KR20200015921A - Anti-VEGFR Antibodies and Uses thereof - Google Patents

Abstract

The present invention relates to an antibody or antigen-binding fragment thereof that binds human vascular endothelial growth factor receptor 2 (VEGFR-2). The invention also provides methods of inhibiting VEGFR-2-mediated signaling in a subject in need thereof, a disease caused by or associated with VEGFR-2 activity and / or signaling in a diseased and diseased subject, and / Or a method of treating a disease, a method of treating a tumor in a subject with a tumor, a method of inhibiting cell proliferation of endothelial cells in a subject in need thereof, and a method of detecting human vascular endothelial growth factor receptor in a sample. .

Description

Anti-VEGFR Antibodies and Uses thereof

The present invention relates to an antibody or antigen-binding fragment thereof specific for human vascular endothelial growth factor receptor (VEGFR) and its use.

The function of the signal transduction pathway by vascular endothelial growth factor (VEGF) promotes angiogenesis. Angiogenesis refers to the formation of new capillaries from the original blood vessels. Malformations of angiogenesis control are associated with pathogenic mechanisms of many diseases and are also characteristic of many types of cancer. The major role involved in the signal transduction pathway is the protein belonging to the vascular endothelial growth factor family and its receptors. Activation of these signal transduction pathways activates complex network downstream and promotes growth, migration and survival of vascular endothelial cells. Such signaling transduction pathways are believed to be directly associated with tumor angiogenesis, so inhibition of signal transduction pathways is important for the regulation of tumor angiogenesis.

The vascular endothelial growth factor family includes a group of homologous dimeric glycoproteins with highly conserved sequences. Members in the vascular endothelial growth factor family are activated by linking two 24 kDa single-stranded molecules through sulfide bonds. Known members within the vascular endothelial growth factor family include EGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PlGF), where VEGF-A plays an important role in angiogenesis Is first discovered and usually fully studied.

When vascular endothelial growth factor is bound to the vascular endothelial growth factor receptor (VEGF receptor, VEGFR), the vascular endothelial growth factor receptor forms dimers and phosphorylates with each other. There are seven Ig-like domains located in the extracellular region of the vascular endothelial growth factor receptor. Generally domains 1-3 are responsible for binding to ligands, and domains 4-7 are responsible for dimerization, phosphorylation and downstream signal transduction.

Vascular endothelial growth factor receptors belong to the tyrosine kinase (TK) family and are key members in the signal transduction pathway. It transduces extracellular signals into cells that induce cell growth, proliferation and anti-apoptosis. There are three types of vascular endothelial growth factor receptors: VEGFR-1 (also known as flt-1), VEGFR-2 (also known as KDR / flk-1) and VEGFR-3 (also known as flt-4) ). VEGF-A binds to VEGFR-1 and VEGFR-2, both receptors are expressed in vascular endothelial cells, and signals from vascular endothelial cells are mainly transduced through VEGFR-2. Although the binding affinity between VEGFR-1 and ligand is 10-fold stronger than that between VEGFR-2 and ligand, the kinase activity of VEGFR-1 is weaker. In another embodiment, VEGFR-3 is mainly expressed in lymphatic endothelial cells.

In addition to intact peripheral vascular endothelial cell proliferation, tumor angiogenesis is formed by attracting vascular endothelial precursor cells to the tumor or its peripheral region under the control of VEGFR-2, and then vascular endothelial precursor cells are vascular endothelial. It is also known to differentiate into cells. Since angiogenesis with respect to vascular endothelial growth factor receptors occurs only in the wound repair and menstrual cycles of women under normal physiological conditions, the effect of blocking signal transduction pathways by vascular endothelial growth factor receptors is normal physiological function. Limited to. As a result, inhibition of signal transduction pathways by vascular endothelial growth factor receptors is an important regulatory inhibition of tumor angiogenesis and eventually leads to tumor cell death. It has been reported that vascular endothelial growth factor is overexpressed in various spheroid tumors such as colorectal cancer, breast cancer, prostate cancer, and lung cancer.

Thus, there is a need to develop new approaches for blocking signal transduction pathways by vascular endothelial growth factor receptors.

The present invention provides antibodies or antigen-binding fragments thereof that bind to human vascular endothelial growth factor receptor 2 or human vascular endothelial growth factor receptor 2 fragments. The antibodies according to the invention are useful for inhibiting VEGFR-2-mediated signaling and for treating diseases and conditions caused by or associated with VEGFR-2 activity and / or signaling. Antibodies of the invention are also useful for inhibiting cell proliferation of endothelial cells.

The present invention provides a method of inhibiting VEGFR-2-mediated signaling in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising an antibody as described above or an antigen-binding fragment thereof to provide.

The present invention relates to VEGFR-2 activity and / or signaling in a diseased and / or diseased subject, comprising administering to the subject a pharmaceutical composition comprising an antibody as described above or an antigen-binding fragment thereof Methods of treating and / or diseases caused by or associated with the same are provided.

The present invention provides a method of treating a tumor in a subject having a tumor, comprising administering to the subject a pharmaceutical composition comprising an antibody as described above or an antigen-binding fragment thereof.

The present invention provides a method of inhibiting cell proliferation of endothelial cells in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising an antibody as described above or an antigen-binding fragment thereof.

The present invention provides a method for detecting human vascular endothelial growth factor receptor in a sample, including contacting the sample with an antibody or antigen-binding fragment thereof as mentioned above.

The invention is described in detail in the following paragraphs. Other features, objects, and advantages of the invention can be found in the description and claims.

1 shows the ELISA results of the binding affinity assay of the antibodies of the invention to mouse VEGFR-2, human VEGFR-1 or VEGFR-3.
Figure 2 shows the ELISA results of the binding domain mapping (domains 1 to 3 and domains 4 to 7) of the antibodies of the present invention.
3A, 3B and 3C show ELISA results of binding domain mapping of antibodies of the invention.
4 shows ELISA results of epitope mapping of antibodies of the invention.
5 shows HUVEC proliferation inhibition assay of the antibodies of the invention.
6 shows the results of antibody internalization of antibodies of the invention into HUVECs by flow cytometry.
7 shows the results of antibody internalization observed by the DeltaVision Microscopy Imaging System.
8 shows the results of size-exclusion chromatography analysis of antibody-drug conjugates (ADCs) according to the present invention.
9 shows the results of electrophoretic analysis of antibody-drug conjugates according to the present invention.
10 shows the results of antibody internalization of ADCs according to the invention into HUVECs by flow cytometry.
11 shows the HUVEC proliferation inhibition assay of the ADC of the present invention.
12 shows quantification analysis of chimeric antigen receptor T cells (CAR-T) according to the present invention by flow cytometry.
Figure 13 shows cytotoxicity analysis of chimeric antigen receptor T cells according to the present invention.
14 shows the efficiency of labeling antibodies according to the invention with radioisotopes.
Figure 15 shows the results of tumor detection in NanoSPECT / CT of HT-29 xenograft mice with radio-labeled antibodies according to the present invention.
16 shows alignment of VL segments of humanized, mouse, and human antibodies. M: 322 A6; Hd: Hu322B1HdH.
17 shows alignment of V _H segments of humanized, mouse, and human antibodies. M: 322 A6; Hu: human template IGHV1-46 ^* 01 F; HuB 1: Hu322B 1 HdH.

The present invention provides antibodies or antigen-binding fragments thereof that bind to human vascular endothelial growth factor receptor 2 or human vascular endothelial growth factor receptor 2 fragments.

In particular, the antibody or antigen-binding fragment thereof according to the invention is specifically bound to an epitope in human vascular endothelial growth factor receptor 2 or a fragment thereof; Wherein the human vascular endothelial growth factor receptor 2 has the amino acid sequence of SEQ ID NO: 1 and the epitope is:

A leucine residue at position 606 of SEQ ID NO: 1, an aspartic acid residue at position 607, an arginine residue at position 647, a lysine residue at position 648, and a threonine residue at position 649; or

A serine residue at position 711 of SEQ ID NO: 1, a lysine residue at position 716, an aspartic acid residue at position 717, and an arginine residue at positions 725 and 726.

Antibodies according to the invention may be full length (eg IgG1 or IgG4 antibodies) or may comprise only antigen-binding portions (eg Fab, F (ab ′) ₂ or scFv fragments), as required Can be modified to affect functionality.

Antibodies or antigen-binding fragments thereof according to the present invention are also known as human VEGFR-2, also known as KDR or flk-1. It specifically binds to VEGFR-2 and is a receptor for vascular endothelial growth factor and is activated by phosphorylating each other dimer when forming a dimer and binding to its ligand. VEGFR-2 comprises seven Ig-like domains located in the extracellular domain, wherein domains 1-3 are responsible for binding to the ligand and domains 4-7 are dimerized, phosphorylated and downstream It is responsible for signal transduction, and preferably, the antibody or antigen-binding fragment thereof according to the invention binds to domains 6 and 7 of the extracellular region of VEGFR-2.

The present invention includes anti-VEGFR-2 antibodies and antigen-binding fragments thereof that bind to monomeric or dimeric VEGFR-2 molecules with high affinity.

In one preferred embodiment of the invention, VEGFR-2 has the amino acid sequence of SEQ ID NO: 1; Domain 6 of the extracellular region of VEGFR-2 has the amino acid sequence of SEQ ID NO: 2; Domain 7 of the extracellular region of VEGFR-2 has the amino acid sequence of SEQ ID NO: 3.

Various techniques known to those skilled in the art can be used to determine whether an antibody specifically binds to one or more amino acids in a polypeptide or protein. Exemplary techniques are described, for example, in conventional cross-blocking assays, such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), alanine Alanine scanning mutational analysis, peptide scanning blot analysis (Reineke, 2004, Methods Mol Biol 248: 443-463), and peptide cleavage analysis. In addition, methods such as epitope ablation, epitope extraction and chemical modification of antigens can be used (Tomer, 2000, Protein Science 9: 487-496). Another method that can be used to identify amino acids in a polypeptide to which an antibody specifically binds is a hydrogen / deuterium exchange detected by mass spectroscopy. In general terms, the hydrogen / deuterium exchange method includes binding the antibody to deuterium-labeled protein after deuterium-labeling the protein of interest. Next, the protein / antibody complex is transferred to water so that hydrogen-deuterium exchange occurs at all residues except residues protected by antibodies (which remain deuterium-labeled). After dissociation of the antibody, the target protein has been subjected to protease cleavage and mass spectrometry analysis to reveal the deuterium-labeled residue corresponding to the specific amino phase with which the antibody interacts. Reference Examples [Ehring (1999) Analytical Biochemistry 267 (2): 252-259; Engen and Smith (2001) Anal. Chem. 73: 256A-265A.

In one preferred embodiment of the invention, the epitope of VEGFR-2 comprises a leucine residue at position 606 of SEQ ID NO: 1, an aspartic acid residue at position 607, an arginine residue at position 647, a lysine residue at position 648 And threonine residues at position 649.

In another preferred embodiment of the invention, the epitope of VEGFR-2 comprises a serine residue at position 711 of SEQ ID NO: 1, a lysine residue at position 716, an aspartic acid residue at position 717, and position 725 and Arginine residue at 726.

The invention further includes anti-VEGFR antibodies that specifically bind to the same epitope. Similarly, the present invention also encompasses anti-VEGFR-2 antibodies that compete for binding to VEGFR-2 with any of the specific exemplary antibodies described herein.

By using conventional methods known in the art, one can readily determine whether an antibody specifically binds to or competes with binding for the same epitope as the reference anti-VEGFR-2 antibody. For example, to determine whether a test antibody binds to the same epitope as the reference anti-VEGFR-2 antibody of the invention, the reference antibody is a VEGFR-2 protein (eg, a VEGFR-2 extracellular domain or cell surface-expressed VEGFR). Soluble portion of -2). Next, the ability of the test antibody to bind VEGFR-2 molecules is analyzed. If the test antibody is able to bind VEGFR-2 after saturation binding with the reference anti-VEGFR-2 antibody, it can be concluded that the test antibody binds to a different epitope from the reference anti-VEGFR-2 antibody. On the other hand, if the test antibody cannot bind to the VEGFR-2 molecule after saturation binding with the reference anti-VEGFR-2 antibody, then the test antibody is the same epitope as the epitope bound by the reference anti-VEGFR-2 antibody of the present invention. Can be combined with Subsequently, to determine if the lack of observed binding of the test antibody is actually due to binding to the same epitope as the reference antibody, or to determine if steric blocking (or another phenomenon) is the cause of the defect of the observed binding. Phosphorus experiments (eg, peptide mutations and binding assays) can be performed. This class of experiments can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody-binding assay available in the art. According to certain embodiments of the invention, for example, when a 1-, 5-, 10-, 20- or 100-fold excess of one antibody is measured in a competitive binding assay, binding of the other is at least If up to 50% but preferably 75%, 90% or also 99% is inhibited, the two antibodies bind to the same (or overlap) epitope. Alternatively, two antibodies are believed to bind to the same epitope if basically all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other. When only a subset of amino acid mutations that reduce or eliminate binding of one antibody reduces or eliminates binding of the other, the two antibodies are considered to have "overlapping epitopes."

In order to determine whether an antibody competes for binding with a reference anti-VEGFR-2 antibody, the above-described binding method is performed in two directions: In the first direction, the VEGFR-2 protein (eg, VEGFR) is subjected to saturation conditions. Binding to the test antibody to the VEGFR-2 molecule after binding to the -2 extracellular domain and the cell surface-expressed soluble portion of VEGFR-2). In the second direction, binding of the reference antibody to the VEGFR-2 molecule is analyzed after allowing the test antibody to bind to the VEGFR-2 molecule under saturation conditions. In both directions, if only the first (saturated) antibody can bind to the VEGFR-2 molecule, it is concluded that the test antibody and the reference antibody compete for binding to VEGFR-2. As will be appreciated by one of ordinary skill in the art, an antibody competing for binding to a reference antibody does not necessarily bind to the same epitope as the reference antibody, but rather three-dimensionally binds the binding of the reference antibody by binding overlapping or adjacent epitopes. You can block.

As used herein, the term “antibody” refers to any antigen-binding molecule or molecule comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (eg, VEGFR-2). Means complex. The term "antibody" includes four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by sulfide bonds, as well as their multimers (eg, IgM). Globulin molecules. Each heavy chain comprises a heavy chain variable region (abbreviated as HCVR or V _H ) and a heavy chain constant region. The heavy chain constant region comprises three domains, C _H1 , C _H2 and C _H3 . Each light chain comprises a light chain variable region (abbreviated as LCVR or V _L ) and a light chain constant region. The light chain constant region comprises one domain (C _L1 ). The V _H and V _L regions may be further subdivided into regions of hypervariability, referred to as complementarity determining regions (CDRs) interspersed with regions that are more conserved, referred to as framework regions (FR). Each VH and VL consists of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention, the FR of the anti-VEGFR antibody (or antigen-binding portion thereof) may be identical to the human germline sequence, or may be naturally or artificially modified. Amino acid consensus sequences can be defined based on side-by-side analysis of two or more CDRs.

The term "antibody" as used herein also includes antigen-binding fragments of the entire antibody molecule. As used herein, the term “antigen-binding portion” of an antibody, and “antigen-binding fragment” of an antibody, and the like, are any naturally occurring, obtainable by enzyme, or synthetic, that specifically bind to an antigen to form a complex. Genetically engineered polypeptides or glycoproteins. Antigen-binding fragments of antibodies can be prepared using any suitable standard technique, including, for example, manipulation and expression of DNA encoding antibody variable and any constant domains, such as proteolytic digestion or recombinant genetic engineering techniques. From the antibody molecule, it can be derived. Such DNA is known and / or can be readily purchased or synthesized, for example, from commercial sources, DNA libraries (including, eg, phage-antibody libraries). DNA can be sequenced and manipulated chemically or by using molecular biology techniques to, for example, arrange one or more variable and / or constant domains in a suitable form, introduce codons, generate cystine residues, or generate amino acids. To modify, add or delete.

Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F (ab ') ₂ fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; And (vii) a minimal recognition unit consisting of amino acid residues representing a high variable region of a restricted FR3-CDR3-FR4 peptide or antibody (eg, an isolated complementarity determining region (CDR), such as a CDR3 peptide). It includes. Other engineered molecules such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies ( minibody, nanobodies (e.g. monovalent nanobodies, divalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains are used herein. Binding Fragments "are also included.

Antigen-binding fragments of antibodies typically comprise at least one variable domain. The variable domain may be of any size or amino acid combination and will generally include at least one CDR in or adjacent to a frame having one or more framework sequences. In antigen-binding fragments having a V _H domain associated with a V _L domain, the V _H and V _L domains can be positioned relative to one another in any suitable arrangement. For example, the variable region may be dimer or contain V _H -V _H , V _H -V _L or V _L -V _L dimers. Alternatively, the antigen-binding fragment of the antibody may contain monomeric V _H or V _L domains.

In certain embodiments, the antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary forms of variable and constant domains that can be found in antigen-binding fragments of antibodies of the invention include: (i) V _H -C _H1 ; (ii) V _H -C _H2 ; (iii) V _H -C _H3 ; (iv) V _H -C _H1 -C _H2 ; (v) V _H -C _H1 -C _H2 -C _H3 , (vi) V _H -C _H2 -C _H3 ; (vii) V _H -C _L ; (viii) V _L -C _H1 ; (ix) V _L -C _H2 ; (x) V _L -C _H3 ; (xi) V _L -C _H1 -C _H2 ; (xii) V _L -C _H1 -C _H2 -C _H3 ; (xiii) V _L -C _H2 -C _H3 ; And (xiv) V _L -C _L. In any form of variable and constant domains, including any of the exemplary forms listed above, the variable and constant domains may be directly linked to another one or linked by a full or partial hinge or linker region. The hinge region is at least two (eg, 5, 10, 15, 20, 40, 60 or more) amino acids that create a flexible or semi-flexible link between adjacent variable and / or constant domains within a single polypeptide molecule. Can be made. Moreover, the antigen-binding fragments of the antibodies of the present invention may comprise the variables listed above in non-covalent association with another and / or one or more monomeric V _H or V _L domains (eg, by sulfide bond (s)) and Homo-dimer or hetero-dimer (or other multimer) of any of the constant domain forms.

Like the entire antibody molecule, the antigen-binding fragment can be monospecific or multispecific (eg bispecific). Multispecific antigen-binding fragments of an antibody will typically comprise at least two different variable domains, where each variable domain can specifically bind to a separate antigen or to different epitopes on the same antigen. . Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adjusted for use in the context of antigen-binding fragments of the antibodies of the invention using conventional techniques available in the art. have.

 Preferably, the antibody or antigen-binding fragment thereof according to the invention is a mammalian antibody.

The term "mammal antibody" as used herein is intended to include antibodies having variable and constant regions derived from mammalian germline immunoglobulin sequences. Mammalian antibodies of the invention are directed to mammalian germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) in CDRs and in particular CDR3. Amino acid residues that are not encoded by

As used herein, the term “recombinant mammalian antibody” uses recombinant expression vectors transfected into any mammalian antibody, such as a host cell (described further below), which is prepared, expressed, produced or isolated by recombinant means. Antibody expressed from a recombinant, combined mammalian antibody library (described further below), an antibody isolated from an animal (eg, a mouse) into which a mammalian immunoglobulin gene is introduced, or a mammal to another DNA sequence. It is intended to include antibodies which have been prepared, expressed, produced or isolated by any other means including splicing of animal immunoglobulin gene sequences. Such recombinant mammalian antibodies have variable and constant regions derived from mammalian germline immunoglobulin sequences. However, in certain embodiments, such recombinant mammalian antibodies are subjected to in vitro mutagenesis (or somatic mutagenesis in vivo when animal transgenic with a human Ig sequence is used), and accordingly, The amino acid sequences of the V _H and V _L regions are sequences that may not naturally exist in the mammalian antibody germline repertoire in vivo, as derived from and associated with the human germline V _H and V _L sequences.

Mammalian antibodies, such as human antibodies, can exist in two forms that are associated with hinge heterogeneity. In one form, the immunoglobulin molecule contains approximately four hundred-160 kDa stable four chain constructs in which the dimers are held together by interchain heavy chain sulfide bonds. In the second form, the dimers are not formed via interchain sulfide bonds, but are formed by light and heavy chains (semi-antibodies) covalently bound to molecules of about 75-80 kDa. This form was extremely difficult to separate even after affinity purification.

The anti-VEGFR-2 antibodies disclosed herein may comprise one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains relative to the corresponding germline sequences from which the antibody is derived. have. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with, for example, germline sequences available from public antibody sequence databases. The present invention includes antibodies derived from any of the amino acid sequences disclosed herein and antigen-binding fragments thereof, wherein one or more amino acids in one or more framework and / or CDR regions of the germline sequence from which the antibody is derived. Mutated to the corresponding residue (s), mutated to the corresponding residue (s) of another mammalian germline sequence, or mutated with conservative amino acid substitutions of the corresponding germline residue (s). Is referred to as "germline mutation"). Starting with the heavy and light chain variable region sequences disclosed herein, one of ordinary skill in the art can readily produce various antibodies and antigen-binding fragments comprising one or more individual germline mutations or combinations thereof. In certain embodiments, framework and / or CDR residues in the V _H and / or V _L domains are mutated back to those residues found in the original germline sequence from which the antibody is derived. In other embodiments, only mutated residues found within certain residues, eg, the first eight amino acids of FR1 or within the last eight amino acids of FR4, or only those found within CDR1, CDR2, or CDR3 The mutated residue is mutated back to the original germline sequence. In other embodiments, one or more of the framework and / or CDR residue (s) are mutated to corresponding residue (s) of a different germline sequence (ie, a germline sequence different from the germline sequence from which the antibody was originally derived). Moreover, an antibody of the invention may contain any combination of two or more germline mutations within the framework and / or CDR regions, eg, where certain individual residues are directed to corresponding residues of a particular germline sequence. While mutated, certain other residues other than the original germline sequence are retained or mutated to corresponding residues of the different germline sequences. Obtained, antibodies and antigen-binding fragments containing one or more germline mutations may have one or more desired properties, such as improved binding specificity, increased binding affinity, improved or improved antagonistic or potent biological properties (if desired). , Reduced immunogenicity and the like can be easily tested. Antibodies and antigen-binding fragments obtained in this general manner are included within the present invention.

The invention also encompasses anti-VEGFR-2 antibodies comprising variants of any of the V _H , V _L , and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the invention provides, for example, 10 or less, 8 or less, 6 or less, 4 compared to any of the V _H , V _L , and / or CDR amino acid sequences disclosed herein. Anti-VEGFR-2 antibodies with V _H , V _L , and / or CDR amino acid sequences having conservative amino acid substitutions, such as or less than that.

The term "substantially homologous" or "substantially identical" when referring to a nucleic acid or fragment thereof is any as discussed below when optimally aligned with an appropriate nucleotide insertion or deletion with another nucleic acid (or complementary strand thereof). Nucleotide at at least about 95%, and more preferably at least about 96%, 97%, 98% or 99% of nucleotide bases, as measured by known sequence homology algorithms of, e.g., FASTA, BLAST or Gap. Indicates that sequence homology is present. A nucleic acid molecule having substantial homology with a reference nucleic acid molecule may, in certain instances, encode a polypeptide having an amino acid sequence that is the same or substantially similar to the polypeptide encoded by the reference nucleic acid molecule.

As applied to a polypeptide, the term “substantial similarity” or “substantially similar” means that at least 95 of the two peptide sequences are optimally aligned by program GAP or BESTFIT using default gap weights. It is meant to share% sequence homology, more preferably at least 98% or 99% sequence homology. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (eg, charge or hydrophobicity). Generally conservative amino acid substitutions will not substantially change the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the degree of percent sequence homology or similarity can be adjusted upward to correct the conservative nature of the substitution. Means for making such adjustments are known to those skilled in the art. Examples of groups of amino acids having side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains: cystine and methionine. Preferred conservative amino acid substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, conservative substitutions are described in Gonnet et al. (1992) Science 256: 1443-1445 Any change with a positive value in the PAM250 log-likelihood matrix. A "medium conservative" substitution is any change with non-negative values in the PAM250 log-probable matrix.

Sequence similarity for polypeptides, also referred to as sequence homology, is typically measured using sequence analysis software. Protein analysis software is comparable to similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software can be used to determine sequence homology or sequence homology between closely related polypeptides, such as cognate polypeptides from different organism species or between wild type proteins and mutants thereof. Programs that can be used together, such as Gap and Bestfit. Polypeptide sequences can also be compared using FASTA using difold or recommended parameters, which is a program in GCG Version 6.1. FASTA (eg, FASTA2 and FASTA3) provide alignment and percent sequence homology of the region of best overlap between the questionable sequence and the search sequence (Pearson (2000) supra). Another preferred algorithm when comparing the sequences of the invention with databases containing large amounts of sequences from different organisms is the computer program BLAST, in particular BLASTP or TBLASTN, using default parameters. See, eg, Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1997) Nucleic Acids Res. 25: 3389-402. Each of which is incorporated herein by reference.

In one preferred embodiment of the invention, the antibody or antigen-binding fragment thereof comprises the complementarity determining region of the heavy chain variable region and the complementarity determining region of the light chain variable region, wherein the complementarity determining region of the heavy chain variable region is CDRH1, A CDRH2 and CDRH3 region, the complementarity determining region of the light chain variable region comprises a CDRL1, CDRL2 and CDRL3 region,

The CDRH1 region comprises the amino acid sequence of SEQ ID NO: 4, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence homology;

The CDRH2 region comprises the amino acid sequence of SEQ ID NO: 5, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence homology;

The CDRH3 region comprises the amino acid sequence of SEQ ID NO: 6, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence homology;

The CDRL1 region comprises the amino acid sequence of SEQ ID NO: 7 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence homology;

The CDRL2 region comprises the amino acid sequence of SEQ ID NO: 8, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence homology;

The CDRL3 region comprises the amino acid sequence of SEQ ID NO: 9, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence homology.

In another preferred embodiment of the invention, the antibody or antigen-binding fragment thereof comprises the complementarity determining region of the heavy chain variable region and the complementarity determining region of the light chain variable region, wherein the complementarity determining region of the heavy chain variable region is Comprising the CDRH1, CDRH2 and CDRH3 regions, the complementarity determining regions of the light chain variable regions comprise the CDRL1, CDRL2 and CDRL3 regions,

The CDRH1 region comprises the amino acid sequence of SEQ ID NO: 10, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence homology;

The CDRH2 region comprises the amino acid sequence of SEQ ID NO: 11 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence homology;

The CDRH3 region comprises the amino acid sequence of SEQ ID NO: 12, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence homology;

The CDRL1 region comprises the amino acid sequence of SEQ ID NO: 13, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence homology;

The CDRL2 region comprises the amino acid sequence of SEQ ID NO: 14, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence homology;

The CDRL3 region comprises the amino acid sequence of SEQ ID NO: 15, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence homology.

In one preferred embodiment of the invention, the antibody 322A6 or antigen-binding fragment thereof has an amino acid sequence of SEQ ID NO: 17 or a substantial portion thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence homology. And heavy chain variable regions comprising similar sequences. Preferably, the heavy chain variable region is encoded by the nucleic acid sequence of SEQ ID NO: 16. 322A6 or an antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 19 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence homology do. Preferably, the light chain variable region is encoded by the nucleic acid sequence of SEQ ID NO: 18.

In one preferred embodiment of the invention, the antibody 12A6 or antigen-binding fragment thereof has an amino acid sequence of SEQ ID NO: 21 or a substantial portion thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence homology. And heavy chain variable regions comprising similar sequences. Preferably, the heavy chain variable region is encoded by the nucleic acid sequence of SEQ ID NO: 20. 12A6 or an antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 23 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence homology do. Preferably, the light chain variable region is encoded by the nucleic acid sequence of SEQ ID NO: 22.

In another embodiment, the antibody according to the invention is preferably a humanized antibody. A “humanized antibody” includes a human framework region (FR) sequence in which an antibody, such as a CDR from a rodent antibody, from one species is transferred from the heavy and light variable chains of the rodent antibody into the human heavy and light variable domains. , Recombinant protein. The constant domains of antibody molecules are derived from those of human antibodies.

In order to improve the binding affinity of the humanized antibodies according to the invention, some amino acid residues in the human framework region may be selected from the species of the CDRs; For example by corresponding amino acid residues in rodents. Preferably, the humanized antibody Hu322B1HdH or antigen-binding fragment thereof has an amino acid sequence of SEQ ID NO: 25 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence homology. Heavy chain variable region comprising. Preferably, the heavy chain variable region is encoded by the nucleic acid sequence of SEQ ID NO: 24. Hu322B1HdH or an antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 27 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence homology do. Preferably, the light chain variable region is encoded by the nucleic acid sequence of SEQ ID NO: 26.

Preferably, the antibody according to the invention is a monoclonal antibody.

Antibodies of the invention can be monospecific, bi-specific, or multispecific. Multispecific antibodies may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. Anti-VEGFR-2 antibodies of the invention are associated with or co-expressed with another functional molecule, eg, another peptide or protein. For example, an antibody or fragment thereof may be operatively linked (eg, by chemical bonding, genetic fusion, and non-covalent association, etc.) to one or more other molecular bodies, such as another antibody or antibody fragment, to provide a second binding specificity. Having bi-specific or multispecific antibodies. For example, in the present invention, one arm of immunoglobulin is specific for human VEGFR-2 or a fragment thereof and the other arm of immunoglobulin is specific for a second therapeutic target or conjugated to a therapeutic moiety. And bispecific antibodies that are gated.

In one preferred embodiment of the invention, the antibody or antigen-binding fragment thereof is conjugated with a therapeutic agent.

As used herein, the term “therapeutic agent” refers to a cytostatic agent or cytotoxic agent or an isotope-chelating agent having a corresponding radioisotope. Examples of cytostatic agents or cytotoxic agents include, but are not limited to, antimetabolites (eg, fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate, Leucovorin, hydroxyurea, thioguanine (6-TG), mercaptopurine (6-MP), cytarabine, pentostatin, fluda Lavaphosphine (fludarabine phosphate), cladribine (2-CDA), asparaginase, asciginase (gemcitabine), capecitibine, azathioprine, cytosine methotrexate (cytosine) methotrexate, trimethoprim, pyrimethamine, or pemetrexed); Alkylating agents (e.g. cmelphalan, chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine, tax Semustine, streptozocin, dacarbazine, mitomycin C, cyclophosphamide, mechlorethamine, uramustine, diblo Dibromomannitol, tetranitrate, procarbazine, altretamine, mitozolomide, or temozolomide); Alkylation-like agents (eg, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, or preplatin); DNA minor groove alkylating agents (eg, duocarmycins such as CC-1065, and any analogs or derivatives thereof; pyrrolobenzodiazapenes, or analogs or derivatives thereof); Anthracyclines (eg, daunorubicin, doxorubicin, epirubicin, idarubicin, or valrubicin); Antibiotics (e.g., dactinomycin, bleomycin, mithramycin, anthramycin, anthracyycin, streptozotocin, gramicidin D, mitomycin (Eg mitomycin C); calicheamicins; antimitotic agents (eg, maytansinoids (eg, DM1, DM3, and DM4)), Auristatin (e.g., monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF)), dolastatins, cryptophycins, vinca alkaloids Vinca alkaloids (e.g. vincristine, vinblastine, vindesine, vinorelbine), taxanes (e.g. paclitaxel, docetaxel, Or noble taxanes, tubulysin, and colchicine); Topoisomerase inhibitors (e.g., irinotecan, topotecan, camptothecin, etoposide, teniposide, amsacrine, or mitoxantrone ( mitoxantrone)); HDAC inhibitors (eg vorinostat, romidepsin, chidamide, panobinostat, or belinostat); Proteasome inhibitors (eg, peptidyl boronic acid); As well as radioactive isotopes such as At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ^{212 or 213} , P ³² and Lu ¹⁷⁷ . Examples of isotope-chelating agents include, without limitation, ethylenediaminetetraacetic acid (EDTA), diethylenetriamine-N, N, N ', N ", N" -pentaacetate (DTPA), 1,4,7, 10-tetraazacyclododecane-N, N ', N ", N"'-tetraacetate (DOTA), 1,4,7,10-tetrakis (2-hydroxypropyl) -1,4,7, 10-tetraazacyclododecane (THP), triethylenetetraamine-N, N, N ', N ", N"', N "'-hexaacetate (TTHA), 1,4,7,10-tetraaza Cyclododecane-N, N ', N ", N"'-tetrakis (methylenephosphonate) (DOTP), and mercaptoacetyltriglycine (MAG3).

In one preferred embodiment of the invention, the antibody or antigen-binding fragment thereof can be produced using any number of expression systems, including prokaryotic and eukaryotic expression systems. In some embodiments, the expression system is mammalian cell expression, such as hybridomas, or CHO cell expression systems. Many such systems are widely available from commercial suppliers. In embodiments in which the antibody comprises both V _H and V _L regions, the V _H and V _L regions can be expressed using a single vector, eg, in a di-cistronic expression unit, or Can be expressed under the control of different promoters. In other embodiments, the V _H and V _L regions can be expressed using separate vectors. The V _H or V _L regions described herein may optionally include methionine at the N-terminus.

Genes encoding the heavy and light chains of the antibody of interest can be cloned from the cell, for example, genes encoding monoclonal antibodies can be cloned from the hybridomas and used to produce recombinant monoclonal antibodies. Can be. Gene libraries encoding the heavy and light chains of monoclonal antibodies can also be prepared from hybridoma or plasma cells. Random combinations of heavy and light chain gene products produce large pools of antibodies with different antigenic specificities (see, eg, Kuby, Immunology (3.sup. Rd ed. 1997)).

Techniques for the production of single chain antibodies or recombinant antibodies (US Pat. No. 4,946,778, US Pat. No. 4,816,567) can be adjusted to produce antibodies to the polypeptides of the invention. In addition, transgenic mice, or other organisms such as other mammals, can be used to express humanized or human antibodies (see, eg, US Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, Marks et al., Bio / Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812 -13 (1994); Fishwild et al., Nature Biotechnology 14: 845-51 (1996); Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg & Huszar, Intern. Rev. Immunol. 13: 65-93 ( 1995)).

In one preferred embodiment of the invention, the antibody or antigen-binding fragment thereof is expressed on the surface of the cell. More preferably, the cell is a T-cell.

The present invention provides a pharmaceutical composition comprising the anti-VEGFR-2 antibody or antigen-binding fragment thereof of the present invention. The pharmaceutical compositions of the present invention are formulated with suitable carriers, excipients and other agents that provide improved transport, delivery, tolerance and the like. Multiple suitable formulations can be found in drug stores known to all pharmaceutical chemists (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa.). These formulations are for example lipids (cationic or anionic) containing powders, pastes, ointments, jelly, waxes, oils, liquids, vesicles (e.g. LIPOFECTIN.TM., Life Technologies, Carlsbad , Calif.), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycols of various molecular weights) , Semi-solid gels, and semi-solid mixtures containing carbowax. See Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52: 238-311.

The dosage of the antibody administered to the patient can vary depending on the age and size of the patient, the target disease, condition, route of administration, and the like. Preferred dosages can typically be calculated according to body weight or body surface area. When the antibodies of the invention are used to treat a condition or disease associated with VEGFR-2 activity in an adult patient, the antibodies of the invention are generally from about 0.01 to about 20 mg / kg body weight, more preferably from about 0.02 to It may be advantageous to administer intravenously in a single dose of about 7, about 0.03 to about 5, or about 0.05 to about 3 mg / kg body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. Effective dosages and schedules for administering anti-VEGFR-2 antibodies can be measured experimentally; For example, patient progress can be monitored by periodic analysis and thus controlled dosages. Moreover, intercalation of dosing flavors can be performed using methods known in the art (eg, Mordenti et al., 1991, Pharmaceut. Res. 8: 1351).

Various delivery systems are known, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor mediated endocytosis, and are used to administer the pharmaceutical compositions of the invention. (See, eg, Wu et al., 1987, J. Biol. Chem. 262: 4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be absorbed by any convenient route, for example by injection or bolus injection, through absorption through the endothelial or mucocutaneous lining (eg, oral mucosa, rectal and small intestinal mucosa, etc.). May be administered together with other physiologically active agents. Administration can be systemic or local.

The pharmaceutical compositions of the invention can be delivered subcutaneously or intravenously with standard needles and syringes. In addition, with regard to subcutaneous delivery, a pen delivery device is readily applied to the delivery of the pharmaceutical composition. Such pen delivery devices may be reusable or disposable. Reusable pen delivery devices generally use replaceable cartridges containing pharmaceutical compositions. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Rather, the disposable pen delivery device is prefilled with a pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied from the pharmaceutical composition, the entire device is discarded.

In certain circumstances, the pharmaceutical composition may be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14: 201). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla.). In another embodiment, a controlled release system may be located in close proximity to the target of the composition, requiring only a fraction of the systemic dosage (see, eg, Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249: 1527-1533.

Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injection, drip infusion, and the like. These injectable preparations can be prepared by methods known to the public. For example, injectable preparations can be prepared, for example, by dissolving, suspending, or emulsifying the antibodies or salts thereof described above in a sterile aqueous medium or an oily medium conventionally used for injection. As aqueous media for injection, for example, suitable stabilizers such as alcohols (eg ethanol), polyalcohols (eg propylene glycol, polyethylene glycol), nonionic surfactants [eg polysorbate 80, HCO- 50 (polyoxyethylene (50 mol) adduct of hardened castor oil)] and the like, and physiological saline, and isotonic solutions containing glucose and other auxiliaries, and the like. As the oily medium, for example, sesame oil, soybean oil and the like which can be used together with stabilizers such as benzyl benzoate, benzyl alcohol and the like are used. The injection solution so prepared is preferably filled in a suitable ampoule.

 Advantageously the oral or parenteral pharmaceutical compositions described above are prepared in dosage form in a single dose suitable for the dosage of the active ingredient. Such dosage forms of unit dosages include, for example, tablets, pills, capsules, injections (ampoules), suppositories, and the like. The amount of the above mentioned antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose; In particular, in the form of injection solutions, the above-mentioned antibodies are preferably contained at about 5 to about 100 mg and about 10 to about 250 mg for other dosage forms.

The antibodies according to the invention are useful for inhibiting VEGFR-mediated signaling and for treating diseases and conditions caused by or associated with VEGFR-2 activity and / or signaling. Antibodies of the invention are also useful for inhibiting cell proliferation of endothelial cells.

The present invention provides a method of inhibiting VEGFR-2-mediated signaling in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising an antibody as described above or an antigen-binding fragment thereof to provide.

The present invention relates to VEGFR-2 activity and / or signaling in a diseased and / or diseased subject, comprising administering to the subject a pharmaceutical composition comprising an antibody as described above or an antigen-binding fragment thereof Provided are methods for treating a disease caused by and / or associated with it.

The present invention provides a method of treating a tumor in a subject having a tumor, comprising administering to the subject a pharmaceutical composition comprising an antibody as described above or an antigen-binding fragment thereof.

The present invention provides a method of inhibiting cell proliferation of endothelial cells in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising an antibody as described above or an antigen-binding fragment thereof.

As used herein, the terms “treating” and “treatment” reduce the severity and / or frequency of symptoms, remove the symptoms and / or their underlying cause, and / or promote improvement or alleviation of loss. For the administration of an agent or formulation to an individual with a condition, disease, or clinical symptom with disease. The terms "preventing" and "prevention" refer to the administration of an agent or composition to an individual with a clinical symptom that is susceptible to certain side effect conditions, diseases or disorders, and thus to preventing the occurrence of symptoms and / or their underlying causes. It is related. As will be understood by one of ordinary skill in the art, preventing or preventing does not have to achieve complete (perfect) blocking or avoidance of the condition. Rather, prevention can achieve substantial (eg greater than about 50%) reduction or avoidance of the disease or condition to be prevented. Unless expressly or implicitly indicated herein, if the term "treatment" (or "treating") is used without a reference to a possible prevention, it is intended that the prevention also be included.

The terms “cancer”, “tumor”, “transformed” and the like include precancerous, neoplastic, transformed and cancerous cells, and can refer to solid tumors or non-solid cancers (see, eg, Edge et). al.AJCC Cancer Staging Manual (7th ed. 2009); Cibas and Ducatman Cytology: Diagnostic principles and clinical correlates (3rd ed. 2009). Cancer includes both benign and malignant neoplasms (abnormal growth). "Transformation" refers to spontaneous or intended phenotypic changes, eg, immortalization of cells, morphological changes, ideal cell growth, reduced contact inhibition and anchorage, and / or malignant tumors (See Freshney, Culture of Animal Cells a Manual of Basic Technique (3rd ed. 1994)). Although transformation occurs from infection by incorporation of the transgenic virus and new genomic DNA, or uptake of exogenous DNA, it can also occur spontaneously or after exposure to carcinogens.

Antibodies of the invention, in particular, are associated with or mediated by VEGFR-2 expression or activity, block interactions between VEGFR-2 and VEGFR-2 ligands, and / or otherwise VEGFR-2 activity and / or signaling It is useful for the treatment, prevention and / or alleviation of any disease or condition treatable by inhibiting and / or promoting receptor internalization and / or reducing the number of cell surface receptors. For example, the antibodies and antigen-binding fragments of the invention are useful for the treatment of tumors that express high levels of VEGFR-2. Antibodies and antigen-binding fragments of the invention include, for example, the brain and meninges, the pharynx, the central pharynx, the lungs and bronchus, the gastrointestinal tract, and the male and female reproductive organs, muscles, bones, skin and appendages, connective tissue, It may be used to treat primary and / or metastatic tumors occurring in the spleen, immune system, angiogenic cells and bone marrow, liver and urinary tract, and special sensory organs such as the eye. In certain embodiments, antibodies and antigen-binding fragments of the invention are used to treat one or more of the following cancers: renal cell carcinoma, colon carcinoma, breast cancer, head and neck cancer, prostate cancer, malignant glioma , Osteosarcoma, colorectal cancer, gastric cancer (e.g. gastric cancer with MET amplification), malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer (e.g. VEGFR-2- Dependent non-small cell lung cancer), synovial sarcoma, thyroid cancer, or melanoma.

The present invention provides a method for detecting human vascular endothelial growth factor receptor in a sample by contacting the sample with an antibody or antigen-binding fragment thereof as mentioned above.

Anti-VEGFR-2 antibodies of the invention can also be used to detect and / or measure VEGFR-2, or VEGFR-2-expressing cells in a sample, for example for diagnostic purposes. For example, anti-VEGFR-2 antibodies, or fragments thereof, can be used to diagnose a condition or disease characterized by aberrant expression (eg, overexpression, lack of expression, lack of expression, etc.) of VEGFR-2. Exemplary diagnostic assays for VEGFR-2 may include, for example, contacting a sample obtained from a patient with an anti-VEGFR-2 antibody of the invention, wherein the anti-VEGFR-2 antibody is detectable Labeled and labeled with a reporter molecule. Alternatively, an unlabeled anti-VEGFR-2 antibody can be used in diagnostic virtue with a secondary antibody that is detectably labeled by itself. Detectable label or reporter molecules include radioisotopes such as 3H, 14C, 32P, 35S, or 125I; Fluorescent or chemiluminescent moieties such as fluorescein isothiocyanate or rhodamine; Or enzymes such as alkaline phosphatase, beta-galactosidase, blush peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure VEGFR-2 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS). .

Samples that can be used in the VEGFR-2 diagnostic assay according to the present invention include any tissue or fluid sample available from a patient containing a detectable amount of VEGFR-2 protein, or fragment thereof, under normal or pathological conditions. In general, the level of VEGFR-2 in certain samples obtained from healthy patients (eg, patients with abnormal VEGFR-2 levels or disease or conditions associated with activity) is preferentially based on the baseline, or standard, of VEGFR-2, It will be measured to establish the level. This baseline level of VEGFR-2 may then be compared against the level of VEGFR-2 measured in a sample obtained from an individual suspected of having a VEGFR-2 related disease or condition.

The following examples are provided to assist those skilled in the art in practicing the present invention.

Example

Construction of the scFv / Fab Antibody Library

A fusion protein containing the sequence of VEGFR2-Fc-His, SEQ ID NO: 1 was used as an antigen for immunizing mice six times every two weeks. After immunization, mice were sacrificed and spleens were obtained. Spleen total RNA was extracted and reverse transcribed into primers in the RT-PCR procedure to construct antibody fragments containing V _H , V _L , V _H -CH 1, V _L -CL. Antibody fragments were assembled into scFv fragments in a polymerase chain reaction and construct scFv libraries. First, Fab libraries were constructed into plasmids with V _L -CL libraries from V _L -CL fragments. Next, the V _H -CH1 fragment was constructed into a plasmid containing the V _L -CL fragment to generate the final Fab library.

Preparation of scFv / Fab Phages for Bio-panning

The resulting library was inoculated in 2 × YT medium containing 100 μg / ml ampicillin and 2% glucose (2YTAG) and incubated with shaking at 37 ° C. until the OD at 600 nm reached 0.5. The culture was infected with helper phage and then incubated in a 37 ° C. water bath for 30 minutes without shaking. Cells were collected and suspended in 2 × YT medium containing 100 μg / ml ampicillin and 25 μg / ml kanamycin (2YTAK) and further incubated with shaking at 30 ° C. overnight. The supernatant of the culture was collected and mixed with 1/5 volume PEG / NaCl (20% polyethylene glycol 8000, 2.5 M NaCl) and maintained at 4 ° C. for at least 1 hour. After centrifugation, the pellets were collected, suspended in 40 mL of PBS and again centrifuged to collect the supernatant.

Selection using the ELISA method

ELISA plates (Nunc) were coated with 1 μg / 100 μL of antigen per well and kept overnight at 4 ° C. in sodium bicarbonate buffer, pH 9.6. Wells were washed three times with PBS and blocked with 300 μL of PBS-5% skim milk (MPBS) per well at 37 ° C. for 1.5 hours. After washing three times with PBS, 100 μL of phage in 5% MPBS containing the fusion protein containing his-tag was added and incubated at 37 ° C. for 90 minutes. After washing 10 times with PBS-0.05% Tween 20 (PBST) and 10 times with PBS, the phage were eluted by adding 100 μL of 100 mM triethylamine (TEA) and reacted at 37 ° C. for 30 minutes. 100 μL of the eluted phage was neutralized with 0 μL of 1 M Tris, pH 7.4. 10 ml of TG1 at exponential growth stage was added to 150 μL of eluted phage. Cultures were incubated for 30 minutes at 37 ° C. without shaking for infection. Infected TG1 bacteria were centrifuged, collected, then suspended in 2 × YT and plated on 2 × YT-AG plates. The bacteria were incubated overnight at 30 ° C.

Selection using the Dynabeads method

In the pre-clean phage stage, Dynabiz was pre-washed three times with 1 ml of PBS and suspended in PBS. 0.3 mL of phage was then mixed with 0.5 ml of 5% MPBS, where the fusion protein contained his-tag, incubated for 30 minutes on the rotor, and then dynabe was removed.

Dynabiz was reacted with biotin-labeled VEGFR2-His for 90 minutes. Dynabiz was washed three times with 1 ml of PBS, suspended in 5% MPBS, incubated for 90 minutes and then washed three times with 1 ml of PBS. Pre-cleaned phage was added to VEGFR2-His containing Dynabiz and incubated on the rotor for another 30 minutes. The dynabe was then washed with 1 ml of 0.05% PBST, 0.2% MPBS, and PBS. Bound phage was eluted with 1 ml of 100 mM TEA. For rapid neutralization, 0.5 ml of 1M Tris, pH 7.4 was added to the eluted phage. Subsequently, 6 ml of exponentially growing culture of TG1 was taken and TEA eluted phage was added. Cultures were incubated at 37 ° C. (water bath) for 30 minutes without shaking. Infected TG1 bacteria were collected and centrifuged to collect pellets. The pelleted bacteria were suspended in 1 ml of 2 × YT and plated on large 2 × YT-AG plates. The bacteria were incubated overnight at 30 ° C.

Preparation of the next round phage

6-6 ml of 2 × YT, 15% glycerol was added to the bacterial plates and the colonies were loosened with a glass spreader. 10 μl of the scraped bacteria were then added to 10 ml of 2 × YT-AG and the bacteria were grown with shaking at 37 ° C. until the OD at 600 nm reached 0.5. 10 ml of culture was infected with M13KO7 helper phage by adding a helper phage at a ratio of 1:20 and the infected culture was incubated for 30 minutes without shaking at 37 ° C. hydrogen. The pellet was collected by centrifugation and the pellet was suspended with 50 mL of 2 × YT-AK and then incubated at 30 ° C. overnight. In addition, 40 ml of bamsan culture was centrifuged at 10,000 rpm for 20 minutes to obtain a supernatant, and 1/5 volume (8 ml) PEG / NaCl was added to the supernatant. Wells were mixed and left at 4 ° C. for at least 1 hour. The mixture was centrifuged at 10,000 rpm for 20 minutes, and the pellets were collected and suspended in 2 ml PBS. The suspension was then centrifuged at 12000 rpm for 10 minutes to remove most residual bacterial fragments.

Selection of VEGFR2-positive Phage by ELISA

Individual colonies from the plates were inoculated into 200 μl 2 × YT-AG 96-well plates and grown overnight at 37 ° C. with shaking, followed by 200 μl of 2 × YT per well to shake 50 ul at 37 ° C. for 2 hours. Transfer to a second 96-well plate containing -AG. Then 50 μl of 2 × YT-AG containing 109 pfu M13KO7 helper phage was added to each well of the second plate. The mixture was left at 37 ° C. for 30 minutes and then shaken at 37 ° C. for 1 hour. After centrifugation at 4000 rpm for 30 minutes, the supernatant was aspirated and the pellet was grown overnight at 30 ° C. with suspension by shaking in 300 μl of 2 × YT-AK. Cultures were centrifuged at 4000 rpm for 30 minutes and 100 μl of culture supernatant was harvested for phage ELISA.

ELISA plates were coated with 1 μg / mL protein antigen per well, then washed three times with PBS and blocked with 300 μl of 2% MPBS per well at 37 ° C. for 2 hours. After an additional three washes with PBS, the 100 μl phage culture supernatant detailed above was added and incubated at 37 ° C. for 90 minutes. The phage solution was discarded and the wells were washed six times with PBST and six times with PBS, followed by the appropriate dilution of HRP-anti-M13 antibody in 5% MPBS. The mixture was incubated at 37 ° C. for 60 minutes and washed six times with PBST and six times with PBS. The wells were developed with substrate solution (TMB) and the reaction was stopped by adding 50 μl of 1 M sulfuric acid. The color turned yellow and the OD at 650 nm and t 450 nm was analyzed.

After screening, a total of 379 clones and 66 types of CDRH3 were identified.

Full length antibody expression

Genes encoding the VH and VL chains of anti-VEGFR2 antibodies were inserted into expression vectors. Free-style 293 cells were transfected with the constructed vector. Suspension Free Style ™ 293 cells were transfected in a 30 ml volume according to the following Zalcha. About 24 hours before transfection, 2 × 10 ⁶ cells / ml of FreeStyle ™ 293 cells were passed against 15 ml. The flask (s) were placed in 37 ° C., 8% CO ₂ incubator. 37.5 μg of plasmid DNA was then diluted to a total volume of 1.5 ml into 1.5 ml of sterile 150 mM NaCl. In a separate tube 37.5 μL of PEI (2.0 mg / ml) was diluted in 1.5 ml of sterile 150 mM NaCl. DNA and PEI solutions were allowed to stand at room temperature for 5 minutes, the tubes were inverted and mixed lightly, and left at room temperature for about 10-20 minutes. Immediately, the DNA-PEI mixture was added into F293 cells and the transfected cells were incubated for 4 hours in a 37 ° C., 8% CO ₂ incubator on an orbital shaker platform rotating at 135-150 rpm. Then equal volume of fresh culture medium was added to the total 30 ml volume and incubated for 5-7 days. Cells were harvested for protein purification and quantification.

A total of 78 colonies were identified, including 322A6 and 12A6.

Binding affinity analysis

ELSA plates were coated overnight at 4 ° C. with 100 μL of human VEGFR-2, mouse VEGFR2, human VEGFR1 or human VEGFR3 per well, then washed three times with PBS and 300 μL of 5% MPBS per well at 37 ° C. for 2 hours. Blocked. Wells were washed three times with PBS, 100 μL of anti-VEGFR2 antibody, 2-fold dilutions were added and incubated at 37 ° C. for 90 minutes. The test solution was discarded and washed three times with PBS. Appropriate dilutions of HRP-anti-human IgG antibody in 5% MPBS (1: 10000) were added, incubated at 37 ° C. for 60 minutes and the wells washed three times with PBS. The wells were developed with 100 μL of substrate solution TMB and the reaction was terminated by adding 50 μL of 1 M sulfuric acid. The color turned yellow and OD at 650 nm and 450 nm was analyzed.

The results of the binding affinity of some antibodies are shown in FIG. 1 and Table 1. 12A6 and 322 A6 antibodies have been shown to have binding affinity for human VEGFR-2. The 322A6 antibody has binding affinity for mouse VEGFR-2, but no human VEGFR-1 and VEGFR-3. Thus, 322A6 is specific for VEGFR-2.

Table 1

The sequences of the CDRs of 322A6 and 12A6 are shown in Table 2. 322A6 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17, which heavy chain variable region is encoded by the nucleic acid sequence of SEQ ID NO: 16. 322A6 comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 19, which light chain variable region is encoded by the nucleic acid sequence of SEQ ID NO: 18. 12A6 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21, which heavy chain variable region is encoded by the nucleic acid sequence of SEQ ID NO: 20. 12A6 comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 23, which light chain variable region is encoded by the nucleic acid sequence of SEQ ID NO: 22.

TABLE 2

Domain mapping analysis

Fragments of human VEGFR-2 were constructed. Fragments were used for binding affinity analysis as mentioned above. The results of domain mapping are shown in FIGS. 2 and 3 and Tables 3 and 3.

Table 3:

Table 4:

In domains 6 (SEQ ID NO: 2) and 7 (SEQ ID NO: 3) of human VEGFR-2, several point mutants were constructed. Mutants were used for binding affinity analysis as mentioned above. The location of the mutation site is shown in FIG. 4. The epitope at 12A6 includes a leucine residue at position 606, an aspartic acid residue at position 607, an arginine residue at position 647, a lysine residue at position 648, and a threonine residue at position 649. The epitope of 322A6 includes a serine residue at position 711, a lysine residue at position 716, an aspartic acid residue at position 717, and an arginine residue at positions 725 and 726.

Anti-VEGF R2 Ab-HUVEC Proliferation Inhibition Assay

Materials: HUVEC (Cascade Biologics, Cat No. C-003-5C), Medium 200 (Cascade Biologics, Cat No. M-200-500), Hybridoma Medium (10% FBS-DMEM), FBS (Hyclone, # SH30071.03), DMEM (GIBCO, # 11995), human CHO VEGF, hVEGF (PROSPEC, Cat No. CYT-260), anti-VEGFR2, WST-1 (Roche, Cat No. 11644807001).

100 μL of antibody sample in 10% FBS-DMEM was inoculated into each well of a 96 well plate and all samples were duplicated. Cells were harvested, suspended in Medium 200 at 8 × 10 ⁴ cells / ml and incubated at 37 ° C., 5% CO ₂ for 30 minutes. hVEGF was diluted in Medium 200 at 50 ng / ml and 50 μL of standard hVEGF was added to the plate and incubated for 96 hours at 37 ° C., 5% CO ₂ . 20 μL of WST-1 was then added to each well and incubated at 37 ° C., 5% CO ₂ for 4 hours. Absorbance (OD450-655 nm) was measured using an ELISA reader.

The results of the proliferation assay are shown in FIG. 5. As shown in FIG. 5, when the concentration of antibody is higher, VEGFR-2 signal The inhibitory effect is stronger and the number of HUVECs (OD450-655 nm) is smaller. Among the antibodies, 12A6 exhibits the strongest effect with IC50 = 3.9 μg / ml.

Internalization of anti-VEGFR-2 antibodies into HUVECs

Internalization of the antibody into HUVECs was analyzed by flow cytometry.

Flow Cytometry (CytoFLEX)

Cell line: HUVEC

First antibody: 322A6

Secondary antibody: goat anti-human Kappa light chain, Bethyl, Cat. A80-115F

Medium 200 (M200), Thermo, Cat. C-003-25P-A, low blood growth supplement (LSGS). Cat.S00310

FBS (Gibco, Cat. 10082-147), FACS buffer (1X PBS, 1% FBS, 0.02% NaN ₃ )

15-mL Falcon Tube (Corning, Cat. CS352096)

T150: Tissue Culture Flask (TPP, 90150)

Cell number of HUVECs were counted, mixed with 322A6 and incubated for 1 hour on ice. Cells were harvested by spinning and washed three times with 10 mL of ice cold FACS buffer. 2 × 10 ⁵ cells / rxn / well (24-well plate) were seeded with RPMI medium w / 2% FBS and then incubated at 37 ° C. or 4 ° C. for a specified period of time. At each time point, cells were washed with 10 mL of ice cold FACS buffer and a second antibody (α-hIgG-FITC, diluted 1: 200 with ice cold FACS buffer 1: 200) was added on ice for 1 hour. After washing three times with 10 ml of ice cold FACS buffer, cells were seeded (2 * 10 ⁵ cells / rxn / tube with FACS buffer) and incubated at 37 ° C. or 4 ° C. for a specified period. At each time point, cells were suspended and analyzed by flow cytometry & CytExpert software.

The results are shown in Table 5 and FIG. 6. Prior to antibody internalization (0 min), HUVEC cells bound to the antibody were measured by flow cytometry. After antibody internalization occurred, the antibody was transferred from the surface of the cell to the endosomes in the cell and the signal of flow cytometry disappeared. Thus, if the antibody has a strong tendency to antibody internalization, the relative binding with the target cell is lower. As can be seen in Table 5 and Figure 6, 322A6 has a stronger tendency to antibody internalization.

Table 5:

Antibody internalization was observed under a microscope with the DeltaVision Microscopy Imaging System and is shown in FIG. 7. Rab5a was taken as a positive control and DAPI was used to label nuclei. By labeling 322A6 and endosome at the same time, 322A6 was shown to successfully internalize into cells.

Conjugation of Anti-VEGFR2 Antibodies by romidepsin

The 322A6 (C45) antibody was conjugated with romidepsin, also known as Istodax, an anticancer agent according to the following scheme:

Conjugation conditions are listed in Table 6.

Table 6:

The obtained antibody-drug conjugate (ADC) (11-0151-00-01) was analyzed by size-exclusion chromatography (SEC):

Column: Superdex 200 Increase 10/300 GL (GE)

Sample: IgG & IgG-ADC, 0.3 mg / mL

Sample volume: 100 μL

Flow rate: 0.5 mL / min

Buffer: 1x PBS

System: AKTA

The results of the chromatography are shown in FIG. 8 and Table 7. ADCs were also given to electrophoresis and are shown in FIG. 9.

Table 7:

As shown in FIG. 8, the obtained antibody-drug conjugate has 95.4% monomer and 95% purity.

Internalization of the ADC was also analyzed as mentioned above. The results are shown in FIG. 10 and the tendency of internalization in the ADC of 322A6 is similar to the internalization in 322 A6 antibody only (c45).

Inhibition of HUVEC proliferation of ADCs was analyzed as mentioned above. The results are shown in FIG. The effect of only 322A6 antibody (c45) on inhibiting HUVEC proliferation was mild. It means that the blocking effect of 322A6 antibody on VEGFR-2 signal transduction is not very strong. However, when treating 322A6 (c45-ADC) conjugated with lomidepsin, the relative cell viability of HUVECs is significantly reduced. Without wishing to be bound by theory, it is believed that the lowering of the relative cell viability of HUVECs results from the cytotoxicity of the ADC of the 322A6 antibody instead of suppressing HUVEC proliferation.

Chimeric Antigen Receptor T-Cell (CAR-T) Immunotherapy

Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors and were treated with Dynabeads® T-Activator CD3 / CD28 (Gibco) in RPMI containing 10% FBS (hyclone) and 100 U / mL recombinant interleukin-2 (Roche). Stimulated for 48 hours. Activated cells were harvested using the Pan T-Cell Isolation Kit, human (Miltenyi Biotec) according to the manufacturer's original. Lentivirus vectors contained single-chain variable fragments of anti-VEGFR-2 antibodies and signaling domains of 4-1BB and CD3-z. Viral supernatants were produced by transient transfection of HEK293T cells by the delivery genomic plasmid and lentiviral packaging helper plasmids pMD2.G and pCMVΔR8.91, and human T cells were transfected onto RetroNectin-coated plates (TaKaRa Bio Inc.). It was used to invert. Transduction efficiency was analyzed by flow cytometry.

The results are shown in FIG. Quantification analysis indicated that 5.1%, 10.4% and 7.79% of 1121, 12A6 and 322A6 CARs were expressed on T cells, respectively.

Cytotoxicity Analysis of Antigen Receptor T-Cells

Cytotoxic activity was measured using a cytotoxicity detection kit (Roche, Indianapolis, IN, USA) according to manufacturer's instructions. Anti-VEGFR-2 CAR T cells were incubated for 16 hours at 37 ° C. with VEGFR2 positive cell lines at the indicated E: T ratio. Percent-specific cytolysis was calculated using the following formula: (test-effector control-low control / high control-low control) * 100. High control was calculated after incubating the target cells at 1% Triton X 100; Effector control is the spontaneous LDH release of T cells alone; Low control is the spontaneous LDH release value of the target cell alone. Effector Cells: Anti-VEGFR-2 CAR Transduced Primary T Cells (5 days post first transduction). Target cell: FS293 (overexpressed VEGFR-2). Effector cells and target cells were co-cultured for 16 hours.

The scFv fragment of CD19 was also used to prepare CAR-T as a control (CD19). Only T cells were also taken as a control (T). The 1121 antibody was a known anti-VEGFR-2 antibody that specifically binds domains 2 and 3 of VEGFR-2, which domain is far from the cell membrane in VEGFR-2. CAR-T of 1121 was used in the analysis for comparison. The results are shown in FIG. CAR-T of both 12A6 and 322A6 have stronger cytotoxicity than CAR-T of 1121. Without wishing to be bound by theory, 12A6 and 322A6 antibodies specifically bind to domains 6 and / or 7 of VEGFR-2, which are close to the cell membrane in VEGFR-2.

Production of Radioactive, Iodine-Bound Antibodies

1 ml of 25 mM Tris-HCl (pH 7.5) and 0.4 M NaCl buffer solution were added to the IODO-gen tube and then decanted. 100 μl of buffer solution was added to the tube and then 1 μl of I-123 or I-131 was added to react with gentle shaking for 6 minutes at room temperature. Anti-VEGFR-2 antibody was added and reacted with gentle shaking for 6-9 minutes at room temperature. The reaction was terminated by transferring the reaction to a new eppendorf tube. The labeling process was analyzed by Radio-TLC with a development solution containing 85% methanol and ITLC / SG paper.

The efficiency of the labeling process ¹³¹ I-anti-VEGFR-2-clone 45 (322A6) is shown in FIG. 14. More than 96.8% of the antibodies were labeled.

Stability of Radioactive Iodine-Bound Antibodies in Serum

Labeled antibodies and serum derived from human or retro were mixed in a volume ratio of 1:19 and reacted at 37 ° C. At 0.25, 0.5, 1, 4, 8, 24, 48, and 72 hours, 800 μl of 10% TCA was added to 10 μl of sample and mixed. The protein contained in the sample was precipitated for 15 minutes by an ice bath. The supernatant was filtered with 0.45 μm PVDF membrane and the radio-activity of the liquid was counted to show dissociated I-131. Stability = (activity before filtration-activity after filtration) / activity before filtration

The results are shown in Table 8, indicating that ¹³¹ I-anti-VEGFR-2-clone-45 did not degrade for 72 hours in serum.

Table 8:

Detection of Tumors by Radioactive Iodine-Bound Antibodies

SPECT / CT was used to detect tumors by the radio-bound antibodies according to the present invention. HT-29 xenograft mice were injected intravenously with radioactivity of 4-8 μCi / μg. Each mouse received 1 to 2 mg / kg BW ¹³¹ I or ¹²³ I labeled anti-VEGFR2 antibody. At 1, 4, 24, and 48 hours, mice were scanned with SPECT / CT.

The results are shown in FIG. The results reveal that radio-bound antibody 322A6 can specifically bind to HT-29 xenograft tumors and radioactive signals accumulate in tumors. The strongest signal appeared at 18 hours in the tumor. Antibodies degraded after 18 hours and almost all of the antibodies degraded after 48 hours.

Combination therapy

Male B6 (C57BL / 6JNarl) mice, 6-8 weeks of age, were taken as animal models. Mice were placed under 12-hour day / night cycles and allowed free access to PMI feed (RMH3000-5P76) and water. Mice were grouped into the following groups.

Group 1: IgG (Mu) (non-functional IgG antibody), 15 mpk / i.v., Twice / week, n = 6

Group 2: αCTLA-4 (Mu), 5 mpk / i.v. + IgG (Mu), 10 mpk / i.v., Twice / week, n = 6

Group 3: αCTLA-4 (Mu), 5 mpk / i.v. + 322 A6, 10 mpk / i.v., Twice / week, n = 6

Group 4: IgG (Mu), 5 mpk / i.v. + 322 A6, 10 mpk / i.v., Twice / week, n = 6

Tumor size was measured and shown in Table 9. The combination of anti-CTLA4 antibody and anti-VEGFR-2 antibody (322A6) shows a synergistic effect.

Table 9:

Body weights were also measured and listed in Table 10, indicating that the worms in all groups were substantially the same.

Table 10:

Selection of human V region framework: Sequence of human V region framework sequences human germ cell V _L and V _H sequences having the highest degree of homology with the 322A6 framework region. The IMGT database (http://www.imgt.org/ ), Generally VH3 / Vk1 (4D5) was used. Finally, framework sequences of VH3 and Vk1 were selected for the VH framework and the VL framework, respectively. The humanized framework labeled Hu is from IMGT and the Hd series is from the 4D5 framework.

Full Length CDR Grafted Ab Construction: 322A6 (HH) consisted of a complete human framework (VL κ subgroup I and VH subgroup III) with six complete murine CDR sequences. 322A6 (HH) and semi-humanized (MH or HM) antibody constructs were assembled by PCR and restriction enzyme digestion for directed sub-cloning into a modified antibody expression vector pTCAE8.3. The plasmid contains DNA fragments encoding human kappa light chain and human IgG1 C region. Full length antibodies were expressed in free-style 293 cells.

Reversion Mutations: Clone 322A6 (HuB1-Hd) was selected for 1 to 3 run reversion mutations from the CDR grafted framework for recovery of binding activity. In summary, we performed an analytical CDR grafting framework by computer modeling to test the upper core region, interface area, and also to apply the previous experience most commonly used in successful cases up to a 5 dB approximation. The inventors of the present invention selected 5 to 11 possible amino acid return mutations in 3 runs. These sites were recognized as important sites for CDR binding and structure. After cloning and antibody expression, binding activity was measured by ELISA and BIAcore. Heavy chain (HuB1) and light chain (Hd) with 7 amino acid return mutation (7 + 0) were combinatorial clones suitable candidates for VEGFR2 binding.

Humanized antibody Hu322B1HdH consisted of a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 25 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 27. The heavy chain variable region is encoded by the nucleic acid sequence of SEQ ID NO: 24 and the light chain variable region is encoded by the nucleic acid sequence of SEQ ID NO: 26. The alignment of the V _L segments is shown in FIG. 16, and the alignment of the V _H segments is shown in FIG. 17.

Although the present invention has been described in conjunction with the specific set of embodiments described above, many alternatives and variations and variations thereof will be apparent to those skilled in the art. All such alternatives, modifications and variations are considered to be within the scope of the present invention.

                         SEQUENCE LISTING <110> DEVELOPMENT CENTER FOR BIOTECHNOLOGY   <120> ANTI-VEGFR ANTIBODY AND USES THEREOF <130> none <160> 27 <170> PatentIn version 3.5 <210> 1 <211> 764 <212> PRT <213> Homo sapiens <400> 1 Met Gln Ser Lys Val Leu Leu Ala Val Ala Leu Trp Leu Cys Val Glu 1 5 10 15 Thr Arg Ala Ala Ser Val Gly Leu Pro Ser Val Ser Leu Asp Leu Pro             20 25 30 Arg Leu Ser Ile Gln Lys Asp Ile Leu Thr Ile Lys Ala Asn Thr Thr         35 40 45 Leu Gln Ile Thr Cys Arg Gly Gln Arg Asp Leu Asp Trp Leu Trp Pro     50 55 60 Asn Asn Gln Ser Gly Ser Glu Gln Arg Val Glu Val Thr Glu Cys Ser 65 70 75 80 Asp Gly Leu Phe Cys Lys Thr Leu Thr Ile Pro Lys Val Ile Gly Asn                 85 90 95 Asp Thr Gly Ala Tyr Lys Cys Phe Tyr Arg Glu Thr Asp Leu Ala Ser             100 105 110 Val Ile Tyr Val Tyr Val Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser         115 120 125 Val Ser Asp Gln His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys     130 135 140 Thr Val Val Ile Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser 145 150 155 160 Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg                 165 170 175 Ile Ser Trp Asp Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile             180 185 190 Ser Tyr Ala Gly Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser         195 200 205 Tyr Gln Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr     210 215 220 Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu 225 230 235 240 Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile                 245 250 255 Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu             260 265 270 Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe         275 280 285 Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu     290 295 300 Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr 305 310 315 320 Phe Val Arg Val His Glu Lys Pro Phe Val Ala Phe Gly Ser Gly Met                 325 330 335 Glu Ser Leu Val Glu Ala Thr Val Gly Glu Arg Val Arg Ile Pro Ala             340 345 350 Lys Tyr Leu Gly Tyr Pro Pro Pro Glu Ile Lys Trp Tyr Lys Asn Gly         355 360 365 Ile Pro Leu Glu Ser Asn His Thr Ile Lys Ala Gly His Val Leu Thr     370 375 380 Ile Met Glu Val Ser Glu Arg Asp Thr Gly Asn Tyr Thr Val Ile Leu 385 390 395 400 Thr Asn Pro Ile Ser Lys Glu Lys Gln Ser His Val Val Ser Leu Val                 405 410 415 Val Tyr Val Pro Pro Gln Ile Gly Glu Lys Ser Leu Ile Ser Pro Val             420 425 430 Asp Ser Tyr Gln Tyr Gly Thr Thr Gln Thr Leu Thr Cys Thr Val Tyr         435 440 445 Ala Ile Pro Pro Pro His His Ile His Trp Tyr Trp Gln Leu Glu Glu     450 455 460 Glu Cys Ala Asn Glu Pro Ser His Ala Val Ser Val Thr Asn Pro Tyr 465 470 475 480 Pro Cys Glu Glu Trp Arg Ser Val Glu Asp Phe Gln Gly Gly Asn Lys                 485 490 495 Ile Glu Val Asn Lys Asn Gln Phe Ala Leu Ile Glu Gly Lys Asn Lys             500 505 510 Thr Val Ser Thr Leu Val Ile Gln Ala Ala Asn Val Ser Ala Leu Tyr         515 520 525 Lys Cys Glu Ala Val Asn Lys Val Gly Arg Gly Glu Arg Val Ile Ser     530 535 540 Phe His Val Thr Arg Gly Pro Glu Ile Thr Leu Gln Pro Asp Met Gln 545 550 555 560 Pro Thr Glu Gln Glu Ser Val Ser Leu Trp Cys Thr Ala Asp Arg Ser                 565 570 575 Thr Phe Glu Asn Leu Thr Trp Tyr Lys Leu Gly Pro Gln Pro Leu Pro             580 585 590 Ile His Val Gly Glu Leu Pro Thr Pro Val Cys Lys Asn Leu Asp Thr         595 600 605 Leu Trp Lys Leu Asn Ala Thr Met Phe Ser Asn Ser Thr Asn Asp Ile     610 615 620 Leu Ile Met Glu Leu Lys Asn Ala Ser Leu Gln Asp Gln Gly Asp Tyr 625 630 635 640 Val Cys Leu Ala Gln Asp Arg Lys Thr Lys Lys Arg His Cys Val Val                 645 650 655 Arg Gln Leu Thr Val Leu Glu Arg Val Ala Pro Thr Ile Thr Gly Asn             660 665 670 Leu Glu Asn Gln Thr Thr Ser Ile Gly Glu Ser Ile Glu Val Ser Cys         675 680 685 Thr Ala Ser Gly Asn Pro Pro Pro Gln Ile Met Trp Phe Lys Asp Asn     690 695 700 Glu Thr Leu Val Glu Asp Ser Gly Ile Val Leu Lys Asp Gly Asn Arg 705 710 715 720 Asn Leu Thr Ile Arg Arg Val Arg Lys Glu Asp Glu Gly Leu Tyr Thr                 725 730 735 Cys Gln Ala Cys Ser Val Leu Gly Cys Ala Lys Val Glu Ala Phe Phe             740 745 750 Ile Ile Glu Gly Ala Gln Glu Lys Thr Asn Leu Glu         755 760 <210> 2 <211> 110 <212> PRT <213> Homo sapiens <400> 2 Pro Glu Ile Thr Leu Gln Pro Asp Met Gln Pro Thr Glu Gln Glu Ser 1 5 10 15 Val Ser Leu Trp Cys Thr Ala Asp Arg Ser Thr Phe Glu Asn Leu Thr             20 25 30 Trp Tyr Lys Leu Gly Pro Gln Pro Leu Pro Ile His Val Gly Glu Leu         35 40 45 Pro Thr Pro Val Cys Lys Asn Leu Asp Thr Leu Trp Lys Leu Asn Ala     50 55 60 Thr Met Phe Ser Asn Ser Thr Asn Asp Ile Leu Ile Met Glu Leu Lys 65 70 75 80 Asn Ala Ser Leu Gln Asp Gln Gly Asp Tyr Val Cys Leu Ala Gln Asp                 85 90 95 Arg Lys Thr Lys Lys Arg His Cys Val Val Arg Gln Leu Thr             100 105 110 <210> 3 <211> 87 <212> PRT <213> Homo sapiens <400> 3 Pro Thr Ile Thr Gly Asn Leu Glu Asn Gln Thr Thr Ser Ile Gly Glu 1 5 10 15 Ser Ile Glu Val Ser Cys Thr Ala Ser Gly Asn Pro Pro Pro Gln Ile             20 25 30 Met Trp Phe Lys Asp Asn Glu Thr Leu Val Glu Asp Ser Gly Ile Val         35 40 45 Leu Lys Asp Gly Asn Arg Asn Leu Thr Ile Arg Arg Val Arg Lys Glu     50 55 60 Asp Glu Gly Leu Tyr Thr Cys Gln Ala Cys Ser Val Leu Gly Cys Ala 65 70 75 80 Lys Val Glu Ala Phe Phe Ile                 85 <210> 4 <211> 10 <212> PRT <213> Mus musculus <400> 4 Gly Tyr Ala Phe Thr Thr Tyr Trp Met His 1 5 10 <210> 5 <211> 17 <212> PRT <213> Mus musculus <400> 5 Met Ile Asp Phe Ser Asp Ser Glu Thr Lys Leu Asn Gln Arg Phe Lys 1 5 10 15 Gly      <210> 6 <211> 8 <212> PRT <213> Mus musculus <400> 6 Asp Val Arg Gly Asn Phe Asp Val 1 5 <210> 7 <211> 15 <212> PRT <213> Mus musculus <400> 7 Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His 1 5 10 15 <210> 8 <211> 7 <212> PRT <213> Mus musculus <400> 8 Leu Ala Ser Asn Leu Glu Ser 1 5 <210> 9 <211> 9 <212> PRT <213> Mus musculus <400> 9 Gln His Ser Arg Glu Leu Pro Trp Thr 1 5 <210> 10 <211> 10 <212> PRT <213> Mus musculus <400> 10 Gly Tyr Ser Phe Thr Asp Tyr Ser Met Tyr 1 5 10 <210> 11 <211> 17 <212> PRT <213> Mus musculus <400> 11 Tyr Ile Asp Pro Tyr Asn Asp Asp Thr Ser Tyr Lys Gln Lys Phe Lys 1 5 10 15 Gly      <210> 12 <211> 8 <212> PRT <213> Mus musculus <400> 12 Gly Tyr Ala Asp Ala Met Asp Tyr 1 5 <210> 13 <211> 11 <212> PRT <213> Mus musculus <400> 13 His Ala Ser Gln Asn Ile Asn Val Trp Leu Ser 1 5 10 <210> 14 <211> 7 <212> PRT <213> Mus musculus <400> 14 Lys Ala Ser Asn Leu His Thr 1 5 <210> 15 <211> 9 <212> PRT <213> Mus musculus <400> 15 Gln Gln Gly Gln Ser Tyr Pro Leu Thr 1 5 <210> 16 <211> 351 <212> DNA <213> Mus musculus <220> <221> CDS (222) (1) .. (351) <400> 16 gag gtg cag ctg cag cag tct ggg cct cag ctg gtt agg cct ggg gct 48 Glu Val Gln Leu Gln Gln Ser Gly Pro Gln Leu Val Arg Pro Gly Ala 1 5 10 15 tca gcg aag ata tcc tgc aag gct tct ggt tac gca ttc acc acc tac 96 Ser Ala Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Thr Tyr             20 25 30 tgg atg cac tgg gtg aaa cag agg cct gga caa ggt ctt gag tgg att 144 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile         35 40 45 ggc atg att gat ttt tcc gat agt gaa act aag tta aat cag agg ttc 192 Gly Met Ile Asp Phe Ser Asp Ser Glu Thr Lys Leu Asn Gln Arg Phe     50 55 60 aag ggc aag gcc aca ttg act gtt gac aaa tcc tcc agc aca gcc tac 240 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 atg caa ctc agc agc ccg aca tct gag gac tct gcg gtc tat tac tgt 288 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys                 85 90 95 gca aga gat gtc aga ggg aac ttc gat gtc tgg ggc gca ggg acc acg 336 Ala Arg Asp Val Arg Gly Asn Phe Asp Val Trp Gly Ala Gly Thr Thr             100 105 110 gtc acc gtc tcc tca 351 Val Thr Val Ser Ser         115 <210> 17 <211> 117 <212> PRT <213> Mus musculus <400> 17 Glu Val Gln Leu Gln Gln Ser Gly Pro Gln Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Ala Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Thr Tyr             20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile         35 40 45 Gly Met Ile Asp Phe Ser Asp Ser Glu Thr Lys Leu Asn Gln Arg Phe     50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Asp Val Arg Gly Asn Phe Asp Val Trp Gly Ala Gly Thr Thr             100 105 110 Val Thr Val Ser Ser         115 <210> 18 <211> 336 <212> DNA <213> Mus musculus <220> <221> CDS (222) (1) .. (336) <400> 18 gat att gtg ttg aca cag tct cct gct tcc tta gct gta tct ctg ggg 48 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 cag agg gcc acc atc tca tgc agg gcc agc aaa agt gtc agt aca tct 96 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser             20 25 30 ggc tat agt tat atg cac tgg tac caa cag aaa cca gga cag cca ccc 144 Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro         35 40 45 aaa ctc ctc atc tat ctt gca tcc aac cta gaa tct ggg gtc cct gcc 192 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala     50 55 60 agg ttc agt ggc agt ggg tct ggg aca gac ttc acc ctc aac atc cat 240 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 cct gtg gag gag gag gat gct gca acc tat tac tgt cag cac agt agg 288 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg                 85 90 95 gag ctt ccg tgg acg ttc ggt gga ggc acc aag ctg gaa atc aaa cgt 336 Glu Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg             100 105 110 <210> 19 <211> 112 <212> PRT <213> Mus musculus <400> 19 Asp Ile Val Leu 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 Thr 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 Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg             100 105 110 <210> 20 <211> 351 <212> DNA <213> Mus musculus <220> <221> CDS (222) (1) .. (351) <400> 20 cag gtc cag ctg cag cag tct gga cct gaa ctg gtg aag cct ggg gct 48 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 tca gtg aag gta tcc tgc aag gct tct ggt tac tca ttc act gac tac 96 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr             20 25 30 agc atg tac tgg gtg aag cag agc cat gga aag agc ctt gag tgg att 144 Ser Met Tyr Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile         35 40 45 gga tat att gat cct tac aat gat gat act agc tac aag cag aag ttc 192 Gly Tyr Ile Asp Pro Tyr Asn Asp Asp Thr Ser Tyr Lys Gln Lys Phe     50 55 60 aag ggc aag gcc aca ttg act gtt gac aag tcc tcc agc aca gcc ttc 240 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Phe 65 70 75 80 atg cat ctc aac agc ctg aca tct gag gac tct gca gtc tat tac tgt 288 Met His Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys                 85 90 95 gca aag ggt tac gcg gat gct atg gac tac tgg ggt caa gga acc tca 336 Ala Lys Gly Tyr Ala Asp Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser             100 105 110 gtc acc gtc tcc tca 351 Val Thr Val Ser Ser         115 <210> 21 <211> 117 <212> PRT <213> Mus musculus <400> 21 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr             20 25 30 Ser Met Tyr Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile         35 40 45 Gly Tyr Ile Asp Pro Tyr Asn Asp Asp Thr Ser Tyr Lys Gln Lys Phe     50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Phe 65 70 75 80 Met His Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys                 85 90 95 Ala Lys Gly Tyr Ala Asp Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser             100 105 110 Val Thr Val Ser Ser         115 <210> 22 <211> 324 <212> DNA <213> Mus musculus <220> <221> CDS (222) (1) .. (324) <400> 22 gat att cag atg att cag tct cca tcc agt ctg tct gca tcc ctt gga 48 Asp Ile Gln Met Ile Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 gac aca att acc atc act tgc cat gcc agt cag aac att aat gtt tgg 96 Asp Thr Ile Thr Ile Thr Cys His Ala Ser Gln Asn Ile Asn Val Trp             20 25 30 tta agc tgg tac cag cag aaa cca gga aat att cct aaa cta ttg atc 144 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile         35 40 45 tat aag gct tcc aac ttg cac aca ggc gtc cca tca agg ttt agt ggc 192 Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 agt gga tct gga aca ggt ttc aca tta acc atc agc agc ctg cag cct 240 Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 gaa gac att gcc acc tac tac tgt caa cag ggt caa agt tat ccg ctc 288 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Ser Tyr Pro Leu                 85 90 95 acg ttc ggt gct ggg acc aag ctg gag ctg aaa cgg 324 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg             100 105 <210> 23 <211> 108 <212> PRT <213> Mus musculus <400> 23 Asp Ile Gln Met Ile Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Thr Ile Thr Ile Thr Cys His Ala Ser Gln Asn Ile Asn Val Trp             20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile         35 40 45 Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Ser Tyr Pro Leu                 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg             100 105 <210> 24 <211> 351 <212> DNA <213> artificial <220> <223> humanized antibody <220> <221> CDS (222) (1) .. (351) <400> 24 caa gtg cag ctg gtg cag tct ggc gcc gaa gtg aag aaa cca ggc gcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 agc gtg aag gtg tcc tgc aag gcc agc ggc tac gcc ttc acc acc tac 96 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Thr Tyr             20 25 30 tgg atg cat tgg gtg cgc cag gcc cct gga cag ggc ctg gaa tgg atc 144 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile         35 40 45 ggc atg atc gac ttc agc gac agc gag aca aag ctg aac cag cgg ttc 192 Gly Met Ile Asp Phe Ser Asp Ser Glu Thr Lys Leu Asn Gln Arg Phe     50 55 60 aag ggc aag gcc acc ctg acc gtg gac aag agc acc agc acc gcc tac 240 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 atg gaa ctg agc agc ctg cgg agc gag gac acc gcc gtg tac tac tgc 288 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 gct aga gat gtg cgg ggc aac ttc gac gtg tgg ggc cag gga aca ctc 336 Ala Arg Asp Val Arg Gly Asn Phe Asp Val Trp Gly Gln Gly Thr Leu             100 105 110 gtg acc gtg tct agc 351 Val Thr Val Ser Ser         115 <210> 25 <211> 117 <212> PRT <213> artificial <220> <223> Synthetic Construct <400> 25 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Thr Tyr             20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile         35 40 45 Gly Met Ile Asp Phe Ser Asp Ser Glu Thr Lys Leu Asn Gln Arg Phe     50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Asp Val Arg Gly Asn Phe Asp Val Trp Gly Gln Gly Thr Leu             100 105 110 Val Thr Val Ser Ser         115 <210> 26 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Humanized antibody <220> <221> CDS (222) (1) .. (336) <400> 26 gac atc cag atg acc cag agc ccc agc agc ctg tct gcc agc gtg ggc 48 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 gac aga gtg acc atc acc tgt cgg gcc agc aag agc gtg tcc acc agc 96 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Val Ser Thr Ser             20 25 30 ggc tac agc tac atg cac tgg tat cag cag aag ccc ggc aag gcc ccc 144 Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro         35 40 45 aag ctg ctg atc tac ctg gcc agc aac ctg gaa agc ggc gtg ccc agc 192 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ser     50 55 60 aga ttt tcc ggc agc ggc tct ggc acc gac ttc acc ctg acc atc agc 240 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 tcc ctg cag ccc gag gac ttc gcc acc tac tac tgc cag cac agc aga 288 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ser Arg                 85 90 95 gag ctg ccc tgg acc ttt ggc cag ggc acc aag gtg gaa atc aag cgg 336 Glu Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg             100 105 110 <210> 27 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 27 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Val Ser Thr Ser             20 25 30 Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro         35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ser     50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ser Arg                 85 90 95 Glu Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg             100 105 110

Claims (22)

An antibody or antigen-binding fragment thereof that specifically binds to the epitope of human vascular endothelial growth factor receptor (VEGFR-2) or fragment thereof,
Human vascular endothelial growth factor receptor 2 has the amino acid sequence of SEQ ID NO: 1 and the epitope is
A leucine residue at position 606 of SEQ ID NO: 1, an aspartic acid residue at position 607, an arginine residue at position 647, a lysine residue at position 648, and a threonine residue at position 649; or
An antibody or antigen-binding fragment thereof comprising a serine residue at position 711 of SEQ ID NO: 1, a lysine residue at position 716, an aspartic acid residue at position 717, and an arginine residue at positions 725 and 726. The method according to claim 1,
An antibody or antigen-binding fragment thereof that is a mammalian antibody. The method according to claim 1,
Complementarity determining region of the heavy chain variable region and complementarity determining region of the light chain variable region, wherein the complementarity determining region of the heavy chain variable region is CDRH1, CDRH2 and CDRH3 region Wherein the complementarity determining region of the light chain variable region comprises a CDRL1, CDRL2 and CDRL3 region, wherein the CDRH1 region comprises the amino acid sequence of SEQ ID NO: 4, or a substantially similar sequence thereof; The CDRH2 region comprises the amino acid sequence of SEQ ID NO: 5, or a substantially similar sequence thereof; The CDRH3 region comprises the amino acid sequence of SEQ ID NO: 6, or a substantially similar sequence thereof; The CDRL1 region comprises the amino acid sequence of SEQ ID NO: 7, or a substantially similar sequence thereof; The CDRL2 region comprises the amino acid sequence of SEQ ID NO: 8, or a substantially similar sequence thereof; The CDRL3 region comprises the amino acid sequence of SEQ ID NO: 9, or a substantially similar sequence thereof, or an antigen-binding fragment thereof. The method according to claim 1,
A complementarity determining region of the heavy chain variable region and a complementarity determining region of the light chain variable region, wherein the complementarity determining region of the heavy chain variable region comprises CDRH1, CDRH2, and CDRH3 regions, and the complementarity determining region of the light chain variable region is CDRL1, CDRL2, and CDRL3 Wherein the CDRH1 region comprises the amino acid sequence of SEQ ID NO: 10, or a substantially similar sequence thereof; The CDRH2 region comprises the amino acid sequence of SEQ ID NO: 11, or a substantially similar sequence thereof; The CDRH3 region comprises the amino acid sequence of SEQ ID NO: 12, or a substantially similar sequence thereof; The CDRL1 region comprises the amino acid sequence of SEQ ID NO: 13, or a substantially similar sequence thereof; The CDRL2 region comprises the amino acid sequence of SEQ ID NO: 14, or a substantially similar sequence thereof; The CDRL3 region comprises the amino acid sequence of SEQ ID NO: 15, or a substantially similar sequence thereof. The method according to claim 1,
A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17 or a substantially similar sequence thereof; And a light chain variable region comprising the amino acid sequence of SEQ ID NO: 19 or a substantially similar sequence thereof. The method according to claim 5,
The heavy chain variable region is encoded by the nucleic acid sequence of SEQ ID NO: 16; The antibody or antigen-binding fragment thereof, wherein the light chain variable region is encoded by the nucleic acid sequence of SEQ ID NO: 18. The method according to claim 1,
A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21 or a substantially similar sequence thereof; And a light chain variable region comprising the amino acid sequence of SEQ ID NO: 23 or a substantially similar sequence thereof. The method according to claim 7,
The heavy chain variable region is encoded by the nucleic acid sequence of SEQ ID NO: 20; The antibody or antigen-binding fragment thereof, wherein the light chain variable region is encoded by the nucleic acid sequence of SEQ ID NO: 22. The method according to claim 1,
A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 25 or a substantially similar sequence thereof; And an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 27 or a substantially similar sequence thereof. The method according to claim 9,
The heavy chain variable region is encoded by the nucleic acid sequence of SEQ ID NO: 24; The antibody or antigen-binding fragment thereof, wherein the light chain variable region is encoded by the nucleic acid sequence of SEQ ID NO: 26. The method according to claim 1,
An antibody or antigen-binding fragment thereof conjugated with a therapeutic agent. The method according to claim 1,
Therapeutic agents include: antimetabolites, alkylating agents, alkylation-like agents, DNA minor groove alkylating agents, anthracycline, antibiotics, calicheamicin, antimitotic agents ), An antibody or antigen-binding fragment thereof, selected from the group consisting of topoisomerase inhibitors, HDAC inhibitors, proteasome inhibitors, and radioisotopes. The method according to claim 1,
An antibody or antigen-binding fragment thereof, expressed on the surface of a cell. The method according to claim 13,
The antibody or antigen-binding fragment thereof, wherein the cell is a T-cell. A method of inhibiting VEGFR-2-mediated signaling in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising the antibody of claim 1 or an antigen-binding fragment thereof. Induced by or associated with VEGFR-2 activity and / or signaling in a diseased and / or diseased subject, comprising administering to the subject a pharmaceutical composition comprising an antibody according to claim 1 or an antigen-binding fragment thereof And a disease associated with the disease. A method of treating a tumor in a subject having a tumor, comprising administering to the subject a pharmaceutical composition comprising an antibody according to claim 1 or an antigen-binding fragment thereof. The method according to claim 17,
The tumor is a solid tumor. The method according to claim 18,
Tumors include renal cell carcinoma, colon carcinoma, breast cancer, head and neck cancer, prostate cancer, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer, malignant mesothelioma, multiple myeloma, ovary Cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, or melanoma. A method of inhibiting cell proliferation of endothelial cells in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising the antibody of claim 1 or an antigen-binding fragment thereof. A method for detecting human vascular endothelial growth factor receptor 2 in a sample, comprising contacting the sample with an antibody according to claim 1 or an antigen-binding fragment thereof. The method according to claim 21,
For detecting domains 6 and 7 of VEGFR-2.

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