CN113512118A

CN113512118A - Anti-human CD146 monoclonal antibody with neutralization activity and application thereof

Info

Publication number: CN113512118A
Application number: CN202010280682.1A
Authority: CN
Inventors: 阎锡蕴; 陈雪晖; 晏荟文; 刘丹; 冯静; 段红霞; 徐庆吉
Original assignee: Institute of Biophysics of CAS
Current assignee: Institute of Biophysics of CAS
Priority date: 2020-04-10
Filing date: 2020-04-10
Publication date: 2021-10-19

Abstract

The invention relates to an anti-human CD146 monoclonal antibody with neutralization activity and application thereof. Specifically, the invention relates to an antibody which is improved on the basis of an AA98 antibody and is targeted to CD146, the antibody can specifically and high-affinity recognize CD146 expressed by neovascular endothelial cells, tumor cells, perivascular cells, lymphocytes and the like, has the capacity of remarkably inhibiting endothelial cell migration and angiogenesis, specifically and efficiently inhibits the growth and metastasis of tumors, can be used for developing broad-spectrum, efficient and low-toxicity antibody medicaments, and is used for treating and early diagnosing tumors, inflammatory diseases and neovascular diseases.

Description

Anti-human CD146 monoclonal antibody with neutralization activity and application thereof

Technical Field

The invention relates to a novel antibody for preventing and/or treating tumor growth and metastasis, in particular to an antibody targeting CD 146.

Background

Biotechnological drugs include antibody drugs, small molecule chemicals, vaccines, protein and polypeptide drugs, gene therapy and cell therapy drugs, and among new research drugs currently in clinical phase III research, monoclonal antibody drugs account for about 50%, and antibody drugs gradually occupy a large market share. Antibody drugs are widely used for the treatment of cancer, autoimmune diseases, infectious diseases, cardiovascular diseases, and the like.

Monoclonal antibodies are highly specific targeting antibodies that act on only a single epitope, and have been widely used in the treatment of many diseases, such as cancer, inflammatory and autoimmune diseases, infectious diseases, etc., and particularly, such specific targeting drugs are important after chemotherapy of cancer has failed.

CD146 is also known as a melanoma cell adhesion molecule,is a single transmembrane glycoprotein receptor, and belongs to the immunoglobulin superfamily (IgSF). CD146 as non-Ca²⁺Cell Adhesion Molecules (CAM) are involved in the adhesion between heterophilic cells and between cells and extracellular matrix, and are closely related to tumorigenesis and metastasis. In normal mature tissues, the expression level of the CD146 molecule is low and angiogenesis is silent. The inducing factors in the tumor microenvironment promote the proliferation and the tube formation of CD146 positive endothelial cells, promote the generation of tumor blood vessels, provide necessary nutrition for the growth of tumor cells and provide convenience for metastasis. CD146 on the surface of tumor cells binds to extracellular matrix and intracellular cytoskeletal proteins, promoting cytoskeletal rearrangement and thus tumor cell migration. In conclusion, CD146 is involved in tumor cell adhesion, metastasis, angiogenesis and new lesion formation. CD146 has been shown to be closely related to the development of melanoma, prostate cancer, lung cancer, liver cancer, breast cancer, chorioepithelioma, osteosarcoma, and the like. In addition, CD146 has been reported to be expressed on perivascular cells, activated lymphocytes, nerve cells, human trophoblast cells and early embryos.

Chinese patent CN1124284C discloses a CD146 antibody for inhibiting tumor growth and metastasis with high efficiency and broad spectrum, which comprises a murine monoclonal antibody AA98, a human-murine chimeric antibody ChiAA98 and Fab and Fv functional fragments thereof, can specifically recognize tumor neovascularization, has the capability of inducing ADCC and CDC, and can specifically and broadly inhibit tumor growth and metastasis. In inflammatory bowel disease, CD146 promotes the development of inflammation by promoting vascular proliferation, assisting in the extravasation of pro-inflammatory lymphocytes. The AA98 antibody can obviously improve the symptoms of enteritis and inhibit colorectal cancer in mice. (Xing et al. targeting Endothelial CD146 patents Colitis an advanced patents Colitis-Associated Cardigenesis, Am J Pathol 2014,184: 1604-.

The antibody has the defects of low affinity with the antigen CD146 and the like, and the need still exists in the field for a more efficient antibody targeting CD 146.

Disclosure of Invention

A first aspect of the invention relates toAn isolated anti-CD 146 antibody, antigen-binding fragment thereof, variant thereof or derivative thereof derived from an AA98 antibody and having at least 8 x 10 binding affinity for CD146^-10Affinity of M, e.g. at least 7X 10^-10M、6.5×10^-10M、6×10^-10M、5.5×10^-10M、5×10^-10M、4.5×10^-10M、4×10^-10M、3.5×10^-10M、3×10^-10M、2.5×10^-10M、2×10^-10M、1.8×10^-10M、1.6×10^-10M、1.5×10^-10M、1.4×10^-10M or higher; optionally, it also has an ability to inhibit lumenization of endothelial cells of greater than 40%, e.g., greater than 45%, 50%, 55%, 60%, 65% or more, and/or an ability to inhibit migration of endothelial cells of greater than 60%, e.g., greater than 65%, 70%, 75%, 80%, 85% or more. The light chain nucleotide sequence of the AA98 antibody is shown as SEQ ID NO.2, the heavy chain nucleotide sequence is shown as SEQ ID NO.3, the heavy chain amino acid sequence is shown as SEQ ID NO.6, the light chain amino acid sequence is shown as SEQ ID NO.7, the heavy chain variable region amino acid sequence is shown as SEQ ID NO.4, the light chain variable region amino acid sequence is shown as SEQ ID NO.5, the light and heavy chain amino acid sequences of Fab are respectively shown as SEQ ID NO.8 and 16, the amino acid sequence of scFv is shown as SEQ ID NO.9, the amino acid sequences of heavy chain CDR1-3 are respectively shown as SEQ ID NO.10-12, and the amino acid sequences of light chain CDR1-3 are respectively shown as SEQ ID NO. 13-15.

In some embodiments, the isolated anti-CD 146 antibody, antigen-binding fragment thereof, variant thereof, or derivative thereof has a substitution mutation of a hydrophilic amino acid residue in I31, S32, and/or L51 of the light chain and/or I28, I57, and/or Y59 of the heavy chain relative to the AA98 antibody; preferably, the hydrophilic amino acid residue is selected from the group consisting of Arg, Asn, Asp, gin, Glu, His, Lys and Tyr; more preferably, the substitution mutation of the light chain is selected from I31E, S32T, L51Y, L51H and/or the substitution mutation of the heavy chain is selected from I28K, I57E, Y59R.

In some embodiments, the CDR sequences of the isolated anti-CD 146 antibody, antigen-binding fragment thereof, variant thereof, or derivative thereof have one or 2 conservative substitutions as shown below or based on the sequence shown below:

heavy chain CDR 1: SGYIFTNYW (SEQ ID No.10), and/or

Heavy chain CDR 2: YPGTDITY (SEQ ID No.11), and/or

Heavy chain CDR 3: SGGYWYFDV (SEQ ID No.12), and/or

Light chain CDR 1: ASKSVSISGYSYM (SEQ ID No.13), and/or

Light chain CDR 2: IYLASNL (SEQ ID No.14), and/or

Light chain CDR 3: HSRELPYTFGG (SEQ ID No.15),

preferably, it comprises said 6 CDRs.

In some embodiments, the isolated anti-CD 146 antibody, antigen-binding fragment thereof, variant thereof, or derivative thereof, has at least 95% but not 100% sequence identity to the sequence set forth in SEQ ID No.4 or at least one amino acid substitution, e.g., at least about 96%, 97%, 98%, or 99%, e.g., at least 2, 3, 4, 5 or more amino acid substitutions, preferably conservative substitutions, compared to the sequence set forth in SEQ ID No.4, and/or has at least 95% but not 100% sequence identity to the sequence set forth in SEQ ID No.5 or at least one amino acid substitution, e.g., at least about 96%, 97%, 98%, or 99%, e.g., at least 2, 3, 4, 5 or more amino acid substitutions, preferably conservative substitutions, in the heavy chain variable region sequence. The amino acid substitution does not disrupt the binding of the isolated anti-CD 146 antibody, antigen-binding fragment thereof, variant thereof, or derivative thereof to its antigen.

In some embodiments, the heavy chain amino acid sequence of the isolated anti-CD 146 antibody, antigen-binding fragment thereof, variant thereof or derivative thereof has at least 95% but not 100% sequence identity to the sequence set forth in SEQ ID No.6 or at least one amino acid substitution, e.g., at least about 96%, 97%, 98%, 99%, 99.3%, 99.6% or 99.8%, such as at least 2, 3, 4, 5,6, 7,8, 9, 10 or more amino acid substitutions, preferably conservative substitutions, as compared to the sequence set forth in SEQ ID No.6, and/or the light chain amino acid sequence of the anti-CD 146 antibody has at least 95% but not 100% sequence identity to the sequence set forth in SEQ ID No.7 or at least one amino acid substitution, e.g., at least about 96%, 97%, 98%, 99% or 99.5%, such as at least 2, 99%, or 99.5%, as compared to the sequence set forth in SEQ ID No.6, 3. 4, 5,6 or more amino acid substitutions, preferably conservative substitutions. The amino acid substitution does not disrupt the binding of the isolated anti-CD 146 antibody, antigen-binding fragment thereof, variant thereof, or derivative thereof to its antigen.

In some embodiments, the anti-CD 146 antibody is selected from IgG, IgA, IgM, or IgE, preferably the anti-CD 146 antibody is IgG, more preferably the anti-CD 146 antibody is of the IgG2 subtype; and/or the antigen binding fragment is selected from the group consisting of SDR, CDR, Fv, dAb, Fab₂Fab ', (Fab')2, Fd, scFv, nanobody, e.g. Fab, having a sequence with at least 95% but not 100% sequence identity to the sequences shown in SEQ ID nos. 8 and 16 or having at least one amino acid substitution compared to the sequences shown in SEQ ID nos. 8 and 16, e.g. at least about 96%, 97%, 98%, 99%, 99.5% sequence identity, e.g. at least 2, 3, 4, 5 or more amino acid substitutions, e.g. scFv having a sequence with at least 95% but not 100% sequence identity to the sequence shown in SEQ ID No.9 or having at least one amino acid substitution compared to the sequence shown in SEQ ID No.9, e.g. at least about 96%, 97%, 98%, 99%, 99.2%, 99.6% sequence identity, e.g. at least 2, 3, 4, 5,6 or more amino acid substitutions; and/or the variant sequence is a variant sequence having at least 80% sequence identity to the above-described antibody or antigen-binding fragment thereof, or a variant sequence which retains the biological activity of the corresponding parent sequence, e.g., at least about 96%, 97%, 98%, 99%, e.g., at least 2, 3, 4, 5,6 or more amino acid substitutions, obtained by deletion, substitution, and/or addition of one or more amino acid residues; and/or the derivative is selected from the group consisting of chimeric antibodies derived from whole antibodies, humanized antibodies, fully human antibodies, recombinant antibodies, bispecific antibodies, products comprising modified amino acids, products conjugated to polymers, products comprising radiolabels, products comprising fluorescent labels, products comprising enzymatic labels, products comprising chemiluminescent substances, products comprising paramagnetic labelsA compound (I) is provided. The antigen binding fragment, variant or derivative of the isolated anti-CD 146 antibody has similar or identical properties as the isolated anti-CD 146 antibody.

In some embodiments, the heavy chain amino acid sequence of the anti-CD 146 antibody is selected from SEQ ID nos. 17, 19, 21, 23, 25, 27, 29, or 31 and the light chain amino acid sequence is selected from SEQ ID nos. 18, 20, 22, 24, 26, 28, 30, or 32. In a further embodiment, the anti-CD 146 antibody is an antibody consisting of the following combination of sequences: SEQ ID Nos. 17 and 18, SEQ ID Nos. 19 and 20, SEQ ID Nos. 21 and 22, SEQ ID Nos. 23 and 24, SEQ ID Nos. 25 and 26, SEQ ID Nos. 27 and 28, SEQ ID Nos. 29 and 30, or SEQ ID Nos. 31 and 32. In still further embodiments, the anti-CD 146 antibody is an antibody consisting of the following combination of sequences: SEQ ID Nos. 17 and 18, SEQ ID Nos. 27 and 28, SEQ ID Nos. 29 and 30, or SEQ ID Nos. 31 and 32. In one embodiment, the anti-CD 146 antibody is an antibody consisting of the sequences shown in SEQ ID nos. 17 and 18.

A second aspect of the invention relates to a nucleotide sequence encoding the isolated anti-CD 146 antibody, antigen-binding fragment thereof, variant thereof, or derivative thereof as described above in the first aspect.

A third aspect of the present invention relates to a recombinant vector comprising the coding nucleotide sequence of the second aspect described above.

The fourth aspect of the invention relates to a cell comprising the coding nucleotide sequence of the second aspect or the recombinant vector of the third aspect.

A fifth aspect of the invention relates to a composition comprising the isolated anti-CD 146 antibody, antigen-binding fragment thereof, variant thereof or derivative thereof of the first aspect, and/or the coding nucleotide sequence of the second aspect, and/or the recombinant vector of the third aspect, and/or the cell of the fourth aspect.

A sixth aspect of the invention relates to a method of producing the isolated anti-CD 146 antibody, antigen-binding fragment thereof, variant thereof or derivative thereof of the first aspect, comprising the steps of: expressing the cell of the fourth aspect under culture conditions suitable for expression of the anti-CD 146 antibody, antigen binding fragment thereof, variant thereof or derivative thereof, and optionally isolating and purifying the resulting product.

A seventh aspect of the invention relates to the use of an isolated anti-CD 146 antibody, an antigen-binding fragment thereof, a variant thereof or a derivative thereof of the first aspect, an encoding nucleotide sequence of the second aspect, a recombinant vector of the third aspect, a cell of the fourth aspect and/or a composition according to the fifth aspect in the manufacture of a medicament for interfering with angiogenesis and development.

An eighth aspect of the invention relates to the use of an isolated anti-CD 146 antibody, an antigen-binding fragment thereof, a variant thereof or a derivative thereof according to the first aspect, a coding nucleotide sequence according to the second aspect, a recombinant vector according to the third aspect, a cell according to the fourth aspect and/or a composition according to the fifth aspect for the manufacture of a medicament for the treatment of a tumor and/or a vascular-related disease and/or an inflammatory disease in a subject.

A ninth aspect of the invention relates to the use of an isolated anti-CD 146 antibody, an antigen-binding fragment thereof, a variant thereof or a derivative thereof of the first aspect, an encoding nucleotide sequence of the second aspect, a recombinant vector of the third aspect, a cell of the fourth aspect and/or a composition according to the fifth aspect in the manufacture of a reagent for diagnosing a tumor and/or a vascular-related disease and/or an inflammatory disease in a subject.

A tenth aspect of the invention relates to a method of interfering with angiogenesis and development, comprising the steps of: administering the isolated anti-CD 146 antibody, antigen-binding fragment thereof, variant thereof or derivative thereof of the first aspect, the coding nucleotide sequence of the second aspect, the recombinant vector of the third aspect, the cell of the fourth aspect and/or the composition according to the fifth aspect to a subject in need thereof, preferably the subject is a mammal or a human.

An eleventh aspect of the invention relates to a method of treating a tumor and/or a vascular-related disease and/or an inflammatory disease in a subject, comprising the steps of: administering the isolated anti-CD 146 antibody, antigen-binding fragment thereof, variant thereof or derivative thereof of the first aspect, the coding nucleotide sequence of the second aspect, the recombinant vector of the third aspect, the cell of the fourth aspect and/or the composition according to the fifth aspect to a subject in need thereof, preferably the subject is a mammal or a human.

A twelfth aspect of the present invention relates to a method of diagnosing a tumor and/or a vascular-related disease and/or an inflammatory disease in a subject, comprising the steps of: contacting the isolated anti-CD 146 antibody, antigen-binding fragment thereof, variant thereof or derivative thereof of the first aspect, the coding nucleotide sequence of the second aspect, the recombinant vector of the third aspect, the cell of the fourth aspect and/or the composition according to the fifth aspect with an ex vivo sample of a subject in need thereof.

A thirteenth aspect of the invention relates to an isolated anti-CD 146 antibody, an antigen-binding fragment thereof, a variant thereof or a derivative thereof according to the first aspect, a coding nucleotide sequence according to the second aspect, a recombinant vector according to the third aspect, a cell according to the fourth aspect and/or a composition according to the fifth aspect for use in interfering with angiogenesis and progression, and/or in treating a tumor and/or a vascular-related disease and/or an inflammatory disease in a subject, and/or in diagnosing a tumor and/or a vascular-related disease and/or an inflammatory disease in a subject.

In some embodiments, the tumor in the above aspects is a benign or malignant tumor, preferably the tumor is selected from melanoma, pancreatic cancer, colorectal cancer, breast cancer, lung cancer, nasopharyngeal cancer, liver cancer, gastric cancer, esophageal cancer, breast cancer, kidney cancer, laryngeal cancer, gallbladder cancer, bladder cancer, prostate cancer, cervical cancer, breast cancer, ovarian cancer, uterine cancer, head and neck cancer, skin cancer, thyroid cancer, tongue cancer, thymus cancer, cystic brain tumor, glioma, lymphoma, preferably selected from melanoma, pancreatic cancer, colorectal cancer, and breast cancer; and/or the vascular-related disease and/or the inflammatory disease is selected from age-related macular degeneration, proliferative diabetic retinopathy, retinopathy of prematurity, glaucoma, macular edema, choroidal neovascular disease, retinal vein occlusion, colorectal inflammation, multiple sclerosis, atherosclerosis, arteriolar sclerosis, systemic vasculitis, gastritis, colitis, pancreatitis, arthritis, diabetic inflammation, inflammatory bowel disease, nephritis, hepatitis, systemic lupus erythematosus, scleroderma, dermatomyositis, thyroid autoimmune disease, alzheimer's disease, parkinson's disease or amyotrophic lateral sclerosis, inflammatory central nervous system disease, diabetes, foot ulcer, pulmonary hypertension, ischemic cardiomyopathy, ischemic brain disease, heart failure and acute hindlimb ischemia, preferably, selected from the group consisting of age related macular degeneration, proliferative diabetic retinopathy, colorectal inflammation, multiple sclerosis, and atherosclerosis.

Compared with the existing antibody, the antibody or the antigen binding fragment thereof has the advantages of broad spectrum, specificity, high efficiency, low toxicity and difficult generation of drug resistance, acts on tumor vascular endothelial cells, specifically recognizes the CD146 expressed by the neovascular endothelial cells, the tumor cells, the perivascular cells, the lymphocytes and the like with high affinity, has the capacity of obviously inhibiting the migration and the vascularization of the endothelial cells, specifically and efficiently inhibits the growth and the metastasis of tumors, can be used for developing broad-spectrum, high-efficiency and low-toxicity antibody medicaments, and is used for treating and early diagnosing tumors, inflammatory diseases and neovascular diseases. Although the specific target molecules of different types of solid tumors differ, the structures of new blood vessels on which they occur and develop are roughly the same. Tumor growth or metastasis is inhibited to varying degrees when tumor blood vessels are completely or incompletely destroyed by the antibodies or antigen-binding fragments thereof of the invention. Meanwhile, the antibody or the antigen binding fragment thereof directly acts on endothelial cells of tumor blood vessels, and does not need to penetrate into the tumor deeply, so that the problems of drug permeation and distribution caused by local high pressure of tumor tissues are avoided. Antibodies can be divided into five classes according to the structural characteristics, most antibodies only have recognition function, and a few antibodies not only have recognition function but also have killing function. The monoclonal antibody of the present invention has a certain ability to induce antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and can directly kill tumor cells. Compared with the AA98 antibody, the invention modifies the related sequences of the original AA98 antibody and the antigen binding fragment thereof, obviously improves the affinity of the antibody and the antigen CD146, and can more effectively inhibit the migration of triple negative breast cancer cells and endothelial cells and the tube forming cavity of the endothelial cells. In addition, the antibody can bind to melanoma cells and liver cancer cells.

Drawings

FIG. 1, Panel A, shows a crystal structure overview of the Fab fragment complex of CD146 and AA98 antibodies. Above the picture are the fourth and fifth domains from CD146, labeled D4 and D5. Immediately adjacent to CD 146D 4-D5 is the V of the light chain of AA98 Fab_LV of Domains and heavy chains_HA domain, the two domains comprising an antigen binding region, each having three CDR loops. Below these two domains is the C of the light chain_LDomains and heavy chain C_H1A domain. Panel B shows a detailed view of the antigen/antibody binding surface in the crystal structure of the Fab fragment complex of the CD146 and AA98 antibodies. The orientation of this figure is the same as the overall crystal structure, and is a partial detail view of the CD 146D 4-D5 and AA98 Fab interaction region. The upper part is the CD 146D 4-D5 part (light grey), the lower left is the light chain of AA98 Fab (middle grey) and the lower right is the heavy chain of AA98 Fab (dark grey), indicated as a ribbon. The interacting residues in the interacting region are shown in a ball and stick model, and the residues of the mutated target are shown in a black ball and stick model, and are S32, I31 and L54 for the light chain and I28, I57 and Y59 for the heavy chain, respectively.

FIG. 2: the change in aggregation state on superdex 75 molecular sieves after binding of the CD 146D 4-D5 protein (dotted line) to AA98 Fab (broken line) (solid line) is shown.

FIG. 3: the maternal AA98 antibody is shown to have an affinity of 1.12X 10 for CD146^-9M (Panel A), the affinity of the engineered antibody H13-112 was 1.44X 10^-10M (panel B), the affinity of the engineered antibody H13-112 was increased nearly 10-fold.

FIG. 4: FACS results for the H13-112 antibody and the human venous endothelial cell line HUVEC (Panel A), the melanoma cell line A375 (Panel B), the liver cancer cell line HepG2 (Panel C), the colon cancer cell line HT-29 (Panel D), the CD146 positive triple negative breast cancer cell line MDA-MB-231 (Panel E), and the CD146 negative/low expressing breast cancer cell line MCF-7 (Panel F) and the H13-112 mutant antibody are shown.

FIG. 5: the migratory capacity of HUVEC cells under the action of antibodies was investigated using the Transwell system. The antibodies used were AA 98-based mutant antibodies such as mIgG, original AA98 antibody, H13-112, H14-114, 106-112 and 106-114, respectively, as negative controls. Panel a shows that the antibody AA98 and AA98 mutants were effective in inhibiting endothelial cell migration compared to control mIgG. And B is the statistical result of the graph A. Compared with AA98, H13-112 has stronger inhibition effect, the inhibition rate is 86%, and the inhibition rate of the original AA98 is 49%.

FIG. 6: the inhibition of the migration ability of the H13-112 antibody against the triple negative breast cancer cell line MDA-MB-231 was investigated using the Transwell system. The antibodies used were mIgG, original AA98 antibody and H13-112 mutant antibody, respectively, as negative controls. Panel a shows that antibodies AA98 and AA98 mutant H13-112 are effective in inhibiting migration of endothelial cells compared to control mIgG. And B is the statistical result of the graph A. Compared with AA98, H13-112 has stronger inhibition effect, the inhibition rate is 82%, and the inhibition rate of the original AA98 is 57%.

FIG. 7 shows that the AA 98-based mutant antibodies H13-112, H14-114, 106-112 and 106-114 can effectively inhibit the endothelial cell lumen formation compared with the negative control mIgG and the control AA98 antibody. Graph A is the actually observed result, and graph B is the statistical result of graph A. Compared with AA98, mutant H3-112 has stronger inhibiting effect on endothelial cell lumen formation.

FIG. 8: it is shown that both antibodies AA98 and H13-112 are effective in inhibiting tumor growth compared to the control mIgG. Compared with AA98, H13-112 has stronger inhibition effect, the inhibition rate reaches 74%, and the original AA98 inhibition rate is only 46%.

Detailed Description

Definition of

The term "CD 146" refers to a melanoma cell adhesion molecule, which may be any CD146 molecule of mammalian origin, in some embodiments, a CD146 molecule of primate origin, and in some embodiments, a CD146 molecule of human origin.

The term "antibody" refers to an immunoglobulin molecule or a fragment of an immunoglobulin molecule according to some embodiments of the invention that has the ability to bind to an epitope of an antigen. Naturally occurring antibodies typically comprise tetramers, which are generally composed of at least two heavy (H) chains and at least two light (L) chains. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region, which is typically composed of 3 domains (CH1, CH2, and CH 3). The heavy chain may be of any isotype, including IgG (IgG1, IgG2, IgG3 and IgG4 subtypes), IgA (IgA1 and IgA2 subtypes), IgM, and IgE. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL). Light chains include kappa and lambda chains. The heavy and light chain variable regions are typically responsible for antigen recognition, while the heavy and light chain constant domains may mediate binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). The VH and VL regions may be further subdivided into hypervariable regions known as "complementarity determining regions" interspersed with more conserved regions known as "framework regions" (FR). Each VH and VL is composed of three CDR domains and four FR domains, arranged from amino-terminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. The heavy and light chain variable regions contain binding regions that interact with antigen.

Meanwhile, unless otherwise indicated, the term "antibody" also includes antibody-like polypeptides, such as chimeric antibodies provided by any known technique (e.g., enzymatic cleavage, peptide synthesis, and recombinant techniques), humanized antibodies, recombinant antibodies, human antibodies produced from transgenic non-human animals, antibodies selected from libraries produced using enrichment techniques available to those skilled in the art, and antibody fragments (antigen-binding fragments) that retain the ability to bind to an antigen. The antibodies so produced may be of any isotype.

The term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of single molecule composition, and refers to antibodies obtained from a population of substantially homogeneous antibodies, i.e., a population comprising individual antibodies that are identical except for possible naturally occurring mutations that may be present in minor amounts. The term "polyclonal antibody" refers to preparations comprising different antibodies directed against different determinants ("epitopes"). Conventional monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope. In certain embodiments, a monoclonal antibody may be composed of more than one Fab domain, thereby increasing specificity for more than one target. The term "monoclonal antibody" or "monoclonal antibody composition" is not limited to any particular method of production (e.g., recombinant, transgenic, hybridoma, etc.).

Monoclonal antibodies herein include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical to or homologous to corresponding sequences of an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to corresponding sequences of antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. The present invention provides variable region antigen binding sequences from human antibodies. Accordingly, chimeric antibodies of primary interest herein include antibodies having one or more human antigen binding sequences (e.g., CDRs) and comprising one or more sequences from a non-human antibody, such as FR or C region sequences. In addition, the chimeric antibodies described herein are antibodies that comprise a human variable region antigen binding sequence of one antibody class or subclass and another sequence, such as an FR or C region sequence, from another antibody class or subclass.

The term "variable" refers to the wide variation in sequence of certain fragments of the variable (V) region between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the variable region of 110 amino acids. In contrast, the V region consists of a relatively invariant fragment called the Framework Region (FR) of 15-30 amino acids, divided by a short region of extreme variability called the "hypervariable region" each 9-12 amino acids in length. The variable regions of native heavy and light chains each comprise four FRs, mostly in a β -sheet configuration, connected by 3 hypervariable regions, which form loops connecting, and in some cases forming part of, the β -sheet structure. The hypervariable regions in each chain are closely linked by the FRs and, together with hypervariable regions from the other chains, contribute to the formation of the antigen-binding site of the antibody.

The term "hypervariable region" herein refers to the amino acid residues of an antibody which are responsible for antigen binding. The hypervariable region typically comprises amino acid residues from a "complementarity determining region" ("CDR").

"Fv" is the smallest antibody fragment that contains the entire antigen recognition and antigen binding site. The fragment comprises a dimer of one heavy chain variable region domain and one light chain variable region domain in tight, non-covalent association. From the folding of these two domains, 6 hypervariable loops (3 loops formed from each of the H and L chains) are generated, which provide amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable region (or half of an Fv, which contains only 3 antigen-specific CDRs) has the ability to recognize and bind antigen, albeit with a lower affinity than the entire binding site.

The term "antigen-binding fragment of an antibody" refers to a fragment, portion, region, or domain of an antibody (e.g., obtainable via cleavage, recombination, synthesis, etc.) that is capable of binding to an epitope, and thus the terms "antigen-binding" and "epitope-binding fragment of an antibody" are the same as "epitope-binding fragment of an antibody". Antigen-binding fragments may contain 1, 2, 3, 4, 5 or all 6 CDR domains of such antibodies and, although capable of binding to the epitope, may exhibit different specificities, affinities or selectivities. Preferably, the antigen binding fragment contains all 6 CDR domains of the antibody. An antigen-binding fragment of an antibody can be a portion of or comprise a single polypeptide chain (e.g., an scFv), or can be two or more polypeptide chains (each having an amino-terminus and a carboxyl-terminus) (e.g., a diabody, a Fab fragment, a Fab)₂Fragments, etc.) or comprise two or more polypeptide chains.

Domain antibodies (dabs) can be produced in a fully human form, being the smallest known antigen-binding fragment of an antibody, ranging from about 11kDa to about 15 kDa. dAbs are the variable regions of the heavy and light chains of immunoglobulins (VH and VL, respectively). It is highly expressed in microbial cell cultures, exhibits good biophysical properties including, for example, but not limited to, solubility and temperature stability, and is well suited for screening and affinity maturation in vitro screening systems such as phage display. dabs are biologically active as monomers and, due to their small size and inherent stability, can form larger molecules to produce drugs with extended serum half-life or other pharmacological activity.

Antibody fragments exhibiting antigen binding capability can be obtained, for example, by protease cleavage of intact antibodies. More preferably, although the two domains of the Fv fragment, VL and VH, are naturally encoded by separate genes or polynucleotides encoding such gene sequences (e.g., which encode cDNA), the two domains can be joined using recombinant methods by a flexible linker that enables the two domains to be a single protein chain in which the VL and VH regions combine to form a monovalent antigen binding molecule (referred to as single chain Fv scFv(s); see, e.g., Bird et al, 1988, Science,242: 423-scion 426; and Huston et al, 1988, Proc. Natl. Acad. Sci. (U.S.A.)85: 5879-5883). Alternatively, bispecific antibodies, diabodies, or similar molecules (in which two such polypeptide chains are joined together to form a bivalent antigen-binding molecule) can be formed by employing a flexible linker that is ultrashort (e.g., less than about 9 residues) such that the VL and VH regions of a single polypeptide chain cannot be joined together (see, e.g., PNAS USA 90(14),6444-8(1993)) for a description of diabodies.

Fv and scFv are the only species that have an intact binding site and lack constant regions. Thus, they are suitable for reducing non-specific binding in vivo use. scFv fusion proteins were constructed to generate fusions of effector proteins at either the amino-or carboxy-terminus of the scFv. Antibody fragments may also be "linear antibodies". Such linear antibody fragments may be monospecific or bispecific.

Examples of antigen binding fragments encompassed by the present invention include (i) Fab' or Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH1 domains, or monovalent antibodies as described in WO 2007059782; (ii) a F (ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at a hinge domain; (iii) an Fd fragment consisting essentially of a VH region and a CHl domain; (iv) an Fv fragment consisting essentially of a VL region and a VH region; (v) dAb fragments (Ward et al, Nature, 341, 544-546(1989)) which essentially consist of a VH domain and are also referred to as domain antibodies (Holt et al, Trends Biotechnol., 2i (ll): 484-90); (vi) camel or nanobody (Revets et al, Expert Opin Biol ther., 5(l):111-24) and (vii) isolated Complementarity Determining Regions (CDRs).

The term "human antibody" ("humAb" or "humAb") includes antibodies having variable and constant domains derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation during gene rearrangement or in vivo).

The antibodies and antigen binding fragments thereof of the invention are preferably humanized or fully human, e.g., for the mouse antibody AA98, it is "humanized". The term "humanized" refers to molecules having an antigen binding site derived from an immunoglobulin from a non-human species and a residual immunoglobulin structure based on the structure and/or sequence of a human immunoglobulin, typically prepared using recombinant techniques. The antigen binding site may comprise fully non-human antibody variable regions fused to human constant regions, or comprise only the Complementarity Determining Regions (CDRs) of such variable regions linked to appropriate human framework regions of the human variable regions. The framework residues of such humanized molecules may be wild-type (e.g., fully human), or they may be modified to include one or more amino acid substitutions found in human antibodies that do not serve as the basis for humanization in their sequence. Humanization reduces or eliminates the possibility that the constant region of the molecule will act as an immunogen In humans (LoBuglio, A.F. et al, (1989), Mouse/Human chiral Monoclonal Antibody In Man: kinetic And Immune Response, Proc. Natl. Acad. Sci. (U.S.A.), 86: 4220-.

Another approach has focused not only on providing constant domains derived from humans, but also on modifying the variable regions so as to modify them as close to human form as possible. It is known that the variable regions of both heavy and light chains contain three Complementarity Determining Regions (CDRs) that are specific for the respective antigens and that alter and determine binding capacity, flanked by four Framework Regions (FRs) that are relatively conserved in a given species and that provide a scaffold for the CDRs. When preparing a non-human antibody against a particular antigen, the variable region can be "engineered" or "humanized" by ligating CDRs derived from the non-human antibody to the FRs present in the human antibody to be modified, see, e.g., Sato, k, et al, (1993), Cancer Res,53: 851-; riechmann, L. et al, (1988), rehaping Human Antibodies for Therapy, Nature,332: 323-; verhoeyen, M. et al, (1988), rehaping Human Antibodies: Grafting An antibody Activity, Science,239: 1534-1536; kettleborough, C.A. et al, (1991), Humanization Of A Mouse Monoclonal Antibody By CDR-Grating, The Import Of Framework resources On Loop conversion, Protein Engineering,4: 773-; maeda, H.et al, (1991), restriction of modified Human Antibodies With HIV-neutralling Activity, Human Antibodies hybrids, 2: 124-; gorman, S.D. et al, (1991), Reshaping A Therapeutic CD4 Antibody, Proc.Natl.Acad.Sci. (U.S.A.),88: 4181-; tempest, P.R. et al, (1991), rehaping A Human Monoclonal Antibody To Inhibit Human respiratory synthetic Virus Infection in vivo), Bio/Technology,9: 266-; co, M.S. et al, (1991), manipulated Antibodies For antibody Therapy, PNAS,88: 2869-2873; carter, P. et al, (1992), Humanization Of An Anti-p185her2Anti For Human Cancer Therapy), PNAS,89: 4285-4289; and, family, M.S. et al, (1992), Chimeric And human Antibodies With Specificity For The CD33 antibody), J.Immunol.,148: 1149-. In some embodiments, the humanized antibody retains all 6 CDR sequences (e.g., a humanized mouse antibody that contains all six CDRs from a mouse antibody). In other embodiments, the humanized antibody has one or more CDRs (one, two, three, four, five, six). The ability to humanize antigens is well known (see, e.g., U.S. Pat. Nos. 5,225,539; 5,530,101; 5,585,089; 5,859,205; 6,407,213; 6,881,557).

Variant antibodies are also included within the scope of the invention. Thus, variations of the sequences recited in the application are also included within the scope of the invention. Other variants of antibody sequences with improved affinity may be obtained by using methods known in the art and are also included within the scope of the present invention. For example, amino acid substitutions can be used to obtain antibodies with further improved affinity. Alternatively, codon optimization of the nucleotide sequence can be used to improve the translation efficiency of the expression system in antibody production.

Such variant antibody sequences have 70% or more (e.g., 80%, 85%, 90%, 95%, 97%, 98%, 99% or more) sequence homology to the sequences recited in the application. Such sequence homology is calculated over the full length of the reference sequence (i.e., the sequence listed in the application).

The numbering of amino acid residues in the regions of the invention is based on

the international ImMunoGeneTics information

Or Kabat, e.a., Wu, t.t., Perry, h.m., Gottesmann, K.S.&Foeller, C., (1991), Sequences of Proteins of Immunological Interest, 5 th edition, NIH Pub. No. 91-3242, U.S. department of health and public service; chothia, C.&Lesk, A.M. (1987), bacterial structures For The hyper domains Of immunology, J.mol.biol.,196, 901-917.

An antibody or antigen-binding fragment thereof "specifically" binds a region of another molecule (i.e., an epitope) means that it reacts or binds to the epitope more frequently, more rapidly, with a longer duration, and/or with greater affinity or avidity than another epitope. In some embodiments, the antibodies or antigen binding fragments thereof of the invention bind to their target (CD146) with at least 10-fold stronger affinity relative to the AA98 antibody; preferably at least 50 times stronger and more preferably at least 100 times stronger. Preferably, the antibody or antigen-binding fragment thereof binds under physiological conditions (e.g., in vivo). Thus, specifically binding to CD146 refers to the ability of the antibody or antigen-binding fragment thereof to bind to CD146 with the specificity described above and/or under such conditions. Methods suitable for determining such binding are known in the art.

The term "binding" in the context of an antibody binding to a specified antigen generally means to correspond to about 10^-9M or less, that is at least 10-fold lower, such as at least 100-fold lower, at least 1,000-fold lower than the affinity of the antibody for binding to a non-specific antigen (e.g., BSA, casein) other than the antigen of interest or a closely related antigen.

As used herein, the term "kd" (sec-1 or 1/s) refers to the off-rate constant for a particular antibody-antigen interaction. Said value is also called koff value.

As used herein, the term "ka" (M-1x sec-1 or 1/Msec) refers to the binding rate constant for a particular antibody-antigen interaction.

As used herein, the term "KD" (M) refers to the dissociation equilibrium constant for a particular antibody-antigen interaction and is obtained by dividing KD by ka.

As used herein, the term "KA" (M-1 or 1/M) refers to the binding equilibrium constant for a particular antibody-antigen interaction and is obtained by dividing KA by kd.

In some embodiments, the invention relates to an antibody or antigen-binding fragment thereof that exhibits one or more of the following properties:

(i) in the range of 0.1-8X 10^-10M (e.g. 0.5-5X 10)^-10M or 0.8-2.5X 10^-10M) binding affinity (K) to CD146_D)；

(ii) Greater than 40% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more, e.g., 40-99%, 50-99%, 60-99%, 70-99%, 80-99%, 90-99%, 85-98%, 85-95%, 90-95%, etc.) of endothelial cells;

(iii) greater than 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more, e.g., 60-99%, 65-99%, 70-99%, 80-99%, 80-95%, 80-92%, 82-90%, etc.) of the ability to inhibit migration of breast cancer cells and endothelial cells.

The term "greater than 40% ability to inhibit lumping of endothelial cells" refers to the reduced luminal length of the antibody of the invention as a percentage of the AA98 group as compared to the AA 98-treated group, e.g., the H13-112-treated group. In some embodiments, the ability to inhibit the lumping of endothelial cells is performed using a method as shown in example 7 below, or a similar method.

The term "an ability to inhibit endothelial cell migration of greater than 60%" means that the antibody of the present invention, e.g., H13-112 treated group, reduced the number of cells migrating as a percentage of the AA98 group compared to the AA98 treated group. In some embodiments, the ability to inhibit endothelial cell migration is performed using a method as set forth in example 5 below or a similar method, and the ability to inhibit breast cancer cell migration is performed using a method as set forth in example 6 below or a similar method.

In some embodiments, an antibody or antigen-binding fragment thereof of the invention requires that only a portion of the CDRs (i.e., a subset of CDR residues required for binding, referred to as SDR) remain bound in the humanized antibody. Epitopes can be identified in Kabat CDR Regions located outside The Chothia Hypervariable loop (see Kabat et al, (1992), Sequences Of Proteins Of Immunological Interest, National Institutes Of Health, published No. 91-3242; Chothia, C.et al, (1987), cancer Structures Of Immunological Interest in The Biogel Of biological Interest, 917. CDR 196) and related epitopes are identified based on previous studies (e.g., residues H60-H65 in CDR H2 are not normally required). In such humanized antibodies, at a position where one or more donor CDR residues are absent or the entire donor CDR is omitted, the amino acid occupying that position may be the amino acid occupying the corresponding position (by Kabat numbering) in the acceptor antibody sequence. Such substitutions are potentially advantageous in reducing the number of mouse amino acids in the humanized antibody and thus reducing potential immunogenicity. However, substitution may also cause a change in affinity, and preferably a significant decrease in affinity is avoided. The substitution position within the CDR and the amino acid to be substituted may also be selected empirically.

Alternative functional CDR sequences can be systematically identified by taking advantage of the fact that Single Amino Acid changes in CDR residues can lead to loss of functional binding (Rudikoff, S. et al, (1982), Single Amino Acid catalysis alteration-binding Specificity, Proc. Natl. Acad. Sci. (USA))79(6): 1979-. In one preferred method for obtaining such variant CDRs, polynucleotides encoding the CDRs are mutagenized (e.g., via random mutagenesis or by site-directed methods) to generate CDRs having substituted amino acid residues. By comparing the identity of the relevant residues in the original (functional) CDR sequence with the identity of the substituted (non-functional) variant CDR sequence, the substituted blosum62.iij substitution score can be identified. The BLOSUM system provides Amino acid substitution matrices created by analyzing sequence databases for use in confident alignments (Eddy, S.R., (2004), Where The BLOSUM62 Alignment Score Matrix From; karlin, S. et al, (1990), Methods For assembling The Statistical signalling Of Molecular Sequence Features By Using General screening Schemes), PNAS,87: 2264-; altschul, S.F. (1991), Amino Acid catalysis materials From An Information therapeutic utility, J.Mol.biol.,219, 555-. Currently, the most advanced BLOSUM database is the BLOSUM62 database (BLOSUM62. iij). Table 1 presents the blosum62.iij substitution scores (the higher the score the more conservative the substitution is and thus more likely the substitution will not affect function). For example, if an antigen-binding fragment containing the resulting CDR is unable to bind to CD146, the blosum62.iij substitution score is deemed to be insufficiently conserved, and a new candidate substitution with a higher substitution score is selected and generated. Thus, for example, if the original residue is glutamic acid (E) and the non-functional substituted residue is histidine (H), the blosum62. ij substitution score will be 0, and more conservative changes (e.g., to aspartic acid, asparagine, glutamine or lysine) are preferred.

The present invention thus contemplates the use of random mutagenesis for the identification of improved CDRs. In the context of the present invention, a conservative substitution may be defined by a substitution within a class of amino acids that is reflected in one or more of the following three tables:

conservative substituted amino acid residue classes:

alternative conservative amino acid residue substitution classes:

physical and functional substitution classifications of amino acid residues:

more conservative groupings of substitutions include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

In some embodiments, the hydrophilic amino acid is selected from Arg, Asn, Asp, gin, Glu, His, Tyr, and Lys.

Additional amino acid groupings can also be made using the principles described in, for example, Creighton, (1984), Proteins: Structure and Molecular Properties, W.H.Freeman and Company).

Thus, the sequences of the CDR variants of the encompassed antibody or antigen-binding fragment thereof can differ from the sequences of the CDRs of the parent antibody by substitutions; for example 4, 3, 2 or 1 amino acid residue. According to an embodiment of the invention, amino acids in the CDR regions may be substituted with conservative substitutions, as defined in the above 3 tables.

"homology" or "sequence identity" refers to the percentage of residues of a variant polynucleotide or polypeptide sequence that are identical to a non-variant sequence after alignment of the sequences and the introduction of gaps. In particular embodiments, the polynucleotide and polypeptide variants have at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% polynucleotide or polypeptide homology to a polynucleotide or polypeptide described herein.

Such variant polypeptide sequences have 70% or more (i.e., 80%, 85%, 90%, 95%, 97%, 98%, 99% or more) sequence identity to the sequences recited in the application. In other embodiments, the invention provides polypeptide fragments comprising contiguous stretches of various lengths of the amino acid sequences disclosed herein. For example, where applicable, peptide sequences provided herein comprise at least about 5,10, 15, 20, 30, 40, 50, 75, 100, 150 or more contiguous peptides of one or more of the sequences disclosed herein and all intermediate lengths therebetween.

The term "treating" refers to ameliorating, slowing, attenuating or reversing the progression or severity of a disease or condition, or ameliorating, slowing, attenuating or reversing one or more symptoms or side effects of such a disease or condition. In the present invention, "treatment" also refers to a method for obtaining a beneficial or desired clinical result, wherein a "beneficial or desired clinical result" includes, but is not limited to, alleviation of symptoms, diminishment of a condition or extent of a disease, stabilized (i.e., not worsening) disease or condition state, delay or slowing of progression of a disease or condition state, amelioration or palliation of a disease or condition state, and remission of a disease or condition, whether partial or total, detectable or undetectable.

The term "effective amount" when applied to an antibody or antigen-binding fragment thereof of the present invention refers to an amount sufficient to achieve the desired biological effect or desired therapeutic result (including but not limited to clinical results) at the desired dosage and for the desired period of time. The term "therapeutically effective amount" when applied to an antibody or antigen-binding fragment thereof of the present invention means an amount of the antibody or antigen-binding fragment thereof sufficient to ameliorate, reduce, stabilize, reverse, slow, attenuate or delay the progression of the condition or disease state, or the progression of symptoms of the condition or disease. In some embodiments, the methods of the invention provide for the administration of a composition of an antibody or antigen-binding fragment thereof and another compound. In such cases, an "effective amount" is an amount of the composition sufficient to cause the desired biological effect.

The therapeutically effective amount of an anti-CD 146 antibody (e.g., H13-112) or antigen binding fragment thereof of the present invention may vary depending on the following factors: the disease state, age, sex, and weight of the individual, and the ability of the anti-CD 146 antibody or antigen-binding fragment thereof to elicit a desired response in the individual. A therapeutically effective amount is also an amount by which the beneficial therapeutic effect of the antibody or antigen-binding fragment thereof exceeds any toxic or detrimental effect thereof.

The antibody of the present invention may be a monoclonal antibody produced by recombinant DNA.

The antibodies of the invention may be of any isotype. The choice of isotype is usually determined by the desired effector function (e.g., ADCC induction). Exemplary isotypes are IgG1, IgG2, IgG3, and IgG 4. Either of the human light chain constant domains κ or λ may be used. The class of the anti-CD 146 antibody of the present invention can be switched by known methods, if necessary. For example, an antibody of the invention that is initially an IgG may be class-switched to an IgM antibody of the invention. In addition, class switching techniques can be used to convert one IgG subclass to another, such as from IgGl to IgG 2. Thus, the effector function of the antibodies of the invention can be changed by isotype switching to, for example, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE or IgM antibodies for various therapeutic uses. In some embodiments, the antibody of the invention is an IgG 2antibody, e.g., IgG2 a. An antibody belongs to a particular isotype if its amino acid sequence is largely homologous to that isotype relative to other isotypes.

In some embodiments, the antibody of the invention is a full length antibody, preferably an IgG antibody. In other embodiments, the antibodies of the invention are antibody antigen-binding fragments or single chain antibodies.

In some embodiments, the anti-CD 146 antibody is a monovalent antibody, preferably a monovalent antibody with a hinge region deletion as described in WO2007059782 (which is incorporated herein by reference in its entirety). Thus, in some embodiments, the antibody is a monovalent antibody, wherein the anti-CD 146 antibody is constructed by: i) providing a nucleic acid construct encoding a light chain of a monovalent antibody, said construct comprising a nucleotide sequence encoding the VL region of a selected antigen-specific anti-CD 146 antibody and a nucleotide sequence encoding the constant CL region of Ig, wherein said nucleotide sequence encoding the VL region of a selected antigen-specific antibody and said nucleotide sequence encoding the CL region of Ig are operatively linked together, and wherein, in the case of the IgG1 subtype, the nucleotide sequence encoding the CL region has been modified such that the CL region does not contain any amino acids capable of forming disulfide or covalent bonds with other peptides comprising the identical amino acid sequence of the CL region in the presence of polyclonal human IgG or when administered to an animal or human; ii) providing a nucleic acid construct encoding a heavy chain of a monovalent antibody, said construct comprising a nucleotide sequence encoding a VH region of a selected antigen-specific antibody and a nucleotide sequence encoding a constant CH region of human Ig, wherein the nucleotide sequence encoding the CH region has been modified such that the region corresponding to the hinge region and other regions of the CH region (as required by the Ig subtype), such as the CH3 region, does not comprise any amino acid residues involved in the formation of disulfide bonds or covalent or stable non-covalent inter-heavy chain bonds with other peptides comprising identical amino acid sequences of the CH region of human Ig in the presence of polyclonal human IgG or when administered to an animal human, wherein said nucleotide sequence encoding a VH region of a selected antigen-specific antibody and said nucleotide sequence encoding a CH region of said Ig are operably linked together; iii) providing a cellular expression system for the production of monovalent antibodies; iv) producing said monovalent antibody by co-expressing the nucleic acid constructs of (i) and (ii) in a cell of the cellular expression system of (iii).

Similarly, in some embodiments, the anti-CD 146 antibody is a monovalent antibody comprising:

(i) the variable region of the antibody of the invention or the antigen-binding portion of said domain as described herein, and

(ii) the CH region of an immunoglobulin or a domain thereof comprising domains CH2 and CH3, wherein the CH region or domain thereof has been modified such that the domain corresponding to the hinge region and, if the immunoglobulin is not of the IgG4 subtype, other domains of the CH region (e.g., the CH3 domain) does not comprise any amino acid residues capable of forming disulfide bonds with the same CH region or other covalent or stable non-covalent inter-heavy chain bonds with the same CH region in the presence of polyclonal human IgG.

In some further embodiments, the heavy chain of the monovalent antibody is modified such that the entire hinge region is deleted.

In further embodiments, the sequence of the monovalent antibody is modified such that it does not contain any acceptor sites for N-linked glycosylation.

The invention also includes "bispecific antibodies" in which the anti-CD 146 binding region (e.g., the CD146 binding region of an anti-CD 146 monoclonal antibody) is part of a bivalent or multivalent bispecific framework that targets more than one epitope (e.g., the second epitope may comprise an epitope of an active transport receptor such that the bispecific antibody will exhibit improved transcytosis across a biological barrier, such as the blood brain barrier). Thus, in further embodiments, a monovalent Fab of an anti-CD 146 antibody can be linked to another Fab or scfv targeting a different protein to produce a bispecific antibody. Bispecific antibodies can have dual functions, such as therapeutic functions conferred by anti-CD 146 binding regions and transport functions that can bind to receptor molecules to enhance transfer across biological barriers, such as the blood-brain barrier.

The antibodies and antigen-binding fragments thereof of the present invention also include single chain antibodies. Single chain antibodies are peptides in which the Fv domains of the heavy and light chains are linked. In some embodiments, the invention provides single chain Fv (scfv), wherein the heavy and light chains in the Fv of the anti-CD 146 antibody of the invention are connected by a flexible peptide linker (typically about 10, 12, 15 or more amino acid residues) into a single peptide chain. Methods for producing such antibodies are described, for example, in US 4,946,778; pluckthun, The Pharmacology of Monoclonal Antibodies, Vol.113, Rosenburg and Moore ed., Springer-Verlag, New York, pages:269-315 (1994); bird et al, Science,242, 423-426 (1988); huston et al, PNAS USA 85, 5879-. Single chain antibodies are monovalent if only a single VH and VL are used; bivalent if two VH and VL are used; or multivalent if more than two VH and VL are used.

Antibodies and antigen-binding fragments thereof of the invention may be modified by inclusion of any "suitable" number of modified amino acids and/or binding to coupling substituents. In this context, "suitable" is generally determined by the ability to at least substantially retain the CD146 selectivity and/or CD146 specificity associated with the non-derivatized parent anti-CD 146 antibody. The inclusion of one or more modified amino acids may be advantageous, for example, in increasing the serum half-life of the polypeptide, reducing the antigenicity of the polypeptide, or increasing the storage stability of the polypeptide. The one or more amino acids are modified, for example, simultaneously with or subsequent to translation during recombinant production (e.g., N-linked glycosylation at the N-X-S/T motif during mammalian cell expression), or by synthetic means. Non-limiting examples of modified amino acids include glycosylated amino acids, sulfated amino acids, prenylated (e.g., farnesylated, geranyl-geranylated) amino acids, acetylated amino acids, acylated amino acids, pegylated amino acids, biotinylated amino acids, carboxylated amino acids, phosphorylated amino acids, and the like. References for amino acid modifications are common in the art, see, e.g., Walker, (1998), Protein Protocols On CD-Rom, Humana Press, Totowa, New Jersey. The modified amino acid may for example be selected from glycosylated amino acids, pegylated amino acids, farnesylated amino acids, acetylated amino acids, biotinylated amino acids, amino acids coupled to a lipid moiety or amino acids coupled to an organic derivatizing agent.

The antibodies and antigen-binding fragments thereof of the present invention may also be chemically modified by covalent coupling to a polymer to increase their circulating half-life. Exemplary polymers and methods for attaching them to peptides are described in, for example, US 4,766,106; US 4,179,337; US 4,495,285 and US 4,609,546. Additional exemplary polymers include polyoxyethylated polyols and polyethylene glycol (PEG) (e.g., PEG having a molecular weight of between about 1,000-40,000D, such as between about 2,000-20,000D, for example, between about 3,000-12,000D).

In some embodiments, the invention provides antibodies and antigen binding fragments thereof comprising one or more radiolabeled amino acids. Radiolabeled anti-CD 146 antibodies may be used for diagnostic and/or therapeutic purposes. Non-limiting examples of such labels include, but are not limited to, bismuth (R), (B), (C), (D) and D) a²¹³Bi), carbon (C: (¹¹C、¹³C、¹⁴C) Chromium (C)⁵¹Cr), cobalt (⁵⁷Co、⁶⁰Co), copper (⁶⁴Cu), dysprosium (¹⁶⁵Dy), erbium (¹⁶⁹Er) fluorine (¹⁸F) Gadolinium (I) and (II)¹⁵³Gd、¹⁵⁹Gd), gallium (⁶⁸Ga、⁶⁷Ga), germanium (⁶⁸Ge), gold (¹⁹⁸Au), holmium (¹⁶⁶Ho), hydrogen (³H) Indium (I) and (II)¹¹¹In、¹¹²In、¹¹³In、¹¹⁵In), iodine (¹²¹I、¹²³I、¹²⁵I、¹³¹I) Iridium (III)¹⁹²Ir), iron (⁵⁹Fe), krypton (^81mKr), lanthanum (¹⁴⁰La), lutetium (¹⁷⁷Lu), manganese (⁵⁴Mn), molybdenum (⁹⁹Mo), nitrogen (¹³N、¹⁵N), oxygen (¹⁵O), palladium (I), palladium (II)¹⁰³Pd), phosphorus (³²P), potassium (⁴²K) Praseodymium (III)¹⁴²Pr), promethium (M)¹⁴⁹Pm), rhenium (¹⁸⁶Re、¹⁸⁸Re), rhodium (II)¹⁰⁵Rh), rubidium (⁸¹Rb、⁸²Rb), ruthenium (⁸²Ru、⁹⁷Ru), samarium (¹⁵³Sm, scandium (⁴⁷Sc), selenium (⁷⁵Se), sodium (²⁴Na), strontium (⁸⁵Sr、⁸⁹Sr、⁹²Sr), sulfur (S: (A)³⁵S), technetium (⁹⁹Tc), thallium (²⁰¹Tl), tin (¹¹³Sn、¹¹⁷Sn), xenon (¹³³Xe), ytterbium (¹⁶⁹Yb、¹⁷⁵Yb、¹⁷⁷Yb), yttrium (b)⁹⁰Y) and zinc (⁶⁵Zn). Methods for preparing radiolabeled amino acid and related peptide derivatives are known in the art (see, e.g., Junghans et al, Cancer chemother and Biotherapy,655 & 686(Version 2, Chafner and Longo, ed. lippincott Raven, (1996)) as well as US 4,681,581, US 4,735,210, US5,101,827, US5,102,990 (US RE35,500), US5,648,471 and US5,697,902). For example, radioisotopes can be coupled by the Chloramine T method (Lindegren, S. et al, (1998), clone-T In High-specificity-Activity radio Of Antibodies Using N-Succinimidyl-3- (trimethylcyanatonyl) Benzoate As Intermediate), Nuclear.Med.biol., 25(7): 659-; kurth, M. et al, (1993), Site-Specific coupling Of A radio coordinated approach To A Monoclonal Antibody Results In incorporated radio activity Localization In Tumor, J.Med.chem.,36(9): 1255-arrangement 1261; rea, D.W. et al, (1990), Site-specific radioactive antigens for targeting tumors, Cancer Res.,50 (super.3): 857s-861 s).

The present invention also provides anti-CD 146 antibodies and antigen-binding fragments thereof labeled with the following detection markers: fluorescent labels (such as rare earth chelates (e.g., europium chelates)), fluorescein-type labels (e.g., fluorescein isothiocyanate, 5-carboxyfluorescein, 6-carboxyfluorescein, dichlorotriazinylamine fluorescein), Roxisene, etcDanmin type markers (e.g., ALEXA)

568(Invitrogen)、

Or dansyl chloride), VIVOTAG 680XL FLUOROCHROME^TM(Perkin Elmer), phycoerythrin; umbelliferone, lissamine; cyanine; phycoerythrin, Texas Red, BODIPY

(Invitrogen) or an analogue thereof, all of which are suitable for optical detection. Antibodies and antigen-binding fragments thereof of the invention may also employ chemiluminescent labels (e.g., luminol, luciferase, luciferin, and aequorin). Such diagnosis and detection may also be accomplished by coupling the diagnostic molecules of the present invention to a detectable substance (including, but not limited to, various enzymes including, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase) or to a prosthetic group complex including, but not limited to, streptavidin/biotin and avidin/biotin.

Paramagnetic labels may also be employed and are preferably detected using Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT). Such paramagnetic labels include, but are not limited to, those containing aluminum (Al), barium (Ba), calcium (Ca), cerium (Ce), dysprosium (Dy), erbium (Er), europium (Eu), gadolinium (Gd), holmium (Ho), iridium (Ir), lithium (Li), magnesium (Mg), manganese (Mn), molybdenum (M), neodymium (Nd), osmium (Os), oxygen (O), palladium (Pd), platinum (Pt), rhodium (Rh), ruthenium (Ru), samarium (Sm), sodium (Na), strontium (Sr), terbium (Tb), thulium (Tm), tin (Sn), titanium (Ti), tungsten (W), and zirconium (Zi) and in particular Co⁺²、CR⁺²、Cr⁺³、Cu⁺²、Fe⁺²、Fe⁺³、Ga⁺³、Mn⁺³、Ni⁺²、Ti⁺³、V⁺³And V⁺⁴The paramagnetic metal ion of (a), positron emitting metals using various positron emission tomography, and non-radioactive paramagnetic metal ions.

Thus, in some embodiments, the anti-CD 146 antibodies of the invention, or CD 146-binding fragments thereof, may be labeled with a fluorescent label, a chemiluminescent label, a paramagnetic label, a radioisotope label, or an enzymatic label. The presence or amount of the CD146 in the subject can be detected or measured using a fragment of the labeled antibody. Such methods may include detection or measurement of in vivo imaging of anti-CD 146 antibodies or CD146 binding fragments that bind to said CD146 and may include ex vivo imaging of anti-CD 146 antibodies or CD146 binding fragments that bind to such CD 146.

Thus, in some embodiments, the anti-CD 146 antibodies and antigen-binding fragments thereof of the present invention include whole antibodies, such as IgG (IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (IgA1 and IgA2 subtypes), IgM, and IgE; antigen binding fragments such as SDR, CDR, Fv, dAb, Fab₂Fab ', (Fab')2, Fd, scFv, nanobody; a variant sequence of an antibody or antigen-binding fragment thereof, such as a variant sequence having at least 80% sequence identity to an antibody or antigen-binding fragment thereof described above. In some embodiments, the invention also includes derivatives comprising anti-CD 146 or antigen binding fragments thereof, such as chimeric antibodies derived from whole antibodies, humanized antibodies, fully human antibodies, recombinant antibodies, bispecific antibodies, products comprising modified amino acids, products conjugated to polymers, products comprising radiolabels, products comprising fluorescent labels, products comprising enzymatic labels, products comprising chemiluminescent labels, products comprising paramagnetic labels.

In a further aspect, the invention relates to an expression vector encoding one or more polypeptide chains of an antibody or antigen-binding fragment thereof of the invention. Such expression vectors can be used for recombinant production of the antibodies and antigen-binding fragments thereof of the invention.

In the present invention, an expression vector may be any suitable DNA or RNA vector, including chromosomal vectors, non-chromosomal vectors, and synthetic nucleic acid vectors (nucleic acid sequences comprising a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculoviruses, yeast plasmids, plasmids derived from a combination of plasmids and phage DNAVectors and viral nucleic acid (RNA or DNA) vectors. In some embodiments, the nucleic acid encoding the anti-CD 146 antibody is contained in a naked DNA or RNA vector, including, for example, a linear expression element (as described, for example, in Sykes and Johnston, Nat Biotech,12, 355-59(1997)), a compact nucleic acid vector (as described, for example, in US 6,077,835 and/or WO 00/70087), a plasmid vector (as described, for example, in pBR322, pUC 19/18 or pUC 118/119), a minimum size nucleic acid vector (as described, for example, in Schakowski et al, MoI Ther,3, 793- "800 (2001)), or as a precipitated nucleic acid vector construct, as in a preferred embodiment, the expression of the anti-CD 146 antibody is carried out in a recombinant vector₄Precipitated constructs (as described, for example, in WO 00/46147; Benveninsty and Reshef, PNAS USA 83, 9551-55 (1986); Wigler et al, Cell,14, 725(1978) and Coraro and Pearson, physical Cell Genetics,2, 603 (1981)). Such nucleic acid vectors and their use are well known in the art (see, e.g., US5,589,466 and US5,973,972).

In some embodiments, the vector is suitable for expressing an anti-CD 146 antibody or antigen-binding fragment thereof in a bacterial cell. Examples of such vectors include, for example, BlueScript (Stratagene), pIN vector (Van Heeke & Schuster, J Biol Chem,264, 5503-.

The expression vector may also be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be used. Suitable vectors include, for example, vectors comprising constitutive or inducible promoters (e.g., alpha factor, alcohol oxidase and PGH) (reviewed in F. Ausubel et al, ed., Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987); Grant et al, Methods in Enzymol,153, 516-garland 544 (1987); Mattanovich, D. et al, Methods in biol. 824, 329-358 (2012); Celik, E. et al, Biotechnol. adv.30 (5), 1108), 1118 (2012); Li, P. et al, appl.biochem. Biotechnol. 142(2), 105-124 (2007);

E. et al, appl. Microbiol. Biotechnol.,77(3), 513-; van der Vaart, j.m., Methods mol. biol.,178, 359-366(2002) and Holliger, p., Methods Mol.Biol.,178，349-357(2002))。

In the expression vectors of the invention, the nucleic acid encoding the anti-CD 146 antibody may comprise or be associated with any suitable promoter, enhancer, and other elements that facilitate expression. Examples of such elements include strongly expressing promoters (e.g., the human CMV IE promoter/enhancer and RSV, SV40, SL3-3, MMTV and HIV LTR promoters), efficient poly (a) termination sequences, origins of replication for plasmid production in e.coli, antibiotic resistance genes as selectable markers, and/or convenient cloning sites (e.g., polylinkers). The nucleic acid may also comprise an inducible promoter as opposed to a constitutive promoter (such as CMV IE).

In a further aspect, the invention relates to a recombinant eukaryotic or prokaryotic host cell (such as a transfectoma) that produces an antibody or antigen-binding fragment thereof of the invention or a bispecific molecule of the invention. Examples of host cells include yeast, bacteria, and mammalian cells (e.g., CHO or HEK cells). For example, in some embodiments, the invention provides a cell comprising a nucleic acid stably integrated into the genome of the cell, the genome comprising a nucleic acid sequence encoding an anti-CD 146 antibody or antigen-binding fragment thereof of the invention. In other embodiments, the invention provides a cell comprising a non-integrating nucleic acid (e.g., a plasmid, cosmid, phagemid, or linear expression element) comprising a sequence encoding an anti-CD 146 antibody or antigen-binding fragment thereof of the invention.

The antibodies and antigen-binding fragments thereof of the present invention can be produced in various cell lines, such as human, non-human mammalian and insect cell lines, e.g., CHO, HEK, BHK-21, murine (e.g., myeloma), fibrosarcoma, PER. C6, HKB-11, CAP and HuH-7 human cell lines (Dumont et al 2015, CrRev Biotechnol., Sep.18,1-13, the contents of which are incorporated herein by reference).

The AA98 antibody refers to the antibody disclosed in Chinese patent CN1124284C, and comprises a murine monoclonal antibody AA98, a human-murine chimeric antibody ChiAA98 and Fab and Fv functional fragments thereof.

The antibody of the invention acts on various tumor neovascularization and can play a role in treating and/or diagnosing various solid tumors depending on tumor angiogenesis, including but not limited to liver cancer, stomach cancer, colorectal cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, cervical cancer, head and neck cancer, colorectal cancer and the like.

The antibodies of the invention may also be used to treat diseases associated with neovascularization and inflammatory diseases, including but not limited to age-related macular degeneration, proliferative diabetic retinopathy, colorectal inflammation, multiple sclerosis, atherosclerosis, and the like.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range and any other stated or intervening value in that stated range, unless the context clearly dictates otherwise, is encompassed within the invention. The upper and lower limits of these smaller ranges, which may independently be included in the smaller ranges, are also encompassed within the invention, subject to any specifically excluded limit in the stated range being excluded. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now disclosed. All publications mentioned herein are incorporated herein by reference in their entirety.

The examples provided herein are for the purpose of describing, on the one hand, the process for preparing the antibody of the present invention, which is illustrative of the relevant methods only and not intended to be limiting, and it will be apparent to those skilled in the art that many modifications can be made thereto without departing from the spirit of the invention, and such modifications are intended to fall within the scope of the invention. The present invention provides examples in another aspect for demonstrating the features and advantages of the antibodies of the invention, but the invention is not limited to these features and advantages.

The following experimental methods are all conventional methods unless otherwise specified, and the experimental materials used are readily available from commercial companies unless otherwise specified. The various antibodies used in the examples of the invention described below were derived from standard antibodies commercially available.

Examples

Example 1 resolution of the Crystal Structure of the CD 146D 4-D5/AA98 Complex

Construction of CD 146D 4-D5 expression plasmid

The DNA sequence (SEQ ID No.1) of CD 146D 4-D5(336-519) with a signal peptide, His-tag, strep-tag and HRV3C recognition sequences at the N end is digested by PCR, XbaI and NotI, is connected between XbaI and NotI on a pcDNA3.1/Hygro (-) vector (Invitrogen), and the sequence is verified to be correct by DNA sequencing.

Construction of cell line stably expressing CD 146D 4-D5

The plasmid obtained above is transferred into CHO Lec 3.2.8.1 cell line (ATCC) by electric shock method, cultured in DMEM medium (Sigma-Aldrich) containing hygromycin, combined with ELISA method taking AA98 as primary antibody, screened out cells with high expression level of CD 146D 4-D5, transferred into a roller bottle for mass culture, and culture supernatant is collected, wherein the culture supernatant contains target protein CD 146D 4-D5.

Expression and purification of CD 146D 4-D5 protein

The culture supernatant containing the target protein is collected, concentrated and then subjected to Ni-NTA affinity (Qiagen) purification and strep-tactin affinity (IBA) purification to obtain the fusion protein containing His-tag and strep-tag with high purity. Performing HRV3C (Sino Biological) protein enzyme digestion, performing Ni-NTA affinity purification to remove HRV3C protease and target protein without fusion N end, and further performing Superdex200 molecular sieve to obtain CD 146D 4-D5 protein with purity of over 95%.

Expression and purification of AA98 antibody and Fab preparation

(A) Culturing AA98 hybridoma HE2A5 (see Chinese patent CN1124284C), collecting culture supernatant, concentrating, adjusting to TBS buffer solution, and purifying by Protein A affinity purification and Superdex200 molecular sieve to obtain AA98 antibody with high purity and good uniformity.

(B) The target antibody is subjected to papain enzyme digestion, Protein A affinity purification and Superdex200 molecular sieve purification, and the intact antibody which is not subjected to cleavage and the Fc part of the antibody are removed, so that the AA98 Fab with high purity and good uniformity is obtained

Purification of CD 146D 4-D5/AA98 Complex protein

The molecular weight ratio of CD 146D 4-D5 AA98 Fab is 1: 4 mixing the two proteins, incubating on ice for 30 minutes, and purifying by a Superdex200 molecular sieve to obtain a compound protein solution.

Growth and optimization of CD 146D 4-D5/AA98 Complex crystals

The obtained CD 146D 4-D5/AA98 Fab complex protein is concentrated to 12mg/ml, possible crystallization conditions are screened by a pendant drop method, and then after multiple rounds of optimization, crystals with higher diffraction resolution which can be used for data collection are obtained.

Collection and structural resolution of CD 146D 4-D5/AA98 Complex Crystal diffraction data

Diffraction data were collected from the cryopreserved crystals of the complex by synchrotron radiation in the Shanghai. After the collected data are processed by HKL-2000 software, a D2.3 Fab crystal structure (1YEC) is used as a model, the phase is cracked by a molecular replacement method, a CD 146D 4-D5 part is manually built, and the 2.8 angstrom compound crystal structure is finally analyzed through multiple rounds of optimization. Panel A of FIG. 1 provides a general view of the crystal structure of the composite. The B diagram of figure 1 provides a detailed view of the antigen/antibody binding surface of the crystal structure of the complex.

When the Superdex 75 molecular sieve is compared with the CD 146D 4-D5 protein and the Fab AA98 binding before and after aggregation, the CD 146D 4-D5 protein is found to be a dimer in a natural state in a solution, and is converted into a monomer after AA98 Fab binding (figure 2), and the resolved crystal structure of the compound also shows that one CD146 molecule is bound with one Fab, and the binding of AA98 breaks CD146 dimerization, so that the monomer structure of the CD146 is stabilized.

In the crystal structure of the complex, AA98 Fab binds between the fourth and fifth domains of CD146, the center of the overall interaction is mainly hydrophobic interactions between aromatic ring residues from the two proteins, and hydrogen bonds and van der waals forces between some hydrophilic residues are around. In order to increase the affinity of the antibody and enhance the interaction between the two, stabilizing the conformation of the complex in solution, the present inventors performed site-directed mutagenesis on the antibody. Analysis revealed that I28, I57 and Y59 of the antibody heavy chain are located in the hydrophilic part of the periphery of the interaction surface, but are hydrophobic residues, and it is envisaged that mutating them to hydrophilic residues may help to increase the stability of the complex and enhance the hydrophilic interaction with the CD146 molecule. Similarly, mutations of I31 to E, S32 to T, and L51 to Y or H of the light chain of the antibody may help to enhance hydrophobic interactions between aromatic ring amino acids in the central portion.

Example 2 method of engineering AA98 antibody

1. Experimental materials, reagents: a plasmid extraction kit (TIANGEN, DP103-3), a DNA purification kit (Transgen,30831), and an agarose gel DNA recovery kit (Transgen, EG 101-02); ex Taq polymerase (Fastpfu polymerase, Transgen, AP221), restriction enzyme (Not I, NEB, R3189S; XbaI, NEB, R0145S), T4 DNA ligase (TaKaRa Co.); peptone, yeast extract, penicillin, streptomycin, RPMI cell culture medium, DMEM cell culture medium, dimethyl sulfoxide (DMSO), fetal bovine serum, trypsin were purchased from Sigma-Aldrich, USA;

2. experimental equipment: carbon dioxide incubator (NBS), shaker, PCR instrument (Applied Biosystem); nanodrop 2000c micro uv spectrophotometer (Thermo Scientific); hermel Z32K refrigerated centrifuge, Sigma SK15 refrigerated centrifuge; gel imager (Bio-Rad);

3. experimental methods

Experiment design:

according to the crystal structure of the CD146/AA98 complex, the action mode of the AA98 antibody and the CD146 molecule is obtained, and the amino acid residues participating in the interaction on the AA98 antibody are mutated (see the following table 5) so as to strengthen the interaction between the AA98 antibody and the CD146 molecule and improve the affinity of the target antibody to the CD146 molecule. The coding sequences of the light chain and the heavy chain of the antibody obtained by the method are respectively cloned to eukaryotic vectors, secreted and expressed in human HEK293T cells (ATCC) and purified to obtain the target antibody.

The technical route is as follows:

(1) construction of plasmids

According to the amino acid sequence of the target antibody Fab, the DNA sequence of the antibody constant region is searched on NCBI website by blast search, and then is directly synthesized by a sequencing company and cloned to a eukaryotic expression vector. The heavy chain of the antibody was cloned between Xba I and Not I of pcDNA3.1/Hygro (-) (Invitrogen company) vector, while the light chain of the antibody was cloned between BamH I and Not I of pEF1/V5-HisA vector, the former containing the selection marker for hygromycin B and the latter containing the selection marker for neomycin.

Based on the nucleotide sequences of the light and heavy chains of the AA98 antibody provided in SEQ ID Nos. 2 and 3, the present invention constructed a number of antibody mutants in which the nucleotide sequence of the light and heavy chains of one mutant antibody involved the replacement of Leu, amino acid 51 of the light chain, to Tyr (L51Y), and Ile, amino acid 28 of the heavy chain, to Lys (I28K). The antibody variant was named H13-112 (heavy and light chain amino acid sequences shown in SEQ ID Nos. 17 and 28, respectively). The other mutants shown in Table 5 can be obtained in the same manner in turn. SEQ ID Nos. 4 and 5 show the amino acid sequences of the variable regions of the light and heavy chains of the AA98 antibody, respectively, SEQ ID Nos. 6 and 7 provide the full-length amino acid sequence of the light and heavy chains of the AA98 antibody, SEQ ID Nos. 8 and 16 provide the amino acid sequence of the Fab of the AA98 antibody, SEQ ID No.9 provides the scFv sequence of the AA98 antibody, and SEQ ID Nos. 10-15 provide the CDR sequences of the light and heavy chains of the AA98 antibody, respectively.

(2) Cell transfection

Expression of the antibody of interest was performed in human HEK293T cells. Plasmids containing the target antibody light chain and heavy chain DNA sequences were prepared in a molar ratio of 1: 1 proportion, the light and heavy chain plasmids of the antibody were transfected into human HEK293T cells according to the operating manual of lipofectamine 2000 from Thermo Fisher, and the transfected cells were cultured for 24 hours and then transferred to HDMEM selective medium (Sigma Aldrich) containing 125ug/ml hygromycin B and 1mg/ml neomycin, during which time the medium containing the antibody of interest was replaced and collected until it was suitable for antibody purification.

(3) Antibody purification

The purification of the target antibody was carried out according to the manual of GE corporation Hitrap rProtein A FF (GE Healthcare, Cat.17-5080-01), and thereafter the target antibody was transferred into a PBS solution (10mM sodium phosphate, 150mM sodium chloride, pH 7.4) suitable for the subsequent test by dialysis. The purity of the purified antibody was determined by SDS-PAGE and the antibody was identified by ELISA (coating antigen with extracellular domain CD146 protein (Sino biological, 10115-H08H-20)).

Example 3 detection of antibody affinity by biofilm interferometry

The method comprises the following steps:

1. 8 Anti-Mouse Fc Capture sensors (Pall FaoteBio) were soaked in PBS buffer (10mM sodium phosphate, 150mM sodium chloride, pH 7.4) supplemented with 0.1% BSA and 0.05% Tween 20 for 10min in advance.

2. sCD146 protein (nano bio., 10115-H08H-20) was diluted with PBS buffer supplemented with 0.1% BSA and 0.05% Tween 20 to: 250nM, 125nM, 62.5nM, 31.25nM, 15.625nM, 7.8125nM, 3.91 nM.

3. The gradient diluted sCD146 of 7 concentrations in step 2 and PBS buffer with 0.1% BSA and 0.05% Tween 20 added were added sequentially to the 10 th column of black 96-well plate (Corning, 655209), 200 μ l/well.

4. The parent AA98 antibody was diluted to 20 μ g/ml with PBS buffer supplemented with 0.1% BSA and 0.05% Tween 20.

5. The antibody described in step 4 was added to

column

2, 200. mu.l/well of the black 96-well plate described in step 2.

6. The H13-112 antibody was diluted to 20. mu.g/ml with PBS buffer supplemented with 0.1% BSA and 0.05% Tween 20.

7. The antibody described in step 6 was added to column 4, 200. mu.l/well of the black 96-well plate described in step 5.

8. To the 1 st, 3 rd, 5 th, 6 th, 7 th, 12 th columns of the black 96-well plate in

step

7, 200. mu.l/well of PBS buffer containing 0.1% BSA and 0.05% Tween 20 was added.

9. To the 11 th column of the black 96-well plate described in

step

8, 200. mu.l/well of Gly-HCl regeneration solution pH 1.5 (50mM, pH 2.5) was added.

10. Placing the 96-well plate in the step 9 into a biomolecule interaction instrument (Pall FaoteBio, Octet Red96), setting a detection program, and sequentially passing the sensor through the 96-well plate in the step 9 in the step 1:

column 1 (equilibrium, 120s) -column 1 (baseline, 60s) -column 2 (load, 360s) -column 6 (baseline, 60s) -column 10 (association, 180s) -column 6 (dissociation, 600s) -column 11 (regeneration, 6s) -column 12 (neutralization, 6s) -column 3 (equilibrium, 120s) -column 4 (load, 360s) -column 7 (baseline, 60s) -column 10 (association, 180s) -column 7 (dissociation, 600s) -column 11 (regeneration, 6s) -column 12 (neutralization, 6s) -column 11 (regeneration, 6s) -12 th column (neutralization, 6s) -11 th column (regeneration, 6s) -12 th column (neutralization, 6 s).

11. After completion of the procedure, data were collected and analyzed, resulting in a parent AA98 antibody affinity of 1.12 × 10 as shown in a and B of fig. 3 and table 5^-9M, the affinity of the engineered antibody H13-112 is 1.44X 10^-10M, the affinity of the engineered antibody H13-112 was increased by nearly 10-fold relative to AA 98. The affinity of other mutants was also increased to varying degrees.

TABLE 5 AA98 antibody series mutants and their K against CD146 extracellular domain protein_DThe value is obtained.

The data indicate that the hydrophobic amino acid residue on the antigen/antibody binding surface has an adverse effect on the binding of the antibody to its antigen, and the activity of binding to its antigen and cell can be significantly improved by mutating the hydrophobic amino acid residue to a hydrophilic amino acid residue.

Example 4 antibody specific detection (FACS)

The human venous endothelial cell line HUVEC, the melanoma cell line A375, the liver cancer cell line HepG2, the colon cancer cell line HT-29, the triple negative breast cancer cell line MDA-MB-231, which is a CD146 positive expression strain, and the breast cancer cell line MCF-7, which is a CD146 negative/low expression strain, were purchased from ATCC.

The experimental steps are as follows:

1. inoculating the above cells into 6-well plate, culturing for 48 hr, trypsinizing adherent cells, collecting cells, centrifuging (4 deg.C, 1000rpm, 5min), washing with pre-cooled PBS once (1000rpm, 5min), resuspending with pre-cooled PBS (containing 0.1% BSA), adjusting cell concentration to 1 × 10⁶The content of the active carbon is one/ml,

2. transferring 100 μ L of cell suspension to a new tube, adding H13-112 primary antibody or isotype control mIgG into each tube, mixing, incubating at 4 deg.C in dark for 40min,

3. washing with precooled PBS (containing 0.1% BSA), centrifuging, removing supernatant, adding 100 μ L PBS (containing 0.1% BSA) to resuspend cells, adding fluorescein labeled secondary antibody Alexa Fluor 555 goat anti-mouse IgG (Invitrogen, A11001), incubating at 4 deg.C in the dark for 30min,

4. cells were washed once with pre-chilled PBS, analyzed and detected using a flow cytometer over 1 hour, and data analyzed using FlowJo software. The results are shown in FIGS. 4A to F.

The above results indicate that the mutated H13-112 antibody retains the affinity effect of the AA98 antibody on CD146 positive cells.

Example 5 cell migration experiment of antibody inhibiting human venous endothelial HUVEC cells

The migratory capacity of HUVEC cells under the action of antibodies was investigated using the Transwell system. The antibodies used were AA 98-based mutant antibodies such as mIgG, original AA98 antibody, H13-112, H14-114, 106-112 and 106-114, respectively, as negative controls.

The experimental procedure was as follows:

1. human umbilical vein endothelial cells HUVEC (ATCC, PCS-100-010) were resuspended in single cell suspension using serum-free RPMI1640 medium (Gibco, C11875500BT) to adjust the cell density to 8X 10⁴Individual cells/ml.

2. RPMI1640 medium containing 10% fetal bovine serum (Gibco, 10091148) was added to the lower chamber of a Corning HTS Transwell-96Cell Migration dish (Corning,3385), at 200. mu.l/well. The upper chamber was filled with 100. mu.l/well of the cell suspension described in step 1.

3. Antibodies mIgG (Abcam, ab37355), AA98 and H13-112 were added to the upper chamber of the dish in step 2 and mixed well to a final concentration of 100. mu.g/ml, with 3 sets of parallel wells for each antibody.

4. Placing the culture dish in step 3 at 37 ℃ and 5% CO₂Incubate in cell incubator for 12 h.

5. The medium in the upper and lower chambers of the dish in step 4 was removed and the cells on the upper layer of the Transwell membrane were wiped off with a cotton swab dipped with 75% ethanol.

6. The Transwell membrane of step 5 was removed from the petri dish and spread on a glass slide (25.4X 76.2mm) with the lower membrane with cells attached facing upwards.

7. The cells on the Transwell membrane in step 6 were fixed with 4% paraformaldehyde in phosphate buffer (10mM sodium phosphate, 150mM sodium chloride, pH 7.4) for 15 min.

8. Remove the 4% paraformaldehyde in step 7 and wash with PBS three times.

9. The cells in the lower layer of the Transwell membrane described in step 7 were stained with 1% crystal violet for 10 min.

10、ddH₂And washing for five times until no purple color exists to remove the crystal violet dye solution.

11. The Transwell membrane in step 10 was placed under an Olympus upright microscope for observation and a photograph was taken of each field.

12. The total number of cells on each Transwell membrane was counted using the software ImageJ (NIH) and subjected to Student's t-test statistical analysis.

13. The results are shown in graph A of FIG. 5, and graph B is a statistical result based on graph A. Both antibody AA98 and antibody AA98 mutants were effective in inhibiting endothelial cell migration compared to control mIgG. Compared with AA98, the H13-112 mutant has stronger inhibition effect, the inhibition rate is 86%, and the inhibition rate of AA98 antibody is only 49%.

Example 6 experiment of antibody inhibiting triple negative breast cancer MDA-MB-231 cell migration

The migratory capacity of MDA-MB-231 cells under the action of antibody was investigated using the Transwell system. The antibodies used were mIgG, the original AA98 antibody and the H13-112 mutant antibody, respectively, as negative controls.

The experimental procedure was as follows:

1. inoculating fineCell to 6-hole plate (5-Pipelo-10 per hole)⁵Cells/well), the medium was H-DMEM containing 2% FBS. mIgG, AA98 and H13-112 antibodies are respectively added into each well for pretreatment for 48 hours, and the final concentration is 100 mu g/ml;

2. after 48 hours, the cells were inoculated into Transwell plates, in which the number of cells in the upper chamber was 1 KHz 10⁴Cells/well, culture medium 50 μ L serum free H-DMEM; simultaneously adding an antibody to block, wherein the final concentration is 100 mu g/ml; the lower chamber was 200. mu.L of H-DMEM containing 10% FBS.

3. After 24h incubation, the cells on the upper layer of the membrane are wiped off by 75% alcohol, the cells on the lower layer of the membrane are fixed by 4% PFA, washed by PBS for 2 times after 15 minutes at room temperature, stained by crystal violet for 15 minutes, washed by excessive staining solution and then placed under a ZEISS Axio scope A1 microscope for photographing, and ImageJ software counts the number of migrated cells

4. The results are shown in graph A of FIG. 6, and graph B is a statistical result based on graph A. Compared with the control mIgG, the antibody AA98 and the antibody AA98 mutant can effectively inhibit the migration of MDA-MB-231 cells of breast cancer cells. Compared with AA98, the H13-112 mutant has stronger inhibition effect, the inhibition rate is 83%, and the inhibition rate of AA98 antibody is only 57%.

Example 7 tube formation experiment

The experimental procedure was as follows:

1. a96-well plate (Corning, 3599) was coated with a 60. mu.l/well ice-bath of Matrigel (Corning, 354248), and left in a cell incubator at 37 ℃ for 30min to allow it to coagulate.

2. Human umbilical vein endothelial cell HUVEC is re-suspended into single cell suspension by serum-free RPMI culture medium, and the cell density is adjusted to 8 x 10⁴Individual cells/ml.

3. The cell suspension described in step 2 was added to the 96-well plate described in step 1 at 100. mu.l/well.

4. And (3) adding the antibodies mIgG, AA98 and H13-112 into the cell suspension in the step 3, mixing uniformly, wherein the final concentration of the antibodies is 100 mu g/ml, and 3 groups of each antibody are subjected to parallel repeated hole.

5. Placing the 96-well plate in step 4 at 37 ℃ and 5% CO₂And incubating for 3-4h in the cell incubator.

6. The 96-well plate in step 5 was placed under an Olympus inverted microscope (IX71) for observation and photographed for each field.

7. Total length of tubular cells per well was counted using software (Image-Pro Plus 7.0) and Student's t-test statistical analysis was performed.

8. The results are shown in a and B graphs of fig. 7, compared with the control group mIgG, the antibodies AA98 and AA98 mutant can effectively inhibit the tube forming cavity of endothelial cells, particularly the H13-112 antibody, the inhibition rate is 93%, the inhibition rate is far more than 25% of that of the AA98 antibody, and the inhibition effect is significantly stronger.

Example 8 triple negative breast cancer tumor suppression assay

Triple negative breast cancer refers to malignant tumors with negative Estrogen Receptor (ER), progestational hormone receptor (PR) and Epidermal Growth Factor Receptor (EGFR), and accounts for about 17-25% of all breast cancers; easy relapse and metastasis, poor prognosis and no effective treatment method at present. According to the results of 505 breast cancer tissue chips, the positive rate of CD146 in the tumors is 66.9%, and the 5-year survival rate of tumor patients with CD146+ is obviously lower than that of tumor patients with CD 146-. The inventor shows that CD146 may be an effective therapeutic target of triple negative breast cancer.

Experimental methods and results: firstly, a humanized triple negative breast cancer tumor model of an immunodeficiency mouse is constructed by the following method: MDA-MB-231 breast cancer cell line 10⁷Inoculated under the skin of the right flank of a BALB/c nude mouse, and subjected to antibody treatment experiments when the tumor grows to 5mm in diameter. The mice were randomly divided into three groups: mIgG group, AA98 group and H13-112 groups, 15 of each group. The injection mode of the antibody is intraperitoneal injection, and the dosage is 200 mug/mouse twice a week. Tumor length and width were measured with a vernier caliper before each injection and tumor volume was calculated according to the following formula: volume length width 2. The results show that, when AA98 antibody treatment was initiated for the second time, tumor growth was delayed, indicating that AA98 treatment was better able to inhibit tumor growth; compared with AA98, the optimized antibody H13-112 has more obvious tumor inhibiting effect. The results are shown in FIG. 8.

Sequence listing

<110> institute of biophysics of Chinese academy of sciences

<120> anti-human CD146 monoclonal antibody with neutralization activity and application thereof

<130> LZ1708600CN01

<160> 32

<170> PatentIn version 3.3

<210> 1

<211> 795

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> CD146 D4-D5

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catgacctga aacgggaggc aggaggcggc tatcgctgcg tggcgtctgt gcccagcata 480

cccggcctga accgcacaca gctggtcaac gtggccattt ttggcccccc ttggatggca 540

ttcaaggaga ggaaggtgtg ggtgaaagag aatatggtgt tgaatctgtc ttgtgaagcg 600

tcagggcacc cccggcccac catctcctgg aacgtcaacg gcacggcaag tgaacaagac 660

caagatccac agcgagtcct gagcaccctg aatgtcctcg tgaccccgga gctgttggag 720

acaggtgttg aatgcacggc ctccaacgac ctgggcaaaa acaccagcat cctcttcctg 780

gagctggtca attta 795

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence of AA98 antibody light chain

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gaactggtga tgacccagag cccggcgagc ctggcggtga gcctgggcca gcgcgcgacc 120

attagctgcc gcgcgagcaa aagcgtgagc attagcggct atagctatat gcattggtat 180

cagcagaaac cgggccagcc gccgaaactg ctgatttatc tggcgagcaa cctggaaagc 240

ggcgtgccgg cgcgctttag cggcagcggc agcggcaccg attttaccct gaacattcat 300

ccggtggaag aagaagatgc ggcgacctat tattgccagc atagccgcga actgccgtat 360

acctttggcg gcggcaccaa actggaaatt aaacgcgcgg atgcggcgcc gaccgtgagc 420

atttttccgc cgagcagcga acagctgacc agcggcggcg cgagcgtggt gtgctttctg 480

aacaactttt atccgaaaga tattaacgtg aaatggaaaa ttgatggcag cgaacgccag 540

aacggcgtgc tgaacagctg gaccgatcag gatagcaaag atagcaccta tagcatgagc 600

agcaccctga ccctgaccaa agatgaatat gaacgccata acagctatac ctgcgaagcg 660

acccataaaa ccagcaccag cccgattgtg aaaagcttta accgcaacga atgc 714

<210> 3

<211> 1398

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

Nucleotide sequence of <223> AA98 antibody heavy chain

<400> 3

atgatggtgt taagtcttct gtacctgttg acagcccttc cgggtatcct gtcagaagtg 60

cagctgctgg aaagcggcgc ggaactggtg cgcccgggcg cgagcgtgaa actgagctgc 120

aaaaccagcg gctatatttt taccaactat tggattcatt gggtgaaaaa ccgcagcggc 180

aacggcctgg aatggattgc gcgcatttat ccgggcaccg atattaccta ttataacgaa 240

aaatttaaag gcaaagcgac cctgaccgtg gataaaagca gcagcagcgc gtatatgctg 300

ctgagcagcc tgaaaagcga agatagcagc gtgtattttt gcgcgcgcag cggcggctat 360

tggtattttg atgtgtgggg cgcgggcacc accgtgaccg tgagcagcgc gaaaaccacc 420

gcgccgagcg tgtatccgct ggcgccggtg tgcggcgata ccaccggcag cagcgtgacc 480

ctgggctgcc tggtgaaagg ctattttccg gaaccggtga ccctgacctg gaacagcggc 540

agcctgagca gcggcgtgca tacctttccg gcggtgctgc agagcgatct gtataccctg 600

agcagcagcg tgaccgtgac cagcagcacc tggccgagcc agagcattac ctgcaacgtg 660

gcgcatccgg cgagcagcac caaagtggat aaaaaaattg aaccgcgcgg cccgaccatt 720

aaaccgtgcc cgccgtgcaa atgcccggcg ccgaacctgc tgggcggccc gagcgtgttt 780

atttttccgc cgaaaattaa agatgtgctg atgattagcc tgagcccgat tgtgacctgc 840

gtggtggtgg atgtgagcga agatgatccg gatgtgcaga ttagctggtt tgtgaacaac 900

gtggaagtgc ataccgcgca gacccagacc catcgcgaag attataacag caccctgcgc 960

gtggtgagcg cgctgccgat tcagcatcag gattggatga gcggcaaaga atttaaatgc 1020

aaagtgaaca acaaagatct gccggcgccg attgaacgca ccattagcaa accgaaaggc 1080

agcgtgcgcg cgccgcaggt gtatgtgctg ccgccgccgg aagaagaaat gaccaaaaaa 1140

caggtgaccc tgacctgcat ggtgaccgat tttatgccgg aagatattta tgtggaatgg 1200

accaacaacg gcaaaaccga actgaactat aaaaacaccg aaccggtgct ggatagcgat 1260

ggcagctatt ttatgtatag caaactgcgc gtggaaaaaa aaaactgggt ggaacgcaac 1320

agctatagct gcagcgtggt gcatgaaggc ctgcataacc atcataccac caaaagcttt 1380

agccgcaccc cgggcaaa 1398

<210> 4

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> AA98 antibody heavy chain variable region

<400> 4

Glu Val Gln Leu Leu Glu Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Ile Phe Thr Asn Tyr

20 25 30

Trp Ile His Trp Val Lys Asn Arg Ser Gly Asn Gly Leu Glu Trp Ile

35 40 45

Ala Arg Ile Tyr Pro Gly Thr Asp Ile Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Ser Ala Tyr

65 70 75 80

Met Leu Leu Ser Ser Leu Lys Ser Glu Asp Ser Ser Val Tyr Phe Cys

85 90 95

Ala Arg Ser Gly Gly Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Lys

115 120

<210> 5

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> AA98 antibody light chain variable region

<400> 5

Glu Leu Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Ile Ser

20 25 30

Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg

85 90 95

Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Ala

<210> 6

<211> 448

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> AA98 antibody heavy chain

<400> 6

Glu Val Gln Leu Leu Glu Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Ile Phe Thr Asn Tyr

20 25 30

Trp Ile His Trp Val Lys Asn Arg Ser Gly Asn Gly Leu Glu Trp Ile

35 40 45

Ala Arg Ile Tyr Pro Gly Thr Asp Ile Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Ser Ala Tyr

65 70 75 80

Met Leu Leu Ser Ser Leu Lys Ser Glu Asp Ser Ser Val Tyr Phe Cys

85 90 95

Ala Arg Ser Gly Gly Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro

115 120 125

Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly

130 135 140

Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn

145 150 155 160

Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr

180 185 190

Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser

195 200 205

Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro

210 215 220

Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu

245 250 255

Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro

260 265 270

Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala

275 280 285

Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val

290 295 300

Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe

305 310 315 320

Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr

325 330 335

Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu

340 345 350

Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys

355 360 365

Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn

370 375 380

Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys

405 410 415

Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly

420 425 430

Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys

435 440 445

<210> 7

<211> 218

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> AA98 antibody light chain

<400> 7

Glu Leu Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Ile Ser

20 25 30

Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg

85 90 95

Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln

115 120 125

Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr

130 135 140

Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln

145 150 155 160

Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg

180 185 190

His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro

195 200 205

Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

210 215

<210> 8

<211> 218

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain sequence of AA98 antibody Fab

<400> 8

Glu Leu Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Ile Ser

20 25 30

Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg

85 90 95

Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln

115 120 125

Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr

130 135 140

Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln

145 150 155 160

Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg

180 185 190

His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro

195 200 205

Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

210 215

<210> 9

<211> 247

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> AA98 antibody scFv

<400> 9

Glu Leu Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Ile Ser

20 25 30

Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg

85 90 95

Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Ser Arg Arg Gly Gly Gly Ser Arg Gly Gly Gly Pro Gly Gly Gly Gly

115 120 125

Ser Glu Val Gln Leu Leu Glu Ser Gly Ala Glu Leu Val Arg Pro Gly

130 135 140

Ala Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Ile Phe Thr Asn

145 150 155 160

Tyr Trp Ile His Trp Val Lys Asn Arg Ser Gly Asn Gly Leu Glu Trp

165 170 175

Ile Ala Arg Ile Tyr Pro Gly Thr Asp Ile Thr Tyr Tyr Asn Glu Lys

180 185 190

Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Ser Ala

195 200 205

Tyr Met Leu Leu Ser Ser Leu Lys Ser Glu Asp Ser Ser Val Tyr Phe

210 215 220

Cys Ala Arg Ser Gly Gly Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly

225 230 235 240

Thr Thr Val Thr Val Ser Ser

245

<210> 10

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> AA98 antibody heavy chain CDR1

<400> 10

Ser Gly Tyr Lys Phe Thr Asn Tyr Trp

1 5

<210> 11

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> AA98 antibody heavy chain CDR2

<400> 11

Tyr Pro Gly Thr Asp Ile Thr Tyr

1 5

<210> 12

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> AA98 antibody heavy chain CDR3

<400> 12

Ser Gly Gly Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly

1 5 10

<210> 13

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> AA98 antibody light chain CDR1

<400> 13

Ala Ser Lys Ser Val Ser Ile Ser Gly Tyr Asp Tyr Met

1 5 10

<210> 14

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> AA98 antibody light chain CDR2

<400> 14

Ser Gly Ser Gly Thr Asp Phe Thr

1 5

<210> 15

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> AA98 antibody light chain CDR3

<400> 15

His Ser Arg Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr

1 5 10

<210> 16

<211> 217

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> AA98 antibody Fab heavy chain sequence

<400> 16

Glu Val Gln Leu Leu Glu Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Ile Phe Thr Asn Tyr

20 25 30

Trp Ile His Trp Val Lys Asn Arg Ser Gly Asn Gly Leu Glu Trp Ile

35 40 45

Ala Arg Ile Tyr Pro Gly Thr Asp Ile Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Ser Ala Tyr

65 70 75 80

Met Leu Leu Ser Ser Leu Lys Ser Glu Asp Ser Ser Val Tyr Phe Cys

85 90 95

Ala Arg Ser Gly Gly Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro

115 120 125

Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly

130 135 140

Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn

145 150 155 160

Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr

180 185 190

Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser

195 200 205

Thr Lys Val Asp Lys Lys Ile Glu Pro

210 215

<210> 17

<211> 448

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> H13-112 heavy chain

<400> 17

Glu Val Gln Leu Leu Glu Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Lys Phe Thr Asn Tyr

20 25 30

Trp Ile His Trp Val Lys Asn Arg Ser Gly Asn Gly Leu Glu Trp Ile

35 40 45

Ala Arg Ile Tyr Pro Gly Thr Asp Ile Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Ser Ala Tyr

65 70 75 80

Met Leu Leu Ser Ser Leu Lys Ser Glu Asp Ser Ser Val Tyr Phe Cys

85 90 95

Ala Arg Ser Gly Gly Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro

115 120 125

Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly

130 135 140

Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn

145 150 155 160

Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr

180 185 190

Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser

195 200 205

Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro

210 215 220

Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu

245 250 255

Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro

260 265 270

Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala

275 280 285

Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val

290 295 300

Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe

305 310 315 320

Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr

325 330 335

Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu

340 345 350

Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys

355 360 365

Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn

370 375 380

Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys

405 410 415

Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly

420 425 430

Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys

435 440 445

<210> 18

<211> 218

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> H13-112 light chain

<400> 18

Glu Leu Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Ile Ser

20 25 30

Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Tyr Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg

85 90 95

Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Ala Asp Ala Lys Pro Thr Val Ser Ile Phe Pro Pro Ser Arg Glu Gln

115 120 125

Leu Asp Ala Gly Gly Ala Thr Val Val Cys Phe Val Asn Asp Phe Tyr

130 135 140

Pro Arg Glu Ile Asn Val Lys Trp Lys Val Asp Gly Ser Glu Lys Gln

145 150 155 160

Asp Gly Val Leu Glu Ser Ile Thr Asp Gln Asp Ser Lys Asp Asn Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Ser Met Thr Lys Ala Asp Tyr Glu Arg

180 185 190

His Ser Leu Tyr Thr Cys Glu Val Thr His Lys Thr Ser Thr Ala Ala

195 200 205

Ile Val Lys Thr Leu Asn Arg Asn Glu Cys

210 215

<210> 19

<211> 448

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 104-112 heavy chain sequence

<400> 19

Glu Val Gln Leu Leu Glu Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Ile Phe Thr Asn Tyr

20 25 30

Trp Ile His Trp Val Lys Asn Arg Ser Gly Asn Gly Leu Glu Trp Ile

35 40 45

Ala Arg Ile Tyr Pro Gly Thr Asp Glu Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Ser Ala Tyr

65 70 75 80

Met Leu Leu Ser Ser Leu Lys Ser Glu Asp Ser Ser Val Tyr Phe Cys

85 90 95

Ala Arg Ser Gly Gly Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro

115 120 125

Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly

130 135 140

Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn

145 150 155 160

Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr

180 185 190

Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser

195 200 205

Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro

210 215 220

Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu

245 250 255

Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro

260 265 270

Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala

275 280 285

Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val

290 295 300

Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe

305 310 315 320

Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr

325 330 335

Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu

340 345 350

Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys

355 360 365

Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn

370 375 380

Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys

405 410 415

Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly

420 425 430

Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys

435 440 445

<210> 20

<211> 218

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 104-112 light chain sequence

<400> 20

Glu Leu Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Ile Ser

20 25 30

Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Tyr Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg

85 90 95

Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Ala Asp Ala Lys Pro Thr Val Ser Ile Phe Pro Pro Ser Arg Glu Gln

115 120 125

Leu Asp Ala Gly Gly Ala Thr Val Val Cys Phe Val Asn Asp Phe Tyr

130 135 140

Pro Arg Glu Ile Asn Val Lys Trp Lys Val Asp Gly Ser Glu Lys Gln

145 150 155 160

Asp Gly Val Leu Glu Ser Ile Thr Asp Gln Asp Ser Lys Asp Asn Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Ser Met Thr Lys Ala Asp Tyr Glu Arg

180 185 190

His Ser Leu Tyr Thr Cys Glu Val Thr His Lys Thr Ser Thr Ala Ala

195 200 205

Ile Val Lys Thr Leu Asn Arg Asn Glu Cys

210 215

<210> 21

<211> 448

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 104-114 heavy chain sequence

<400> 21

Glu Val Gln Leu Leu Glu Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Ile Phe Thr Asn Tyr

20 25 30

Trp Ile His Trp Val Lys Asn Arg Ser Gly Asn Gly Leu Glu Trp Ile

35 40 45

Ala Arg Ile Tyr Pro Gly Thr Asp Glu Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Ser Ala Tyr

65 70 75 80

Met Leu Leu Ser Ser Leu Lys Ser Glu Asp Ser Ser Val Tyr Phe Cys

85 90 95

Ala Arg Ser Gly Gly Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro

115 120 125

Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly

130 135 140

Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn

145 150 155 160

Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr

180 185 190

Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser

195 200 205

Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro

210 215 220

Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu

245 250 255

Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro

260 265 270

Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala

275 280 285

Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val

290 295 300

Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe

305 310 315 320

Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr

325 330 335

Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu

340 345 350

Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys

355 360 365

Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn

370 375 380

Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys

405 410 415

Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly

420 425 430

Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys

435 440 445

<210> 22

<211> 218

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 104-114 light chain sequence

<400> 22

Glu Leu Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Glu Ser

20 25 30

Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg

85 90 95

Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Ala Asp Ala Lys Pro Thr Val Ser Ile Phe Pro Pro Ser Arg Glu Gln

115 120 125

Leu Asp Ala Gly Gly Ala Thr Val Val Cys Phe Val Asn Asp Phe Tyr

130 135 140

Pro Arg Glu Ile Asn Val Lys Trp Lys Val Asp Gly Ser Glu Lys Gln

145 150 155 160

Asp Gly Val Leu Glu Ser Ile Thr Asp Gln Asp Ser Lys Asp Asn Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Ser Met Thr Lys Ala Asp Tyr Glu Arg

180 185 190

His Ser Leu Tyr Thr Cys Glu Val Thr His Lys Thr Ser Thr Ala Ala

195 200 205

Ile Val Lys Thr Leu Asn Arg Asn Glu Cys

210 215

<210> 23

<211> 448

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> H13-111 heavy chain sequence

<400> 23

Glu Val Gln Leu Leu Glu Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Lys Phe Thr Asn Tyr

20 25 30

Trp Ile His Trp Val Lys Asn Arg Ser Gly Asn Gly Leu Glu Trp Ile

35 40 45

Ala Arg Ile Tyr Pro Gly Thr Asp Ile Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Ser Ala Tyr

65 70 75 80

Met Leu Leu Ser Ser Leu Lys Ser Glu Asp Ser Ser Val Tyr Phe Cys

85 90 95

Ala Arg Ser Gly Gly Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro

115 120 125

Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly

130 135 140

Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn

145 150 155 160

Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr

180 185 190

Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser

195 200 205

Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro

210 215 220

Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu

245 250 255

Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro

260 265 270

Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala

275 280 285

Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val

290 295 300

Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe

305 310 315 320

Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr

325 330 335

Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu

340 345 350

Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys

355 360 365

Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn

370 375 380

Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys

405 410 415

Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly

420 425 430

Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys

435 440 445

<210> 24

<211> 218

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> H13-111 light chain sequence

<400> 24

Glu Leu Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Ile Ser

20 25 30

Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu His Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg

85 90 95

Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Ala Asp Ala Lys Pro Thr Val Ser Ile Phe Pro Pro Ser Arg Glu Gln

115 120 125

Leu Asp Ala Gly Gly Ala Thr Val Val Cys Phe Val Asn Asp Phe Tyr

130 135 140

Pro Arg Glu Ile Asn Val Lys Trp Lys Val Asp Gly Ser Glu Lys Gln

145 150 155 160

Asp Gly Val Leu Glu Ser Ile Thr Asp Gln Asp Ser Lys Asp Asn Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Ser Met Thr Lys Ala Asp Tyr Glu Arg

180 185 190

His Ser Leu Tyr Thr Cys Glu Val Thr His Lys Thr Ser Thr Ala Ala

195 200 205

Ile Val Lys Thr Leu Asn Arg Asn Glu Cys

210 215

<210> 25

<211> 448

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> H13-115 heavy chain sequence

<400> 25

Glu Val Gln Leu Leu Glu Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Lys Phe Thr Asn Tyr

20 25 30

Trp Ile His Trp Val Lys Asn Arg Ser Gly Asn Gly Leu Glu Trp Ile

35 40 45

Ala Arg Ile Tyr Pro Gly Thr Asp Ile Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Ser Ala Tyr

65 70 75 80

Met Leu Leu Ser Ser Leu Lys Ser Glu Asp Ser Ser Val Tyr Phe Cys

85 90 95

Ala Arg Ser Gly Gly Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro

115 120 125

Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly

130 135 140

Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn

145 150 155 160

Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr

180 185 190

Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser

195 200 205

Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro

210 215 220

Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu

245 250 255

Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro

260 265 270

Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala

275 280 285

Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val

290 295 300

Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe

305 310 315 320

Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr

325 330 335

Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu

340 345 350

Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys

355 360 365

Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn

370 375 380

Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys

405 410 415

Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly

420 425 430

Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys

435 440 445

<210> 26

<211> 218

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> H13-115 light chain sequence

<400> 26

Glu Leu Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Glu Thr

20 25 30

Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg

85 90 95

Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Ala Asp Ala Lys Pro Thr Val Ser Ile Phe Pro Pro Ser Arg Glu Gln

115 120 125

Leu Asp Ala Gly Gly Ala Thr Val Val Cys Phe Val Asn Asp Phe Tyr

130 135 140

Pro Arg Glu Ile Asn Val Lys Trp Lys Val Asp Gly Ser Glu Lys Gln

145 150 155 160

Asp Gly Val Leu Glu Ser Ile Thr Asp Gln Asp Ser Lys Asp Asn Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Ser Met Thr Lys Ala Asp Tyr Glu Arg

180 185 190

His Ser Leu Tyr Thr Cys Glu Val Thr His Lys Thr Ser Thr Ala Ala

195 200 205

Ile Val Lys Thr Leu Asn Arg Asn Glu Cys

210 215

<210> 27

<211> 448

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 106-112 heavy chain sequence

<400> 27

Glu Val Gln Leu Leu Glu Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Ile Phe Thr Asn Tyr

20 25 30

Trp Ile His Trp Val Lys Asn Arg Ser Gly Asn Gly Leu Glu Trp Ile

35 40 45

Ala Arg Ile Tyr Pro Gly Thr Asp Ile Thr Arg Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Ser Ala Tyr

65 70 75 80

Met Leu Leu Ser Ser Leu Lys Ser Glu Asp Ser Ser Val Tyr Phe Cys

85 90 95

Ala Arg Ser Gly Gly Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro

115 120 125

Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly

130 135 140

Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn

145 150 155 160

Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr

180 185 190

Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser

195 200 205

Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro

210 215 220

Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu

245 250 255

Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro

260 265 270

Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala

275 280 285

Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val

290 295 300

Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe

305 310 315 320

Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr

325 330 335

Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu

340 345 350

Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys

355 360 365

Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn

370 375 380

Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys

405 410 415

Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly

420 425 430

Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys

435 440 445

<210> 28

<211> 218

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 106-112 light chain sequence

<400> 28

Glu Leu Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Ile Ser

20 25 30

Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Tyr Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg

85 90 95

Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Ala Asp Ala Lys Pro Thr Val Ser Ile Phe Pro Pro Ser Arg Glu Gln

115 120 125

Leu Asp Ala Gly Gly Ala Thr Val Val Cys Phe Val Asn Asp Phe Tyr

130 135 140

Pro Arg Glu Ile Asn Val Lys Trp Lys Val Asp Gly Ser Glu Lys Gln

145 150 155 160

Asp Gly Val Leu Glu Ser Ile Thr Asp Gln Asp Ser Lys Asp Asn Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Ser Met Thr Lys Ala Asp Tyr Glu Arg

180 185 190

His Ser Leu Tyr Thr Cys Glu Val Thr His Lys Thr Ser Thr Ala Ala

195 200 205

Ile Val Lys Thr Leu Asn Arg Asn Glu Cys

210 215

<210> 29

<211> 448

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 106-114 heavy chain sequence

<400> 29

Glu Val Gln Leu Leu Glu Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Ile Phe Thr Asn Tyr

20 25 30

Trp Ile His Trp Val Lys Asn Arg Ser Gly Asn Gly Leu Glu Trp Ile

35 40 45

Ala Arg Ile Tyr Pro Gly Thr Asp Ile Thr Arg Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Ser Ala Tyr

65 70 75 80

Met Leu Leu Ser Ser Leu Lys Ser Glu Asp Ser Ser Val Tyr Phe Cys

85 90 95

Ala Arg Ser Gly Gly Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro

115 120 125

Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly

130 135 140

Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn

145 150 155 160

Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr

180 185 190

Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser

195 200 205

Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro

210 215 220

Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu

245 250 255

Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro

260 265 270

Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala

275 280 285

Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val

290 295 300

Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe

305 310 315 320

Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr

325 330 335

Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu

340 345 350

Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys

355 360 365

Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn

370 375 380

Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys

405 410 415

Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly

420 425 430

Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys

435 440 445

<210> 30

<211> 218

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 106-114 light chain sequence

<400> 30

Glu Leu Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Glu Ser

20 25 30

Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg

85 90 95

Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Ala Asp Ala Lys Pro Thr Val Ser Ile Phe Pro Pro Ser Arg Glu Gln

115 120 125

Leu Asp Ala Gly Gly Ala Thr Val Val Cys Phe Val Asn Asp Phe Tyr

130 135 140

Pro Arg Glu Ile Asn Val Lys Trp Lys Val Asp Gly Ser Glu Lys Gln

145 150 155 160

Asp Gly Val Leu Glu Ser Ile Thr Asp Gln Asp Ser Lys Asp Asn Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Ser Met Thr Lys Ala Asp Tyr Glu Arg

180 185 190

His Ser Leu Tyr Thr Cys Glu Val Thr His Lys Thr Ser Thr Ala Ala

195 200 205

Ile Val Lys Thr Leu Asn Arg Asn Glu Cys

210 215

<210> 31

<211> 448

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> H14-114 heavy chain sequence

<400> 31

Glu Val Gln Leu Leu Glu Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Lys Phe Thr Asn Tyr

20 25 30

Trp Ile His Trp Val Lys Asn Arg Ser Gly Asn Gly Leu Glu Trp Ile

35 40 45

Ala Arg Ile Tyr Pro Gly Asn Asp Glu Thr Arg Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Ser Ala Tyr

65 70 75 80

Met Leu Leu Ser Ser Leu Lys Ser Glu Asp Ser Ser Val Tyr Phe Cys

85 90 95

Ala Arg Ser Gly Gly Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro

115 120 125

Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly

130 135 140

Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn

145 150 155 160

Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr

180 185 190

Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser

195 200 205

Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro

210 215 220

Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu

245 250 255

Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro

260 265 270

Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala

275 280 285

Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val

290 295 300

Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe

305 310 315 320

Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr

325 330 335

Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu

340 345 350

Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys

355 360 365

Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn

370 375 380

Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys

405 410 415

Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly

420 425 430

Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys

435 440 445

<210> 32

<211> 218

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> H14-114 light chain sequence

<400> 32

Glu Leu Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Glu Ser

20 25 30

Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg

85 90 95

Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Ala Asp Ala Lys Pro Thr Val Ser Ile Phe Pro Pro Ser Arg Glu Gln

115 120 125

Leu Asp Ala Gly Gly Ala Thr Val Val Cys Phe Val Asn Asp Phe Tyr

130 135 140

Pro Arg Glu Ile Asn Val Lys Trp Lys Val Asp Gly Ser Glu Lys Gln

145 150 155 160

Asp Gly Val Leu Glu Ser Ile Thr Asp Gln Asp Ser Lys Asp Asn Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Ser Met Thr Lys Ala Asp Tyr Glu Arg

180 185 190

His Ser Leu Tyr Thr Cys Glu Val Thr His Lys Thr Ser Thr Ala Ala

195 200 205

Ile Val Lys Thr Leu Asn Arg Asn Glu Cys

210 215

Claims

1. An isolated anti-CD 146 antibody, antigen-binding fragment thereof, variant thereof or derivative thereof derived from an AA98 antibody and having at least 8 x 10 binding affinity for CD146^-10Affinity of M; optionally, it also has an ability to inhibit the lumenization of endothelial cells of greater than 40%, and/or an ability to inhibit endothelial cell migration of greater than 60%.

2. The isolated anti-CD 146 antibody, antigen-binding fragment thereof, variant thereof or derivative thereof of claim 1, having a substitution mutation of a hydrophilic amino acid residue in I31, S32 and/or L51 of the light chain and/or I28, I57 and/or Y59 of the heavy chain relative to the AA98 antibody; preferably, the hydrophilic amino acid residue is selected from the group consisting of Arg, Asn, Asp, gin, Glu, His, Lys and Tyr; more preferably, the substitution mutation of the light chain is selected from I31E, S32T, L51Y, L51H and/or the substitution mutation of the heavy chain is selected from I28K, I57E, Y59R.

3. The isolated anti-CD 146 antibody, antigen-binding fragment thereof, variant thereof, or derivative thereof of claim 1 or 2, having a CDR sequence with one or 2 conservative substitutions as shown below or based on the sequence shown below:

heavy chain CDR 1: SGYIFTNYW (SEQ ID No.10), and/or

Heavy chain CDR 2: YPGTDITY (SEQ ID No.11), and/or

Heavy chain CDR 3: SGGYWYFDV (SEQ ID No.12), and/or

Light chain CDR 1: ASKSVSISGYSYM (SEQ ID No.13), and/or

Light chain CDR 2: IYLASNL (SEQ ID No.14), and/or

Light chain CDR 3: HSR ELPYTFGG (SEQ ID No.15),

preferably, it comprises said 6 CDRs.

4. The isolated anti-CD 146 antibody, antigen-binding fragment thereof, variant thereof or derivative thereof according to any one of claims 1-3, which heavy chain variable region sequence has at least 95% but not 100% sequence identity to the sequence set forth in SEQ ID No.4 or at least one amino acid substitution compared to the sequence set forth in SEQ ID No.4, and/or which light chain variable region sequence has at least 95% but not 100% sequence identity to the sequence set forth in SEQ ID No.5 or at least one amino acid substitution compared to the sequence set forth in SEQ ID No. 5.

5. The isolated anti-CD 146 antibody, antigen-binding fragment thereof, variant thereof or derivative thereof of any of claims 1-4, wherein the heavy chain amino acid sequence of said anti-CD 146 antibody has at least 95% but not 100% sequence identity to the sequence set forth in SEQ ID No.6 or has at least one amino acid substitution as compared to the sequence set forth in SEQ ID No.6, and/or the light chain amino acid sequence of said anti-CD 146 antibody has at least 95% but not 100% sequence identity to the sequence set forth in SEQ ID No.7 or has at least one amino acid substitution as compared to the sequence set forth in SEQ ID No. 7.

6. The isolated anti-CD 146 antibody, antigen-binding fragment thereof, variant thereof, or derivative thereof of any of claims 1-5, wherein said anti-CD 146 antibodySelected from IgG, IGA, IgM, or IgE, preferably said anti-CD 146 antibody is IgG, more preferably said anti-CD 146 antibody is of the IgG2 subtype; and/or the antigen binding fragment is selected from the group consisting of SDR, CDR, Fv, dAb, Fab₂Fab ', (Fab')2, Fd, scFv, nanobody; and/or the variant sequence is a variant sequence having at least 80% sequence identity to the above antibody or antigen-binding fragment thereof, or a variant sequence which retains the biological activity of the corresponding parent sequence obtained by deletion, substitution and/or addition of one or more amino acid residues; and/or the derivative is selected from the group consisting of chimeric antibodies derived from whole antibodies, humanized antibodies, fully human antibodies, recombinant antibodies, bispecific antibodies, products comprising modified amino acids, products conjugated to polymers, products comprising radiolabels, products comprising fluorescent labels, products comprising enzymatic labels, products comprising chemiluminescent labels, products comprising paramagnetic labels.

7. The isolated anti-CD 146 antibody, antigen-binding fragment thereof, variant thereof, or derivative thereof of any of claims 1-6, wherein the heavy chain amino acid sequence of the anti-CD 146 antibody is selected from SEQ ID nos. 17, 19, 21, 23, 25, 27, 29, or 31 and the light chain amino acid sequence is selected from SEQ ID nos. 18, 20, 22, 24, 26, 28, 30, or 32.

8. An isolated anti-CD 146 antibody, antigen-binding fragment thereof, variant thereof, or derivative thereof of any of claims 1-7.

9. A recombinant vector comprising the coding nucleotide sequence according to claim 8.

10. A cell comprising the coding nucleotide sequence according to claim 8 or the recombinant vector according to claim 8.

11. A composition comprising the isolated anti-CD 146 antibody, antigen-binding fragment thereof, variant thereof or derivative thereof of any one of claims 1-7, and/or the encoding nucleotide sequence of claim 8, and/or the recombinant vector of claim 9, and/or the cell of claim 10.

12. A method of producing the isolated anti-CD 146 antibody, antigen-binding fragment thereof, variant thereof, or derivative thereof of any of claims 1-7, comprising the steps of: expressing the cell of claim 10 under culture conditions suitable for expression of the anti-CD 146 antibody, antigen binding fragment thereof, variant thereof or derivative thereof, optionally isolating, purifying the resulting product.

13. Use of the isolated anti-CD 146 antibody, antigen-binding fragment thereof, variant thereof or derivative thereof according to any one of claims 1-7, the coding nucleotide sequence according to claim 8, the recombinant vector according to claim 9, the cell according to claim 10 and/or the composition according to claim 11 for the manufacture of a medicament for interfering with angiogenesis and progression.

14. Use of the isolated anti-CD 146 antibody, antigen-binding fragment thereof, variant thereof or derivative thereof according to any one of claims 1-7, the coding nucleotide sequence according to claim 8, the recombinant vector according to claim 9, the cell according to claim 10 and/or the composition according to claim 11 for the manufacture of a medicament for treating a tumor and/or a vascular-related disease and/or an inflammatory disease in a subject.

15. Use of the isolated anti-CD 146 antibody, antigen-binding fragment thereof, variant thereof or derivative thereof according to any one of claims 1-7, the coding nucleotide sequence according to claim 8, the recombinant vector according to claim 9, the cell according to claim 10 and/or the composition according to claim 11 for the preparation of an agent for diagnosing a tumor and/or a vascular-related disease and/or an inflammatory disease in a subject.

16. The use according to any one of claims 13-15, wherein the tumor is a benign or malignant tumor, preferably the tumor is selected from melanoma, pancreatic cancer, colorectal cancer, breast cancer, lung cancer, nasopharyngeal cancer, liver cancer, gastric cancer, esophageal cancer, breast cancer, kidney cancer, laryngeal cancer, gallbladder cancer, bladder cancer, prostate cancer, cervical cancer, breast cancer, ovarian cancer, uterine cancer, head and neck cancer, skin cancer, thyroid cancer, tongue cancer, thymus cancer, cystic brain tumor, glioma, lymphoma; and/or the vascular-related disease and/or inflammatory disease is selected from age-related macular degeneration, proliferative diabetic retinopathy, retinopathy of prematurity, glaucoma, macular edema, choroidal neovascular disease, retinal vein occlusion, colorectal inflammation, multiple sclerosis, atherosclerosis, arteriolar sclerosis, arteriolar inflammation, systemic vasculitis, gastritis, colitis, pancreatitis, arthritis, diabetic inflammation, inflammatory bowel disease, nephritis, hepatitis, systemic lupus erythematosus, scleroderma, dermatomyositis, thyroid autoimmune disease, alzheimer's disease, parkinson's disease or amyotrophic lateral sclerosis, inflammatory central nervous system disease, diabetes, foot ulcer, pulmonary hypertension, ischemic cardiomyopathy, ischemic brain disease, heart failure and acute hindlimb ischemia.