct 315Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 1 5
10 15 Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr Ala Met 20 25
30 Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val Ser Ala 35 40 45
Ile Asn Ala Asp Gly Asn Arg Lys Tyr Tyr Ala Asp Ser Val Lys Gly
50 55 60 Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65
70 75 80 Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85
90 95 Gly Lys Gly Asn Thr His Lys Pro Tyr Gly Tyr
Val Arg Tyr Phe Asp 100 105
110 Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 316372DNAartificial sequencesynthetic
construct 316caattggtgg aaagcggcgg cggcctggtg caaccgggcg gcagcctgcg
tctgagctgc 60gcggcctccg gatttacctt tagcagctat gcgatgagct gggtgcgcca
agcccctggg 120aagggtctcg agtgggtgag cgctattaat gctaatggtt ataagaagta
ttatgctgat 180tctgttaagg gtcgttttac catttcacgt gataattcga aaaacaccct
gtatctgcaa 240atgaacagcc tgcgtgcgga agatacggcc gtgtattatt gcgcgcgtgg
taagggtaat 300actcataagc cttatggtta tgttcgttat tttgatgttt ggggccaagg
caccctggtg 360acggttagct ca
372317124PRTartificial sequencesynthetic construct 317Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 1 5
10 15 Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr Ala Met 20 25
30 Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val Ser Ala 35 40 45
Ile Asn Ala Asn Gly Tyr Lys Lys Tyr Tyr Ala Asp Ser Val Lys Gly
50 55 60 Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65
70 75 80 Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85
90 95 Gly Lys Gly Asn Thr His Lys Pro Tyr Gly Tyr
Val Arg Tyr Phe Asp 100 105
110 Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 318372DNAartificial sequencesynthetic
construct 318caattggtgg aaagcggcgg cggcctggtg caaccgggcg gcagcctgcg
tctgagctgc 60gcggcctccg gatttacctt tagcagctat gcgatgagct gggtgcgcca
agcccctggg 120aagggtctcg agtgggtgag cgctattaat gctaatggtt ataagaagta
ttatgctgat 180tctgttaagg gtcgttttac catttcacgt gataattcga aaaacaccct
gtatctgcaa 240atgaacagcc tgcgtgcgga agatacggcc gtgtattatt gcgcgcgtgg
taagggtaat 300actcataagc cttatggtta tgttcgttat tttgatgttt ggggccaagg
caccctggtg 360acggttagct ca
372319124PRTartificial sequencesynthetic construct 319Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 1 5
10 15 Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr Ala Met 20 25
30 Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val Ser Ala 35 40 45
Ile Asn Ala Asn Gly Tyr Lys Lys Tyr Tyr Ala Asp Ser Val Lys Gly
50 55 60 Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65
70 75 80 Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85
90 95 Gly Lys Gly Asn Thr His Lys Pro Tyr Gly Tyr
Val Arg Tyr Phe Asp 100 105
110 Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 320372DNAartificial sequencesynthetic
construct 320caattggtgg aaagcggcgg cggcctggtg caaccgggcg gcagcctgcg
tctgagctgc 60gcggcctccg gatttacctt tagcagctat gcgatgagct gggtgcgcca
agcccctggg 120aagggtctcg agtgggtgag cgctattaat gctaatggtt ataagaagta
ttatgctgat 180tctgttaagg gtcgttttac catttcacgt gataattcga aaaacaccct
gtatctgcaa 240atgaacagcc tgcgtgcgga agatacggcc gtgtattatt gcgcgcgtgg
taagggtaat 300actcataagc cttatggtta tgttcgttat tttgatgttt ggggccaagg
caccctggtg 360acggttagct ca
372321124PRTartificial sequencesynthetic construct 321Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 1 5
10 15 Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr Ala Met 20 25
30 Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val Ser Ala 35 40 45
Ile Asn Ala Asn Gly Tyr Lys Lys Tyr Tyr Ala Asp Ser Val Lys Gly
50 55 60 Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65
70 75 80 Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85
90 95 Gly Lys Gly Asn Thr His Lys Pro Tyr Gly Tyr
Val Arg Tyr Phe Asp 100 105
110 Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 322366DNAartificial sequencesynthetic
construct 322caattggtgg aaagcggcgg cggcctggtg caaccgggcg gcagcctgcg
tctgagctgc 60gcggcctccg gatttacctt tagcagctat gcgatgagct gggtgcgcca
agcccctggg 120aagggtctcg agtgggtgag cgctatttct cgttctggtt ctaatattta
ttatgctgat 180tctgttaagg gtcgttttac catttcacgt gataattcga aaaacaccct
gtatctgcaa 240atgaacagcc tgcgtgcgga agatacggcc gtgtattatt gcgcgcgtct
tctttctcgt 300ggttataatg gttattatca taagtttgat gtttggggcc aaggcaccct
ggtgacggtt 360agctca
366323122PRTartificial sequencesynthetic construct 323Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 1 5
10 15 Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr Ala Met 20 25
30 Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val Ser Ala 35 40 45
Ile Ser Arg Ser Gly Ser Asn Ile Tyr Tyr Ala Asp Ser Val Lys Gly
50 55 60 Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 65
70 75 80 Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys Ala Arg 85
90 95 Leu Leu Ser Arg Gly Tyr Asn Gly Tyr Tyr His
Lys Phe Asp Val Trp 100 105
110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 324330DNAartificial sequencesynthetic construct
324gatatcgtgc tgacccagag cccggcgacc ctgagcctgt ctccgggcga acgtgcgacc
60ctgagctgca gagcgagccg gcgtattcat gtttattatc tggcgtggta ccagcagaaa
120ccaggtcaag caccgcgtct attaatttat ggcgcgagca gccgtgcaac tggggtcccg
180gcgcgtttta gcggctctgg atccggcacg gattttaccc tgaccattag cagcctggaa
240cctgaagact ttgcgactta ttattgccag cagacttatg attatcctcc tacctttggc
300cagggtacga aagttgaaat taaacgtacg
330325110PRTartificial sequencesynthetic construct 325Asp Ile Val Leu Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5
10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser
Arg Arg Ile His Val Tyr 20 25
30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu 35 40 45 Ile
Tyr Gly Ala Ser Ser Arg Ala Thr Gly Val Pro Ala Arg Phe Ser 50
55 60 Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu 65 70
75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Thr Tyr Asp Tyr Pro 85 90
95 Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr
100 105 110 326330DNAartificial
sequencesynthetic construct 326gatatcgtgc tgacccagag cccggcgacc
ctgagcctgt ctccgggcga acgtgcgacc 60ctgagctgca gagcgagccg gcgtattcat
gtttattatc tggcgtggta ccagcagaaa 120ccaggtcaag caccgcgtct attaatttat
ggcgcgagca gccgtgcaac tggggtcccg 180gcgcgtttta gcggctctgg atccggcacg
gattttaccc tgaccattag cagcctggaa 240cctgaagact ttgcgactta ttattgccag
cagacttatg attatcctcc tacctttggc 300cagggtacga aagttgaaat taaacgtacg
330327110PRTartificial
sequencesynthetic construct 327Asp Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly 1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Arg Arg Ile His Val
Tyr 20 25 30 Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35
40 45 Ile Tyr Gly Ala Ser Ser
Arg Ala Thr Gly Val Pro Ala Arg Phe Ser 50 55
60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Glu 65 70 75
80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Asp Tyr Pro
85 90 95 Pro Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 100
105 110 328330DNAartificial sequencesynthetic
construct 328gatatcgtgc tgacccagag cccggcgacc ctgagcctgt ctccgggcga
acgtgcgacc 60ctgagctgca gagcgagcca gcgtcttggt cgtctttatc tggcgtggta
ccagcagaaa 120ccaggtcaag caccgcgtct attaatttat ggcgcgagca gccgtgcaac
tggggtcccg 180gcgcgtttta gcggctctgg atccggcacg gattttaccc tgaccattag
cagcctggaa 240cctgaagact ttgcgactta ttattgccag cagacttatg attatcctcc
tacctttggc 300cagggtacga aagttgaaat taaacgtacg
330329110PRTartificial sequencesynthetic construct 329Asp Ile
Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5
10 15 Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Arg Leu Gly Arg Leu 20 25
30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala
Pro Arg Leu Leu 35 40 45
Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Val Pro Ala Arg Phe Ser
50 55 60 Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu 65
70 75 80 Pro Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Thr Tyr Asp Tyr Pro 85
90 95 Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg Thr 100 105 110
330330DNAartificial sequencesynthetic construct 330gatatcgtgc tgacccagag
cccggcgacc ctgagcctgt ctccgggcga acgtgcgacc 60ctgagctgca gagcgagccg
gcgtattcat gtttattatc tggcgtggta ccagcagaaa 120ccaggtcaag caccgcgtct
attaatttat ggcgcgagca gccgtgcaac tggggtcccg 180gcgcgtttta gcggctctgg
atccggcacg gattttaccc tgaccattag cagcctggaa 240cctgaagact ttgcgactta
ttattgccag cagacttatg attatcctcc tacctttggc 300cagggtacga aagttgaaat
taaacgtacg 330331110PRTartificial
sequencesynthetic construct 331Asp Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly 1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Arg Arg Ile His Val
Tyr 20 25 30 Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35
40 45 Ile Tyr Gly Ala Ser Ser
Arg Ala Thr Gly Val Pro Ala Arg Phe Ser 50 55
60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Glu 65 70 75
80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Asp Tyr Pro
85 90 95 Pro Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 100
105 110 332330DNAartificial sequencesynthetic
construct 332gatatcgtgc tgacccagag cccggcgacc ctgagcctgt ctccgggcga
acgtgcgacc 60ctgagctgca gagcgagccg gcgtattcat gtttattatc tggcgtggta
ccagcagaaa 120ccaggtcaag caccgcgtct attaatttat ggcgcgagca gccgtgcaac
tggggtcccg 180gcgcgtttta gcggctctgg atccggcacg gattttaccc tgaccattag
cagcctggaa 240cctgaagact ttgcgactta ttattgccag cagacttatg attatcctcc
tacctttggc 300cagggtacga aagttgaaat taaacgtacg
330333110PRTartificial sequencesynthetic construct 333Asp Ile
Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5
10 15 Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Arg Arg Ile His Val Tyr 20 25
30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala
Pro Arg Leu Leu 35 40 45
Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Val Pro Ala Arg Phe Ser
50 55 60 Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu 65
70 75 80 Pro Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Thr Tyr Asp Tyr Pro 85
90 95 Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg Thr 100 105 110
334330DNAartificial sequencesynthetic construct 334gatatcgtgc tgacccagag
cccggcgacc ctgagcctgt ctccgggcga acgtgcgacc 60ctgagctgca gagcgagcca
gcgtcttggt cgtctttatc tggcgtggta ccagcagaaa 120ccaggtcaag caccgcgtct
attaatttat ggcgcgagca gccgtgcaac tggggtcccg 180gcgcgtttta gcggctctgg
atccggcacg gattttaccc tgaccattag cagcctggaa 240cctgaagact ttgcgactta
ttattgccag cagacttatg attatcctcc tacctttggc 300cagggtacga aagttgaaat
taaacgtacg 330335110PRTartificial
sequencesynthetic construct 335Asp Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly 1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Arg Leu Gly Arg
Leu 20 25 30 Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35
40 45 Ile Tyr Gly Ala Ser Ser
Arg Ala Thr Gly Val Pro Ala Arg Phe Ser 50 55
60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Glu 65 70 75
80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Asp Tyr Pro
85 90 95 Pro Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 100
105 110 336330DNAartificial sequencesynthetic
construct 336gatatcgtgc tgacccagag cccggcgacc ctgagcctgt ctccgggcga
acgtgcgacc 60ctgagctgca gagcgagcca gtttattcag cgtttttatc tggcgtggta
ccagcagaaa 120ccaggtcaag caccgcgtct attaatttat ggcgcgagca gccgtgcaac
tggggtcccg 180gcgcgtttta gcggctctgg atccggcacg gattttaccc tgaccattag
cagcctggaa 240cctgaagact ttgcggttta ttattgccag cagacttata attatcctcc
tacctttggc 300cagggtacga aagttgaaat taaacgtacg
330337110PRTartificial sequencesynthetic construct 337Asp Ile
Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5
10 15 Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Phe Ile Gln Arg Phe 20 25
30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala
Pro Arg Leu Leu 35 40 45
Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Val Pro Ala Arg Phe Ser
50 55 60 Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu 65
70 75 80 Pro Glu Asp Phe Ala Val Tyr
Tyr Cys Gln Gln Thr Tyr Asn Tyr Pro 85
90 95 Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg Thr 100 105 110
338330DNAartificial sequencesynthetic construct 338gatatcgtgc tgacccagag
cccggcgacc ctgagcctgt ctccgggcga acgtgcgacc 60ctgagctgca gagcgagcca
gtatgttgat cgtacttatc tggcgtggta ccagcagaaa 120ccaggtcaag caccgcgtct
attaatttat ggcgcgagca gccgtgcaac tggggtcccg 180gcgcgtttta gcggctctgg
atccggcacg gattttaccc tgaccattag cagcctggaa 240cctgaagact ttgcgactta
ttattgccag cagatttatt cttttcctca tacctttggc 300cagggtacga aagttgaaat
taaacgtacg 330339110PRTartificial
sequencesynthetic construct 339Asp Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly 1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Tyr Val Asp Arg
Thr 20 25 30 Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35
40 45 Ile Tyr Gly Ala Ser Ser
Arg Ala Thr Gly Val Pro Ala Arg Phe Ser 50 55
60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Glu 65 70 75
80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ile Tyr Ser Phe Pro
85 90 95 His Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 100
105 110 340330DNAartificial sequencesynthetic
construct 340gatatcgtgc tgacccagag cccggcgacc ctgagcctgt ctccgggcga
acgtgcgacc 60ctgagctgca gagcgagcca gcgttttttt tataagtatc tggcgtggta
ccagcagaaa 120ccaggtcaag caccgcgtct attaatttct ggttcttcta accgtgcaac
tggggtcccg 180gcgcgtttta gcggctctgg atccggcacg gattttaccc tgaccattag
cagcctggaa 240cctgaagact ttgcggttta ttattgcctt cagctttata atattcctaa
tacctttggc 300cagggtacga aagttgaaat taaacgtacg
330341110PRTartificial sequencesynthetic construct 341Asp Ile
Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5
10 15 Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Arg Phe Phe Tyr Lys 20 25
30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala
Pro Arg Leu Leu 35 40 45
Ile Ser Gly Ser Ser Asn Arg Ala Thr Gly Val Pro Ala Arg Phe Ser
50 55 60 Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu 65
70 75 80 Pro Glu Asp Phe Ala Val Tyr
Tyr Cys Leu Gln Leu Tyr Asn Ile Pro 85
90 95 Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg Thr 100 105 110
342330DNAartificial sequencesynthetic construct 342gatatcgtgc tgacccagag
cccggcgacc ctgagcctgt ctccgggcga acgtgcgacc 60ctgagctgca gagcgagcca
gtatgttgat cgtacttatc tggcgtggta ccagcagaaa 120ccaggtcaag caccgcgtct
attaatttat ggcgcgagca gccgtgcaac tggggtcccg 180gcgcgtttta gcggctctgg
atccggcacg gattttaccc tgaccattag cagcctggaa 240cctgaagact ttgcgactta
ttattgccag cagatttatt cttttcctca tacctttggc 300cagggtacga aagttgaaat
taaacgtacg 330343110PRTartificial
sequencesynthetic construct 343Asp Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly 1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Tyr Val Asp Arg
Thr 20 25 30 Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35
40 45 Ile Tyr Gly Ala Ser Ser
Arg Ala Thr Gly Val Pro Ala Arg Phe Ser 50 55
60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Glu 65 70 75
80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ile Tyr Ser Phe Pro
85 90 95 His Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 100
105 110 344330DNAartificial sequencesynthetic
construct 344gatatcgtgc tgacccagag cccggcgacc ctgagcctgt ctccgggcga
acgtgcgacc 60ctgagctgca gagcgagcca gtatgttttt cgtcgttatc tggcgtggta
ccagcagaaa 120ccaggtcaag caccgcgtct attaatttct ggttcttcta accgtgcaac
tggggtcccg 180gcgcgtttta gcggctctgg atccggcacg gattttaccc tgaccattag
cagcctggaa 240cctgaagact ttgcggttta ttattgcctt cagctttata atattcctaa
tacctttggc 300cagggtacga aagttgaaat taaacgtacg
330345110PRTartificial sequencesynthetic construct 345Asp Ile
Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5
10 15 Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Tyr Val Phe Arg Arg 20 25
30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala
Pro Arg Leu Leu 35 40 45
Ile Ser Gly Ser Ser Asn Arg Ala Thr Gly Val Pro Ala Arg Phe Ser
50 55 60 Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu 65
70 75 80 Pro Glu Asp Phe Ala Val Tyr
Tyr Cys Leu Gln Leu Tyr Asn Ile Pro 85
90 95 Asn Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg Thr 100 105 110
346330DNAartificial sequencesynthetic construct 346gatatcgtgc tgacccagag
cccggcgacc ctgagcctgt ctccgggcga acgtgcgacc 60ctgagctgca gagcgagcca
gtatgttgat cgtacttatc tggcgtggta ccagcagaaa 120ccaggtcaag caccgcgtct
attaatttat ggcgcgagca gccgtgcaac tggggtcccg 180gcgcgtttta gcggctctgg
atccggcacg gattttaccc tgaccattag cagcctggaa 240cctgaagact ttgcgactta
ttattgccag cagatttatt cttttcctca tacctttggc 300cagggtacga aagttgaaat
taaacgtacg 330347110PRTartificial
sequencesynthetic construct 347Asp Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly 1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Tyr Val Asp Arg
Thr 20 25 30 Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35
40 45 Ile Tyr Gly Ala Ser Ser
Arg Ala Thr Gly Val Pro Ala Arg Phe Ser 50 55
60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Glu 65 70 75
80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ile Tyr Ser Phe Pro
85 90 95 His Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 100
105 110 348330DNAartificial sequencesynthetic
construct 348gatatcgtgc tgacccagag cccggcgacc ctgagcctgt ctccgggcga
acgtgcgacc 60ctgagctgca gagcgagcca gcgtctttct cctcgttatc tggcgtggta
ccagcagaaa 120ccaggtcaag caccgcgtct attaatttat ggcgcgagca gccgtgcaac
tggggtcccg 180gcgcgtttta gcggctctgg atccggcacg gattttaccc tgaccattag
cagcctggaa 240cctgaagact ttgcgactta ttattgcctt cagatttata atatgcctat
tacctttggc 300cagggtacga aagttgaaat taaacgtacg
330349110PRTartificial sequencesynthetic construct 349Asp Ile
Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5
10 15 Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Arg Leu Ser Pro Arg 20 25
30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala
Pro Arg Leu Leu 35 40 45
Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Val Pro Ala Arg Phe Ser
50 55 60 Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu 65
70 75 80 Pro Glu Asp Phe Ala Thr Tyr
Tyr Cys Leu Gln Ile Tyr Asn Met Pro 85
90 95 Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg Thr 100 105 110
350330DNAartificial sequencesynthetic construct 350gatatcgtgc tgacccagag
cccggcgacc ctgagcctgt ctccgggcga acgtgcgacc 60ctgagctgca gagcgagcca
gtatgttttt cgtcgttatc tggcgtggta ccagcagaaa 120ccaggtcaag caccgcgtct
attaatttct ggttcttcta accgtgcaac tggggtcccg 180gcgcgtttta gcggctctgg
atccggcacg gattttaccc tgaccattag cagcctggaa 240cctgaagact ttgcggttta
ttattgcctt cagctttata atattcctaa tacctttggc 300cagggtacga aagttgaaat
taaacgtacg 330351110PRTartificial
sequencesynthetic construct 351Asp Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly 1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Tyr Val Phe Arg
Arg 20 25 30 Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35
40 45 Ile Ser Gly Ser Ser Asn
Arg Ala Thr Gly Val Pro Ala Arg Phe Ser 50 55
60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Glu 65 70 75
80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Leu Gln Leu Tyr Asn Ile Pro
85 90 95 Asn Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 100
105 110 352330DNAartificial sequencesynthetic
construct 352gatatcgtgc tgacccagag cccggcgacc ctgagcctgt ctccgggcga
acgtgcgacc 60ctgagctgca gagcgagcca gcgtgtttct ggtcgttatc tggcgtggta
ccagcagaaa 120ccaggtcaag caccgcgtct attaatttat ggcgcgagca gccgtgcaac
tggggtcccg 180gcgcgtttta gcggctctgg atccggcacg gattttaccc tgaccattag
cagcctggaa 240cctgaagact ttgcgactta ttattgccag cagctttctt cttatcctcc
tacctttggc 300cagggtacga aagttgaaat taaacgtacg
330353110PRTartificial sequencesynthetic construct 353Asp Ile
Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5
10 15 Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Arg Val Ser Gly Arg 20 25
30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala
Pro Arg Leu Leu 35 40 45
Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Val Pro Ala Arg Phe Ser
50 55 60 Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu 65
70 75 80 Pro Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Leu Ser Ser Tyr Pro 85
90 95 Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg Thr 100 105 110
35439DNAartificial sequencesynthetic construct 354cttactcatt atgctcgtta
ttatcgttat tttgatgtt 3935513PRTartificial
sequencesynthetic construct 355Leu Thr His Tyr Ala Arg Tyr Tyr Arg Tyr
Phe Asp Val 1 5 10
35639DNAartificial sequencesynthetic construct 356cttactcatt atgctcgtta
ttatcgttat tttgatgtt 3935713PRTartificial
sequencesynthetic construct 357Leu Thr His Tyr Ala Arg Tyr Tyr Arg Tyr
Phe Asp Val 1 5 10
35839DNAartificial sequencesynthetic construct 358cttactcatt atgctcgtta
ttatcgttat tttgatgtt 3935913PRTartificial
sequencesynthetic construct 359Leu Thr His Tyr Ala Arg Tyr Tyr Arg Tyr
Phe Asp Val 1 5 10
36039DNAartificial sequencesynthetic construct 360cttactcatt atgctcgtta
ttatcgttat tttgatgtt 3936113PRTartificial
sequencesynthetic construct 361Leu Thr His Tyr Ala Arg Tyr Tyr Arg Tyr
Phe Asp Val 1 5 10
36239DNAartificial sequencesynthetic construct 362cttactcatt atgctcgtta
ttatcgttat tttgatgtt 3936313PRTartificial
sequencesynthetic construct 363Leu Thr His Tyr Ala Arg Tyr Tyr Arg Tyr
Phe Asp Val 1 5 10
36439DNAartificial sequencesynthetic construct 364cttactcatt atgctcgtta
ttatcgttat tttgatgtt 3936513PRTartificial
sequencesynthetic construct 365Leu Thr His Tyr Ala Arg Tyr Tyr Arg Tyr
Phe Asp Val 1 5 10
36639DNAartificial sequencesynthetic construct 366cttactcatt atgctcgtta
ttatcgttat tttgatgtt 3936713PRTartificial
sequencesynthetic construct 367Leu Thr His Tyr Ala Arg Tyr Tyr Arg Tyr
Phe Asp Val 1 5 10
36851DNAartificial sequencesynthetic construct 368ggtaagggta atactcataa
gccttatggt tatgttcgtt attttgatgt t 5136917PRTartificial
sequencesynthetic construct 369Gly Lys Gly Asn Thr His Lys Pro Tyr Gly
Tyr Val Arg Tyr Phe Asp 1 5 10
15 Val 37051DNAartificial sequencesynthetic construct
370ggtaagggta atactcataa gccttatggt tatgttcgtt attttgatgt t
5137117PRTartificial sequencesynthetic construct 371Gly Lys Gly Asn Thr
His Lys Pro Tyr Gly Tyr Val Arg Tyr Phe Asp 1 5
10 15 Val 37251DNAartificial
sequencesynthetic construct 372ggtaagggta atactcataa gccttatggt
tatgttcgtt attttgatgt t 5137317PRTartificial
sequencesynthetic construct 373Gly Lys Gly Asn Thr His Lys Pro Tyr Gly
Tyr Val Arg Tyr Phe Asp 1 5 10
15 Val 37451DNAartificial sequencesynthetic construct
374ggtaagggta atactcataa gccttatggt tatgttcgtt attttgatgt t
5137517PRTartificial sequencesynthetic construct 375Gly Lys Gly Asn Thr
His Lys Pro Tyr Gly Tyr Val Arg Tyr Phe Asp 1 5
10 15 Val 37651DNAartificial
sequencesynthetic construct 376ggtaagggta atactcataa gccttatggt
tatgttcgtt attttgatgt t 5137717PRTartificial
sequencesynthetic construct 377Gly Lys Gly Asn Thr His Lys Pro Tyr Gly
Tyr Val Arg Tyr Phe Asp 1 5 10
15 Val 37851DNAartificial sequencesynthetic construct
378ggtaagggta atactcataa gccttatggt tatgttcgtt attttgatgt t
5137917PRTartificial sequencesynthetic construct 379Gly Lys Gly Asn Thr
His Lys Pro Tyr Gly Tyr Val Arg Tyr Phe Asp 1 5
10 15 Val 38051DNAartificial
sequencesynthetic construct 380ggtaagggta atactcataa gccttatggt
tatgttcgtt attttgatgt t 5138117PRTartificial
sequencesynthetic construct 381Gly Lys Gly Asn Thr His Lys Pro Tyr Gly
Tyr Val Arg Tyr Phe Asp 1 5 10
15 Val 38245DNAartificial sequencesynthetic construct
382cttctttctc gtggttataa tggttattat cataagtttg atgtt
4538315PRTartificial sequencesynthetic construct 383Leu Leu Ser Arg Gly
Tyr Asn Gly Tyr Tyr His Lys Phe Asp Val 1 5
10 15 38424DNAartificial sequencesynthetic construct
384cagcagactt atgattatcc tcct
243858PRTartificial sequencesynthetic construct 385Gln Gln Thr Tyr Asp
Tyr Pro Pro 1 5 38624DNAartificial
sequencesynthetic construct 386cagcagactt atgattatcc tcct
243878PRTartificial sequencesynthetic
construct 387Gln Gln Thr Tyr Asp Tyr Pro Pro 1 5
38824DNAartificial sequencesynthetic construct 388cagcagactt
atgattatcc tcct
243898PRTartificial sequencesynthetic construct 389Gln Gln Thr Tyr Asp
Tyr Pro Pro 1 5 39024DNAartificial
sequencesynthetic construct 390cagcagactt atgattatcc tcct
243918PRTartificial sequencesynthetic
construct 391Gln Gln Thr Tyr Asp Tyr Pro Pro 1 5
39224DNAartificial sequencesynthetic construct 392cagcagactt
atgattatcc tcct
243938PRTartificial sequencesynthetic construct 393Gln Gln Thr Tyr Asp
Tyr Pro Pro 1 5 39424DNAartificial
sequencesynthetic construct 394cagcagactt atgattatcc tcct
243958PRTartificial sequencesynthetic
construct 395Gln Gln Thr Tyr Asp Tyr Pro Pro 1 5
39624DNAartificial sequencesynthetic construct 396cagcagactt
ataattatcc tcct
243978PRTartificial sequencesynthetic construct 397Gln Gln Thr Tyr Asn
Tyr Pro Pro 1 5 39824DNAartificial
sequencesynthetic construct 398cagcagattt attcttttcc tcat
243998PRTartificial sequencesynthetic
construct 399Gln Gln Ile Tyr Ser Phe Pro His 1 5
40024DNAartificial sequencesynthetic construct 400cttcagcttt
ataatattcc taat
244018PRTartificial sequencesynthetic construct 401Leu Gln Leu Tyr Asn
Ile Pro Asn 1 5 40224DNAartificial
sequencesynthetic construct 402cagcagattt attcttttcc tcat
244038PRTartificial sequencesynthetic
construct 403Gln Gln Ile Tyr Ser Phe Pro His 1 5
40424DNAartificial sequencesynthetic construct 404cttcagcttt
ataatattcc taat
244058PRTartificial sequencesynthetic construct 405Leu Gln Leu Tyr Asn
Ile Pro Asn 1 5 40624DNAartificial
sequencesynthetic construct 406cagcagattt attcttttcc tcat
244078PRTartificial sequencesynthetic
construct 407Gln Gln Ile Tyr Ser Phe Pro His 1 5
40824DNAartificial sequencesynthetic construct 408cagcagattt
attcttttcc tcat
244098PRTartificial sequencesynthetic construct 409Leu Gln Ile Tyr Asn
Met Pro Ile 1 5 41024DNAartificial
sequencesynthetic construct 410cttcagcttt ataatattcc taat
244118PRTartificial sequencesynthetic
construct 411Leu Gln Leu Tyr Asn Ile Pro Asn 1 5
41224DNAartificial sequencesynthetic construct 412cagcagcttt
cttcttatcc tcct
244138PRTartificial sequencesynthetic construct 413Gln Gln Leu Ser Ser
Tyr Pro Pro 1 5 41452PRTartificial
sequencesynthetic construct 414Ile Ser Glu Val Lys Met Asp Ala Glu Phe
Arg His Asp Ser Gly Tyr 1 5 10
15 Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly
Ser 20 25 30 Asn
Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala 35
40 45 Thr Val Ile Val 50
4156PRTArtificialSynthetic Construct 415Ala Glu Phe Arg His Asp 1
5 4167PRTArtificialSynthetic Construct 416Glu Phe Arg
His Asp Ser Gly 1 5 4175PRTArtificialSynthetic
Construct 417Glu Phe Arg His Asp 1 5
4184PRTArtificialSynthetic Construct 418His Asp Ser Gly 1
4195PRTArtificialSynthetic Construct 419His His Gln Lys Leu 1
5 4206PRTArtificialSynthetic Construct 420Leu Val Phe Phe Ala Glu 1
5 4216PRTArtificialSynthetic Construct 421Val Phe Phe
Ala Glu Asp 1 5 4224PRTArtificialSynthetic Construct
422Val Phe Phe Ala 1 4236PRTArtificialSynthetic Construct
423Phe Phe Ala Glu Asp Val 1 5
424378DNAArtificialSynthetic Construct 424caggtggaat tggtggaaag
cggcggcggc ctggtgcaac cgggcggcag cctgcgtctg 60agctgcgcgg cctccggatt
tacctttagc agctatgcga tgagctgggt gcgccaagcc 120cctgggaagg gtctcgagtg
ggtgagcgct attaatgctt ctggtactcg tacttattat 180gctgattctg ttaagggtcg
ttttaccatt tcacgtgata attcgaaaaa caccctgtat 240ctgcaaatga acagcctgcg
tgcggaagat acggccgtgt attattgcgc gcgtggtaag 300ggtaatactc ataagcctta
tggttatgtt cgttattttg atgtttgggg ccaaggcacc 360ctggtgacgg ttagctca
378425126PRTArtificialSynthetic Construct 425Gln Val Glu 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
Thr Phe Ser Ser Tyr 20 25
30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ser
Ala Ile Asn Ala Ser Gly Thr Arg Thr Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Lys Gly Asn Thr His Lys Pro Tyr Gly Tyr Val Arg Tyr
100 105 110 Phe Asp
Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 125
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