Anti-Carbonic Anhydrase IX Antibody

Abstract

The present invention relates to antigenic determinants located in CAIX and anti-CAIX antibodies having binding activity with cancer cells. The present invention also relates to compositions and the use of these CAIX antigenic determinants and these anti-CAIX antibodies in the diagnosis, prevention and / or treatment of cancer.

Description

Technical Field

[0001] The present invention generally relates to the field of cancer treatment. Specifically, the present invention relates to antigenic peptides of carbonic anhydrase IX (CAIX), anti-CAIX antibodies and their use in preventing and / or treating cancer. Background Art

[0002] Hypoxia is a prominent feature of rapidly growing malignancies and their metastases. In the early stages of tumor development, tissue hypoxia occurs due to insufficient blood supply. Hypoxia and acidification of the extratumoral environment are associated with aggressive tumor growth, metastasis formation, and adverse responses to radiation therapy, surgery, and / or anticancer chemotherapy. Carbonic anhydrase (CA) is involved in a variety of physiological functions, including pH regulation, ion transport, bone resorption, and secretion of gastric, cerebrospinal, and pancreatic juices. The expression of CAIX in normal tissues is restricted to the basolateral surface of the gastric, intestinal (proliferating crypt enterocytes of the duodenum, jejunum, and intestinal mucosa) and gallbladder epithelium in humans. CAIX is overexpressed in many solid tumors but not in their corresponding normal tissues. Previous studies have reported that CAIX expression is upregulated in lung, breast, liver, cervical, colon, ovarian, bladder, head and neck, brain, and oral cancers and is associated with poor prognosis in these cancers.

[0003] US 20050158809 is about methods for aiding the prognosis of renal cell carcinoma, the methods comprising quantifying the expressed carbonic anhydrase in a sample derived from a patient with renal cell carcinoma. US 20130274305 is about CAIX inhibitors comprising a nitroimidazole moiety, including their use in cancer treatment. US 20130336923 provides scFv antibodies and monoclonal antibodies that bind to CAIX and reduce the activity of CAIX.

[0004] There is a need in the art for new antibodies directed against CAIX for use in medical applications including cancer treatment. Summary of the invention

[0005] The present invention provides an antigenic peptide comprising an antigenic determinant of CAIX having an amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33 or an amino acid sequence of SEQ ID NO: 32 and SEQ ID NO: 33. Therefore, the present invention provides an immunogenic composition comprising the antigenic peptide of the present invention and a pharmaceutically acceptable adjuvant selected as appropriate. An antibody that specifically binds to the antigenic determinant of the present invention is also provided.

[0006] The present invention also provides an isolated anti-CAIX antibody or an antigen-binding portion thereof, which comprises at least one of the following: a light chain complementary determining region 1 (L-CDR1) of SEQ ID NO: 1, 4 or 6 or a variant thereof; a light chain CDR2 (L-CDR2) of SEQ ID NO: 2, 5 or 7 or a variant thereof; and a light chain CDR3 (L-CDR3) of SEQ ID NO: 3 or 8 or a variant thereof; and a heavy chain CDR1 (H-CDR1) of SEQ ID NO: 9, 12, 15 or 18 or a variant thereof; a heavy chain CDR2 (H-CDR2) of SEQ ID NO: 10, 13, 16 or 19 or a variant thereof; and a heavy chain CDR3 (H-CDR3) of SEQ ID NO: 11, 14, 17 or 20 or a variant thereof; such that the isolated antibody or antigen-binding portion thereof binds to CAIX.

[0007] Related embodiments of the isolated anti-CAIX antibody include constant regions and one or more heavy and light chain variable framework regions of human antibody sequences. In related embodiments, the isolated anti-CAIX antibody comprises a framework region that is at least 70% identical to Avastin.

[0008] Related embodiments include an isolated antibody that specifically binds CAIX as disclosed herein, comprising up to 10 amino acid substitutions (such as up to 1, 2, 3, 4, 5, 6, 7, 8, or up to 9 amino acid substitutions) in the framework regions.

[0009] Relevant embodiments of the framework region include: L-FR1, which has an amino acid sequence selected from SEQ ID NOs: 34 to 37; L-FR2, which has an amino acid sequence selected from SEQ ID NOs: 38 to 40; L-FR3, which has an amino acid sequence selected from SEQ ID NOs: 41 to 43; and L-FR4, which has an amino acid sequence selected from SEQ ID NOs: 44 to 45; and H-FR1, which has an amino acid sequence selected from SEQ ID NOs: 46 to 52; H-FR2, which has an amino acid sequence selected from SEQ ID NOs: 53 to 56; H-FR3, which has an amino acid sequence selected from SEQ ID NOs: 57 to 62; and H-FR4, which has an amino acid sequence selected from SEQ ID NOs: 63 to 65.

[0010] The present invention also provides a light chain comprising an amino acid sequence having a sequence selected from the group consisting of as set forth in SEQ ID NOs: 21 to 23.

[0011] The present invention also provides a heavy chain comprising an amino acid sequence having a sequence selected from the group consisting of as set forth in SEQ ID NOs: 24 to 28.

[0012] The present invention further provides an isolated antibody or antigen binding portion thereof comprising (i) a light chain having an amino acid sequence as set forth in a sequence selected from the group consisting of SEQ ID NOs: 21 to 23 or a variant thereof, and (ii) a heavy chain having an amino acid sequence as set forth in a sequence selected from the group consisting of SEQ ID NOs: 24 to 28 or a variant thereof. In one embodiment, the variant has at least 80% sequence identity to the corresponding sequence. Certain embodiments of the isolated antibodies include: a light chain having an amino acid sequence as set forth in SEQ ID NO:21 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:24; a light chain having an amino acid sequence as set forth in SEQ ID NO:22 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:25; a light chain having an amino acid sequence as set forth in SEQ ID NO:21 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:26; a light chain having an amino acid sequence as set forth in SEQ ID NO:23 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:27; or a light chain having an amino acid sequence as set forth in SEQ ID NO:21 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:28.

[0013] Certain embodiments relate to cells expressing an antibody, or antigen-binding portion thereof, of the invention.

[0014] Certain embodiments relate to a pharmaceutical composition comprising an antibody against CAIX of the invention or a cell expressing an antibody of the invention or an antigen-binding portion thereof and a pharmaceutically acceptable carrier or excipient.

[0015] Certain embodiments are directed to a method for preventing and / or treating cancer in a subject, comprising expressing an antibody of the invention, or an antigen-binding portion thereof, in a cell in vitro, and administering the cell to the subject.

[0016] Simple diagram description

[0017] Figure 1 (A) to Figure 1 (C) Shows the purification of (A) anti-CAIX scFv (NAVICA9S1, NAVICA9S2, NAVICA9L1, NAVICA9L2, NAVICA9L3) and (B) CAIX protein (ECD, F3) and (C) the size of the full length, extracellular domain (ECD), fragment 1 (F1), fragment 2 (F2) and fragment 3 (F3) of CAIX. Lane 1: Ni 2+Supernatant after agarose gel binding; Lane 2: Collection wash buffer; Lane 3: Ni after elution 2+ Agarose gel; Lane 4: pooled eluted protein; ECD: extracellular domain; and F3: fragment 3.

[0018] Figure 2 (A) shows the location of CAIX protein and A549 cell lysate on SDS-PAGE. Panels (B) to (I) show the binding ability of anti-CAIX antibodies or scFv by Western blot. (B), mouse anti-CAIX Ab; (C), mouse anti-CAIX Ab; (D), NAVICA9S1; (E), NAVICA9S2; (F), IgY; (G), NAVICA9L1; (H), NAVICA9L2; and (I), NAVICA9L3.

[0019] Figure 3 (A) to Figure 3 (E) Binding affinities of anti-CAIX monoclonal scFvs displaying (A) NAVICA9S1, (B) NAVICA9S2, (C) NAVICA9L1, (D) NAVICA9L2, and (E) NAVICA9L3.

[0020] Figure 4 (A) and Figure 4 (B) shows that (A) cloned NAVICA9S1, NAVICA9S2 and NAVICA9L3 bind to CAIX FL, ECD, F2 and F3 fragments, and cloned NAVICA9L2 binds to CAIX FL and ECD, but not to F2 and F3 fragments, and (B) the two antigenic determinants are located between 38 and 153 amino acids and between 228 and 353 amino acids, respectively.

[0021] Figure 5 Flow cytometry of anti-CAIX scFv and anti-CAIX (abcam, ab107257) against A549 cells or THP-1 cells is shown. DETAILED DESCRIPTION

[0022] The present invention is based on the discovery of antigenic determinants located in CAIX and anti-CAIX antibodies having the activity of binding to cancer cells.

[0023] In the following description, a number of terms are used and the following definitions are provided to aid in understanding the claimed subject matter. Terms not expressly defined herein are used according to their ordinary and general meanings.

[0024] Unless otherwise specified, "a" or "an" means "one or more".

[0025] As used herein, the term "antigenic determinant" refers to the site where an antibody binds to an antigen.

[0026] As used herein, the term "specifically binds" means that the antibody does not cross-react to a significant extent with other antigenic determinants.

[0027] As used herein, the term "antibody" refers to single-chain, double-chain and multi-chain proteins and polypeptides belonging to the classes of polyclonal, monoclonal, chimeric and humanized antibodies; it also includes synthetic and genetically engineered variants of these antibodies. "Antibody fragments" include Fab, Fab', F(ab') 2 and Fv fragments, as well as any portion of an antibody that has specificity for one or more desired target antigenic determinants.

[0028] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. In other words, a monoclonal antibody is composed of homogeneous antibodies grown from a single cell line (e.g., a hybridoma, a eukaryotic host cell transfected with a DNA molecule encoding the homogeneous antibody, or a prokaryotic host cell transfected with a DNA molecule encoding the homogeneous antibody). These antibodies are directed against a single antigenic determinant and are therefore highly specific.

[0029] As used herein, the term "complementarity determining region" (CDR) refers to non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. CDRs have been described by Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196: 901-917 (1987); and MacCallum et al., J. Mol. Biol. 262: 732-745 (1996), where definitions include overlaps or subsets of amino acid residues when compared against each other.

[0030] As used herein, the term "framework" or FR residues are those variable domain residues other than the hypervariable region residues.

[0031] As used herein, the term "humanized antibody" refers to a recombinant protein in which the CDRs are from an antibody of one species (e.g., a murine or chicken antibody) and are transferred from the heavy variable chain and light variable chain of the antibody of the species to human heavy variable domains and light variable domains (framework regions). The constant domains of the antibody molecule are those constant domains derived from human antibodies. In some cases, specific residues of the framework region of the humanized antibody, especially those specific residues that contact or approach the CDR sequence, can be modified, for example, by replacement with corresponding residues from the original murine, rodent, humanoid monkey or other antibody. Humanized antibodies can be obtained using a variety of methods, including (a) only non-human CDRs are transplanted (grafted) to human framework regions and constant regions while retaining or not retaining key framework residues, or (b) transplanting (transplant) the entire non-human variable domain, but "covering" the entire non-human variable domain with a humanoid portion by replacing surface residues. Such methods suitable for practicing the present invention include those disclosed in Padlan, Mol. Immunol., 31(3): 169-217 (1994).

[0032] As used herein, the term "chimeric antibody" refers to a recombinant protein containing the variable domains of the heavy and light antibody chains, including the complementarity determining regions (CDRs) derived from antibodies of one species (preferably rodent antibodies or chicken antibodies, more preferably hamster antibodies), while the constant domains of the antibody molecule are derived from those of human antibodies.

[0033] As used herein, the term "antigen binding" refers to the ability to specifically bind to an antigen. It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment, consisting of the VH and CH1 domains; (iv) a Fv fragment, consisting of the VL and VH domains of a single arm of an antibody; (v) a single domain or dAb (domain antibody) fragment, consisting of a VH domain; and (vi) isolated complementary determining regions (CDRs), or (vii) a combination of two or more isolated CDRs, which may be optionally joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker that enables them to be produced as a single protein chain, in which the VL and VH regions pair to form a monovalent molecule known as single-chain Fv (scFv). These antibody fragments are obtained using well-known techniques known to those skilled in the art, and the fragments are screened for use in the same manner as intact antibodies.

[0034] As used herein, single-chain antibodies ("SCAs") comprise single-chain Fv fragments ("scFv") in which the variable light ("VL") and variable heavy ("VH") domains are linked by a peptide bridge or by a disulfide bond.

[0035] As used herein, the term "linker" refers to a bifunctional molecule that can be used to connect two molecules into one continuous molecule. In some cases, the linker is a peptide within an antibody binding fragment (such as an Fv fragment) that indirectly binds the variable heavy chain to the variable light chain.

[0036] As used herein, the term K D Refers to the dissociation constant for a given interaction, such as a polypeptide-ligand interaction or an antibody-antigen interaction. For example, for a bimolecular interaction of an antibody (such as those disclosed herein) and an antigen, it is the concentration of the individual components of the bimolecular interaction divided by the concentration of the complex.

[0037] As used herein, the terms "treatment" and "treating" and the like encompass any treatment of a disease in mammals, especially humans, and include: (a) preventing the disease from occurring in an individual susceptible to the disease but not yet diagnosed with the disease; (b) inhibiting the disease, i.e., curbing its development; and (c) ameliorating the disease, i.e., causing regression of the disease.

[0038] As used interchangeably herein, the terms "individual," "subject," "host," and "patient" refer to mammals, including but not limited to, murines (rats, mice), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, sheep, porcines, goats), and the like.

[0039] As used herein, the term "therapeutically effective amount" or "effective amount" refers to the amount of an anti-CAIX antibody of a subject that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease.

[0040] In one aspect, the present invention provides an antigenic peptide comprising an antigenic determinant of CAIX having an amino acid sequence:

[0041] or

[0042] or

[0043] The amino acid sequences of SEQ ID NO:32 and SEQ ID NO:33.

[0044] Therefore, the present invention provides an immunogenic composition comprising the antigenic peptide of the present invention and a pharmaceutically acceptable adjuvant selected as appropriate. Examples of pharmaceutically acceptable adjuvants may include aluminum hydroxide, alum, aluminum colloid (aluminum trihydrate) or other aluminum containing salts, virus particles, nucleic acids containing CpG motifs, squalene, oil, MF59, QS21, various saponins, virus-like particles, monophosphoryl-lipid A / trehalose dimycolate, toll-like receptor agonists, copolymers (such as polypropylene oxide and polyethylene oxide), or the like.

[0045] The present invention also provides an antibody that specifically binds to the antigenic determinant of the present invention. Antibodies that can bind to the novel antigenic determinant can be used as diagnostic agents, therapeutic agents and vaccines.

[0046] In one embodiment, the present invention provides an isolated anti-CAIX antibody or an antigen-binding portion thereof, comprising at least one of the following: a light chain complementary determining region 1 (L-CDR1) of SEQ ID NO: 1, 4 or 6 or a variant thereof; a light chain CDR2 (L-CDR2) of SEQ ID NO: 2, 5 or 7 or a variant thereof; and a light chain CDR3 (L-CDR3) of SEQ ID NO: 3 or 8 or a variant thereof; and

[0047] At least one of the following: heavy chain CDR1 (H-CDR1) of SEQ ID NO: 9, 12, 15 or 18 or a variant thereof; heavy chain CDR2 (H-CDR2) of SEQ ID NO: 10, 13, 16 or 19 or a variant thereof; and heavy chain CDR3 (H-CDR3) of SEQ ID NO: 11, 14, 17 or 20 or a variant thereof; such that the isolated antibody or antigen-binding portion thereof binds to CAIX.

[0048] In one embodiment, the variant has an amino acid sequence having at least 80% sequence identity with its corresponding CDR. Preferably, the sequence identity as mentioned above is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0049] The amino acid sequences of the complementarity determining regions in the heavy and light chains are listed in the table below.

[0050] CDR of light chain

[0051]

[0052] Heavy chain CDR

[0053]

[0054] In some embodiments, the isolated anti-CAIX antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a human antibody.

[0055] Each heavy and light chain comprises a constant region and a variable region (these regions are also referred to as "domains"). In some embodiments, the heavy chain variable domain and the light chain variable domain combine to specifically bind to an antigen. The light chain variable domain and the heavy chain variable domain contain a "framework" region, also referred to as a "complementarity determining region" or "CDR" (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991), interspersed with three hypervariable regions. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody (i.e., the combined framework regions of the component light and heavy chains) provides a position and aligns the CDRs in three-dimensional space. The CDRs are primarily responsible for binding to the antigenic determinants of an antigen. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. ("Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991; "Kabat" numbering scheme), Al-Lazikani et al. (JMB 273, 927-948, 1997; "Chothia" numbering scheme), and Lefranc et al. ("IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev. Comp. Immunol., 27:55-77, 2003; "IMGT" numbering scheme).

[0056] In some embodiments, the isolated anti-CAIX antibody comprises a constant region and one or more heavy and light chain variable framework regions of a human antibody sequence. In a related embodiment, the antibody comprises a modified or unmodified constant region of human IgG1, IgG2, IgG3, or IgG4. In a preferred embodiment, the constant region is human IgG1 or IgG4, which may be modified to enhance or reduce certain properties as appropriate. In a related embodiment, the isolated anti-CAIX antibody comprises a framework region that is at least 70% identical to Avastin.

[0057] In some embodiments, an isolated antibody that specifically binds CAIX as disclosed herein comprises up to 10 amino acid substitutions (such as up to 1, 2, 3, 4, 5, 6, 7, 8, or up to 9 amino acid substitutions) in the heavy chain of the antibody, the light chain of the antibody, or the framework regions of the heavy and light chains of the antibody (e.g., according to the Kabat, Clothia, or IMGT numbering systems). In some embodiments, the framework region comprises: L-FR1 having an amino acid sequence selected from SEQ ID NOs: 34 to 37; L-FR2 having an amino acid sequence selected from SEQ ID NOs: 38 to 40; L-FR3 having an amino acid sequence selected from SEQ ID NOs: 41 to 43; and L-FR4 having an amino acid sequence selected from SEQ ID NOs: 44 to 45; and H-FR1 having an amino acid sequence selected from SEQ ID NOs: 46 to 52; H-FR2 having an amino acid sequence selected from SEQ ID NOs: 53 to 56; H-FR3 having an amino acid sequence selected from SEQ ID NOs: 57 to 62; and H-FR4 having an amino acid sequence selected from SEQ ID NOs: 63 to 65.

[0058] Light chain

[0059] L-FR1

[0060]

[0061] L-FR2

[0062]

[0063] L-FR3

[0064]

[0065] L-FR4

[0066]

[0067] Heavy chain

[0068] H-FR1

[0069]

[0070] H-FR2

[0071]

[0072] H-FR3

[0073]

[0074] H-FR4

[0075]

[0076] According to the present invention, examples of amino acids of the light chain and heavy chain of the antibody of the present invention are listed below.

[0077]

[0078]

[0079] In some embodiments, the present invention provides a light chain comprising an amino acid sequence having a sequence selected from the group consisting of as set forth in SEQ ID NOs: 21-23.

[0080] In some embodiments, the present invention provides a heavy chain comprising an amino acid sequence having a sequence selected from the group consisting of as set forth in SEQ ID NOs: 24-28.

[0081] In another embodiment, the present invention comprises an isolated antibody or antigen-binding portion thereof comprising (i) a light chain having an amino acid sequence as set forth in a sequence selected from the group consisting of SEQ ID NOs: 21 to 23 or a variant thereof, and (ii) a heavy chain having an amino acid sequence as set forth in a sequence selected from the group consisting of SEQ ID NOs: 24 to 28 or a variant thereof. In one embodiment, the variant has at least 80% sequence identity with the corresponding sequence. In one embodiment, the variant has at least 80% sequence identity with the framework of the light chain or heavy chain. Preferably, the sequence identity as mentioned above is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0082] In another embodiment, the invention comprises an isolated antibody, or an antigen-binding portion thereof, comprising a light chain having an amino acid sequence as set forth in SEQ ID NO:21 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:24; a light chain having an amino acid sequence as set forth in SEQ ID NO:22 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:25; a light chain having an amino acid sequence as set forth in SEQ ID NO:21 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:26; a light chain having an amino acid sequence as set forth in SEQ ID NO:23 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:27; or a light chain having an amino acid sequence as set forth in SEQ ID NO:21 and a heavy chain having an amino acid sequence as set forth in SEQ ID NO:28.

[0083] In another embodiment, the present invention provides a single-chain variable fragment (scFv) that binds CAIX, comprising a) a light chain variable region of a light chain, the light chain having an amino acid sequence as set forth in a sequence selected from the group consisting of SEQ ID NOs: 21 to 23; b) a linker; and c) a heavy chain variable region of a heavy chain, the heavy chain having an amino acid sequence as set forth in a sequence selected from the group consisting of SEQ ID NOs: 24 to 28. In one embodiment, the linker is a peptide linker. In some embodiments, the linker comprises 4-20 amino acids. In some embodiments, the linker comprises a peptide having an amino acid sequence of GQSSRSS (SEQ ID NO: 29), GQSSRSSSGGGSSGGGGS (SEQ ID NO: 30), or GQSSRSSGGGGSSGGGGS (SEQ ID NO: 31).

[0084] Techniques for preparing monoclonal antibodies against virtually any target antigen are well known in the art. See, for example, Kohler and Milstein, Nature 256:495 (1975), and Coligan et al. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, Vol. 1, pp. 2.5.1-2.6.7 (John Wiley & Sons 1991). In brief, monoclonal antibodies can be obtained by injecting mice or chickens with a composition comprising an antigen; removing the spleen to obtain B-lymphocytes; fusing B-lymphocytes with myeloma cells to produce fusion tumors; selecting fusion tumors; selecting positive pure lines that produce antibodies against the antigen; culturing these pure lines that produce antibodies against the antigen; and isolating antibodies from fusion tumor cultures. In another example, phage display technology for preparing antibodies is also known in the art. Phage display of antibody libraries has become a powerful method for studying immune responses and a method for rapidly selecting and evolving desired antibodies. This technique relies on the utilization of phage displayed libraries in a screening procedure known as biopanning (George K. et al., Affinity Chromatography pp. 195-208).

[0085] Various techniques such as producing chimeric or humanized antibodies may involve the process of antibody cloning and construction. The antigen-binding variable light chain and variable heavy chain sequences of the antibody of interest can be obtained by a number of molecular cloning procedures, such as RT-PCR, 5'-RACE, and cDNA library screening. The variable heavy chain or light chain sequence genes of antibodies from cells expressing mouse antibodies can be cloned by PCR amplification and sequencing. To confirm their authenticity, the cloned V L and V HThe genes can be expressed in cell culture as chimeric antibodies as described by Orlandi et al. (Proc. Natl. Acad. Sci., USA, 86:3833 (1989)). Based on the variable heavy or light chain gene sequences, humanized antibodies can then be designed and constructed as described by Leung et al. (Mol. Immunol., 32:1413 (1995)).

[0086] Chimeric antibodies are recombinant proteins in which the variable regions of a human antibody have been replaced by, for example, the variable regions of a mouse antibody, including the complementarity determining regions (CDRs) of the mouse antibody. When administered to a subject, chimeric antibodies exhibit reduced immunogenicity and increased stability. Methods for constructing chimeric antibodies are well known in the art (e.g., Leung et al., 1994, Hybridoma 13:469).

[0087] Chimeric monoclonal antibodies can be humanized by transferring the mouse CDRs of the heavy and light variable chains of mouse immunoglobulins into the corresponding variable domains of human antibodies. The mouse framework regions (FRs) in chimeric monoclonal antibodies are also replaced with human FR sequences. To preserve the stability and antigenic specificity of the humanized monoclonal, one or more human FR residues can be replaced with mouse counterpart residues. Humanized monoclonal antibodies can be used for therapeutic treatment of individuals. The techniques for producing humanized monoclonal antibodies are well known in the art. (See, e.g., Jones et al., 1986, Nature, 321:522; Riechmann et al., Nature, 1988, 332:323; Verhoeyen et al., 1988, Science, 239:1534; Carter et al., 1992, Proc. Nat'l Acad. Sci. USA, 89:4285; Sandhu, Crit. Rev. Biotech., 1992, 12:437; Tempest et al., 1991, Biotechnology 9:266; Singer et al., J. Immun., 1993, 150:2844.

[0088] The nucleic acids encoding the polypeptides described herein may be modified without reducing their biological activity. Some modifications may facilitate cloning, expression, or incorporation of the target molecule into a fusion protein. Such modifications are well known to those skilled in the art and include, for example, stop codons, methionine added at the amino terminus to provide a start, site, additional amino acids placed at either end to create a restriction site for convenient localization, or additional amino acids (such as poly-His) to aid in purification steps. In addition to recombinant methods, standard peptide synthesis known in the art may also be used to construct the antibodies of the present invention in whole or in part.

[0089] As a modification of the double-chain antibody purification protocol, the heavy chain region and the light chain region can be dissolved and reduced separately and then combined in the refolding solution. Exemplary yields can be produced when these two proteins are mixed in a molar ratio such that the molar number of one protein does not exceed 5 times that of the other. After redox shuffling is completed, glutathione peroxide or other low molecular weight oxidizing compounds can be added to the refolding solution.

[0090] In addition to recombinant methods, the antibodies and variants thereof disclosed herein may also be constructed in whole or in part using standard peptide synthesis. Solid phase synthesis of polypeptides can be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support, followed by the continuous addition of the remaining amino acids in the sequence. Solid phase synthesis techniques are described by Barany & Merrifield, The Peptides: Analysis, Synthesis Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A. pp. 3-284; Merrifield et al., J. Am. Chem. Soc. 85: 2149-2156, 1963, and Stewart et al., Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, Ill., 1984. Proteins of greater length may be synthesized by condensation of the amino and carboxyl termini of shorter fragments.

[0091] Antibody composition, cell expressing antibody and administration method

[0092] Certain embodiments relate to cells expressing antibodies or antigen binding portions thereof of the invention. Cells can be produced by culturing polynucleotides encoding antibodies or antigen binding portions thereof in a culture medium for expressing antibodies or antigen binding portions thereof, and harvesting antibodies or antigen binding portions thereof of the invention from the culture medium. Alternatively, these cells can be produced by transfection with viral vectors expressing the antibodies.

[0093] Certain embodiments relate to a pharmaceutical composition comprising an antibody to CAIX of the present invention or a cell expressing an antibody or antigen-binding portion thereof of the present invention and a pharmaceutically acceptable carrier or excipient. "Pharmaceutically acceptable carrier" is intended to be, but not limited to, any type of non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation aid known to those skilled in the art. Diluents such as polyols, polyethylene glycols and polydextrose can be used to increase the biological half-life of the conjugate.

[0094] The pharmaceutical composition of the present invention can be formulated according to known methods (e.g., Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA), and may also contain pharmaceutically acceptable carriers and additives. Examples include, but are not limited to, surfactants, excipients, colorants, flavoring agents, preservatives, stabilizers, buffers, suspending agents, isotonic agents, binders, disintegrants, lubricants, fluidity promoters, and corrective agents, and other commonly used carriers may be used appropriately. Specific examples of carriers include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carboxymethylcellulose calcium, carboxymethylcellulose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinylacetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain triglycerides, polyoxyethylene hardened castor oil 60, sucrose, carboxymethyl cellulose, corn starch, inorganic salts, and the like.

[0095] Certain embodiments are directed to a method for preventing and / or treating cancer in a subject, comprising administering to the subject an anti-CAIX antibody of the invention or a cell expressing an antibody of the invention or an antigen-binding portion thereof. The method also comprises administering an anti-CAIX antibody of the invention or a cell expressing an antibody of the invention or an antigen-binding portion thereof simultaneously with or after other standard therapeutic agents.

[0096] Certain embodiments are directed to a method for preventing and / or treating cancer in a subject, comprising expressing an antibody of the invention, or an antigen-binding portion thereof, in a cell in vitro, and administering the cell to the subject.

[0097] In a preferred embodiment, the subject is a mammal. Exemplary mammals include humans, pigs, sheep, goats, horses, mice, dogs, cats, cattle, etc. Cancers that can be treated with antibodies or their pharmaceutical compositions include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias or lymphoid malignancies. More specific examples of such cancers include: squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, multiple myeloma and B-cell lymphoma, brain cancer and head and neck cancer, and related cancer metastases. In some embodiments, the cancer is lung cancer or lung adenocarcinoma.

[0098] The anti-CAIX antibodies or cells or pharmaceutical compositions thereof as disclosed herein may be administered intravenously, topically, intraperitoneally, intra-arterially, intrathecally, intravesically or intratumorally. One of ordinary skill in the art will appreciate that the effective amount of an anti-cancer antibody or composition thereof may be determined empirically. It will be understood that when administered to a human patient, the total daily dosage of the anti-CAIX antibody or composition thereof may be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dosage amount for any particular patient will depend on a variety of factors: the type and extent of the cellular response to be achieved; the activity of the specific anti-CAIX antibody or composition thereof employed; the patient's age, weight, general health, sex and diet; the time of administration, route of administration and excretion rate of the anti-CAIX antibody or composition thereof; the duration of treatment; drugs used in combination or concomitantly with the anti-CAIX antibody or composition thereof; and similar factors well known in the medical art.

[0099] Each of the above identified compositions and treatment methods may further include the administration of additional anti-cancer drugs and additional one or more anti-cancer drugs. In the treatment methods, the anti-CAIX antibodies of the invention or the cells of the invention may be administered simultaneously, sequentially or separately with the additional one or more anti-cancer drugs.

[0100] The following examples are provided to illustrate certain aspects of the invention and should not be construed as limiting the invention in any way.

[0101] Examples

[0102] Materials and Methods

[0103] Expression and purification of recombinant CAIX protein

[0104] Briefly, the CAIX gene was constructed in pET-21a(+) and pET-32a(+) plasmids, and the resulting vectors were transformed into E. coli BL21 cells. Bacterial cultures from individual colonies were grown overnight at 37°C in 5 ml LB medium containing ampicillin (50 μg / ml), diluted 100-fold in the same LB medium, and further cultured until OD 600 To induce CAIX protein expression, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the culture to a final concentration of 0.5 mM. The cell pellet was resuspended in His-binding buffer and lysed by sonication. After centrifugation, the resulting cell lysate was mixed with Ni 2+ -agarose gel was incubated to purify the recombinant CAIX fusion protein according to the manufacturer's instructions (GE Healthcare, IL, USA).

[0105] Animal immunization

[0106] Female white Leghorn (Gallus domesticus) chickens were immunized by intramuscular injection with 0.5 ml Freund's complete adjuvant (sigma, USA) containing 60 μg of purified his-CAIX. Five additional immunizations were performed every 7 days with Freund's incomplete adjuvant containing his-CAIX. After each immunization, polystrain IgY antibodies in egg yolk were partially purified and titrated by enzyme-linked immunosorbent assay (ELISA) to determine the presence of humoral anti-CAIX immune response. IgY antibodies were purified from egg yolk using 10% sulfated polydextrose. The purified IgY antibodies were dissolved in 5 ml TBS containing 0.05% sodium azide and stored at -20°C.

[0107] Construction and panning of scFv antibody library

[0108] The antibody library was established based on the reported technology and method (Lee et al., Appl Environ Microbiol., 82 (23): 6973-6982, 2016). In brief, the spleen collected from the chicken after the final immunization was immediately placed in Trizol (Invitrogen, CA, USA) for homogenization. Twenty micrograms of total RNA were reverse transcribed into first-strand cDNA using the SuperScript RT kit (Invitrogen, USA). After amplification using chicken-specific primers, the PCR products of the heavy and light chain variable (VH and VL) regions were subjected to a second round of PCR to form full-length scFv fragments with short linkers (such as SEQ ID NO.29) or long linkers (SEQ ID NO: 30 or 31), which were further digested with SfiI and cloned into the pComb3X vector. The recombinant phage DNA was transformed into the E. coli ER2738 strain by electroporation (MicroPulser from Bio-Rad). The production of recombinant phage was initiated by adding wild-type VCS-M13 helper phage, which was then precipitated with 4% polyethylene glycol 8000 (Sigma, MO, USA) and 3% NaCl (w / v), and finally resuspended in 1× phosphate-buffered saline (PBS). 10 -10 11 Plaque forming units (pfu) were added to wells pre-coated with purified CAIX protein (0.25 μg / well) and incubated at 37°C for 2 hours. After removal of unbound phage, bound phage were eluted with His-elution buffer (pH=2.2), neutralized with 2M Tris base buffer (pH=9.1) and used to infect E. coli ER2738 strain. Amplified phages were precipitated and recovered for the next round of selection as described above. After the 4th biopanning, total phagemid DNA from E. coli ER2738 was purified. A set of randomly selected clones were cultured overnight in a 1 mM MgCl 2 The cells were diluted 100× in supernatant containing 100 μg / ml of ampicillin and grown for another 8 h. After induction with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) overnight, the bacteria were harvested by centrifugation, resuspended in histidine (His)-binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM sodium phosphate, 6 M urea, pH 7.4), and lysed by sonication. The scFv antibody was lysed using a Ni-conjugated 2+ Charged agarose gel (GE Healthcare Bio-Sciences AB, Sweden) was purified according to the manufacturer's instructions.

[0109] Western blot

[0110] Purified recombinant CAIX protein or cell lysate was subjected to SDS-PAGE analysis and transferred to nitrocellulose membrane (Amersham Biosciences, UK). After blocking with TBST containing 5% skim milk for 1 hour, anti-CAIX IgY from the fifth immunized chicken (1:5,000-8,000) or bacterial supernatant containing anti-CAIX scFv antibody (1:10) or purified anti-CAIX scFv antibody (10 μg / ml) was added and incubated for 1 hour at room temperature. After vigorous washing, horseradish peroxidase (HRP)-conjugated polystrain donkey anti-chicken IgY antibody (1:10,000) (Jackson Immuno Research, PA, USA) was added and incubated for an additional 1 hour for detection of bound IgY antibody. In addition, mouse anti-HA tag mAb (1:3,000-5,000) followed by HRP-conjugated rabbit anti-mouse antibody (1:5,000-10,000) was used to detect bound scFv antibodies. After washing as above, the membrane was developed with diaminobenzidine (DAB) or ECL substrate. ImageQuant LAS4500 was used for ECL intensity detection.

[0111] ELISA and competitive ELISA

[0112] To examine its binding reactivity, a series of diluted IgY antibodies (500-256,000-fold) or anti-CAIX scFv antibodies (1:10) or purified anti-scFv antibodies (1 μg / ml) purified from chickens after the fifth immunization were incubated with purified CAIX protein (0.25 μg / well) immobilized on the wells of ELISA culture plates. After vigorous washing, bound IgY antibodies were detected by adding HRP-conjugated polyclonal donkey anti-chicken IgY antibodies (1:10,000) (Jackson ImmunoResearch, PA, USA), while bound CAIX scFv antibodies were detected by mouse anti-HA mAb (1:3,000-5,000) and then by HRP-conjugated rabbit anti-mouse (1:5,000-10,000). After washing as above, tetramethylbenzidine (TMB) substrate solution (Sigma, USA) was added to the wells for color development. The reaction was stopped with 1N HCl and the optical density was measured at 450 nm using an ELISA plate reader (BioTek Synergy HT). The dissociation constant (K) was calculated according to the published protocol (Friguet et al., J. Immunol. Methods, 77: 305-319, 1985) d ).

[0113] For competitive ELISA, CAIX fragment protein (200-0 μg / ml) was first mixed with an equal volume of CAIX scFv antibody (1 μg / ml) at 37°C for 1 hour or at 4°C overnight and added to a culture plate coated with purified CAIX protein to detect binding specificity. The bound CAIX scFv antibody was detected by mouse anti-HA mAb (1:5,000) followed by HRP-conjugated rabbit anti-mouse IgG (1:10,000). After washing as above, tetramethylbenzidine (TMB) substrate solution (Sigma, USA) was added to the wells for color development. The reaction was stopped with HCl, and the optical density was measured at 450 nm using an ELISA plate reader (BioTek Synergy HT). Two wells were tested in duplicate for each sample with ELISA. ELISA data are presented as the mean ± SD of replicate experiments.

[0114] Example 1 Analysis of purified anti-CAIX scFvs (NAVICA9S1, NAVICA9S2, NAVICA9L1, NAVICA9L2, and NAVICA9L3) and recombinant CAIX proteins.

[0115] SDS-PAGE was stained with Coomassie blue dye and is shown below. NAVICA9S1 (lane 1), NAVICA9S2 (lane 2), NAVICA9L1 (lane 3), NAVICA9L2 (lane 4), and NAVICA9L3 (lane 5) were purified according to the above method ( Figure 1 (A)). Recombinant CAIX protein (ECD) and F3 were also purified and displayed. Figure 1 (B) Chinese. Figure 1 (C) The full length, extracellular domain (ECD), fragment 1 (F1), fragment 2 (F2), and fragment 3 (F3) of CAIX are shown.

[0116] Example 2 Analysis of the Binding Ability of Anti-CAIX scFv by Western Blot

[0117] Recombinant CAIX ECD (lane 1), F3 fragment (lane 2), and A549 cell lysate (lane 3) are visible on SDS-PAGE ( Figure 2A). Therefore, the same amount of protein on the replicate SDS-PAGE was transferred to a nitrocellulose membrane (NC). After blocking for one hour, mouse anti-CAIX IgG (1:2,000) (ab107257) or chicken anti-CAIX IgY (1:5,000) was added to the NC as the primary antibody. After one hour of reaction, the NC was washed 3 times with PBST. After washing, HRP-conjugated rabbit anti-mouse IgG (1:10,000) or HRP-conjugated donkey anti-IgY (1:10,000) was added to the NC. After one hour of reaction, the NC was washed with PBST and incubated with ECL solution. Similarly, anti-CAIX scFv monoclonal antibody (10 μl / ml) was added to the NC as the primary antibody. Then, mouse anti-HA IgG and HRP-conjugated rabbit anti-mouse IgG were added sequentially as the secondary and tertiary antibodies. After incubation with ECL, binding activity was detected and displayed. Figure 2 middle.

[0118] Example 3 Analysis of the binding ability of anti-CAIX scFv by ELISA

[0119] Recombinant CAIX-FL, ECD, ECD, F2 and F3 (expressed in pET32a or pET21a) were attached to 96 half-well ELISA plates at a concentration of 0.25 μg / well. After one hour of incubation, unbound proteins were removed and 5% PBS-milk was added for blocking. After one hour of reaction, PBS-milk was removed and each of the anti-CAIX antibodies (1 μg / ml) was added. After washing with PBST, mouse anti-HA IgG (1:5,000) and HRP-conjugated rabbit anti-mouse IgG (1:10,000) were added to the plate in sequence. Next, 3,3',5,5'-tetramethylbenzidine (TMB) was added for color development, which was stopped by HCl and detected using an ELISA plate reader. The results are shown in Figure 3 middle.

[0120] Example 4 Antigenic determinant mapping

[0121] To define the antigenic epitopes on the CAIX protein, purified anti-CAIX antibodies were examined for binding to recombinant CAIX fragments. The results showed that NAVICA9S1, NAVICA9S2 and NAVICA9L3 recognized CAIX FL, ECD, F2 and F3 fragments, indicating that the antigenic determinant is located between amino acid residues 228 to 353. The cloned NAVICA9L2 recognized CAIX FL and ECD, but not F2 and F3 fragments, indicating that the antigenic determinant is located between amino acids 38 to 153. Example 5 Estimation of the dissociation constant of anti-CAIX scFv by competitive ELISA

[0122] To determine the binding affinity of anti-CAIX scFv, a competitive ELISA was performed. CAIX ECD fragment protein (100-13 μg / ml) serially diluted 2-fold was incubated with anti-CAIX scFv (NAVICA9S1: 0.62 μg / ml; NAVICA9S2: 0.32 μg / ml; NAVICA9L2: 0.16 μg / ml; NAVICA9L3: 0.32 μg / ml) at room temperature in a 1:1 volume ratio for 1 hour. The mixture was then added to an ELISA plate coated with CAIX ECD fragment protein (0.25 or 0.5 μg / well). After incubation for 1 hour, mouse anti-HA IgG (1:5,000) and HRP-conjugated rabbit anti-mouse IgG (1:10,000) were added as secondary and tertiary antibodies. After incubation and washing, 3,3',5,5'-tetramethylbenzidine (TMB) was added for color development, which was stopped by HCl and detected using an ELISA plate reader. K of scFv d Values ​​were estimated and expressed by molar concentration (M) as described by Friguet et al. (J. Immunol. Methods, 77:305-319, 1985). N / A: Not available. The dissociation constants of anti-CAIX monoclonal scFvs are shown in Table 1 below.

[0123] Table 1

[0124] Anti-CAIX scFv Dissociation constant NAVICA9S1 <![CDATA[6.43×10 -8 M]]> NAVICA9S2 <![CDATA[1.15×10 -8 M]]> NAVICA9L1 N / A NAVICA9L2 <![CDATA[1.40×10 -7 M]]> NAVICA9L3 <![CDATA[9.26×10 -9 M]]>

[0125] Example 6 Flow cytometry analysis

[0126] Anti-CAIX scFv and anti-CAIX (abcam, ab107257) were incubated with 2×10 5 A549 cells (CAIX+) or THP-1 cells (CAIX-) were incubated together for 20 minutes. Mouse anti-HA IgG and PE-conjugated anti-mouse IgG were added as the secondary and tertiary antibodies; PE-conjugated anti-mouse IgG was also added as the secondary antibody of anti-CAIX antibody (abcam, ab107257). After washing, the cells were subjected to flow cytometry analysis and the results are shown in Figure 5 middle. Sequence Listing <110> Navi Recycling Technology Co., Ltd. <120> Anti-Carbonic Anhydrase IX Antibody <130> N96150 / US6667 <160> 66 <170> PatentIn Version 3.5 <210> 1 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 1 Ser Gly Ser Ser Gly Ser Tyr Gly 1 5 <210> 2 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 2 Tyr Asn Asp Lys Arg Pro Ser 1 5 <210> 3 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 3 Gly Ser Ala Asp Arg Ser Gly Ala Gly Ile 1 5 10 <210> 4 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 4 Ser Gly Ser Arg Gly Ser Tyr Gly 1 5 <210> 5 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 5 Tyr Asn Val Lys Arg Pro Ser 1 5 <210> 6 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 6 Ser Gly Gly Gly Lys Asn Tyr Gly 1 5 <210> 7 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 7 Leu Asn Asp Lys Arg Pro Ser 1 5 <210> 8 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 8 Gly Ser Arg Asp Ser Ser Pro Thr 1 5 <210> 9 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 9 Ser His Gly Met Ala 1 5 <210> 10 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 10 Gly Ile Ser Asn Thr Gly Arg Tyr Thr Asn Tyr Gly Ser Ala Val Lys 1 5 10 15 Gly <210> 11 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 11 Ala Ala Val Asn Cys Val Tyr Gly Cys Pro Gly Ser Ile Asp Ala 1 5 10 15 <210> 12 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 12 Ser Tyr Ala Ile Gln 1 5 <210> 13 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 13 Gly Ile Ser Asp Asp Gly Ser Trp Thr Gly Tyr Gly Ala Ala Val Lys 1 5 10 15 Gly <210> 14 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 14 Gly Ala Gly Thr Gly Tyr Cys Ala Asn Arg Ser Phe Gly Cys Ala Ser 1 5 10 15 Thr Ile Asp Ala 20 <210> 15 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 15 Ser Phe Asn Met Phe 1 5 <210> 16 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 16 Ala Ile Ser Ser Asp Gly Ser Arg Ser Arg Tyr Gly Ser Ala Val Gln 1 5 10 15 Gly <210> 17 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 17 Arg Pro Tyr Ser Gly Cys Thr Ser Asn Asn Phe Cys Phe Asn Ala Ala 1 5 10 15 Tyr Ile Asp Val 20 <210> 18 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 18 Ser Tyr Ala Met Asn 1 5 <210> 19 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 19 Gly Ile Thr Asn Thr Gly Ser Ser Ala Gly Tyr Gly Ala Ala Val Lys 1 5 10 15 Gly <210> 20 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 20 Ser Tyr Gly Gly Trp Cys Asp His Ala Cys Ala Pro Asp Asp Ile Asp 1 5 10 15 Thr <210> twenty one <211> 103 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> twenty one Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Leu Gly Glu Thr Val 1 5 10 15 Glu Ile Thr Cys Ser Gly Ser Ser Gly Ser Tyr Gly Trp Tyr Gln Gln 20 25 30 Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr Tyr Asn Asp Lys 35 40 45 Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser Lys Ser Gly Ser 50 55 60 Thr Gly Thr Leu Thr Ile Thr Gly Val Gln Ala Glu Asp Glu Ala Val 65 70 75 80 Tyr Tyr Cys Gly Ser Ala Asp Arg Ser Gly Ala Gly Ile Phe Gly Ala 85 90 95 Gly Thr Thr Leu Thr Val Leu 100 <210> 22 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 22 Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Leu Gly Glu Thr Val 1 5 10 15 Glu Ile Thr Cys Ser Gly Ser Arg Gly Ser Tyr Gly Trp Tyr Gln Gln 20 25 30 Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr Tyr Asn Val Lys 35 40 45 Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser Gly Ser Lys Ser Gly Ser 50 55 60 Thr Gly Thr Leu Thr Ile Thr Gly Val Gln Ala Glu Asp Glu Ala Val 65 70 75 80 Tyr Tyr Cys Gly Ser Ala Asp Arg Ser Gly Ala Gly Ile Phe Gly Ala 85 90 95 Gly Thr Thr Leu Thr Val Leu 100 <210> 23 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 23 Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu Thr Val 1 5 10 15 Lys Ile Thr Cys Ser Gly Gly Gly Lys Asn Tyr Gly Trp Tyr Gln Gln 20 25 30 Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr Leu Asn Asp Lys 35 40 45 Arg Pro Ser Asn Ile Pro Ser Arg Phe Ser Gly Ser Lys Ser Gly Ser 50 55 60 Thr His Thr Leu Thr Ile Thr Gly Val Gln Ala Glu Asp Glu Ala Val 65 70 75 80 Tyr Phe Cys Gly Ser Arg Asp Ser Ser Pro Thr Phe Gly Ala Gly Thr 85 90 95 Thr Leu Thr Val Leu 100 <210> 24 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 24 Ala Val Thr Leu Asp Glu Pro Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Thr Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Asp Ile Ser Ser His 20 25 30 Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ala Gly Ile Ser Asn Thr Gly Arg Tyr Thr Asn Tyr Gly Ser Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Val Arg 65 70 75 80 Leu Gln Leu Asn Asp Leu Arg Ala Glu Asp Ala Gly Thr Tyr Phe Cys 85 90 95 Ala Arg Ala Ala Val Asn Cys Val Tyr Gly Cys Pro Gly Ser Ile Asp 100 105 110 Ala Trp Gly Leu Gly Thr Glu Val Ile Val Ser Ser 115 120 <210> 25 <211> 129 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 25 Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Gly Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Ser Asp Asp Gly Ser Trp Thr Gly Tyr Gly Ala Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Met Arg 65 70 75 80 Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr Tyr Cys 85 90 95 Ala Lys Gly Ala Gly Thr Gly Tyr Cys Ala Asn Arg Ser Phe Gly Cys 100 105 110 Ala Ser Thr Ile Asp Ala Trp Gly His Gly Thr Glu Val Ile Val Ser 115 120 125 Ser <210> 26 <211> 129 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 26 Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Asn Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ala Ala Ile Ser Ser Asp Gly Ser Arg Ser Arg Tyr Gly Ser Ala Val 50 55 60 Gln Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Val Arg 65 70 75 80 Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys 85 90 95 Ala Lys Arg Pro Tyr Ser Gly Cys Thr Ser Asn Asn Phe Cys Phe Asn 100 105 110 Ala Ala Tyr Ile Asp Val Trp Gly His Gly Thr Glu Val Ile Val Ser 115 120 125 Ser <210> 27 <211> 126 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 27 Ala Val Thr Leu Asp Glu Ser Glu Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val 35 40 45 Ala Gly Ile Thr Asn Thr Gly Ser Ser Ala Gly Tyr Gly Ala Ala Val 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Val Arg 65 70 75 80 Leu Gln Leu Asn Asn Leu Arg Ala Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Thr Lys Ser Tyr Gly Gly Trp Cys Asp His Ala Cys Ala Pro Asp Asp 100 105 110 Ile Asp Thr Trp Gly His Gly Thr Glu Val Ile Val Ser Ser 115 120 125 <210> 28 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 28 Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Arg Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Asn Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ala Ala Ile Ser Ser Asp Gly Ser Arg Ser Arg Tyr Gly Ser Ala Val 50 55 60 Gln Gly Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Val Arg 65 70 75 80 Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys 85 90 95 Ala Lys Arg Pro Tyr Ser Gly Cys Thr Ser Asn Asn Phe Cys Phe Asn 100 105 110 Ala Ala Tyr Ile Asp Val Trp Gly His Gly Thr Glu Val Ile Val Ser 115 120 125 Ser <210> 29 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 29 Gly Gln Ser Ser Arg Ser Ser 1 5 <210> 30 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 30 Gly Gln Ser Ser Arg Ser Ser Ser Gly Gly Gly Ser Ser Gly Gly Gly 1 5 10 15 Gly Ser <210> 31 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 31 Gly Gln Ser Ser Arg Ser Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly 1 5 10 15 Gly Ser <210> 32 <211> 116 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 32 Gln Arg Leu Pro Arg Met Gln Glu Asp Ser Pro Leu Gly Gly Gly Ser 1 5 10 15 Ser Gly Glu Asp Asp Pro Leu Gly Glu Glu Asp Leu Pro Ser Glu Glu 20 25 30 Asp Ser Pro Arg Glu Glu Asp Pro Pro Gly Glu Glu Asp Leu Pro Gly 35 40 45 Glu Glu Asp Leu Pro Gly Glu Glu Asp Leu Pro Glu Val Lys Pro Lys 50 55 60 Ser Glu Glu Glu Gly Ser Leu Lys Leu Glu Asp Leu Pro Thr Val Glu 65 70 75 80 Ala Pro Gly Asp Pro Gln Glu Pro Gln Asn Asn Ala His Arg Asp Lys 85 90 95 Glu Gly Asp Asp Gln Ser His Trp Arg Tyr Gly Gly Asp Pro Pro Trp 100 105 110 Pro Arg Val Ser 115 <210> 33 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 33 His Trp Gly Ala Ala Gly Arg Pro Gly Ser Glu His Thr Val Glu Gly 1 5 10 15 His Arg Phe Pro Ala Glu Ile His Val Val His Leu Ser Thr Ala Phe 20 25 30 Ala Arg Val Asp Glu Ala Leu Gly Arg Pro Gly Gly Leu Ala Val Leu 35 40 45 Ala Ala Phe Leu Glu Glu Gly Pro Glu Glu Asn Ser Ala Tyr Glu Gln 50 55 60 Leu Leu Ser Arg Leu Glu Glu Ile Ala Glu Glu Gly Ser Glu Thr Gln 65 70 75 80 Val Pro Gly Leu Asp Ile Ser Ala Leu Leu Pro Ser Asp Phe Ser Arg 85 90 95 Tyr Phe Gln Tyr Glu Gly Ser Leu Thr Thr Pro Pro Cys Ala Gln Gly 100 105 110 Val Ile Trp Thr Val Phe Asn Gln Thr Val Met Leu Ser Ala 115 120 125 <210> 34 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 34 Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Leu Gly Glu Thr Val 1 5 10 15 Glu Ile Thr Cys 20 <210> 35 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 35 Ala Leu Thr Gln Pro Ser Ser Val Ser Ala Asn Pro Gly Glu Thr Val 1 5 10 15 Lys Ile Thr Cys 20 <210> 36 <211> 26 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 36 Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 20 25 <210> 37 <211> 26 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 37 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser 20 25 <210> 38 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 38 Trp Tyr Gln Gln Lys Ser Pro Gly Ser Ala Pro Val Thr Val Ile Tyr 1 5 10 15 <210> 39 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 39 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 1 5 10 15 Tyr <210> 40 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 40 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 1 5 10 15 Tyr <210> 41 <211> 32 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 41 Asp Ile Pro Ser Arg Phe Ser Gly Ser Lys Ser Gly Ser Thr Gly Thr 1 5 10 15 Leu Thr Ile Thr Gly Val Gln Ala Glu Asp Glu Ala Val Tyr Tyr Cys 20 25 30 <210> 42 <211> 32 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 42 Asn Ile Pro Ser Arg Phe Ser Gly Ser Lys Ser Gly Ser Thr His Thr 1 5 10 15 Leu Thr Ile Thr Gly Val Gln Ala Glu Asp Glu Ala Val Tyr Phe Cys 20 25 30 <210> 43 <211> 32 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 43 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25 30 <210> 44 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 44 Phe Gly Ala Gly Thr Thr Leu Thr Val Leu 1 5 10 <210> 45 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 45 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 1 5 10 <210> 46 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 46 Ala Val Thr Leu Asp Glu Pro Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Thr Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Asp Ile Ser 20 25 30 <210> 47 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 47 Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Gly Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser 20 25 30 <210> 48 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 48 Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser 20 25 30 <210> 49 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 49 Ala Val Thr Leu Asp Glu Ser Glu Gly Gly Leu Gln Thr Pro Gly Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser 20 25 30 <210> 50 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 50 Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Arg Gly 1 5 10 15 Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser 20 25 30 <210> 51 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 51 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ile 20 25 30 <210> 52 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 52 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr 20 25 30 <210> 53 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 53 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val Ala 1 5 10 <210> 54 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 54 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 1 5 10 <210> 55 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 55 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val Ala 1 5 10 <210> 56 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 56 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly 1 5 10 <210> 57 <211> 32 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 57 Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Val Arg Leu Gln 1 5 10 15 Leu Asn Asp Leu Arg Ala Glu Asp Ala Gly Thr Tyr Phe Cys Ala Arg 20 25 30 <210> 58 <211> 32 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 58 Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Met Arg Leu Gln 1 5 10 15 Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr Tyr Cys Ala Lys 20 25 30 <210> 59 <211> 32 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 59 Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Val Arg Leu Gln 1 5 10 15 Leu Asn Ser Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys Ala Lys 20 25 30 <210> 60 <211> 32 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 60 Arg Ala Thr Ile Ser Arg Asp Asn Gly Gln Ser Thr Val Arg Leu Gln 1 5 10 15 Leu Asn Asn Leu Arg Ala Glu Asp Thr Ala Thr Tyr Phe Cys Thr Lys 20 25 30 <210> 61 <211> 32 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 61 Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys 20 25 30 <210> 62 <211> 32 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 62 Arg Phe Thr Ile Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys 20 25 30 <210> 63 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 63 Trp Gly Leu Gly Thr Glu Val Ile Val Ser Ser 1 5 10 <210> 64 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 64 Trp Gly His Gly Thr Glu Val Ile Val Ser Ser 1 5 10 <210> 65 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 65 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 66 <211> 459 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 66 Met Ala Pro Leu Cys Pro Ser Pro Trp Leu Pro Leu Leu Ile Pro Ala 1 5 10 15 Pro Ala Pro Gly Leu Thr Val Gln Leu Leu Leu Ser Leu Leu Leu Leu 20 25 30 Val Pro Val His Pro Gln Arg Leu Pro Arg Met Gln Glu Asp Ser Pro 35 40 45 Leu Gly Gly Gly Ser Ser Gly Glu Asp Asp Pro Leu Gly Glu Glu Asp 50 55 60 Leu Pro Ser Glu Glu Asp Ser Pro Arg Glu Glu Asp Pro Pro Gly Glu 65 70 75 80 Glu Asp Leu Pro Gly Glu Glu Asp Leu Pro Gly Glu Glu Asp Leu Pro 85 90 95 Glu Val Lys Pro Lys Ser Glu Glu Glu Gly Ser Leu Lys Leu Glu Asp 100 105 110 Leu Pro Thr Val Glu Ala Pro Gly Asp Pro Gln Glu Pro Gln Asn Asn 115 120 125 Ala His Arg Asp Lys Glu Gly Asp Asp Gln Ser His Trp Arg Tyr Gly 130 135 140 Gly Asp Pro Pro Trp Pro Arg Val Ser Pro Ala Cys Ala Gly Arg Phe 145 150 155 160 Gln Ser Pro Val Asp Ile Arg Pro Gln Leu Ala Ala Phe Cys Pro Ala 165 170 175 Leu Arg Pro Leu Glu Leu Leu Gly Phe Gln Leu Pro Pro Leu Pro Glu 180 185 190 Leu Arg Leu Arg Asn Asn Gly His Ser Val Gln Leu Thr Leu Pro Pro 195 200 205 Gly Leu Glu Met Ala Leu Gly Pro Gly Arg Glu Tyr Arg Ala Leu Gln 210 215 220 Leu His Leu His Trp Gly Ala Ala Gly Arg Pro Gly Ser Glu His Thr 225 230 235 240 Val Glu Gly His Arg Phe Pro Ala Glu Ile His Val Val His Leu Ser 245 250 255 Thr Ala Phe Ala Arg Val Asp Glu Ala Leu Gly Arg Pro Gly Gly Leu 260 265 270 Ala Val Leu Ala Ala Phe Leu Glu Glu Gly Pro Glu Glu Asn Ser Ala 275 280 285 Tyr Glu Gln Leu Leu Ser Arg Leu Glu Glu Ile Ala Glu Glu Gly Ser 290 295 300 Glu Thr Gln Val Pro Gly Leu Asp Ile Ser Ala Leu Leu Pro Ser Asp 305 310 315 320 Phe Ser Arg Tyr Phe Gln Tyr Glu Gly Ser Leu Thr Thr Pro Pro Cys 325 330 335 Ala Gln Gly Val Ile Trp Thr Val Phe Asn Gln Thr Val Met Leu Ser 340 345 350 Ala Lys Gln Leu His Thr Leu Ser Asp Thr Leu Trp Gly Pro Gly Asp 355 360 365 Ser Arg Leu Gln Leu Asn Phe Arg Ala Thr Gln Pro Leu Asn Gly Arg 370 375 380 Val Ile Glu Ala Ser Phe Pro Ala Gly Val Asp Ser Ser Pro Arg Ala 385 390 395 400 Ala Glu Pro Val Gln Leu Asn Ser Cys Leu Ala Ala Gly Asp Ile Leu 405 410 415 Ala Leu Val Phe Gly Leu Leu Phe Ala Val Thr Ser Val Ala Phe Leu 420 425 430 Val Gln Met Arg Arg Gln His Arg Arg Gly Thr Lys Gly Gly Val Ser 435 440 445 Tyr Arg Pro Ala Glu Val Ala Glu Thr Gly Ala 450 455

Claims

1. An isolated anti-CAIX antibody or an antigen-binding portion thereof, comprising: a light chain complementary determining region 1 (L-CDR1) of SEQ ID NO: 4; a light chain CDR2 (L-CDR2) of SEQ ID NO: 5; and a light chain CDR3 (L-CDR3) of SEQ ID NO: 3; and The heavy chain CDR1 (H-CDR1) of SEQ ID NO: 12; the heavy chain CDR2 (H-CDR2) of SEQ ID NO: 13; and the heavy chain CDR3 (H-CDR3) of SEQ ID NO: 14; so that the isolated antibody or antigen-binding portion thereof binds to CAIX.

2. The isolated anti-CAIX antibody or antigen-binding portion thereof of claim 1, which is a monoclonal antibody, a chimeric antibody, a humanized antibody or a human antibody.

3. The isolated anti-CAIX antibody or antigen-binding portion thereof of claim 1, comprising a constant region and one or more heavy and light chain variable framework regions of a human antibody sequence.

4. The isolated anti-CAIX antibody, or antigen-binding portion thereof, of claim 3, comprising up to 10 amino acid substitutions in the framework regions.

5. The isolated anti-CAIX antibody or antigen-binding portion thereof of claim 3, comprising the framework region of Avastin.

6. The isolated anti-CAIX antibody, or antigen-binding portion thereof, of claim 3, wherein the framework regions comprise: L-FR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 34 to 37; L-FR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 38 to 40; L-FR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 41 to 43; and L-FR4 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 44 to 45; and H-FR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 46 to 52; H-FR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 56; H-FR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 57 to 62; and H-FR4 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 63 to 65.

7. The isolated anti-CAIX antibody or antigen-binding portion thereof of claim 1, comprising a light chain comprising the amino acid sequence of SEQ ID NO:

22.

8. The isolated anti-CAIX antibody or antigen-binding portion thereof of claim 1, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:

25.

9. The isolated anti-CAIX antibody or antigen-binding portion thereof of claim 1, comprising: (i) a light chain having an amino acid sequence as set forth in SEQ ID NO: 22 or a variant thereof that is at least 80% identical to SEQ ID NO: 22, and (ii) a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 25 or a variant thereof that is at least 80% identical to SEQ ID NO:

25.

10. The isolated anti-CAIX antibody, or antigen-binding portion thereof, of claim 1, comprising a light chain having the amino acid sequence set forth in SEQ ID NO: 22 and a heavy chain having the amino acid sequence set forth in SEQ ID NO:

25.

11. The isolated anti-CAIX antibody or antigen binding portion thereof according to any one of claims 1 to 10, wherein the antigen binding portion is Fab, F(ab')2, Fv or scFv.

12. A single-chain variable fragment (scFv) that binds to CAIX, comprising a) a light chain variable region of a light chain, wherein the light chain has an amino acid sequence such as SEQ ID NO: 22; b) a linker; and c) a heavy chain variable region of a heavy chain, wherein the heavy chain has an amino acid sequence such as selected from SEQ ID NO: 25, wherein the linker is a peptide consisting of an amino acid sequence of SEQ ID NO: 29, 30 or 31.

13. A pharmaceutical composition comprising the antibody or antigen-binding portion thereof according to any one of claims 1 to 11 or the scFv according to claim 12, and a pharmaceutically acceptable additive.

14. The pharmaceutical composition of claim 13, wherein the additive is a carrier.

15. The pharmaceutical composition of claim 13, further comprising one or more additional anti-cancer drugs.

16. A cell expressing the antibody or antigen binding portion thereof according to any one of claims 1 to 11 or the scFv according to claim 12.

17. A pharmaceutical composition comprising the cell according to claim 16 and a pharmaceutically acceptable additive.

18. The pharmaceutical composition of claim 17, wherein the additive is a carrier.

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