Avian Derived Antibodies

Abstract

The invention is drawn to a composition comprising an isolated mixture of cytotoxic anti-CD20 antibody molecules produces in a transgenic avian. The antibody molecules have a heavy chain and a light chain and exhibit an increased level of antibody-dependent cell-mediated cytotoxicity (ADCC) as compared to that of anti-CD20 antibody molecules produced by CHO cells.

Description

RELATED APPLICATIONS

[0001] This application is Continuation of U.S. patent application Ser. No. 12/800,989, filed May 27, 2010, which claims the benefit of U.S. provisional application No. 61/217,138, filed May 27, 2009. The disclosures of the U.S. patent application and the provisional application are expressly incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to the expression of exogenous genetic material in avian cells. The invention also relates to transgenic avian species, including chicken, quail and turkey, and to avians which lay eggs containing exogenous proteins, for example pharmaceutical proteins including antibodies such as cytotoxic antibodies (e.g., anti-CD20) and to the exogenous proteins produced.

BACKGROUND

[0003] Recent developments in avian transgenesis have allowed the modification of avian genomes for exogenous protein production. Germ-line transgenic chickens can be produced by injecting replication-defective retrovirus into the subgerminal cavity of chick blastoderms in freshly laid eggs. See, for example, U.S. Pat. No. 7,511,120, issued Mar. 31, 2009, the disclosure of which is incorporated in its entirety herein by reference; issued U.S. Pat. No. 7,338,654, issued Mar. 4, 2008, the disclosure of which is incorporated in its entirety herein by reference; and US patent publication No. 2008/0064862 published Mar. 13, 2008, the disclosure of which is incorporated in its entirety herein by reference.

[0004] By weight, approximately 60% of an avian egg is composed of albumen which is composed of four major protein components; ovalbumin, ovomucoid, lysozyme and ovotransferrin with ovalbumin and ovomucoid being present in the greatest quantities. Use of regulatory sequences of genes which encode these proteins allows for expression of a heterologous gene product in the oviduct of a transgenic avian which is typically significantly advantageous over ubiquitous expression in the bird. That is; the consequences of ubiquitous expression of a bioactive gene product throughout the host animal is often undesirable. For example, in certain instances the ubiquitous presence of recombinant protein may be harmful to the development of the avian leading to death of the bird. Alternatively, the bird's health may be negatively effected leading to reduced levels of protein production.

[0005] Many currently accepted methods of producing therapeutic cytotoxic antibodies result in a less than optimum antibody dependent cellular cytotoxicity (ADCC) level of the antibody. What is needed are improved therapeutic antibodies including novel and improved forms of cytotoxic antibodies such as anti-CD20 antibodies.

SUMMARY OF THE INVENTION

[0006] The invention encompasses novel antibodies (e.g., cytotoxic antibodies) such as anti-CD20 antibodies produced in an avian, e.g., in an avian oviduct. In addition, the invention includes transgenic avians which produce eggs containing the recombinant antibody, progeny of the transgenic avians, methods of making the avians, the eggs containing the antibodies and isolating the antibodies.

[0007] In one particular aspect, the antibodies of the invention (e.g., cytotoxic antibodies) such as anti-CD20 are produced and glycosylated in an oviduct cell of the avian. For example, the antibody can be produced and glycosylated in a quail, chicken and turkey oviduct cell. In one embodiment, the antibody is produced and glycosylated in a tubular gland cell of the avian. The invention includes the avians (e.g., chicken, turkey and quail) that lay the eggs containing egg white which contains therapeutic protein molecules of the invention comprising one or more of the glycosylation structures disclosed herein.

[0008] Representative glycosylation structures have been determined for the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules (Anti-CD20) of the invention and are shown in FIGS. 1 and 2.

[0009] In one important aspect, the invention relates to an isolated mixture of antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules comprising an antibody molecule of the invention glycosylated with at least one of the structures shown in FIGS. 1A, 1B and 2.

[0010] In one embodiment, the invention is directed to antibodies of the invention, such as anti-CD20 antibodies, glycosylated with a particular oligosaccharide structure chosen from those shown in FIGS. 1A, 1B and 2. For example, the invention can be directed to an anti-CD20 antibody glycosylated with the oligosaccharide structure shown at 1906.7 m/z of FIG. 2. In another example, the invention can be directed to an anti-CD20 antibody glycosylated with one or both of the oligosaccharide structures shown at 1620.7 m/z of FIG. 2. In one embodiment, the invention is directed to antibodies of the invention, such as anti-CD20 antibodies of the invention, glycosylated with two specific oligosaccharide structures chosen from those shown in FIGS. 1A, 1B and 2. In one embodiment, the invention is directed to antibodies of the invention, such as anti-CD20 antibodies of the invention, glycosylated with three specific oligosaccharide structures chosen from those shown in FIGS. 1A, 1B and 2. In one embodiment, the invention is directed to antibodies of the invention, such as anti-CD20 antibodies of the invention, glycosylated with four specific oligosaccharide structures chosen from those shown in FIGS. 1A, 1B and 2. In one embodiment, the invention is directed to antibodies of the invention, such as anti-CD20 antibodies of the invention, glycosylated with five or more specific oligosaccharide structures chosen from those shown in FIGS. 1A, 1B and 2, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more structures (e.g., all the structures) shown in FIGS. 1A, 1B and 2.

[0011] In one embodiment, the anti-CD20 molecules of an isolated mixture have the amino acid sequence shown in FIG. 3.

[0012] In one embodiment, the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules are in a pharmaceutical composition.

[0013] A potential glycosylation site for an anti-CD20 antibody of the invention is shown in FIG. 3. However, the invention is not limited to glycosylation at any particular site of the anti-CD20 antibody.

[0014] The invention is also directed to methods of treatment using the antibody molecules of the invention, (e.g., cytotoxic antibody molecules) such as anti-CD20, as is understood in the art. See, for example, U.S. Pat. No. 7,381,560, issued Jun. 3, 2008; U.S. Pat. No. 5,736,137, issued Apr. 7, 1998; U.S. Pat. No. 5,677,180, issued Oct. 14, 1997; and U.S. Pat. No. 7,422,739, issued Sep. 9, 2008. The disclosure of each of these four patents is incorporated in its entirety herein by reference.

[0015] Anti-CD20 molecules of the invention are useful to destroy both normal and malignant B cells that have CD20 on their surfaces, and are therefore useful to treat diseases which are characterized by excess B cells, overactive B cells or dysfunctional B cells. Examples of indications that may be treated by administration of anti-CD20 antibodies of the invention include, but are not limited to, rheumatoid arthritis, multiple sclerosis, B cell lymphoma, polychondritis, mononeuritis multiplex, Alzheimer's disease, inflammatory bowel disease, systemic lupus erythematosis and anemias (such as autoimmune anemias) progressive multifocal leukoencephalopathy (PML) infection in SLE patients, idiopathic autoimmune hemolytic anemia, pure red cell aplasia, idiopathic thrombocytopenic purpura (ITP), Evans syndrome, vasculitis (for example Wegener's Granulomatosis), bullous skin disorders (for example pemphigus, pemphigoid), type 1 diabetes mellitus, Sjogren's syndrome and Devic's disease. It is also contemplated that antibodies of the invention can be used for anti-rejection treatment in organ transplant such as kidney transplant.

[0016] In one aspect, the invention is directed to antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules obtained from a transgenic avian, for example, a transgenic chicken, which contains a transgene encoding the antibody. In one embodiment, the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules are produced in an avian oviduct cell, for example, a tubular gland cell. In one embodiment, the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules are contained in a hard shell egg, for example, a hard shell egg laid by an avian, for example, a chicken, which contains a transgene encoding the antibody molecules. For example, the antibody molecules may be present in the contents of an intact hard shell egg (e.g., in the egg white). The invention also includes egg white containing an antibody of the invention.

[0017] In one aspect, the invention is drawn to compositions containing antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules produced in an avian (e.g., a transgenic chicken) which contains a transgene encoding the antibody molecules. In one embodiment, the antibody molecules in the composition are produced in an oviduct cell (e.g., a tubular gland cell) of a transgenic avian (e.g., transgenic chicken) and the molecules are isolated from egg white produced by the transgenic avian.

[0018] It is contemplated that the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules in a composition of the invention are N-glycosylated and/or O-glycosylated. In one embodiment, the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules in the composition are N-glycosylated and/or O-glycosylated in the oviduct cell (e.g., tubular gland cell) of the bird, for example, a chicken.

[0019] In one aspect, the invention relates to a composition, for example, a pharmaceutical composition, containing isolated antibody molecules of the invention having an avian derived glycosylation pattern. In one aspect, the invention relates to a composition, for example, a pharmaceutical composition, containing isolated antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules molecules, having an avian or poultry derived glycosylation pattern. In one aspect, the invention relates to a composition, for example, a pharmaceutical composition, containing isolated and glycosylated antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules, produced in accordance with the invention.

[0020] In one embodiment, antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules in compositions of the invention contain a glycosylation pattern other than that of antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules produced in a mammalian cell. In one embodiment, antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules in compositions of the invention contain a glycosylation pattern other than that of antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules produced in a CHO cell.

[0021] In one embodiment, antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention are attached to one or more N-linked oligosaccharide structures disclosed herein (e.g., those shown in Example 5). In one embodiment, antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention are attached to one or more O-linked oligosaccharide structures disclosed in US patent publication No. 2009/0074718, published Mar. 19, 2009, the disclosure of which is incorporated in its entirety herein by reference.

[0022] One aspect of the present invention relates to avian hard shell eggs (e.g., chicken hard shell eggs) which contain an antibody of the invention including, but not limited to, a pharmaceutical antibody. The antibody in the egg is encoded by a transgene of a transgenic avian. The antibody may be present in an egg laid by the avian in any useful amount. In one embodiment, the antibody of the invention is present in an amount in a range of between about 0.01 μg per hard-shell egg and about 1 gram per hard-shell egg. In another embodiment, the antibody is present in an amount in a range of between about 1 μg per hard-shell egg and about 1 gram per hard-shell egg. For example, the antibody may be present in an amount in a range of between about 10 pig per hard-shell egg and about 1 gram per hard-shell egg (e.g., a range of between about 10 μg per hard-shell egg and about 400 mg per hard-shell egg).

[0023] In one embodiment, the antibody of the invention, for example, the pharmaceutical antibody (e.g., a cytotoxic antibody such as anti-CD20) is present in the egg white of the egg. In one embodiment, the antibody is present in an amount in a range of between about 1 ng per ml of egg white and about 0.2 gram per ml of egg white. For example, the antibody may be present in an amount in a range of between about 0.1 μg per ml of egg white and about 0.2 gram per ml of egg white (e.g., the antibody may be present in an amount in a range of between about 1 μg per ml of egg white and about 100 mg per ml of egg white. In one embodiment, the antibody is present in an amount in a range of between about 1 μg per ml of egg white and about 50 mg per ml of egg white. For example, the antibody may be present in an amount in a range of between about 1 μg per ml of egg white and about 10 mg per ml of egg white (e.g., the antibody may be present in an amount in a range of between about 1 μg per ml of egg white and about 1 mg per ml of egg white). In one embodiment, the antibody is present in an amount of more than 0.1 μg per ml of egg white. In one embodiment, the antibody is present in an amount of more than 0.5 μg per ml of egg white. In one embodiment, the antibody is present in an amount of more than 1 μg per ml of egg white. In one embodiment, the antibody is present in an amount of more than 1.5 μg per ml of egg white.

[0024] The avians developed from the blastodermal cells into which a vector containing a transgene encoding an antibody of the invention has been introduced are the G0 generation and can be referred to as "founders". Founder birds are typically chimeric for each inserted transgene. That is, only some of the cells of the G0 transgenic bird contain the transgene(s). The G0 generation typically is also hemizygous for the transgene(s). The G0 generation may be bred to non-transgenic animals to give rise to G1 transgenic offspring which are also hemizygous for the transgene and contain the transgene(s) in essentially all of the bird's cells. The G1 hemizygous offspring may be bred to non-transgenic animals giving rise to G2 hemizygous offspring or may be bred together to give rise to G2 offspring homozygous for the transgene. Substantially all of the cells of birds which are positive for the transgene that are derived from G1 offspring will contain the transgene(s). In one embodiment, hemizygotic G2 offspring from the same line can be bred to produce G3 offspring homozygous for the transgene. In one embodiment, hemizygous G0 animals are bred together to give rise to homozygous G1 offspring containing two copies of the transgene(s) in each cell of the animal. These are merely examples of certain useful breeding methods and the present invention contemplates the employment of any useful breeding method such as those known to individuals of ordinary skill in the art.

[0025] The invention also provides for compositions which contain isolated mixtures of an individual type of useful antibody molecule, such as those antibodies disclosed herein, where one or more of the antibody molecules contained in the mixture has a specific oligosaccharide structure attached, in particular, an oligosaccharide structure disclosed herein which may be produced by a transgenic avian.

[0026] Amino acids sequences within the scope of the invention include those sequences having a nucleotide sequence 80% identical and 85% identical and 90% identical and 91% identical and 92% identical and 93% identical and 94% identical and 95% identical and 96% identical and 97% identical and 98% identical and 99% identical to each of the sequences disclosed herein such as the sequences disclosed in FIGS. 3, 4 and 5. Nucleotide coding sequences for such amino acid sequences are also within the scope of the invention.

[0027] Nucleotide sequences also within the scope of the invention include those sequences having a nucleotide sequence 80% identical and 85% identical and 90% identical and 91% identical and 92% identical and 93% identical and 94% identical and 95% identical and 96% identical and 97% identical and 98% identical and 99% identical to each of the nucleotide sequences disclosed herein, such as those in the accompanying Sequence Listing.

[0028] Any useful combination of features described herein is included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art.

[0029] Additional objects and aspects of the present invention will become more apparent upon review of the detailed description set forth below when taken in conjunction with the accompanying figures, which are briefly described as follows.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIGS. 1A and 1B show N-glycans present on anti-CD20 antibodies produced in accordance with the invention identified by nanospray ionization (NSI). Square=N-acetylglucosamine; Circle=Galactose; Filled Triangle=Fucose; and Filled Circle=Mannose.

[0031] FIG. 2 shows N-glycans present on anti-CD20 antibodies produced in accordance with the invention identified by MALDI-TOF-MS. Square=N-acetylglucosamine; Circle=Galactose; Filled Triangle=Fucose; and Filled Circle=Mannose.

[0032] FIG. 3 shows the light chain (SEQ ID NO: 1) and heavy chain (SEQ ID NO: 2) amino acid sequences of anti-CD20 antibody made in accordance with the invention. The signal sequences which are cleaved are in italics and underlined. Glycosylation site is underlined and in bold. Terminal arginine of the heavy chain is encoded but may be removed from some or all of the antibodies during post-translational processing.

[0033] FIG. 4 shows the amino acid sequence of a single chain anti-CD20 molecule (SEQ ID NO: 3) that can be produced in accordance with the invention.

[0034] FIG. 5 shows a heavy chain (SEQ ID NO: 4) and light chain (SEQ ID NO: 5) of C2H7 antibody contemplated for production in accordance with the invention.

[0035] FIG. 6 shows the CDC activity of the anti-CD20 produced in accordance with the invention as described in Examples 1 to 3 (filled triangle) compared to the same anti-CD20 antibody produced in CHO cells (filled diamond).

[0036] FIG. 7 shows the ADCC activity of the anti-CD20 produced in accordance with the invention as described in Examples 1 to 3 (filled circle) compared to the same anti-CD20 antibody produced in CHO cells (filled triangle).

[0037] FIG. 8 shows the PK of the anti-CD20 produced in accordance with the invention as described in Examples 1 to 3 (filled diamond) compared to the same anti-CD20 antibody produced in CHO cells (filled square).

[0038] FIG. 9 shows a map of vector pSIN-OV-1.1-I-SBC201-I3. The sequence of the vector is shown in SEQ ID NO: 6. Some of the features of pSIN-OV-1.1-I-SBC201-I3 are specified by the following nucleotide sequences: CDS--9206.10618; promoter--2835.3966; 5'UTR--1182.1198; exon--2788.2834; LTR--7610.7955; LTR--4335.4507; intron--1199.2787; 5'UTR--1182.1198; CDS--474.1181; 3'UTR--8518.9191; and glycosil site--9659.9661.

DEFINITIONS

[0039] Certain definitions are set forth herein to illustrate and define the meaning and scope of the various terms used to describe the invention herein.

[0040] A "nucleic acid or polynucleotide sequence" includes, but is not limited to, eukaryotic mRNA, cDNA, genomic DNA, and synthetic DNA and RNA sequences, comprising the natural nucleoside bases adenine, guanine, cytosine, thymidine, and uracil. The term also encompasses sequences having one or more modified bases.

[0041] The term "avian" as used herein refers to any species, subspecies or race of organism of the taxonomic class ayes, such as, but not limited to chickens, quails, turkeys, ducks, geese, pheasants, parrots, finches, hawks, crows and ratites including ostrich, emu and cassowary. The term includes the various known strains of Gallus gallus, or chickens, (for example, White Leghorn, Brown Leghorn, Barred-Rock, Sussex, New Hampshire, Rhode Island, Australorp, Minorca, Amrox, California Gray), as well as strains of turkeys, pheasants, quails, duck, ostriches and other poultry commonly bred in commercial quantities. Avian also includes an individual avian organism in all stages of development, including embryonic and fetal stages.

[0042] A "cytotoxic antibody" after targeting and binding to an antigen triggers lysis and/or death of the cell expressing the antigen to which the cytotoxic antibody has bound.

[0043] "Anti-CD20" is an antibody that selectivity binds to the extracellular domain of the human CD20 antigen. RITUXAN® is a commercially available anti-CD20.

[0044] Nucleic acid "control sequences" or "regulatory sequences" refer to promoter sequences, translational start and stop codons, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers, and the like, as necessary and sufficient for the transcription and translation of a given coding sequence in a defined host cell. Examples of control sequences suitable for eukaryotic cells are promoters, polyadenylation signals, and enhancers. All of these control sequences need not be present in a recombinant vector so long as those necessary and sufficient for the transcription and translation of the desired coding sequence are present.

[0045] "Operably or operatively linked" refers to the configuration of the coding and control sequences so as to perform the desired function. Thus, control sequences operably linked to a coding sequence are capable of effecting the expression of the coding sequence. A coding sequence is operably linked to or under the control of transcriptional regulatory regions in a cell when DNA polymerase will bind the promoter sequence and transcribe the coding sequence into mRNA that can be translated into the encoded protein. The control sequences need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0046] The terms "heterologous" and "exogenous" as they relate to nucleic acid sequences such as coding sequences and control sequences, denote sequences that are not normally associated with a region of a recombinant construct or with a particular chromosomal locus, and/or are not normally associated with a particular cell. Thus, an "exogenous" region of a nucleic acid construct is an identifiable segment of nucleic acid within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature. For example, an exogenous region of a construct could include a coding sequence flanked by sequences not found in association with the coding sequence in nature. Another example of an exogenous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., synthetic sequences having codons different from the native gene). Similarly, a host cell transformed with a construct or nucleic acid which is not normally present in the host cell would be considered exogenous to the cell.

[0047] As used herein the terms "oligosaccharide", "oligosaccharide pattern", "oligosaccharide structure", "carbohydrate chain", "glycosylation pattern" and "glycosylation structure" can have essentially the same meaning and refer to one or more structures which are formed from sugar residues and are attached to proteins of the invention.

[0048] "Exogenous protein" as used herein refers to a protein not naturally present in a particular tissue or cell and is the expression product of an exogenous expression construct or transgene, and/or a protein not naturally present in a given quantity in a particular tissue or cell. A protein that is exogenous to an egg is a protein that is not normally found in the egg. For example, a protein exogenous to an egg may be a protein that is present in the egg as a result of the expression of an exogenous or heterologous coding sequence present in a transgene of the animal laying the egg.

[0049] "Endogenous nucleotide sequence" refers to a naturally occurring nucleotide sequence or fragment thereof normally associated with a particular cell.

[0050] Each of the glycosylation structures shown in FIGS. 1 and 2 is an "oligosaccharide structure type" of the invention.

[0051] "Vector" means a polynucleotide comprised of single strand, double strand, circular, or supercoiled DNA or RNA. A typical vector may be comprised of one or more the following elements operatively linked at appropriate distances for allowing functional gene expression: replication origin, promoter, enhancer, 5' mRNA leader sequence, ribosomal binding site, nucleic acid cassette, termination and polyadenylation sites, and selectable marker sequences. The nucleic acid cassette can include a restriction site for insertion of the nucleic acid sequence to be expressed. In a functional vector the nucleic acid cassette typically contains the nucleic acid sequence to be expressed including translation, initiation and termination sites. An intron optionally may be included in the construct, for example, 5' to the coding sequence. A vector is constructed so that the particular coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the control sequences being such that the coding sequence is transcribed under the "control" of the control sequences or regulatory sequences. Modification of the sequences encoding the particular protein of interest may be desirable to achieve this end. For example, in some cases it may be necessary to modify the sequence so that it may be attached to the control sequences with the appropriate orientation; or to maintain the reading frame. The control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector. In one embodiment, the coding sequence is cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site which is in reading frame with and under regulatory control of the control sequences.

[0052] A "promoter" is a site on the DNA to which RNA polymerase binds to initiate transcription of a gene. In some embodiments the promoter will be modified by the addition or deletion of sequences, or replaced with alternative sequences, including natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences. Many eukaryotic promoters contain two types of recognition sequences: the TATA box and the upstream promoter elements. The former, located upstream of the transcription initiation site, is involved in directing RNA polymerase to initiate transcription at the correct site, while the latter appears to determine the rate of transcription and is upstream of the TATA box. Enhancer elements can also stimulate transcription from linked promoters, but many function exclusively in a particular cell type. Many enhancer/promoter elements derived from viruses, e.g., the SV40 promoter, the cytomegalovirus (CMV) promoter, the rous-sarcoma virus (RSV) promoter, and the murine leukemia virus (MLV) promoter are all active in a wide array of cell types, and are termed "ubiquitous". In one embodiment, non-constitutive promoters such as the mouse mammary tumor virus (MMTV) promoter are used in the present invention. The nucleic acid sequence inserted in the cloning site may have any open reading frame encoding a polypeptide of interest, with the proviso that where the coding sequence encodes a polypeptide of interest, it should preferably lack cryptic splice sites which can block production of appropriate mRNA molecules and/or produce aberrantly spliced or abnormal mRNA molecules.

[0053] The term "poultry derived" refers to a composition or substance produced by or obtained from poultry. "Poultry" refers to birds that can be kept as livestock, including but not limited to, chickens, duck, turkey, quail and ratites. For example, "poultry derived" may refer to chicken derived, turkey derived and/or quail derived.

[0054] A "retroviral particle", "transducing particle", or "transduction particle" refers to a replication-defective or replication-competent virus capable of transducing non-viral DNA or RNA into a cell.

[0055] The terms "transformation", "transduction" and "transfection" all denote the introduction of a polynucleotide into a cell such as an avian cell.

[0056] "Magnum" is that part of the oviduct between the infundibulum and the isthmus containing tubular gland cells that synthesize and secrete the egg white proteins of the egg. The term "oviduct cell" or "oviduct cells" as used herein can refer to magnum cell(s) and/or tubular gland cell(s).

[0057] Various methods of cloning, amplification, expression, and purification will be apparent to the skilled artisan. Representative methods are disclosed in Sambrook, Fritsch, and Maniatis, Molecular Cloning, a Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989).

DETAILED DESCRIPTION

[0058] The invention is directed to antibodies, such as anti-CD20 antibodies, produced in transgenic avian oviduct tissue. For example, FIG. 3 shows the amino acid sequence of an anti-CD20 antibody of the invention. Other examples of anti-CD20 antibodies which are included within the scope of the invention include, without limitation, ZEVALIN® (Ibritumomab tiuxetan), BEXXAR® (Tositumomab) and C2H7, the sequence of which is shown in FIG. 5.

[0059] Shown in FIGS. 1A and 1B are oligosaccharide structures identified by NSI full-MS spectrum of permethylated N-glycans released, as is known in the art, from isolated anti-CD20 antibodies produced as disclosed herein, the heavy chain and light chain amino acid sequences of which are shown in FIG. 3.

[0060] As can be deduced from the relative peak heights of the spectrums shown in FIGS. 1A and 1B, the structures shown at 801.4 (structure 1), 821.9 (structure 2), 842.4 (structure 3), 924.0 (structure 4), 944.5 (structure 5), 965.0 (structure 6) and 1047.0 (structure 7) represent prevalent oligosaccharide structures. Accordingly, the invention includes compositions (e.g., egg white, pharmaceutical formulations) containing an antibody having one of structures 1 to 7. The invention also includes compositions containing 2 antibodies, each having a different attached oligosaccharide structure selected from structures 1 to 7. The invention also includes compositions containing 3 antibodies, each having a different attached oligosaccharide structure selected from structures 1 to 7. The invention also includes compositions containing 4 antibodies, each having a different attached oligosaccharide structure selected from structures 1 to 7. The invention also includes compositions containing 5 antibodies, each having a different attached oligosaccharide structure selected from structures 1 to 7. The invention also includes compositions containing 6 antibodies, each having a different attached oligosaccharide structure selected from structures 1 to 7. The invention also includes compositions containing 7 antibodies, each having a different attached oligosaccharide structure selected from structures 1 to 7.

[0061] FIG. 2 shows at least two additional oligosaccharide structures which were identified by MALDI/TOF-MS (1906.7 m/z and 2110.8 m/z) and which were not identified in the NSI-full MS spectrum analysis.

[0062] The invention encompasses compositions (e.g., egg white, pharmaceutical formulations) which contain anti-CD20 antibodies each having a single oligosaccharide attachment site in the Fc region of each polypeptide chain wherein the antibodies are glycosylated with oligosaccharide structures selected from FIGS. 1 and 2. The invention includes compositions (e.g., egg white, pharmaceutical formulations) containing an antibody having an attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 2 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 3 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 4 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 5 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 6 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 7 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 8 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 9 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 10 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 11 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 12 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 13 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 14 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 15 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 16 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 17 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 18 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 19 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 20 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 21 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 22 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 23 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 24 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 25 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 26 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 27 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 28 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 29 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 30 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 31 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 32 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 33 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 34 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 35 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 36 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2. The invention also includes compositions containing 37 antibodies of the invention, each having a different attached oligosaccharide structure selected from the group shown in FIGS. 1 and 2.

[0063] In one aspect, the invention includes antibodies (e.g., cytotoxic antibodies) such as anti-CD20 antibodies wherein the antibodies have a poultry derived glycosylation pattern (e.g., poultry oviduct cell derived glycosylation pattern) such as a chicken, turkey or quail derived glycosylation pattern. In one aspect, the invention includes antibodies (e.g., cytotoxic antibodies) such as anti-CD20 antibodies wherein the antibodies have a transgenic avian derived glycosylation pattern (e.g., oviduct cell derived glycosylation pattern).

[0064] In one embodiment, the glycosylation pattern is other than that of the same antibody produced in a CHO cell. For example, the compositions can include an antibody (e.g., cytotoxic antibody) such as an anti-CD20 antibody with a poultry or avian derived carbohydrate chain (i.e., glycosylation structure) and that carbohydrate chain or glycosylation structure is not found on that antibody obtained from CHO cell production. However, the composition may also include an antibody (e.g., cytotoxic antibody) such as anti-CD20 that has one or more glycosylation structures that are the same as that found on the antibody when produced in CHO cells. That is, the mixture of antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules may contain one or more antibody molecules having an oligosaccharide pattern which is disclosed herein and is not present when produced in CHO cells plus one or more antibody molecules having an oligosaccharide pattern which could be obtained in CHO cell production.

[0065] In one embodiment, the glycosylation pattern of an antibody (e.g., cytotoxic antibody) such as anti-CD20 produced in accordance with the invention is other than that of the antibody produced in mammalian cells. For example, the compositions can include an antibody (e.g., cytotoxic antibody) such as anti-CD20 molecule with a poultry or avian derived carbohydrate chain (i.e., glycosylation structure) and that carbohydrate chain or glycosylation structure is not found on that antibody obtained from mammalian cells. However, the composition may also include an antibody (e.g., cytotoxic antibody) such as anti-CD20 that has one or more glycosylation structures that are the same as that found on the antibody produced in mammalian cells. That is, the mixture of antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules may contain one or more antibody molecules having an oligosaccharide pattern which is disclosed herein and is not present when produced in CHO cells plus one or more antibody molecules having an oligosaccharide pattern which could be obtained in mammalian cell production.

[0066] In one embodiment, provided for are antibodies of the invention (e.g., cytotoxic antibodies) such as anti-CD20 which are isolated. In one embodiment, the antibodies of the invention are contained in a composition are isolated. For example, the antibodies (e.g., cytotoxic antibodies) such as anti-CD20 may be isolated from egg white. The isolated antibodies may be antibody molecules that do not all have the same glycosylation structures among the antibody molecules or the isolated antibodies may be an isolated individual species of antibody molecules having only one particular glycosylation structure at a particular glycosylation site among the species of antibody molecules.

[0067] In one embodiment, at least about 5% of the N-linked oligosaccharides present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention contain fucose. In one embodiment, at least about 4% of the N-linked oligosaccharides present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention contain fucose. In one embodiment, at least about 3% of the N-linked oligosaccharides present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention contain fucose. In one embodiment, at least about 2% of the N-linked oligosaccharides present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention contain fucose. In one embodiment, at least about 1% of the N-linked oligosaccharides present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention contain fucose.

[0068] In one embodiment, some of the N-linked oligosaccharides present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention do not contain fucose. In one embodiment, about 90% or more of the N-linked oligosaccharides present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention do not contain fucose. In another embodiment, about 95% or more of the N-linked oligosaccharides present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention do not contain fucose. In another embodiment, about 96% or more of the N-linked oligosaccharides present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention do not contain fucose. In another embodiment, about 97% or more of the N-linked oligosaccharides present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention do not contain fucose. In another embodiment, about 98% or more of the N-linked oligosaccharides present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention do not contain fucose. In another embodiment, about 99% or more of the N-linked oligosaccharides present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention do not contain fucose. In one embodiment, the percentages in this paragraph refer specifically to the percentage of N-linked oligosaccharide structure present only on the Fc region that do not contain fucose.

[0069] In one embodiment, essentially none of the N-linked oligosaccharide structure types present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention contain fucose. In another embodiment, about 70% or more of the N-linked oligosaccharide structure types present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention do not contain fucose. In another embodiment, about 75% or more of the N-linked oligosaccharide structure types present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention do not contain fucose. In another embodiment, about 80% or more of the N-linked oligosaccharide structure types present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention do not contain fucose. In another embodiment, about 85% or more of the N-linked oligosaccharide structure types present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention do not contain fucose. In another embodiment, about 90% or more of the N-linked oligosaccharide structure types present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention do not contain fucose. In another embodiment, about 95% or more of the N-linked oligosaccharide structure types present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention do not contain fucose.

[0070] In one embodiment, some of the N-linked oligosaccharides present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention contain a bisecting GlcNAc. In one embodiment, about 2% or more of the N-linked oligosaccharides present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention contain a bisecting GlcNAc. In another embodiment, about 5% or more of the N-linked oligosaccharides present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention contain a bisecting GlcNAc. In another embodiment, about 10% or more of the N-linked oligosaccharides present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention contain a bisecting GlcNAc. In another embodiment, about 15% or more of the N-linked oligosaccharides present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention contain a bisecting GlcNAc. In another embodiment, about 20% or more of the N-linked oligosaccharides present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention contain a bisecting GlcNAc. In another embodiment, about 30% or more of the N-linked oligosaccharides present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention contain a bisecting GlcNAc. In one embodiment, the percentages in this paragraph refer specifically to the percentage of N-linked oligosaccharide structure present only on the Fc region that contain a bisecting GlcNAc.

[0071] In one embodiment, about 1% or more of the N-linked oligosaccharide structure types present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention contain a bisecting GlcNAc. In another embodiment, about 5% or more of the N-linked oligosaccharide structure types present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention contain a bisecting GlcNAc. In another embodiment, about 10% or more of the N-linked oligosaccharide structure types present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention contain a bisecting GlcNAc. In another embodiment, about 15% or more of the N-linked oligosaccharide structure types present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention contain a bisecting GlcNAc. In another embodiment, about 20% or more of the N-linked oligosaccharide structure types present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention contain a bisecting GlcNAc. In another embodiment, about 30% or more of the N-linked oligosaccharide structure types present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention contain a bisecting GlcNAc.

[0072] Without wishing to limit the invention to any particular theory or mechanism of operation it is believed that the presence of bisecting GlcNAc increases receptor binding (e.g., CFC receptor binding) providing for an increased activity or efficacy of the antibody.

[0073] In one embodiment, some of the N-linked oligosaccharides present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention are terminated partially or exclusively with N-acetyl glucosamine. In one embodiment, about 95% or more of the N-linked oligosaccharides present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention are terminated partially or exclusively with N-acetyl glucosamine. In another embodiment, about 90% or more of the N-linked oligosaccharides present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention are terminated partially or exclusively with N-acetyl glucosamine. In another embodiment, about 80% or more of the N-linked oligosaccharides present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention are terminated partially or exclusively with N-acetyl glucosamine. In another embodiment, about 70% or more of the N-linked oligosaccharides present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention are terminated partially or exclusively with N-acetyl glucosamine. In another embodiment, about 60% or more of the N-linked oligosaccharides present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention are terminated partially or exclusively with N-acetyl glucosamine. In another embodiment, about 50% or more of the N-linked oligosaccharides present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention are terminated partially or exclusively with N-acetyl glucosamine.

[0074] In one embodiment, all of the N-linked oligosaccharides structure types present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention are terminated partially or exclusively with N-acetyl glucosamine. In another embodiment, about 95% or more of the N-linked oligosaccharide structure types present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention are terminated partially or exclusively with N-acetyl glucosamine. In another embodiment, about 90% or more of the N-linked oligosaccharide structure types present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention are terminated partially or exclusively with N-acetyl glucosamine. In another embodiment, about 80% or more of the N-linked oligosaccharide structure types present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention are terminated partially or exclusively with N-acetyl glucosamine. In another embodiment, about 70% or more of the N-linked oligosaccharide structure types present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention are terminated partially or exclusively with N-acetyl glucosamine. In another embodiment, about 60% or more of the N-linked oligosaccharide structure types present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention are terminated partially or exclusively with N-acetyl glucosamine. In another embodiment, about 50% or more of the N-linked oligosaccharide structure types present on the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention are terminated partially or exclusively with N-acetyl glucosamine.

[0075] In one embodiment, the antibody (e.g., cytotoxic antibody) such as anti-CD20 is present in a hard shell egg. For example, the antibody may be present in the egg white of a hard shell egg laid by a transgenic avian of the invention. That is, in one embodiment, the invention is directed to avian (e.g., chicken) egg white containing an antibody of the invention. In one embodiment, the antibody (e.g., cytotoxic antibody) such as anti-CD20 is present in the egg white in an amount in excess of about 1 microgram per ml of egg white (e.g., present in an amount of about 1 microgram to about 0.5 gram per ml of egg white). For example, the antibody (e.g., cytotoxic antibody) such as anti-CD20 can be present in an amount greater than about 2 micrograms per ml of egg white (e.g., present in an amount of about 2 micrograms to about 200 micrograms per ml of egg white).

[0076] In one embodiment, the antibody, (e.g., cytotoxic antibody) such as anti-CD20 antibody, produced in accordance with the invention is produced as a single chain antibody, as is understood in the art. See, for example, Lee et al (1999) Molecular Immunology vol 36, p 61-71, the disclosure of which is incorporated in its entirety herein by reference, which discloses exemplary methodology useful for the design of a single chain antibody.

[0077] It is understood that though the reported method of making compositions of the invention is in avians, the compositions are not limited thereto. For example, certain of the glycosylated protein molecules of the invention may be produced in other organisms such as transgenic fish, transgenic plants, such as tobacco and duck weed (Lemna minor) or certain strains of yeast.

[0078] While it is possible that, for use in therapy, antibodies produced in accordance with this invention may be administered in raw form, it is preferable to administer the antibodies as part of a pharmaceutical composition.

[0079] One aspect of the invention relates to compositions containing antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules produced in accordance with the invention. In a particularly useful embodiment, the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules are purified or isolated (e.g., isolated form egg white). For example, the antibody molecules can be removed from the contents of a hard shell egg (e.g., from the egg white) laid by a transgenic avian. In one embodiment, the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules of the invention have a glycosylation pattern resulting from the molecules being produced in an oviduct cell of an avian.

[0080] Another aspect of the invention relates to compositions containing antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules produced in an avian oviduct cell (e.g., a tubular gland cell) that have a glycosylation pattern other than that of antibody molecules produced in a mammalian cell such as a CHO cell. In one aspect, the invention provides for compositions that contain isolated antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules having an avian or poultry derived glycosylation pattern. For example, the compositions can contain a mixture of antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules produced in avians, for example, chickens, in accordance with the invention and isolated from egg white. In one useful embodiment, the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules are in pharmaceutical compositions.

[0081] The invention provides for pharmaceutical compositions comprising poultry or avian derived glycosylated antibodies (e.g., cytotoxic antibodies) such as anti-CD20 together with one or more pharmaceutically acceptable carriers thereof and, optionally, other therapeutic and/or prophylactic ingredients and methods of administering such pharmaceutical compositions. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Methods of treating a patient (e.g., quantity of pharmaceutical protein administered, frequency of administration and duration of treatment period) using pharmaceutical compositions of the invention can be determined using standard methodologies known to physicians of skill in the art.

[0082] Pharmaceutical compositions include those suitable for oral, rectal, nasal, topical (including buccal and sub-lingual), vaginal or parenteral. The pharmaceutical compositions include those suitable for administration by injection including intramuscular, sub-cutaneous and intravenous administration. The pharmaceutical compositions also include those for administration by inhalation or insufflation. The compositions or formulations may, where appropriate, be conveniently presented in discrete dosage units and may be prepared by any of the methods well known in the art of pharmacy. The methods of producing the pharmaceutical compositions typically include the step of bringing the antibodies into association with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.

[0083] Pharmaceutical compositions suitable for oral administration may conveniently be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution; as a suspension; or as an emulsion. The active ingredient may also be presented as a bolus, electuary or paste. Tablets and capsules for oral administration may contain conventional excipients such as binding agents, fillers, lubricants, disintegrants, or wetting agents. The tablets may be coated according to methods well known in the art. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils) or preservatives.

[0084] Antibodies of the invention may also be formulated for parenteral administration (e.g., by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. The antibodies may be injected by, for example, subcutaneous injections, intramuscular injections, and intravenous infusions or injections.

[0085] The antibodies may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. It is also contemplated that the antibodies may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

[0086] For topical administration to the epidermis, the antibodies produced according to the invention may be formulated as ointments, creams or lotions, or as a transdermal patch. Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents or coloring agents.

[0087] Formulations suitable for topical administration in the mouth include lozenges comprising active ingredient in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

[0088] Pharmaceutical compositions suitable for rectal administration wherein the carrier is a solid are most preferably represented as unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art, and the suppositories may be conveniently formed by a mixture of the active compound with the softened or melted carrier(s) followed by chilling and shaping in molds.

[0089] Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient, such carriers as are known in the art to be appropriate.

[0090] For intra-nasal administration the antibodies of the invention may be used as a liquid spray or dispersible powder or in the form of drops.

[0091] Drops may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing agents or suspending agents. Liquid sprays are conveniently delivered from pressurized packs.

[0092] For administration by inhalation, antibodies according to the invention may be conveniently delivered from an insufflator, nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray. Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.

[0093] For administration by inhalation or insufflation, the antibodies according to the invention may take the form of a dry powder composition, for example a powder mix of the compound and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form in, for example, capsules or cartridges or, e.g., gelatin or blister packs from which the powder may be administered with the aid of an inhalator or insufflator.

[0094] When desired, the above described formulations adapted to give sustained release of the active ingredient, may be employed.

[0095] The pharmaceutical compositions according to the invention may also contain other active ingredients such as antimicrobial agents, or preservatives.

[0096] It is contemplated that the antibodies of the invention may be used in combination with other therapeutic agents.

[0097] Compositions or compounds of the invention can be used to treat a variety of conditions. For example, there are many conditions for which treatment therapies are known to practitioners of skill in the art in which antibodies obtained from cell culture (e.g., CHO cells) are employed. The present invention contemplates that the glycosylated antibodies produced in an avian system can be employed to treat such conditions. That is, the invention contemplates the treatment of conditions known to be treatable by conventionally produced antibodies by using antibodies produced in accordance with the invention. For example, antibodies (e.g., cytotoxic antibodies) such as anti-CD20 produced in accordance with the invention can be used to treat human conditions, as understood in the art.

[0098] Generally, the dosage administered will vary depending upon known factors such as age, health and weight of the recipient, type of concurrent treatment, frequency of treatment, and the like. Usually, a dosage of active ingredient can be between about 0.0001 mg and about 10 mg per kilogram of body weight. Precise dosage, frequency of administration and time span of treatment can be determined by a physician skilled in the art of administration of the respective therapeutic protein.

[0099] By the methods of the present invention, transgenes can be introduced into avian embryonic blastodermal cells to produce a transgenic chicken, transgenic turkey, transgenic quail and other avian species, that carry a transgene in the genetic material of its germ-line tissue to produce proteins of the invention. The blastodermal cells are typically stage VII-XII cells, or the equivalent thereof, and in one embodiment are near stage X. The cells useful in the present invention include embryonic germ (EG) cells, embryonic stem (ES) cells & primordial germ cells (PGCs). The embryonic blastodermal cells may be isolated freshly, maintained in culture, or in a particularly useful embodiment, reside within an embryo.

[0100] Some vectors useful in carrying out the methods of the present invention are described herein. These vectors can be used for stable introduction of an exogenous coding sequence into the genome of an avian. The vectors may be used to produce proteins of the invention such as antibodies in specific tissues of an avian, for example, in the oviduct tissue of an avian. The vectors may also be used in methods to produce avian eggs which contain exogenous protein. In one embodiment, the coding sequence and the promoter are both positioned between 5' and 3' LTRs before introduction into blastodermal cells. In one embodiment, the vector is retroviral and the coding sequence and the promoter are both positioned between the 5' and 3' LTRs of the retroviral SIN vector. In one useful embodiment, the LTRs or retroviral vector is derived from the avian leukosis virus (ALV), murine leukemia virus (MLV), or lentivirus.

[0101] Useful retroviruses for introducing a transgene into the avian genome are the replication-deficient avian leucosis virus (ALV), the replication-deficient murine leukemia virus (MLV) and the lentivirus. Any of the vectors of the present invention may include a coding sequence encoding a signal peptide that will direct secretion of the protein expressed by the vector's coding sequence from the tubular gland cells of the oviduct. Where an exogenous protein would not otherwise be secreted, the vector containing the coding sequence is modified to comprise a DNA sequence encoding a useful signal peptide. The DNA sequence encoding the signal peptide is inserted in the vector such that it is located at the N-terminus of the protein encoded by the DNA. The signal peptide can direct secretion of the exogenous protein expressed by the vector into the egg white of a hard shell egg. The vector may include a marker gene, wherein the marker gene is operably linked to a promoter.

[0102] Any useful promoter can be employed. For example, the promoter can be a constitutive promoter such as a cytomegalovirus (CMV) promoter, a nous-sarcoma virus (RSV) promoter, a murine leukemia virus (MLV) promoter, a beta-actin promoter. The promoter can also be a magnum specific promoter such as an ovalbumin promoter, a lysozyme promoter, a conalbumin promoter, an ovomucoid promoter, an ovomucin promoter or an ovotransferrin promoter. Both constitutive and magnum specific promoters have proven suitable for expression of exogenous protein in the oviduct.

[0103] The methods of the invention which provide for the production of protein of the invention in the avian oviduct and the production of eggs which contain the exogenous protein involve an additional step subsequent to providing a suitable vector and introducing the vector into embryonic blastodermal cells so that the vector is integrated into the avian genome. The subsequent step involves deriving a mature transgenic avian from the transgenic blastodermal cells produced. Deriving a mature transgenic avian from the blastodermal cells typically involves transferring the vector into an embryo and allowing that embryo to develop fully, so that the transduced cells become incorporated into the avian as the embryo is allowed to develop. The resulting chick is then grown to maturity. In one embodiment, the cells of a blastodermal embryo are transfected or transduced with the vector directly within the embryo. The resulting embryo is allowed to develop and the chick allowed to mature.

[0104] The transgenic avian so produced from the transgenic blastodermal cells is known as a founder. Some founders will carry the transgene in the tubular gland cells in the magnum of their oviducts. These avians will express the exogenous protein encoded by the transgene in their oviducts. The exogenous protein may also be expressed in other tissues (e.g., blood) in addition to the oviduct. If the exogenous protein contains the appropriate signal sequence(s), it will be secreted into the lumen of the oviduct and into the egg white of the egg. Some founders are germ-line founders. A germ-line founder is a founder that carries the transgene in genetic material of its germ-line tissue, and may also carry the transgene in oviduct magnum tubular gland cells that express the exogenous protein. Therefore, in accordance with the invention, the transgenic avian will have tubular gland cells expressing the exogenous protein, and the offspring of the transgenic avian will also have oviduct magnum tubular gland cells that express the exogenous protein.

[0105] Other specific examples of therapeutic proteins which may be produced as disclosed herein include, without limitation, factor VIII, b-domain deleted factor VIII, factor VIIa, factor IX, anticoagulants, hirudin, alteplase, reteplase, tPA, tPA--3 of 5 domains deleted, insulin, insulin lispro, insulin aspart, insulin glargine, long-acting insulin analogs, hgh, glucagons, tsh, follitropin-beta, fsh, gm-csf, pdgh, IFN alpha2, IFN alpha2a, IFN alpha2b, IFN-alpha, IFN-beta 1b, IFN-beta, IFN-gamma1b, IL-2, IL-11, hbsag, ospa, murine mab directed against t-lymphocyte antigen, murine mab directed against tag-72, tumor-associated glycoprotein, Fab fragments derived from chimeric mab directed against platelet surface receptor gpII(b)/III(a), murine mab fragment directed against tumor-associated antigen CA 125, murine mab fragment directed against human carcinoembryonic antigen, cea, murine mab fragment directed against human cardiac myosin, murine mab fragment directed against tumor surface antigen psma, murine mab fragments (Fab/Fab2 mix) directed against hmw-maa, murine mab fragment (Fab) directed against carcinoma-associated antigen, mab fragments (Fab) directed against NCA-90, a surface granulocyte nonspecific cross reacting antigen, humanized mab directed against the alpha chain of the IL-2 receptor, chimeric mab directed against the alpha chain of the IL-2 receptor, chimeric mab directed against TNF-alpha, humanized mab directed against an epitope on the surface of respiratory synctial virus, humanized mab directed against HER2, human epidermal growth factor receptor 2, human mab directed against cytokeratin tumor-associated antigen anti-CTLA4, dornase-alpha DNase, beta glucocerebrosidase, TNF-alpha, IL-2-diptheria toxin antibody, TNFR-IgG fragment antibody laronidase, alefacept, darbepoetin alfa (colony stimulating factor), tositumomab, murine mab, alemtuzumab, rasburicase, agalsidase beta, teriparatide, parathyroid hormone derivatives, adalimumab (IgG1), anakinra, biological modifier, nesiritide, human b-type natriuretic peptide (hbnp), colony stimulating factors, pegvisomant, human growth hormone receptor antagonist, recombinant activated protein c, omalizumab, immunoglobulin e (IgE) blocker, lbritumomab tiuxetan, ACTH, glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone, pigmentary hormones, somatomedin, erythropoietin, luteinizing hormone, chorionic gonadotropin, hypothalmic releasing factors, etanercept, antidiuretic hormones, prolactin and thyroid stimulating hormone.

[0106] The invention also includes the production of lysosomal acid lipase (LAL) produced in accordance with the invention. The amino acid sequence for human LAL is well known in the art, see, for example, Anderson, R. A. and Sando, G. N., "Cloning and Expression of cDNA Encoding Human Lysosomal Acid Lipase/Cholesteryl Ester Hydrolase", Journal of Biological Chemistry, Vol. 266, No. 33, Issue of November 25, pp. 22479-22484 (1991).

[0107] The invention also includes the production of glucocerebrosidase produced in accordance with the invention. Sequence information for human glucocerebrosidase is well known in the art, see, and, for example, Sorge, et al. "Molecular cloning and nucleotide sequence of human glucocerebrosidase cDNA", Proc. Natl. Acad. Sci, Vol 82, pp 7289-7293 (1985).

[0108] Certain antibodies which may be produced in accordance with the invention include, without limitation, Muromonab; Satumomab pendetide; mAb=B72.3, conjugate of B72.3 and radioligand=CYT 103; Abciximab; Edrecolomab, Mab 17-1A; murine Mab fragment directed against tumor-associated antigen CA 125; Arcitumomab; Imciromab pentetate Capromab pendetide; murine Mab fragments (Fab/Fab2 mix) directed against HMW-MAA; Nofetumomab; Sulesomab; chimeric Mab directed against CD20 antigen found on surface of B lymphocytes; Daclizumab; Basiliximab; Palivizumab; Trastuzumab; human Mab directed against cytokeratin tumor-associated antigen; Rituximab; Infliximab; Gemtuzumab ozogamicin; Alemtuzumab; Tositumomab (conjugated to 131I); Omalizumab; Ibritumomab tiuxetan (conjugated to 90Y); Efalizumab; Cetuximab; Bevacizumab; Adalimumab (IgG1); Technetium (99mTc) fanolesomab; Natalizumab; Ranibizumab; Panitumumab; Eculizumab.

[0109] In one particularly useful embodiment, antibodies produced in accordance with the invention are produced in a single chain form. See, for example, Lee et al, Molecular Immunology (1999) vol 36, p 61-71 which discloses the production of single chain antibodies, the disclosure of which is incorporated in its entirety herein by reference. For example, any antibody which can be produced in accordance with the invention in single chain form, including but not limited to each of the antibodies specifically disclosed herein, is contemplated for production in a single chain form in a transgenic avian oviduct.

[0110] Certain enzymes, such as human enzymes, which can be produced in accordance with the invention include Rasburicase; Asparaginase; Urokinase; Tenecteplase; adenosin deaminase; Glucocerebrosidase; lysosomal acid lipase (Cholestrase); Palmitoyl-protein thioesterase 1; PPT1, B-Galactosidase; Neuraminidase; heparan sulfamidase; N-acetylglucosaminidase; alpha-N-acetylglucosaminidase; alpha-glucosaminide N-acetyltransferase; N-acetylglucosamine-6-sulfate sulfatase; galactosylceramidase (GALC); Glucoronidase; NPC1; NPC2; Agalsidase alfa; Agalsidase beta; alpha-glucosidase; Acid Sphingomyelinase (ASM); N-acetylgalactosamine 6-sulfatase (GALNS or galactose 6-sulfatase); beta-galactosidase; Idursulfase; alpha-L-duronidase; Galsulfase: arylsulfatase B, BM 102, arylsulfatase B, N-acetylgalactosamine-4-sulfatase, ASB.; lysosomal alpha-mannosidase (LAMAN); beta-hexosaminidase; alglucosidase alfa; beta-hexosaminidase A; tripeptidyl peptidase 1 (TPP1).

[0111] Other protein therapeutics which can be produced in accordance with the invention include, without limitation, Factor VIII; B-domain deleted Factor VIII; Factor VIIa; Factor IX; anticoagulant; recombinant hirudin; anticoagulant; recombinant hirudin; Alteplase, tPA; Reteplase, human tPA--3 of 5 domains deleted; Factor XI; Factor XII (Hageman factor); Factor XIII; Alpha2-antiplasmin; Microplasmin; insulin lispro; Bio Lysprol, an insulin analog; insulin aspart; insulin glargine; long-acting insulin analog; hGH; glucagons; TSH; follitropin-beta FSH; salmon calcitonin; (Teriparatide) Parathyroid hormone derivative; nesiritide, B-type natriuretic peptide (BNP); PDGH; Lutropin alfa; Choriogonadotropin alfa; Somatropin Pegvisomant, human growth hormone receptor antagonist; platelet derived growth factor (PDGF); Keratinocyte growth factor; fibroblast growth factor 23; insulin-like growth factor-1, IGF-1 complexed with IGFBP-3; HBsAg; vaccine containing HBsAgn as one component; OspA, a lipoprotein found on the surface of B burgorferi; Hep B-IPV HIB vaccine; Hep B-IPV vaccine; Comb vaccine; Pneumococcal conjugate vaccine; Influenza virus vaccine live, intranasal; Alefacept, Immunosuppressive agent; TNF-alpha; TNFR-IgG fragment antibody; Abatacept; recombinant activated protein C; dornase-alpha DNAse; Enfuvirtide (HIV fusion inhibitor) Anakinra, Botulinum Toxins, e.g., Type A; Samarium [153 m] lexidronam; Perfultren; Cetrorelix; Eptifibatide; Insulin Glargine; Insulin Aspart; Hepatitis B virus small surface antigen (HbsAg); Eptoterminalfa; Protein C; Inactivated hepatitis A virus hepatitis B surface antigen; Dibotermin alfa; IL-2-diptheria toxin antibody that targets cells displaying a surface IL-2 receptor; Endostatin; Human insulin-like growth factor binding protein-6.

[0112] The therapeutic proteins of the invention can be produced by methods such as those disclosed herein or by other such methods including those disclosed in US patent publication No. 2008/0064862, published Mar. 13, 2008.

[0113] The invention encompasses glycosylated antibody compositions of matter such as cytotoxic antibodies. For example, the invention includes the glycosylated composition of matter for anti-CD20; TNFR-Fc (e.g., TNF receptor type II-IgG, e.g., Enbrel); EPO-Fc (e.g., erythropoietin-Fc); GIRT-Fc (e.g., glucocorticoid induced tumor necrosis factor); cytotoxic IL-2/Fc as well as other cytotoxic antibodies.

[0114] The invention includes methods for producing multimeric proteins including immunoglobulins, such as antibodies, and antigen binding fragments thereof. Thus, in one embodiment of the present invention, the multimeric protein is an immunoglobulin, wherein the first and second heterologous polypeptides are immunoglobulin heavy and light chains respectively.

[0115] In certain embodiments, an immunoglobulin polypeptide encoded by the transcriptional unit of at least one expression vector may be an immunoglobulin heavy chain polypeptide comprising a variable region or a variant thereof, and may further comprise a D region, a J region, a C region, or a combination thereof. An immunoglobulin polypeptide encoded by an expression vector may also be an immunoglobulin light chain polypeptide comprising a variable region or a variant thereof, and may further comprise a J region and a C region. The present invention also contemplates multiple immunoglobulin regions that are derived from the same animal species, or a mixture of species including, but not only, human, mouse, rat, rabbit and chicken. In certain embodiments, the antibodies are human or humanized.

[0116] In other embodiments, the immunoglobulin polypeptide encoded by at least one expression vector comprises an immunoglobulin heavy chain variable region, an immunoglobulin light chain variable region, and a linker peptide thereby forming a single-chain antibody capable of selectively binding an antigen.

[0117] Some other examples of therapeutic antibodies that may be produced in methods of the invention include, but are not limited, to HERCEPTIN® (Trastuzumab) (Genentech, CA) which is a humanized anti-HER2 monoclonal antibody for the treatment of patients with metastatic breast cancer; REOPRO® (abciximab) (Centocor) which is an anti-glycoprotein IIb/IIIa receptor on the platelets for the prevention of clot formation; ZENAPAX® (daclizumab) (Roche Pharmaceuticals, Switzerland) which is an immunosuppressive, humanized anti-CD25 monoclonal antibody for the prevention of acute renal allograft rejection; PANOREX® which is a murine anti-17-IA cell surface antigen IgG2 an antibody (Glaxo Wellcome/Centocor); BEC2 which is a murine anti-idiotype (GD3 epitope) IgG antibody (ImClone System); IMC-C225 which is a chimeric anti-EGFR IgG antibody (ImClone System); VITAXINT® which is a humanized anti-αVβ3 integrin antibody (Applied Molecular Evolution/MedImmune); Campath; Campath 1H/LDP-03 which is a humanized anti-CD52 IgG1 antibody (Leukosite); Smart M195 which is a humanized anti-CD33 IgG antibody (Protein Design Lab/Kanebo); RITUXAN® which is a chimeric anti-CD2O IgG1 antibody (IDEC Pharm/Genentech, Roche/Zettyaku); LYMPHOCIDE® which is a humanized anti-CD22 IgG antibody (Immunomedics); ICM3 is a humanized anti-ICAM3 antibody (ICOS Pharm); IDEC-114 is a primate anti-CD80 antibody (IDEC Pharm/Mitsubishi); ZEVALIN® is a radiolabelled murine anti-CD20 antibody (IDEC/Schering AG); IDEC-131 is a humanized anti-CD40L antibody (IDEC/Eisai); IDEC-151 is a primatized anti-CD4 antibody (IDEC); IDEC-152 is a primatized anti-CD23 antibody (IDEC/Seikagaku); SMART anti-CD3 is a humanized anti-CD3 IgG (Protein Design Lab); 5G1.1 is a humanized anti-complement factor 5 (CS) antibody (Alexion Pharm); D2E7 is a humanized anti-TNF-α antibody (CATIBASF); CDP870 is a humanized anti-TNF-α Fab fragment (Celltech); IDEC-151 is a primatized anti-CD4 IgG1 antibody (IDEC Pharm/SmithKline Beecham); MDX-CD4 is a human anti-CD4 IgG antibody (Medarex/Eisai/Genmab); CDP571 is a humanized anti-TNF-α IgG4 antibody (Celltech); LDP-02 is a humanized anti-α4β7 antibody (LeukoSite/Genentech); OrthoClone OKT4A is a humanized anti-CD4 IgG antibody (Ortho Biotech); ANTOVA® is a humanized anti-CD40L IgG antibody (Biogen); ANTEGREN® is a humanized anti-VLA-4 IgG antibody (Elan); CAT-152, a human anti-TGF-132 antibody (Cambridge Ab Tech); Cetuximab (BMS) is a monoclonal anti-EGF receptor (EGFr) antibody; Bevacizuma (Genentech) is an anti-VEGF human monoclonal antibody; Infliximab (Centocore, JJ) is a chimeric (mouse and human) monoclonal antibody used to treat autoimmune disorders; Gemtuzumab ozogamicin (Wyeth) is a monoclonal antibody used for chemotherapy; and Ranibizumab (Genentech) is a chimeric (mouse and human) monoclonal antibody used to treat macular degeneration.

[0118] Proteins produced in transgenic avians in accordance with the invention can be purified from egg white by any useful procedure such as those apparent to a practitioner of ordinary skill in the art of protein purification. For example, the antibody molecules (e.g., cytotoxic antibody molecules) such as anti-CD20 molecules produced in transgenic avians in accordance with the invention can be purified from egg white by methods apparent to practitioners of ordinary skill in the art of protein purification. For example, cytotoxic containing antibodies of the invention may be isolated using a Protein A column.

[0119] The contents of all references, published patents and patents cited throughout the present application are hereby incorporated by reference in their entireties.

[0120] The following specific examples are intended to illustrate the invention and should not be construed as limiting the scope of the claims.

Example 1

Construction of pSIN-OV-1.1-I-SBC201-I3

[0121] The vector pSIN-OV-3.5-I-CTLA4-inv disclosed in US patent publication No. 2008/0064862, filed Mar. 13, 2008, the disclosure of which was incorporated in its entirety herein by reference, was digested with AflII, filled in with Klenow and then cut with NruI. The resulting 9170 bp fragment was isolated and self-ligated to create pSIN-OV-1.1-I-CTLA4-inv.

[0122] Coding sequences and flanking sequences for SBC201 were synthesized by IDT (Coralville, Iowa) and cloned into a PUC-based plasmid (pUC57) resulting in SBC201 B (SEQ ID NO: 7) and SBC201 A (SEQ ID NO: 8). The coding sequence of SBC201 B is represented by nucleotides 990 to 2402 of SEQ ID NO: 7. The coding sequence of SBC201 A is represented by nucleotides 425 to 1132 of SEQ ID NO: 8.

[0123] pSIN-OV-1.1-I-SBC201 was generated by ligation of the 7795 bp NcoI/AflII fragment of pSIN-OV-1.1-I-CTLA4-inv to the 2234 bp EcoRI/AflII fragment of SBC201B and the 733 bp EcoRI/NcoI fragment of SBC201A.

[0124] A 8517 bp KpnI/KpnI fragment of pSIN-OV-1.1-I-SBC201 was ligated to a 1919 bp KpnI/SexAI fragment of pSIN-OV-1.1-I-SBC201 and 326 bp KpnI/SexAI fragment of syn IRES3 090508 (SEQ ID NO: 9) to produce pSIN-OV-1.1-I-SBC201-I3 shown in FIG. 9 and in SEQ ID NO: 6. Alternative cloning strategies are available to produce pSIN-OV-1.1-I-SBC201-I3, as is understood by a practitioner of skill in the art.

Example 2

Production Of Transgenic Chickens Using pSIN-OV-1.1-I-SBC201-I3

[0125] Retroviral particles pseudotyped with the VSV envelope protein and containing pSIN-OV-1.1-I-SBC201-I3 were produced as described in US patent publication No. 2007/0077650, published Apr. 5, 2007, the disclosure of which is incorporated in its entirety herein by reference. Virus was harvested at 48 hours post-transfection, concentrated and on the same day approximately 7 microliters injected into the subgerminal cavity of stage X eggs. Eggs were resealed and incubated until hatch. The transgenesis level in these hens is estimated at 5% or less.

[0126] G1 hens were obtained by crossing G0 transgenic roosters to wild type hens and screening for transgenic offspring. Egg white from G1 hens produced substantially more antibody than produced in the chimeric G0 hens, as expected. The amino acid sequence of the anti-CD20 antibody produced is shown in FIG. 2.

Example 3

Carbohydrate Analysis of Transgenic Poultry Derived Anti-CD20

[0127] Anti-CD20 was prepared from egg white obtained from eggs laid by G1 transgenic chickens produced as described in Example 2 using a Protein A column, as is understood in the art.

[0128] MALDI-TOF-MS (Matrix assisted laser desorption ionization time-of-flight mass spectrometry) analysis and ESI MS/MS (electrospray ionization tandem mass spectrometry) were performed on the oligosaccharides after release from the peptide backbone of the purified avian derived anti-CD20. Permethylated N-glycan structures identified by MALDI-TOF-MS are shown in FIG. 2. Approximate m/z are indicated for each of the structures.

[0129] The permethlyated N-glycans were also analyzed by nanospray ionization (NSI), as is understood in the art. The NSI method of analysis revealed additional oligosaccharide structures not detected using MALDI-TOF-MS, some of which have a fucose residue. For example, the fucose residue may be attached through an N-acetylglucosamine of the reducing terminus. It has been estimated that approximately 1% to 3% of the oligosaccharides attached to the antibodies have an attached fucose. Approximate m/z are indicated in FIGS. 1 and 2 for each of the structures.

Example 4

Anti-CD20 CDC (Complement Dependent Cytotoxicity) Determination

[0130] Daudi cells were washed and resuspended in a serum free medium to a concentration of about 5×10⁴ cells per ml and 5 μl aliquots of the cells were added to the wells of a 96 well microtiter plate. 50 μl aliquots of anti-CD20 antibody produced as described in Examples 1 and 2 above were prepared having concentrations shown in FIG. 6 (concentration ranges from 0.3 ng/ml to 1000 ng/ml). The antibody aliquots were added to wells of the 96 well microtiter plate containing the Daudi cells. 50 μl of 1:3 diluted normal human serum complement solution was added to each sample well, the solution was gently mixed then incubated at 37° C. After 2 hours of incubation, 30 μl of CellTiter-Blue Reagent® was gently mixed into each well followed by 16 hours of incubation at 37° C. The optical density was determined using a fluorometer at 560_Ex/590_EM nm and percentage of cellular lysis was calculated. The assay was repeated using commercially available anti-CD20 made in CHO cells having the same heavy chain and light chain amino acid sequences as the avian derived anti-CD20. Results are shown in FIG. 6.

Example 5

Anti-CD20 ADCC Antibody Dependent Cellular Cytotoxicity) Determination

[0131] PBMCs (Peripheral blood mononuclear cells) to be used as human effecter cells were prepared from blood of a healthy donor by separation in Ficoll-Hypaque density gradient. After separation, the cells were washed three times in 1×PBS.

[0132] Daudi cells were harvested, washed, and resuspended in RPMI-1640 plus 10% HIA FBS, 1× pen/strep, 1×L-glutamin to a concentration of 1×10⁶ cells per ml. Calcein-AM (Calcein acetoxymethyl ester) was added to the Daudi cells to a final concentration of 15 μM and the mixture was incubated at 37° C. for 30 min. The cells were then washed twice with 1×PBS and were resuspended in RPMI-1640 plus 10% HIA FBS, 1× pen/strep, 1×L-glutamin. Equal volumes of labeled target and PBMC suspensions were mixed, resulting in a ratio of PBMC Effector:Daudi Target Cells of about 50:1. 100 μl of the cell mixture was added to each well of a 96-well plate. 50 μl aliquots of antibody (concentrations ranging from 0.01 ng/ml to 10,000 ng/ml) produced as described in Examples 1 and 2 were added to the wells. The samples were incubated for 4 h at 37° C. in a 5% CO₂ incubator and then centrifuged at 300×g for 3 min. 75 μl of supernatant were removed from each well and percentage of cytolysis was determined by analyzing optical density. The assay was repeated using commercially available anti-CD20 made in CHO cells having the same heavy chain and light chain amino acid sequences as the avian derived anti-CD20. Results are shown in FIG. 7. It can be seen the anti-CD20 antibody produced in accordance with the invention has a surprisingly high ADCC.

Example 6

Anti-CD20 PK Determination

[0133] A healthy rat was injected with 0.1 mg of anti-CD20 produced as described in Examples 1 and 2 above per kilogram of body weight. A control rat was injected with commercially available anti-CD20 made in CHO cells having the same heavy chain and light chain amino acid sequences as the avian derived anti-CD20.

[0134] The antibody was assayed for by Elisa to determine serum concentration at time points over a four day period. The results are shown in FIG. 8.

[0135] All documents (e.g., U.S. patents, U.S. patent applications, publications) cited in the above specification are incorporated herein by reference. Various modifications and variations of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the following claims.

Sequence CWU 1

1

91235PRTArtificialLC 1Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile 20 25 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 Ser Ser Val Ser Tyr Ile His Trp Phe Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 85 90 95 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Thr Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 115 120 125 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 130 135 140 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 145 150 155 160 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 165 170 175 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 180 185 190 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 195 200 205 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 210 215 220 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 2470PRTArtificialHC 2Met Gly Trp Ser Leu Ile Leu Leu Phe Leu Val Ala Val Ala Thr Arg 1 5 10 15 Val Leu Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys 20 25 30 Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu 50 55 60 Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn 65 70 75 80 Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn 115 120 125 Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro 225 230 235 240 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 355 360 365 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 370 375 380 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 Ser Leu Ser Pro Gly Lys 465 470 3716PRTArtificialSingle Chain Anti CD-20 3Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile 20 25 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 Ser Ser Val Ser Tyr Ile His Trp Phe Gln Gln Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 85 90 95 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Thr Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 115 120 125 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 130 135 140 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 145 150 155 160 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 165 170 175 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 180 185 190 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 195 200 205 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 210 215 220 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser 225 230 235 240 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 245 250 255 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Pro 260 265 270 Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys 275 280 285 Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asn Met His Trp Val Lys Gln 290 295 300 Thr Pro Gly Arg Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn 305 310 315 320 Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr 325 330 335 Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr 340 345 350 Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Ser Thr Tyr Tyr Gly 355 360 365 Gly Asp Trp Tyr Phe Asn Val Trp Gly Ala Gly Thr Thr Val Thr Val 370 375 380 Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 385 390 395 400 Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 405 410 415 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 420 425 430 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 435 440 445 Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 450 455 460 Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val 465 470 475 480 Asp Lys Lys Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 485 490 495 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 500 505 510 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 515 520 525 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 530 535 540 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 545 550 555 560 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 565 570 575 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 580 585 590 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 595 600 605 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 610 615 620 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 625 630 635 640 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 645 650 655 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 660 665 670 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 675 680 685 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 690 695 700 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 705 710 715 4226PRTArtificialC2H7 Anti-CD20 HC 4Gln Ala Tyr Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asn Met His Trp Val Lys Gln Thr Pro Arg Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp 100 105 110 Gly Thr Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp 225 5213PRTArtificialC2H7 Anti-CD20 LC 5Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Ala Pro Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 610762DNAArtificialpSIN-OV-1.1-I-SBC201-I3 6cttctgggca tccttcagcc ccttgttgaa tacgcttgag gagagccatt tgactctttc 60cacaactatc caactcacaa cgtggcactg gggttgtgcc gcctttgcag gtgtatctta 120tacacgtggc ttttggccgc agaggcacct gtcgccaggt ggggggttcc gctgcctgca 180aagggtcgct acagacgttg tttgtcttca agaagcttcc agaggaactg cttccttcac 240gacattcaac agaccttgca ttcctttggc gagaggggaa agacccctag gaatgctcgt 300caagaagaca gggccaggtt tccgggccct cacattgcca aaagacggca atatggtgga 360aaataacata tagacaaacg cacaccggcc ttattccaag cggcttcggc cagtaacgtt 420aggggggggg gagggagagg ggcggaattc caccacactg gactagtgga tcctcaacac 480tctcccctgt tgaagctctt tgtgacgggc gagctcaggc cctgatgggt gacttcgcag 540gcgtagactt tgtgtttctc gtagtctgct ttgctcagag tcagggtgct gctgaggctg 600taggtgctgt ccttgctgtc ctgctctgtg acactctcct gggagtttcc cgattggagg 660gcgttatcaa ccttccactg tactttggcc tctctgggat agaagttatt cagcaggcac 720acaacagagg cagttccaga tttcaactgc tcatcagatg gcgggaagat gaagacagat 780ggtgcagcca ccgtacgttt gatttccagc ttggtccccc ctccgaacgt gggtgggtta 840ctagtccact gctggcagta ataagtggca gcatcttcag cctccactct gctgattgtg 900agagagtaag aagtcccaga cccactgcca ctgaagcgaa cagggactcc agaagccagg 960ttggatgtgg cataaatcca aggttttggg gaggatcctg gcttttgctg gaaccagtgg 1020atgtaactga cacttgagct ggccctgcaa gtcattgtga ccttctcccc gggagatgca 1080gacaggattg ccggagattg ggagagaaca atttgtcctc tggacattat gactgaagca 1140ctgattagca ggaagctgat aatctgcacc tgaaaatcca tggtgaactc tgagttgtct 1200agagcaaaca gcagaacagt gaaaatgtaa ggatggaatg ctgtacatag taccatgcag 1260ggtactctat ggtaggctac aacagtaaat tacgagcagt ttttaggcaa ttaaatgtta 1320acaagtagtt ttaaagtaat tctgtggtaa tgtgtctgtt gctatatcca cctctcatgt 1380gcatgttcaa aaccatattc ataaatctat ttatgtattt gcattcagtt gtcttttggg 1440tagcaaactg tcccagaagc cagttgcctc tacatatttt tgttcagtga aagctagaat 1500tcattgatac ttttcagtac ctctgattaa aacacaatct gataggcttg caaaactgga 1560aattcaaaga gcaaatttca gtaaacttta ggtttggaca gatatatgag aaagcagagg 1620cttgctgact attttatttc ttatttttat tccctaaaaa taaatgtaga gaaatatctg 1680tttgttgcac actacttgct atgagtagat cttcaaaagt atttttacct ttgttttggt 1740gatggcagaa tagataagga atgtaattta tatggggtca tgtagtctag gagaaagaca 1800cgcatgtaat tcatattctg ctctattgca ctttcaggta tggtttgctt tgctcaaaga 1860tatgcatgtg tactgtagta taaactttct gtggagttaa attttagtgg tgacattcag 1920acagaagaga aatgcagaca tgataaaata gcaatgttta ctataaaaca gagccactga 1980atgaattctt gttcatgaca tagaccaata gaagatttat acttgttctg tctgtttcta 2040ttataaagag ctgaactgta caactattgt atagccagtg tgcttatata aagcacagct 2100tttggagcca gcatgaatct agttgctttc ctgagattta tataatctgt gaaagtcaga 2160agtccttcag agcccagccc tttatatgcg tactgagtgc tggggcctca ggattggatt 2220ttctgtatta aacccctcaa aagtttttac tgaccacgtg tgtgagtata cacacacaca 2280tttttctcat tttcttttct gtatataagt tcacatgtat ctattattgt aagaatatac 2340gtttatgcac cccccacatt tttatcttgt gtagtgatca gcagctgcac tttgcaggaa 2400ttaaacttct agagaatttt cacattaaaa taactcccca gaattcactg aacaccatga 2460ttttgctctc tgtgcactct gtagggctag aagttaatca agcaaactgc aaagcatatc 2520agatagtgaa cgacaggata agatgttctg aaattaaaaa catattttaa gcacaaagaa 2580taagcctcct gaaaacaaac acaaagcttt tacacataat aaaatagtgc agaatgcata 2640cacaggtgag aagtttttat agggggtatc acgcaggtac ttcaccctta aagatacaac 2700acatagcaca ataattgtta

attttttaaa gtttaggtgc aagtaagagc taatatagag 2760agaaggtaat tccagagagt tgcttacctt tcgagcttga ctgctaaagg caatacagct 2820ttctagctgt atgtacagac actggctgag ccctggggaa tatatagtct gaattgtgac 2880ccacccacag gttcccttca gaagtttgac ctttgacacc atagaaatca tttaatggga 2940ttgggttaga ttttagtttc aataggtcca ttttggattg aatggagagc aaatattagt 3000ttttaattct gggtaacaat gtgttttctg cctgttctgc taatccatca ggactgttgg 3060atgggagaga agactgggaa atattgctca tgttccattg agcttcagtt acaaccagat 3120aatgggatct ttaagaaaac agaaaaatgt gggaaccttg gagatggaaa acataattag 3180caattattag ttagtgtgct tattactatg gttgtagtaa cagaccagaa gtctgtttca 3240tttgatcctt cttgtatgta caatgtgcat ctgagccacg ctagacagga cataaatgag 3300aacaagactt gacctattat tttcttgaca aaataggaga aataaagaag cgtgcatgtg 3360aaggagccaa ctgagactag agtgaagagc agacacactt tctttcctat agttggaata 3420tttaaatcta tctttttatg ggtgtgaatg ctttataaca aacttttatt ctgaggatac 3480agcaaaacat agctccatac aatgcaaaac aatactcaat ttcaaatgtg tttatgatat 3540gaacttgcag tgttcctcaa agatcttcca tgaataactt aatggcctgg cagatgacag 3600aggaattgtg aaattcagct ggaggagtgt tcatggttcg agggacaatc ataatataca 3660atagcaaata tatttcagtt atagaagcta ttgttctgta ttgaaataat agaattgaca 3720aacagtaaag aaaccattct gacctctgta aagcactgtt tgatttaaaa atgggggaaa 3780aaagtacaac ataattcttc aggacataca tagagatcac tgcaatctct gttaagcaga 3840attactttcc tataccacta gctgaagttt agtcagtgcc attttctttt gtttctctcc 3900ttccttttgt gaaaacatat atactgtgga aatctacatt ctccttgcca agtctgagga 3960cttaacgagg aatataaaaa aattacagga ggcttataag cagcccgaaa gaagagcgta 4020ggcgagttct tgtattccgt gtgatagctg gttggattgg taattgatcg gctggcacgc 4080ggaatatagg aggtcgctga atagtaaact tgtagacttg gctacagcat agagtatctt 4140ctgtagctct gatgactgct aggaaataat gctacggata atgtggggag ggcaaggctt 4200gcgaatcggg ttgtaacggg caaggcttga ctgaggggac aatagcatgt ttaggcgaaa 4260agcggggctt cggttgtacg cggttaggag tcccctcagg atatagtagt ttcgcttttg 4320catagggagg gggacggatt ggacgaacca ctgaattccg cattgcagag atattgtatt 4380taagtgccta gctcgataca ataaacgcca tttgaccatt caccacattg gtgtgcacct 4440gggttgatgg ccggaccgtt gattccctga cgactacgag cacatgcatg aagcagaagg 4500cttcatttgg tgaccccgac gtgatcgtta gggaatacgc gctcactggc cgtcgtttta 4560caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc 4620cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg 4680cgcagcctga atggcgaatg gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa 4740atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata 4800aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac 4860tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc 4920cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa 4980atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg 5040cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg 5100tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag 5160gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 5220caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 5280ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 5340gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 5400tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 5460ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 5520tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 5580atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 5640gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 5700caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 5760ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 5820ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 5880ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 5940ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 6000gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 6060ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 6120taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 6180agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 6240atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 6300aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 6360caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 6420ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 6480cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 6540tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 6600gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 6660ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 6720gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 6780caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 6840ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 6900tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 6960ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 7020agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 7080aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 7140gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 7200tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 7260tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 7320ccaagcgcgc attggtaatt gatcggctgg cacgcggaat ataggaggtc gctgaatagt 7380aaacttgtag acttggctac agcatagagt atcttctgta gctctgatga ctgctaggaa 7440ataatgctac ggataatgtg gggagggcaa ggcttgcgaa tcgggttgta acgggcaagg 7500cttgactgag gggacaatag catgtttagg cgaaaagcgg ggcttcggtt gtacgcggtt 7560aggagtcccc tcaggatata gtagtttcgc ttttgcatag ggagggggaa atgtagtctt 7620atgcaatact cttgtagtct tgcaacatgc ttatgtaacg atgagttagc aacatgcctt 7680ataaggagag aaaaagcacc gtgcatgccg attggtggga gtaaggtggt atgatcgtgg 7740tatgatcgtg ccttgttagg aaggcaacag acgggtctaa cacggattgg acgaaccact 7800gaattccgca ttgcagagat attgtattta agtgcctagc tcgatacaat aaacgccatt 7860tgaccattca ccacattggt gtgcacctgg gttgatggcc ggaccgttga ttccctgacg 7920actacgagca catgcatgaa gcagaaggct tcatttggtg accccgacgt gatcgttagg 7980gaatagtggt cggccacagg cggcgtggcg atcctgtcct catccgtctc gcttattcgg 8040ggagcggacg atgaccctag tagagggggc tgcggcttag gagggcagaa gctgagtggc 8100gtcggaggga gccctactgc agggggccaa cataccctac cgagaactca gagagtcgtt 8160ggaagacggg aaggaagccc gacgactgag cggtccaccc caggcgtgat tccggttgct 8220ctgcgtgatt ccggtcgccc ggtggatcaa gcatggaagc cgtcataaag gtgatttcgt 8280ccgcgtgtaa gacctattgc gggaaaacct ctccttctaa gaaggaaata ggggctatgt 8340tgtccctgtt acaaaaggaa gggttgctta cgtccccctc agacttatat tccccggggt 8400cctgggatcc cattaccgcg gcgctctctc agcgggctat ggtacttgga aaatcgggag 8460agttaaaaac ctggggattg gttttggggg cattgaaggc ggctcgagat ccggtacctt 8520caaatactac aagtgaaaag tgtttgctta aacatgtttt tattatgatt aaaggaacaa 8580aagagcacat tcacaagacc cattacatat gggtacaagg aaaacaattt gaatagtaat 8640ataccatatt tgccaacata ccatgattga gtcaaagttt agggagaaat gtgaattata 8700agatttttat aatgcatctt taggaagtca ggaagagcct tgtagtatca ggaacacaga 8760gaacaagcaa ttgccttgtc agcataggaa tggttggtga cagttgataa tttaatctga 8820gagattttga gtgactaatt ctggagcagc ttggtcatac agatatctgg cttaattgga 8880aggctgcatt tttcccccat aaaccttctg ctgatgtatc aggttgcatt tttcagtgtg 8940atgactcagt actgtgagtc caatttcatt cccttaagcc ttcatccatg agttaccagt 9000attactctgt gtaaaggaaa agtgaattgc acctgttctc acagtgtaat ttctttctga 9060ttttttttct agattaagct ccagctttta tgaagtctgg atgcagcaga taacatactt 9120ttcattttac ccctgatact acagtgctct gggtcttgtt ggaagggaca gagtttttca 9180gctttcttct tgcggccgct tcgaatcatt tgcccggaga cagggagagg ctcttctgcg 9240tgtagtggtt gtgcagagcc tcatgcatca cggagcatga gaagacgttc ccctgctgcc 9300atctgctctt gtccacggtc agtttggagt agaggaagaa ggagccgtcg gagtccagca 9360cgggaggcgt ggtcttgtag ttgttctccg gctgcccatt gctctcccac tccacggcga 9420tgtcgctggg atagaagcct ttgaccaggc aggtcaggct gacttggttc ttggtcagct 9480catcccggga tgggggcagg gtgtaaacct gtggttctcg gggctgccct ttggctttgg 9540agatggtttt ctcgatgggg gctgggaggg ctttgttgga gaccttgcac ttgtactcct 9600tgccattcag ccagtcttgg tgcaggacgg tcaggacgct gaccacacga tacgtgctgt 9660tgtactgctc ctcccgcggc tttgtcttgg cattatgaac ctccacgccg tccacgtacc 9720agttgaactt gacctcaggg tcttcgtggc taacgtccac caccacgcat gtgacctcag 9780gggtccggga gatcatgagg gtgtccttgg gttttggggg gaagaggaag actgacggtc 9840cccccaggag ttcaggtgct gggcacggtg ggcatgtgtg agttttgtca caagatttgg 9900gctctgcttt cttgtccacc ttggtgttgc tgggcttgtg attcacgttg cagatgtagg 9960tctgggtgcc caagctgctg gagggcacgg tcaccacgct gctgagggag tagagtcctg 10020aggattgtag gacagccggg aaagtatgca cgccgctggt cagggcgcct gagttccacg 10080acaccgtcac cggttcgggg aagtagtcct tgaccaggca gcccagggcc gctgtgcccc 10140cagaggtgct cttggaggag ggtgccaggg ggaagaccga tgggcccttg gtgctagctg 10200cagagacggt gaccgtggtc cctgcgcccc agacattgaa gtaccagtca ccgccgtagt 10260aagtcgatct tgcacagtaa tagaccgcag agtcctcaga tgtcaggctg ctgagttgca 10320tgtaggctgt gctggaggat ttgtctgcag tcaatgtggc cttgcctttg aacttctgat 10380tgtaggaagt atcaccattt ccgggataaa tagctccaat ccattccagg ccccgaccag 10440gtgtctgttt tacccagtgc atattgtaac tggtaaatgt gtagccagaa gccttgcagg 10500acatcttcac tgaggcccca ggcttcacca gctccgcccc cggctgctgc agttgtactt 10560gggacaggac acgcgtagca acagcgacaa ggaagagcaa gatgaggctc caacccatgg 10620ttgtggccat attatcatcg tgtttttcaa aggaaaacca cgtccccgtg gttcgggggg 10680cctagacgtt ttttaacctc gactaaacac atgtaaagca tgtgcaccga ggccccagat 10740cagatcccat acaatggggt ac 1076275109DNAArtificialSBC201 B in pUC57 (IDT) 7tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ccgcccctct ccctcccccc 420cccctaacgt tactggccga agccgcttgg aataaggccg gtgtgcgttt gtctatatgt 480tattttccac catattgccg tcttttggca atgtgagggc ccggaaacct ggccctgtct 540tcttgacgag cattcctagg ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga 600atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag acaaacaacg tctgtagcga 660ccctttgcag gcagcggaac cccccacctg gcgacaggtg cctctgcggc caaaagccac 720gtgtataaga tacacctgca aaggcggcac aaccccagtg ccacgttgtg agttggatag 780ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg aaggatgccc 840agaaggtacc ccattgtatg ggatctgatc tggggcctcg gtgcacatgc tttacatgtg 900tttagtcgag gttaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg 960aaaaacacga tgataagctt gccacaacca tgggttggag cctcatcttg ctcttccttg 1020tcgctgttgc tacgcgtgtc ctgtcccaag tacaactgca gcagccgggg gcggagctgg 1080tgaagcctgg ggcctcagtg aagatgtcct gcaaggcttc tggctacaca tttaccagtt 1140acaatatgca ctgggtaaaa cagacacctg gtcggggcct ggaatggatt ggagctattt 1200atcccggaaa tggtgatact tcctacaatc agaagttcaa aggcaaggcc acattgactg 1260cagacaaatc ctccagcaca gcctacatgc aactcagcag cctgacatct gaggactctg 1320cggtctatta ctgtgcaaga tcgacttact acggcggtga ctggtacttc aatgtctggg 1380gcgcagggac cacggtcacc gtctctgcag ctagcaccaa gggcccatcg gtcttccccc 1440tggcaccctc ctccaagagc acctctgggg gcacagcggc cctgggctgc ctggtcaagg 1500actacttccc cgaaccggtg acggtgtcgt ggaactcagg cgccctgacc agcggcgtgc 1560atactttccc ggctgtccta caatcctcag gactctactc cctcagcagc gtggtgaccg 1620tgccctccag cagcttgggc acccagacct acatctgcaa cgtgaatcac aagcccagca 1680acaccaaggt ggacaagaaa gcagagccca aatcttgtga caaaactcac acatgcccac 1740cgtgcccagc acctgaactc ctggggggac cgtcagtctt cctcttcccc ccaaaaccca 1800aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg gacgttagcc 1860acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtt cataatgcca 1920agacaaagcc gcgggaggag cagtacaaca gcacgtatcg tgtggtcagc gtcctgaccg 1980tcctgcacca agactggctg aatggcaagg agtacaagtg caaggtctcc aacaaagccc 2040tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga gaaccacagg 2100tttacaccct gcccccatcc cgggatgagc tgaccaagaa ccaagtcagc ctgacctgcc 2160tggtcaaagg cttctatccc agcgacatcg ccgtggagtg ggagagcaat gggcagccgg 2220agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc ttcctctact 2280ccaaactgac cgtggacaag agcagatggc agcaggggaa cgtcttctca tgctccgtga 2340tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct ccgggcaaat 2400gattcgaagc ggccgcaaga agaaagctga aaaactctgt cccttccaac aagacccaga 2460gcactgtagt atcaggggta aaatgaaaag tatgttatct gctgcatcca gacttcataa 2520aagctggagc ttaatctaga aaaaaaatca gaaagaaatt acactgtgag aacaggtgca 2580attcactttt cctttacaca gagtaatact ggtaactcat ggatgaaggc ttaagggaat 2640gaaattggac tcacagtact gagtcatcac actgaaaaat gcaacctgat acatcagcag 2700aaggtttatg ggggaaaaat gcagccttcc aattaagcca gatatctgta tgaccaagct 2760gctccagaat tagtcactca aaatctctca gattaaatta tcaactgtca ccaaccattc 2820ctatgctgac aaggcaattg gggcccgtcg actgcagagg cctgcatgca agcttggcgt 2880aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca 2940tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat 3000taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt 3060aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct 3120cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 3180aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 3240aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 3300tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 3360caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 3420cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 3480ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 3540gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 3600agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 3660gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 3720acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 3780gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 3840gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 3900cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 3960caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 4020gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 4080cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 4140cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 4200caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 4260gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 4320gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 4380cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 4440catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 4500gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 4560ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 4620gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 4680cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 4740tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 4800gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 4860atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 4920ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 4980gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 5040acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc 5100cctttcgtc 510984091DNAArtificialSBC201 A in pUC57 (IDT) 8tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ccaccacact ggactagtgg 420atcctcaaca ctctcccctg ttgaagctct ttgtgacggg cgagctcagg ccctgatggg 480tgacttcgca ggcgtagact ttgtgtttct cgtagtctgc tttgctcaga gtcagggtgc 540tgctgaggct gtaggtgctg tccttgctgt cctgctctgt gacactctcc tgggagtttc 600ccgattggag ggcgttatca accttccact gtactttggc ctctctggga tagaagttat 660tcagcaggca cacaacagag gcagttccag atttcaactg ctcatcagat ggcgggaaga 720tgaagacaga tggtgcagcc accgtacgtt tgatttccag cttggtcccc cctccgaacg 780tgggtgggtt actagtccac tgctggcagt aataagtggc agcatcttca gcctccactc 840tgctgattgt gagagagtaa gaagtcccag acccactgcc actgaagcga acagggactc 900cagaagccag gttggatgtg gcataaatcc aaggttttgg ggaggatcct ggcttttgct 960ggaaccagtg gatgtaactg acacttgagc tggccctgca agtcattgtg accttctccc 1020cgggagatgc agacaggatt gccggagatt gggagagaac aatttgtcct ctggacatta 1080tgactgaagc actgattagc aggaagctga taatctgcac ctgaaaatcc atggtgaact 1140ctgagttgtc tttcgagctt gactgctaaa ggcaatacag ctttctagct gtatgtacag 1200acactggctg agccctgggg aatatatagt ctgaattgtg acccacccac aggttccctt 1260cagaagtttg acctttgaca ccatagaaat catttaatgg gattgggtta gattttagtt 1320tcaataggtc cattttggat tgaatggaga gcaaatatta gtttttaatt ctgggtaaca 1380atgtgttttc tgcctgttct gctaatccat caggactgtt ggatgggaga gaagactggg 1440aaatattgct catgttccat tgagcttcag ttacaaccag ataatgggat ctttaagaaa 1500acagaaaaat gtgggaacct tggagatgga aaacataatt agcaattatt agttagtgtg 1560cttattacta tggttgtagt aacagaccag aagtctgttt catttgatcc ttcttgtatg 1620tacaatgtgc atctgagcca cgctagacag gacataaatg agaacaagac ttgacctatt 1680attttcttga caaaatagga gaaataaaga agcgtgcatg tgaaggagcc

aactgagact 1740agagtgaaga gcagacacac tttctttcct atagttggaa tatttaaatc tatcttttta 1800tgggtgtgaa tgctttataa cagggcccgt cgactgcaga ggcctgcatg caagcttggc 1860gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa 1920catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac 1980attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca 2040ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc 2100ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 2160aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 2220aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 2280gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 2340gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 2400tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 2460ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 2520ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 2580tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 2640tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 2700ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 2760aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 2820ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 2880tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 2940atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 3000aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 3060ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 3120tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 3180ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 3240tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 3300aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 3360gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 3420tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 3480cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 3540tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 3600ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 3660cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 3720actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 3780ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 3840aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 3900ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 3960atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 4020tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag 4080gccctttcgt c 40919329DNAArtificialsynIRES3 090508 9ggtaccccat tgtatgggat ctgatctggg gcctcggtgc acatgcttta catgtgttta 60gtcgaggtta aaaaacgtct aggccccccg aaccacgggg acgtggtttt cctttgaaaa 120acacgatgat aatatggcca caaccatggg ttggagcctc atcttgctct tccttgtcgc 180tgttgctacg cgtgtcctgt cccaagtaca actgcagcag ccgggggcgg agctggtgaa 240gcctggggcc tcagtgaaga tgtcctgcaa ggcttctggc tacacattta ccagttacaa 300tatgcactgg gtaaaacaga cacctggtc 329

Claims

1. A composition comprising an isolated mixture of cytotoxic anti-CD20 antibody molecules produced in a transgenic avian, wherein the antibody molecules comprise a heavy chain and a light chain whose amino acid sequences set forth from position 23 to position 235 of SEQ ID NO:1 and from position 20 to position 470 of SEQ ID NO: 2, respectively, wherein the isolated mixture of cytotoxic anti-CD20 antibody molecules exhibit an increased level of antibody-dependent cell-mediated cytotoxicity (ADCC) as compared to that of anti-CD20 antibody molecules produced in CHO cells and having the same amino acid sequence.

2. The composition of claim 1, wherein the isolated mixture of the anti-CD20 antibody molecules are produced in tubular gland cells of the transgenic avian.

3. The composition of claim 2, wherein the transgenic avian is selected from the group consisting of a chicken, a quail and a turkey.

4. The composition of claim 3, wherein the transgenic avian is a chicken.

5. The composition of claim 4, wherein the cytotoxic anti-CD20 antibody molecules are isolated from egg white of the transgenic chicken.

6. The composition of claim 1, wherein about 95% or more of the N-linked oligosaccharide structures present on the antibody molecules do not contain fucose.

Images