Antibodies that specifically bind to chemokine beta-4   

This application is a divisional of U.S. application Ser. No. 12/123,419, filed May 19, 2008, which is a divisional of U.S. application Ser. No. 10/513,705, filed Nov. 8, 2004 (now U.S. Pat. No. 7,375,192, issued May 20, 2008) and accorded a 371 filing date of Sep. 6, 2005, which is the National Stage of International Application No. PCT/US03/13414, filed Apr. 30, 2003, which claims the benefit of 60/376,561, filed May 1, 2002.

This application refers to a “Sequence Listing” listed below, which is provided as a text file. The text file contains a document entitled “PF598USD1-SequenceListing.txt” (67,673 bytes, created Apr. 2, 2008), which is incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to antibodies and related molecules that specifically bind to chemokine beta-4. Such antibodies have uses, for example, in wound healing and in the diagnosis, prevention, and treatment of cancers, as well as immune system diseases and disorders including autoimmune disease, inflammatory disorders, immunodeficiencies, infections, HIV, arthritis, allergy, psoriasis, dermatitis, and inflammatory bowel disease. The invention also relates to nucleic acid molecules encoding anti-chemokine beta-4 antibodies, vectors and host cells containing these nucleic acids, and methods for producing the same. The present invention relates to methods and compositions for preventing, detecting, diagnosing, treating or ameliorating a disease or disorder including cancers, as well as immune system diseases and disorders including autoimmune disease, inflammatory disorders, immunodeficiencies, infections, HIV, arthritis, allergy, psoriasis, dermatitis, and inflammatory bowel disease, comprising administering to an animal, preferably a human, an effective amount of one or more antibodies or fragments or variants thereof, or related molecules, that specifically bind to chemokine beta-4.

BACKGROUND OF THE INVENTION

Chemokines, also referred to as intercrine cytokines, are a subfamily of structurally and functionally related cytokines. These molecules are 8-14 kd in size. In general chemokines exhibit 20% to 75% homology at the amino acid level and are characterized by four conserved cysteine residues that form two disulfide bonds. Based on the arrangement of the first two cysteine residues, chemokines have been classified into two subfamilies, alpha and beta. In the alpha subfamily, the first two cysteines are separated by one amino acid and hence are referred to as the “CXC” subfamily. In the beta subfamily, the two cysteines are in an adjacent position and are, therefore, referred to as the “CC” subfamily. Recently, a new chemokine-like molecule, lymphotactin/SCM-1, which lacks the first and the third conserved cysteine residues, has been isolated and may represent a third subfamily (Kelner et al., Science 266:1395-1399 (1994)).

The intercrine cytokines exhibit a wide variety of functions. A hallmark feature is their ability to elicit chemotactic migration of distinct cell types, including monocytes, neutrophils, T lymphocytes, basophils and fibroblasts. Many chemokines have proinflammatory activity and are involved in multiple steps during an inflammatory reaction. These activities include stimulation of histamine release, lysosomal enzyme and leukotriene release, increased adherence of target immune cells to endothelial cells, enhanced binding of complement proteins, induced expression of granulocyte adhesion molecules and complement receptors, and respiratory burst. In addition to their involvement in inflammation, certain chemokines have been shown to exhibit other activities. For example, macrophage inflammatory protein I (MIP-1) is able to suppress hematopoietic stem cell proliferation, platelet factor-4 (PF-4) is a potent inhibitor of endothelial cell growth, Interleukin-8 (IL-8) promotes proliferation of keratinocytes, and GRO is an autocrine growth factor for melanoma cells.

Chemokine beta-4 (CK-β4; also known as CCL20, MIP-3α, and LARC) represents a novel, divergent beta-chemokine. CK-β4 contains the four cysteine residues characteristic of CC chemokines and shows sequence similarity with other human CC chemokines. The highest homology (28%) is with chemokine MIP-1β (Hieshima, et al. J. Biol. Chem. 272:5846-5853 (1997)). CK-β4 is expressed preferentially in lymphocytes and monocytes, and its expression is markedly upregulated by mediators of inflammation such as tumor necrosis factor (TNF) and lipopolysaccharide. The CK-β4 gene has been mapped between the bands of q33 and q37 of chromosome 2 (Ibid.). The only receptor identified for CK-β4, thus far, is CCR6 (Greaves, et al., J. Exp. Med. 186:837-844).

The immune cells that are responsive to the chemokines have a vast number of in vivo functions and therefore their regulation by such chemokines is an important area in the treatment of disease. For example, eosinophils destroy parasites to lessen parasitic infection. Eosinophils are also responsible for chronic inflammation in the airways of the respiratory system. Macrophages are responsible for suppressing tumor formation in vertebrates. Further, basophils release histamine, which may play an important role in allergic inflammation.

Accordingly, promoting and inhibiting such cells, has wide therapeutic application. There is a clear need, therefore, for identification and characterization of compositions, such as antibodies, that influence the biological activity of chemokines, both normally and in disease states. In particular, there is a need to isolate and characterize antibodies that modulate the biological activities of chemokine beta-4 for the treatment of proliferative disorders, as well as immune system diseases and disorders including autoimmune disease, inflammatory disorders, immunodeficiencies, infections, HIV, arthritis, allergy, psoriasis, dermatitis, and inflammatory bowel disease.

SUMMARY

The present invention encompasses antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that specifically bind to a chemokine beta-4 polypeptide (CK-β4; also known as macrophage inflammatory protein 3-α (MIP-3α), CCL20, Exodus-1, and liver and activation-regulated chemokine (LARC); described in International Publication Nos. WO 96/05856, WO 97/31098, and U.S. Pat. No. 5,981,230 each of which are hereby incorporated by reference in their entireties) or a polypeptide fragment or variant of CK-β4. In particular, the invention encompasses antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that specifically bind to a CK-β4 polypeptide or polypeptide fragment or variant of human CK-β4 such as SEQ ID NO:2.

The present invention relates to methods and compositions for preventing, treating or ameliorating a disease or disorder comprising administering to an animal, preferably a human, an effective amount of one or more antibodies or fragments or variants thereof, or related molecules, that specifically bind to CK-β4 or a fragment or variant thereof. In specific embodiments, the present invention relates to methods and compositions for preventing, treating or ameliorating a disease or disorder associated with CK-β4 function or aberrant CK-β4 expression, comprising administering to an animal, preferably a human, an effective amount of one or more antibodies or fragments or variants thereof, or related molecules, that specifically bind to CK-β4 or a fragment or variant thereof.

In highly preferred embodiments, the present invention encompasses methods for using the antibodies of the present invention to treat, prevent, diagnose and/or prognose a disease or disorder of the immune system. In highly preferred embodiments, the present invention encompasses methods for using antibodies of the invention to treat, prevent, diagnose and/or prognose an inflammatory disorder (e.g., psoriasis, dermatitis, Langerhans cell histocytosis, inflammatory bowel syndrome, allergy, Crohn\'s disease). In additional preferred embodiments, the present invention relates to antibody-based methods and compositions for preventing, treating or ameliorating infectious disorders (e.g., human immunodeficiency virus (HIV) infection). In other preferred embodiments, the present invention relates to antibody-based methods and compositions for preventing, treating or ameliorating autoimmune disorders (e.g., rheumatoid arthritis and autoimmune encephalitis), graft-vs.-host reaction, and/or immunodeficiencies.

In other highly preferred embodiments, the invention encompasses methods for using the antibodies of the invention to inhibit B cell, T cell and/or dendritic cell chemotaxis. In specific embodiments, the invention encompasses methods for using the antibodies of the present invention to inhibit memory B cell or T cell (e.g., CD4 positive T cell) chemotaxis.

In other highly preferred embodiments, the invention encompasses methods for using the antibodies of the invention to stimulate B cell, T cell and/or dendritic cell chemotaxis. In specific embodiments, the invention encompasses methods for using the antibodies of the present invention to stimulate memory B cell or T cell (e.g., CD4 positive T cell) chemotaxis.

In other preferred embodiments, the invention encompasses methods for using the antibodies of the invention to inhibit proliferation of cells expressing a CK-B4 receptor (e.g., T cell, dendritic cell, or B cell).

In other preferred embodiments, the invention encompasses methods for using the antibodies of the invention to stimulate proliferation of cells expressing a CK-β4 receptor (e.g., T cell, dendritic cell, or B cell).

In highly preferred embodiments, the present invention relates to antibody-based methods and compositions for preventing, treating or ameliorating cancers (e.g., leukemia, T-cell lymphoma, B-cell lymphoma, prostate cancer, breast cancer, lung cancer, colon cancer, urinary cancer, non-Hodgkin\'s lymphoma, renal cell carcinoma, and myeloproliferative disorders).

In a further highly preferred embodiment of the invention, antibodies of the present invention may be used to promote wound healing.

Another embodiment of the present invention includes the use of the antibodies of the invention as a diagnostic tool to monitor the expression of CK-β4.

The present invention encompasses single chain Fv\'s (scFvs) that specifically bind CK-β4 polypeptides (e.g., SEQ ID NOs:20-36). Thus, the invention encompasses these scFvs, listed in Table 1. In addition the invention encompasses cell lines engineered to express antibodies corresponding to these scFvs which have been deposited with the American Type Culture Collection (“ATCC™”) as of the dates listed in Table 1 and given the ATCC™ Deposit Numbers identified in Table 1. The ATCC™ is located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA. The ATCC™ deposit was made pursuant to the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Purposes of Patent Procedure.

Further, the present invention encompasses the polynucleotides encoding the scFvs, as well as the amino acid sequences encoding the scFvs. Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs (e.g., VH domains, VH CDRs, VL domains, or VL CDRs having an amino acid sequence of any one of the scFvs referred to in Table 1), that specifically bind to CK-β4 or fragments or variants thereof are also encompassed by the invention, as are nucleic acid molecules that encode these antibodies and/or molecules.

The present invention also provides anti-CK-β4 antibodies which are coupled to a detectable label, such as an enzyme, a fluorescent label, a luminescent label, or a bioluminescent label. The present invention also provides anti-CK-β4 antibodies which are coupled to a therapeutic or cytotoxic agent. The present invention also provides anti-CK-β4 antibodies which are coupled to a radioactive material.

The present invention also provides antibodies that specifically bind one or more CK-β4 polypeptides and that act as either CK-β4 agonists or CK-β4 antagonists. In specific embodiments, the antibodies of the invention inhibit CK-β4 binding to a CK-β4 receptor (e.g., CCR6; GenBank ID: U68030).

In specific embodiments, the antibodies of the invention inhibit chemotaxis of cells that express a CK-β4 receptor (e.g., CCR6). In specific embodiments, the antibodies of the invention inhibit chemotaxis of lymphocytes (e.g., T cells, B cells). In specific embodiments, the antibodies of the invention inhibit chemotaxis of dendritic cells.

In specific embodiments, the antibodies of the invention stimulate chemotaxis of cells that express a CK-β4 receptor (e.g., CCR6). In specific embodiments, the antibodies of the invention stimulate chemotaxis of lymphocytes (e.g., T cells, B cells). In specific embodiments, the antibodies of the invention stimulate chemotaxis of dendritic cells.

In further embodiments, the antibodies of the invention have a dissociation constant (KD) of 10−7 M or less. In preferred embodiments, the antibodies of the invention have a dissociation constant (KD) of 10−9 M or less.

In further embodiments, antibodies of the invention have an off rate (koff) of 10−3/sec or less. In preferred embodiments, antibodies of the invention have an off rate (koff) of 10−4/sec or less. In other preferred embodiments, antibodies of the invention have an off rate (koff) of 10−5/sec or less.

The present invention also provides panels of antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants) wherein the panel members correspond to one, two, three, four, five, ten, fifteen, twenty, or more different antibodies of the invention (e.g., whole antibodies, Fabs, F(ab′)2 fragments, Fd fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies, and scFvs). The present invention further provides mixtures of antibodies, wherein the mixture corresponds to one, two, three, four, five, ten, fifteen, twenty, or more different antibodies of the invention (e.g., whole antibodies, Fabs, F(ab′)2 fragments, Fd fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies, and scFvs)). The present invention also provides for compositions comprising, or alternatively consisting of, one, two, three, four, five, ten, fifteen, twenty, or more antibodies of the present invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof). A composition of the invention may comprise, or alternatively consist of, one, two, three, four, five, ten, fifteen, twenty, or more amino acid sequences of one or more antibodies or fragments or variants thereof. Alternatively, a composition of the invention may comprise, or alternatively consist of, nucleic acid molecules encoding one or more antibodies of the invention.

The present invention also provides for fusion proteins comprising an antibody (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) of the invention, and a heterologous polypeptide (i.e., a polypeptide unrelated to an antibody or antibody domain). Nucleic acid molecules encoding these fusion proteins are also encompassed by the invention. A composition of the present invention may comprise, or alternatively consist of, one, two, three, four, five, ten, fifteen, twenty or more fusion proteins of the invention. Alternatively, a composition of the invention may comprise, or alternatively consist of, nucleic acid molecules encoding one, two, three, four, five, ten, fifteen, twenty or more fusion proteins of the invention.

The present invention also provides for a nucleic acid molecule(s), generally isolated, encoding an antibody (including molecules, such as scFvs, VH domains, or VL domains, that comprise, or alternatively consist of, an antibody fragment or variant thereof) of the invention. The present invention also provides a host cell transformed with a nucleic acid molecule of the invention and progeny thereof. The present invention also provides a method for the production of an antibody (including a molecule comprising, or alternatively consisting of, an antibody fragment or variant thereof) of the invention. The present invention further provides a method of expressing an antibody (including a molecule comprising, or alternatively consisting of, an antibody fragment or variant thereof) of the invention from a nucleic acid molecule. These and other aspects of the invention are described in further detail below.

DETAILED DESCRIPTION

The term “antibody,” as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen. As such, the term antibody encompasses not only whole antibody molecules, but also antibody multimers and antibody fragments as well as variants (including derivatives) of antibodies, antibody multimers and antibody fragments. Examples of molecules which are described by the term “antibody” herein include, but are not limited to: single chain Fvs (scFvs), Fab fragments, Fab′ fragments, F(ab′)2, disulfide linked Fvs (sdFvs), Fvs, and fragments comprising or alternatively consisting of, either a VL or a VH domain. The term “single chain Fv” or “scFv” as used herein refers to a polypeptide comprising a VH domain of antibody linked to a VL domain of an antibody. Antibodies that specifically bind to CK-β4 may have cross-reactivity with other antigens. Preferably, antibodies that specifically bind to CK-β4 do not cross-react with other antigens (e.g., other members of the chemokine superfamily). Antibodies that specifically bind to CK-β4 can be identified, for example, by immunoassays or other techniques known to those of skill in the art.

Antibodies of the invention include, but are not limited to, monoclonal, multispecific, human or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′) fragments, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), intracellularly-made antibodies (i.e., intrabodies), and epitope-binding fragments of any of the above. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. Preferably, an antibody of the invention comprises, or alternatively consists of, a VH domain, VH CDR, VL domain, or VL CDR having an amino acid sequence of any one of those referred to in Table 1, or a fragment or variant thereof. In a preferred embodiment, the immunoglobulin is an IgG1 isotype. In another preferred embodiment, the immunoglobulin is an IgG4 isotype. Immunoglobulins may have both a heavy and light chain. An array of IgG, IgE, IgM, IgD, IgA, and IgY heavy chains may be paired with a light chain of the kappa or lambda forms.

Antibodies of the invention may also include multimeric forms of antibodies. For example, antibodies of the invention may take the form of antibody dimers, trimers, or higher-order multimers of monomeric immunoglobulin molecules. Dimers of whole immunoglobulin molecules or of F(ab′)2 fragments are tetravalent, whereas dimers of Fab fragments or scFv molecules are bivalent. Individual monomers within an antibody multimer may be identical or different, i.e., they may be heteromeric or homomeric antibody multimers. For example, individual antibodies within a multimer may have the same or different binding specificities.

Multimerization of antibodies may be accomplished through natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art. For example, some percentage of purified antibody preparations (e.g., purified IgG1 molecules) spontaneously form protein aggregates containing antibody homodimers, and other higher-order antibody multimers. Alternatively, antibody homodimers may be formed through chemical linkage techniques known in the art. For example, heterobifunctional crosslinking agents including, but not limited to, SMCC [succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate] and SATA [N-succinimidyl S-acethylthio-acetate] (available, for example, from Pierce Biotechnology, Inc. (Rockford, Ill.)) can be used to form antibody multimers. An exemplary protocol for the formation of antibody homodimers is given in Ghetie et al., Proceedings of the National Academy of Sciences USA (1997) 94:7509-7514, which is hereby incorporated by reference in its entirety. Antibody homodimers can be converted to Fab′2 homodimers through digestion with pepsin. Another way to form antibody homodimers is through the use of the autophilic T15 peptide described in Zhao and Kohler, The Journal of Immunology (2002) 25:396-404, which is hereby incorporated by reference in its entirety.

Alternatively, antibodies can be made to multimerize through recombinant DNA techniques. IgM and IgA naturally form antibody multimers through the interaction with the J chain polypeptide. Non-IgA or non-IgM molecules, such as IgG molecules, can be engineered to contain the J chain interaction domain of IgA or IgM, thereby conferring the ability to form higher order multimers on the non-IgA or non-IgM molecules. (see, for example, Chintalacharuvu et al., (2001) Clinical Immunology 101:21-31. and Frigerio et al., (2000) Plant Physiology 123:1483-94, both of which are hereby incorporated by reference in their entireties.) ScFv dimers can also be formed through recombinant techniques known in the art; an example of the construction of scFv dimers is given in Goel et al., (2000) Cancer Research 60:6964-6971 which is hereby incorporated by reference in its entirety. Antibody multimers may be purified using any suitable method known in the art, including, but not limited to, size exclusion chromatography.

By “isolated antibody” is intended an antibody removed from its native environment. Thus, an antibody produced by, purified from and/or contained within a hybridoma and/or a recombinant host cell is considered isolated for purposes of the present invention.

Unless otherwise defined in the specification, specific binding or immunospecific binding by an anti-CK-β4 antibody means that the anti-CK-β4 antibody binds CK-β4 but does not significantly bind to (i.e., cross-react with) proteins other than CK-β4, such as other proteins in the same family of proteins). An antibody that binds CK-β4 protein and does not cross-react with other proteins is not necessarily an antibody that does not bind said other proteins in all conditions; rather, the CK-β4-specific antibody of the invention preferentially binds CK-β4 compared to its ability to bind said other proteins such that it will be suitable for use in at least one type of assay or treatment, i.e., give low background levels or result in no unreasonable adverse effects in treatment. It is well known that the portion of a protein bound by an antibody is known as the epitope. An epitope may either be linear (i.e., comprised of sequential amino acids residues in a protein sequences) or conformational (i.e., comprised of one or more amino acid residues that are not contiguous in the primary structure of the protein but that are brought together by the secondary, tertiary or quaternary structure of a protein). Given that CK-β4-specific antibodies bind to epitopes of CK-β4, an antibody that specifically binds CK-β4 may or may not bind fragments of CK-β4 and/or variants of CK-β4 (e.g., proteins that are at least 90% identical to CK-β4) depending on the presence or absence of the epitope bound by a given CK-β4-specific antibody in the CK-β4 fragment or variant. Likewise, CK-β4-specific antibodies of the invention may bind species orthologues of CK-β4 (including fragments thereof) depending on the presence or absence of the epitope recognized by the antibody in the orthologue. Additionally, CK-β4-specific antibodies of the invention may bind modified forms of CK-β4, for example, CK-β4 fusion proteins. In such a case when antibodies of the invention bind CK-β4 fusion proteins, the antibody must make binding contact with the CK-β4 moiety of the fusion protein in order for the binding to be specific. Antibodies that specifically bind to CK-β4 can be identified, for example, by immunoassays or other techniques known to those of skill in the art, e.g., the immunoassays described in the Examples below.

The term “variant” as used herein refers to a polypeptide that possesses a similar or identical amino acid sequence as a CK-β4 polypeptide, a fragment of a CK-β4 polypeptide, an anti-CK-β4 antibody or antibody fragment thereof. A variant having a similar amino acid sequence refers to a polypeptide that satisfies at least one of the following: (a) a polypeptide comprising, or alternatively consisting of, an amino acid sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence of a CK-β4 polypeptide, a fragment thereof, an anti-CK-β4 antibody or antibody fragment thereof (including a VH domain, VHCDR, VL domain, or VLCDR having an amino acid sequence of any one or more scFvs referred to in Table 1) described herein; (b) a polypeptide encoded by a nucleotide sequence, the complementary sequence of which hybridizes under stringent conditions to a nucleotide sequence encoding a CK-β4 polypeptide (e.g., SEQ ID NO:2), a fragment of a CK-β4 polypeptide, an anti-CK-β4 antibody or antibody fragment thereof (including a VH domain, VHCDR, VL domain, or VLCDR having an amino acid sequence of any one of those referred to in Table 1), described herein, of at least 5 amino acid residues, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, or at least 150 amino acid residues; and (c) a polypeptide encoded by a nucleotide sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99%, identical to the nucleotide sequence encoding a CK-β4 polypeptide, a fragment of a CK-β4 polypeptide, an anti-CK-β4 antibody or antibody fragment thereof (including a VH domain, VHCDR, VL domain, or VLCDR having an amino acid sequence of any one or more scFvs referred to in Table 1), described herein. A polypeptide with similar structure to a CK-β4 polypeptide, a fragment of a CK-β4 polypeptide, an anti-CK-β4 antibody or antibody fragment thereof, described herein refers to a polypeptide that has a similar secondary, tertiary or quaternary structure of a CK-β4 polypeptide, a fragment of a CK-β4 polypeptide, an anti-CK-β4 antibody, or antibody fragment thereof, described herein. The structure of a polypeptide can determined by methods known to those skilled in the art, including but not limited to, X-ray crystallography, nuclear magnetic resonance, and crystallographic electron microscopy. Preferably, a variant CK-β4 polypeptide, a variant fragment of a CK-β4 polypeptide, or a variant anti-CK-β4 antibody and/or antibody fragment possesses similar or identical function and/or structure as the reference CK-β4 polypeptide, the reference fragment of a CK-β4 polypeptide, or the reference anti-CK-β4 antibody and/or antibody fragment, respectively.

To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical overlapping positions/total number of positions×100%). In one embodiment, the two sequences are the same length.

The determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993). The BLASTn and BLASTx programs of Altschul, et al. J. Mol. Biol. 215:403-410 (1990) have incorporated such an algorithm. BLAST nucleotide searches can be performed with the BLASTn program (score=100, wordlength=12) to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTx program (score=50, wordlength=3) to obtain amino acid sequences homologous to a protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. Nucleic Acids Res. 25:3589-3402 (1997). Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used.

Another example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0) which is part of the GCG sequence alignment software package has incorporated such an algorithm. Other algorithms for sequence analysis known in the art include ADVANCE and ADAM as described in Torellis and Robotti Comput. Appl. Biosci., 10 :3-5 (1994); and FASTA described in Pearson and Lipman Proc. Natl. Acad. Sci. 85:2444-8 (1988). Within FASTA, ktup is a control option that sets the sensitivity and speed of the search.

The term “derivative” as used herein, refers to a variant polypeptide of the invention that comprises, or alternatively consists of, an amino acid sequence of a CK-β4 polypeptide, a fragment of a CK-β4 polypeptide, or an antibody of the invention that specifically binds to a CK-β4 polypeptide, which has been altered by the introduction of amino acid residue substitutions, deletions or additions. The term “derivative” as used herein also refers to a CK-β4 polypeptide, a fragment of a CK-β4 polypeptide, or an antibody that specifically binds to a CK-β4 polypeptide which has been modified, e.g., by the covalent attachment of any type of molecule to the polypeptide. For example, but not by way of limitation, a CK-β4 polypeptide, a fragment of a CK-β4 polypeptide, or an anti-CK-β4 antibody, may be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. A derivative of a CK-β4 polypeptide, a fragment of a CK-β4 polypeptide, or an anti-CK-β4 antibody, may be modified by chemical modifications using techniques known to those of skill in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Further, a derivative of a CK-β4 polypeptide, a fragment of a CK-β4 polypeptide, or an anti-CK-β4 antibody, may contain one or more non-classical amino acids. A polypeptide derivative possesses a similar or identical function as a CK-β4 polypeptide, a fragment of a CK-β4 polypeptide, or an anti-CK-β4 antibody, described herein.

The term “fragment” as used herein refers to a polypeptide comprising an amino acid sequence of at least 5 amino acid residues, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues, at least 35 amino acid residues, at least 40 amino acid residues, at least 45 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino acid residues, or at least 90 amino acid residues, of the amino acid sequence of CK-β4, or an anti-CK-β4 antibody (including molecules such as scFv\'s, that comprise, or alternatively consist of, antibody fragments or variants thereof) that specifically binds to CK-β4.

The term “host cell” as used herein refers to the particular subject cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.

The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody\'s isotype as IgM, IgD, IgG, IgA, and IgE, respectively. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes). The variable regions of each light/heavy chain pair form the antibody binding site.

Thus, an intact IgG antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are the same.

The chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the heavy and the light chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J Mol. Biol. 196:901-917 (1987); Chothia et al. Nature 342:878-883 (1989).

A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab′ fragments. See, e.g., Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321 (1990), Kostelny et al. J Immunol. 148:1547 1553 (1992). In addition, bispecific antibodies may be formed as “diabodies” (Holliger et al. “‘Diabodies’: small bivalent and bispecific antibody fragments” PNAS USA 90:6444-6448 (1993)) or “Janusins” (Traunecker et al. “Bispecific single chain molecules (Janusins) target cytotoxic lymphocytes on HIV infected cells” EMBO J 10:3655-3659 (1991) and Traunecker et al. “Janusin: new molecular design for bispecific reagents” Int. J. Cancer Suppl. 7:51-52 (1992)).

Production of bispecific antibodies can be a relatively labor intensive process compared with production of conventional antibodies and yields and degree of purity are generally lower for bispecific antibodies. Bispecific antibodies do not exist in the form of fragments having a single binding site (e.g., Fab, Fab′, and Fv).

Using phage display technology, single chain antibody molecules (“scFvs”) have been identified that specifically bind to CK-β4 (or fragments or variants thereof). Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs (e.g., including VH domains, VH CDRs, VL domains, or VL CDRs having an amino acid sequence of any one of those referred to in Table 1), that specifically bind to CK-β4 (or fragments or variants thereof) are also encompassed by the invention, as are nucleic acid molecules that encode these scFvs, and/or molecules.

In particular, the invention relates to scFvs comprising, or alternatively consisting of the amino acid sequence of any one of SEQ ID NOs:20-36, referred to in Table 1 below. Molecules comprising, or alternatively consisting of, fragments or variants (e.g., including VH domains, VH CDRs, VL domains, or VL CDRs identified in Table 1) of the scFvs referred to in Table 1, that specifically bind to CK-β4 are also encompassed by the invention, as are nucleic acid molecules that encode these scFvs, and/or molecules (e.g., SEQ ID NOs:3-19).

ScFvs corresponding to SEQ ID NOs: 20-36 were selected for their ability bind CK-β4 polypeptide.

The present invention provides antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that specifically bind to a polypeptide or a polypeptide fragment of CK-β4. In particular, the invention provides antibodies corresponding to the scFvs referred to in Table 1, such scFvs may routinely be “converted” to immunoglobulin molecules by inserting, for example, the nucleotide sequences encoding the VH and/or VL domains of the scFv into an expression vector containing the constant domain sequences and engineered to direct the expression of the immunoglobulin molecule, as described in more detail in Example 2 below.

Cell lines that express IgG1 antibodies that comprise the VH and VL domains of scFvs of the invention have been deposited with the American Type Culture Collection (“ATCC™”) on the dates listed in Table 1 and given the ATCC™ Deposit Numbers identified in Table 1. The ATCC™ is located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA. The ATCC™ deposit was made pursuant to the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Purposes of Patent Procedure.

Accordingly, in one embodiment, the invention provides antibodies that comprise the VH and VL domains of scFvs of the invention.

In a preferred embodiment, an antibody of the invention is an antibody expressed by any one of the cell lines disclosed in Table 1.

TABLE 1 scFvs that Specifically bind to CK-β4 scFv scFv DNA Protein AAs of AAs of AAs of AAs of AAs of AAs of AAs of AAs of Cell Line ATCC ™ ATCC ™ SEQ ID SEQ ID VH VH VH VH VL VL VL VL Expressing Deposit Deposit ScFv NO: NO: Domain CDR1 CDR2 CDR3 Domain CDR1 CDR2 CDR3 antibody Number Date F003A09 3 20 1-121 26-35 50-66 99-110 138-250 159-172 188-194 227-239 F081C09 4 21 1-119 26-35 50-66 99-108 135-245 157-169 185-191 224-234 F076F10 5 22 1-123 26-35 50-66 99-112 140-250 162-174 190-196 229-239 F002D07 6 23 1-118 26-35 50-66 99-107 136-245 156-169 185-191 224-234 F076C06 7 24 1-125

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