CN110305213A

CN110305213A - A kind of anti-B7-H3 antibody and preparation method thereof, its conjugate and application

Info

Publication number: CN110305213A
Application number: CN201811333430.XA
Authority: CN
Inventors: 郭青松; 杨彤; 沈毅珺
Original assignee: FUDAN ZHANGJIANG BIOLOGICAL MEDICINE Co Ltd SHANGHAI
Current assignee: SHANGHAI FUDAN-ZHANGJIANG BIO-PHARMACEUTICAL CO LTD; Taizhou Fudan Zhangjiang Pharmaceutical Co Ltd
Priority date: 2018-11-09
Filing date: 2018-11-09
Publication date: 2019-10-08
Anticipated expiration: 2038-11-09
Also published as: CN110305213B

Abstract

The invention discloses a kind of anti-B7-H3 antibody and preparation method thereof, its conjugate and applications.It includes complementary determining regions for the anti-B7-H3 antibody, and the sequence of the complementary determining region is as recorded in the present invention.Anti- B7-H3 antibody of the invention is the human antibody of phage library screening, has unique antigen binding epitope；The B7-H3 antigen on tumour cell can be specifically bound, and in conjunction with tumour cell after can rapid internalization into cell, can be used for ADC drug development and be expected to obtain more preferably anti-tumor activity and effect, to reach the purposes for the treatment of cancer.

Description

A kind of anti-B7-H3 antibody and preparation method thereof, its conjugate and application

Technical field

The invention belongs to antibody arts, and in particular to specific binding mammal, particularly people B7-H3 antibody and Preparation method, its conjugate and application；Especially it is used for the human antibody and human antibody conjugate for the treatment of cancer.

Background technique

B7-H3 also known as CD276, most reported earlier than 2001 (Chapoval AI etc., Nat Immmunol 2001,2 (3): 269-274), albumen is because it lacks a heptad structure and B30.2 structural domain so thinking to be not belonging to butyrophilin And myelin oligodendrocyte glycoprotein, and identify and belong to B7 family contactin member (Chapoval AI etc., Nat Immmunol 2001,2 (3): 269-274), with family other members such as PD-L1, B7-H4, CD80, CD86 etc. Unlike: it is 2IgB7-H3 and 4IgB7-H3 that B7-H3, which exists in human body with two different variant forms, wherein 4IgB7- The exon that H3 is 2IgB7-H3 replicates, and mainly there is (Sun M etc., The Journal in human body in the form of 4Ig B7-H3 of Immunology 2002,168(12):6294-6297；Ling V etc., Genomics 2003,82 (3): 365-377； Steinberger P etc., J IMMUNOL 2004,172 (4): 2352-2359) 2IgB7-H3 structure, is contained only in mouse (Sun M etc., The Journal of Immunology 2002,168 (12): 6294-6297).Result of study shows natural small The 2IgB7-H3 of the mouse and 4IgB7-H3 of people shows similar function and nonfunctional difference (Ling V etc., Genomics 2003,82(3):365-377；Hofmeyer KA etc., Proc Natl Acad Sci U S A 2008,105 (30): 10277- 10278.), crystal structure shows that the FG loop in the area protein I gV is the important epitope (Vigdorovich that B7-H3 is functioned V etc., Structure 2013,21 (5): 707-717).

Although B7-H3 mRNA level in-site expression it is relatively broad, such as can include in many histoorgans of human body heart, Liver, placenta, prostate, testis, uterus, pancreas, small intestine and the medium mRNA level in-site for checking high-caliber B7-H3 of colon, But it is nonimmune that protein expression level is confined to fibroblast, endothelial cell, osteoblast, amniotic fluid stem cell of tranquillization etc. relatively Cell, and the antigen presenting cell, NK cell surface (Hofmeyer KA etc., Proc Natl Acad Sci the U S that are induced A 2008,105(30):10277-10278；Yi KH etc., Immunol Rev 2009,229 (1): 145-151；Picarda E Deng CLIN CANCER RES 2016,22 (14): 3425-3431).B7-H3 protein level low table in normal healthy tissues It reaches, for example can be detected in the liver of normal human, lung, bladder, testis, prostate, breast, placenta, lymphatic organ official rank tissue The lower protein level of B7-H3, but B7-H3 albumen is overexpressed in a large amount of malignant tumour, is a mark of tumour cell Property antigen, research shows that B7-H3 can be in prostate cancer, oophoroma, colorectal cancer, clear-cell carcinoma, non-small cell lung cancer, pancreas High expression in many cancers such as cancer, melanoma, gastric cancer, bladder cancer, glioblastoma and osteosarcoma, especially in head Overexpression (Roth TJ etc., CANCER RES in the kinds cancers such as neck cancer, kidney, glioma and thyroid cancer 2007,67(16):7893-7900；Zang X etc., MODERN PATHOL 2010,23 (8): 1104-1112； Ingebrigtsen VA etc., INT J CANCER 2012,131 (11): 2528-2536；Sun J etc., Cancer Immunology,Immunotherapy 2010,59(8):1163-1171；Crispen PL etc., CLIN CANCER RES 2008,14(16):5150-5157；Zhang G etc., LUNG CANCER 2009,66 (2): 245-249；Yamato I etc., Br J Cancer 2009,101(10):1709-1716；Tekle C etc., INT J CANCER 2012,130 (10): 2282- 2290；Katayama A etc., INT J ONCOL 2011,38 (5): 1219-1226；Wu CP etc., World J Gastroenterol 2006,12(3):457-459；Wu D etc., ONCOL LETT 2015,9 (3): 1420-1424), B7-H3 It not only expresses on tumour cell, the same high expression on endothelial cells in tumor neogenetic blood vessels, is the tumour mark of a unusual wide spectrum Will antigen.The expression of B7-H3 albumen height can promote cancer progression, related with the poor prognosis of patient and poor existence benefit.

Although early stage result of study shows B7-H3 and can stimulate activating T cell function, promote the proliferation of CD4 and cd8 cell with And the secretion of IFN-γ, but as research is deeply it has been shown that B7-H3 mainly plays inhibition T as a kind of immunologic test point The effect of cell function lowers T cell activity, is the negative regulatory molecule of T cell.Woong-Kyung Suh and Durbaka The research of V.R.Prasad shows that the B7-H3 albumen of mouse dose-dependent can significantly inhibit the proliferation of CD4, cd8 cell (Suh W etc., NAT IMMUNOL 2003,4 (9): 899-906；Prasad DVR etc., The Journal of Immunology 2004,173(4):2500-2506).The research of Judith Leitner et al. again show that people 4Ig-B7-H3Ig and 2Ig-B7-H3Ig can inhibit the proliferation of T cell in vitro, and inhibit the relevant cell factor (IFN- of CD4, cd8 cell γ, IL-2, IL-10, IL-13) secretion (Leitner J etc., EUR J IMMUNOL2009,39 (7): 1754-1764), into The analysis of one step points out that B7-H3 is mainly to pass through to inhibit the generation of IL-2 to mediate and inhibit T cell proliferation.And the target in Mice Body It can significantly promote the progress of experimental autoimmune encephalomyelitis (EAE) to the antibody for neutralizing B7-H3 and promote cd4 cell Proliferation, it is objective to illustrate that B7-H3 inhibits T cell function (Prasad DVR etc., The Journal of in vivo Immunology 2004,173(4):2500-2506).The B7-H3 deficient mice phase in the research of Woong-Kyung Suh Equally show that generation experimental autoimmune encephalomyelitis earlier is (thin by Th1 under the conditions of immune EAE than wild-type mice Born of the same parents cause), illustrate that B7-H3 mainly inhibits Th1 cell (Suh W etc., NAT IMMUNOL 2003,4 (9): 899-906).As above There is arguement to the function of T cell in the B7-H3, but B7-H3 promotes T cell function to be only present in the research of mouse at present, And human B 7-H 3 promotes T cell function not have been reported that temporarily, although the receptor of B7-H3 does not determine, current educational circles main points of view thinks B7-H3 is the negative regulator of T cell.

Based on B7-H3 can inhibit T cell activity to mediate tumor cell escape immunosurveillance, therefore block B7-H3 with The combination of unknown receptor is effective to mediating T-cell activation and inhibition activity of tumor cells, such as The existing clinical effectiveness of Enoblituzumab (US2018134790A1) shows that it has different degrees of delay to different tumours Solution, tool have a better effect, but still have several patients that progression of disease occurs, therefore simple exploitation is anti-for the monoclonal of B7-H3 Body still has biggish clinic not meet；And above-mentioned antibody be screened by hybridoma, then carried out it is humanization modified, it is miscellaneous Although handing over tumor screening by humanization modified, but still contain sequence of mouse source, there is potential immunogenicity risk, and existing faces Bed is the result shows that its antitumous effect remains to be further improved.

The high expression in kinds of tumors in view of B7-H3, and its antigen kurtosis is higher in different tumours, is to be suitble to exploitation The target spot of antibody coupling drug.

Summary of the invention

The technical problem to be solved by the present invention is to lack for anti-B7-H3 antibody levels in the prior art and be mostly to contain There is the defect of the antibody of sequence of mouse source and a kind of anti-B7-H3 antibody is provided and preparation method thereof, its conjugate and application.The present invention Using the full people's library screening of bacteriophage, the sequence of the antibody of acquisition is full human source sequence, and potential immunogenicity risk is lower, tool There is certain clinical application safety, and have the function of internalization, can be used for ADC drug development and be expected to obtain more preferably antitumor work Property and effect, to reach the purposes for the treatment of cancer.

The present invention mainly solves above-mentioned technical problem by following technological means.

The present invention provides a kind of anti-B7-H3 antibody, and it includes complementary determining regions: heavy chain CDR1, heavy chain CDR2 and heavy chain One of CDR3 or a variety of, and/or, one of light chain CDR1, light chain CDR2 and light chain CDR3 or a variety of；The heavy chain The amino acid sequence of CDR1 is as shown in SEQ ID NO.7,16,25 or 34 in sequence table；The amino acid sequence of the heavy chain CDR2 As shown in SEQ ID NO.8,17,26 or 35 in sequence table；SEQ ID in the amino acid sequence of the heavy chain CDR3 such as sequence table Shown in NO.9,18,27,36,43 or 49；SEQ ID NO.11 in the amino acid sequence of the light chain CDR1 such as sequence table, 20, 29, shown in 38,45 or 51；SEQ ID NO.12,21,30,39 or 52 in the amino acid sequence of the light chain CDR2 such as sequence table It is shown；The amino acid sequence of the light chain CDR3 is as shown in SEQ ID NO.13,22,31,40,46 or 53 in sequence table.

Preferably, in above-mentioned B7-H3 antibody:

The amino acid sequence of the heavy chain CDR1 is as shown in SEQ ID NO.7, the amino acid sequence of the heavy chain CDR2 Column are as shown in SEQ ID NO.8, and the amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID NO.9；

Alternatively, the amino acid sequence of the heavy chain CDR1 is as shown in SEQ ID NO.16, the ammonia of the heavy chain CDR2 Base acid sequence is as shown in SEQ ID NO.17, and the amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID NO.18；

Alternatively, the amino acid sequence of the heavy chain CDR1 is as shown in SEQ ID NO.25, the ammonia of the heavy chain CDR2 Base acid sequence is as shown in SEQ ID NO.26, and the amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID NO.27；

Alternatively, the amino acid sequence of the heavy chain CDR1 is as shown in SEQ ID NO.34, the ammonia of the heavy chain CDR2 Base acid sequence is as shown in SEQ ID NO.35, and the amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID NO.36；

Alternatively, the amino acid sequence of the heavy chain CDR1 is as shown in SEQ ID NO.7, the amino of the heavy chain CDR2 Acid sequence is as shown in SEQ ID NO.8, and the amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID NO.43；

Alternatively, the amino acid sequence of the heavy chain CDR1 is as shown in SEQ ID NO.7, the amino of the heavy chain CDR2 Acid sequence is as shown in SEQ ID NO.8, and the amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID NO.49.

The amino acid sequence of the light chain CDR1 is as shown in SEQ ID NO.11, the amino acid sequence of the light chain CDR2 Column are as shown in SEQ ID NO.12, and the amino acid sequence of the light chain CDR3 is as shown in SEQ ID NO.13；

Alternatively, the amino acid sequence of the light chain CDR1 is as shown in SEQ ID NO.20, the ammonia of the light chain CDR2 Base acid sequence is as shown in SEQ ID NO.21, and the amino acid sequence of the light chain CDR3 is as shown in SEQ ID NO.22；

Alternatively, the amino acid sequence of the light chain CDR1 is as shown in SEQ ID NO.29, the ammonia of the light chain CDR2 Base acid sequence is as shown in SEQ ID NO.30, and the amino acid sequence of the light chain CDR3 is as shown in SEQ ID NO.31；

Alternatively, the amino acid sequence of the light chain CDR1 is as shown in SEQ ID NO.38, the ammonia of the light chain CDR2 Base acid sequence is as shown in SEQ ID NO.39, and the amino acid sequence of the light chain CDR3 is as shown in SEQ ID NO.40；

Alternatively, the amino acid sequence of the light chain CDR1 is as shown in SEQ ID NO.45, the ammonia of the light chain CDR2 Base acid sequence is as shown in SEQ ID NO.12, and the amino acid sequence of the light chain CDR3 is as shown in SEQ ID NO.46；

Alternatively, the amino acid sequence of the light chain CDR1 is as shown in SEQ ID NO.51, the ammonia of the light chain CDR2 Base acid sequence is as shown in SEQ ID NO.52, and the amino acid sequence of the light chain CDR3 is as shown in SEQ ID NO.53.

More preferably, the anti-B7-H3 antibody includes heavy chain variable region (alternatively referred to as VH structural domain) and/or light chain variable Area's (alternatively referred to as VL structural domain), or the equivalent and their homologue that replace with one or more conservative amino acids (homologs)；SEQ ID NO.6,15,24,33,42 or 48 in the amino acid sequence of the heavy chain variable region such as sequence table It is shown, either have at least 90% with the amino acid sequence as shown in SEQ ID NO.6,15,24,33,42 or 48 in sequence table Homology；The amino acid sequence of the light chain variable region such as the institute of SEQ ID NO.10,19,28,37,44 or 50 in sequence table Show either there is at least 90% with such as the amino acid sequence as shown in SEQ ID NO.10,19,28,37,44 or 50 in sequence table Homology.The amino acid sequence of heretofore described " at least 90% homology " is by institute in foregoing sequences table The amino acid sequence shown is inserted into, is lacked or is replaced acquisition, and the replacement can are as follows: for example, carrying out computer to sequence Structural simulation analysis, to that may be present, particularly CDR region posttranscriptional modification (Potential post- Translational modifications, PTMs) Locus Analysis in Shoots, aggregation, demidizate including antibody are sensitive (asparagine deamidation, site (NG, NS, NH etc.), aspartic acid isomery (DG, DP) sensitivity site, N glycosylation The analysis of (N- { P } S/T) sensitivity site and oxidation-sensitive site etc. and replacement.

The anti-B7-H3 antibody includes or as having from any shown in SEQ ID NO.6,15,24,33,42 and 48 The amino acid sequence of VH structural domain and ammonia from any VL structural domain shown in SEQ ID NO.10,19,28,37,44 and 50 A kind of polypeptide of base acid sequence forms, and to provide one VH/VL pairs, indicates the antigen binding site of antibody.

Further more preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.6 in sequence table, and The amino acid sequence of the light chain variable region is as shown in SEQ ID NO.10 in sequence table；

Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.15 in sequence table, and described The amino acid sequence of light chain variable region is as shown in SEQ ID NO.19 in sequence table；

Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.24 in sequence table, and described The amino acid sequence of light chain variable region is as shown in SEQ ID NO.28 in sequence table；

Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.33 in sequence table, and described The amino acid sequence of light chain variable region is as shown in SEQ ID NO.37 in sequence table；

Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.42 in sequence table, and described The amino acid sequence of light chain variable region is as shown in SEQ ID NO.44 in sequence table；

Alternatively, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.48 in sequence table, and described The amino acid sequence of light chain variable region is as shown in SEQ ID NO.50 in sequence table.

Further more preferably, the anti-B7-H3 antibody further includes that heavy chain constant region and/or antibody light chain are constant Area；The preferred source of people of the heavy chain constant region or small source of mouse heavy chain constant region；The antibody light chain constant region is excellent It chooses source or small source of mouse antibody light chain constant region.

In a preferred embodiment of the present invention, in the amino acid sequence of the heavy chain of the anti-B7-H3 antibody such as sequence table SEQ ID NO.58,60,62,64,66 perhaps shown in SEQ ID NO.68 or with SEQ ID NO.58 in such as sequence table, 60, 62, amino acid sequence shown in 64,66 or SEQ ID NO.68 has at least 90% homology；The anti-B7-H3 antibody Light chain amino acid sequence as shown in the perhaps SEQ ID NO.69 of SEQ ID NO.59,61,63,65,67 in sequence table or Have at least with the amino acid sequence as shown in SEQ ID NO.59,61,63,65,67 or SEQ ID NO.69 in sequence table 90% homology；Preferably, SEQ ID NO.58 in the amino acid sequence of the heavy chain of the anti-B7-H3 antibody such as sequence table The amino acid sequence of the light chain of shown and described anti-B7-H3 antibody is as shown in SEQ ID NO.59 in sequence table；Or it is described Anti- B7-H3 antibody heavy chain amino acid sequence as shown in SEQ ID NO.60 in sequence table, and the anti-B7-H3 antibody Light chain amino acid sequence as shown in SEQ ID NO.61 in sequence table；Or the ammonia of the heavy chain of the anti-B7-H3 antibody Base acid sequence is as shown in SEQ ID NO.62 in sequence table, and the amino acid sequence such as sequence of the light chain of the anti-B7-H3 antibody In list shown in SEQ ID NO.63；Or the heavy chain of the anti-B7-H3 antibody amino acid sequence such as sequence table in SEQ Shown in ID NO.64, and the amino acid sequence of the light chain of the anti-B7-H3 antibody is as shown in SEQ ID NO.65 in sequence table； Or the amino acid sequence of the heavy chain of the anti-B7-H3 antibody is as shown in SEQ ID NO.66 in sequence table, and described anti- The amino acid sequence of the light chain of B7-H3 antibody is as shown in SEQ ID NO.67 in sequence table；Or the anti-B7-H3 antibody The amino acid sequence of heavy chain is as shown in SEQ ID NO.68 in sequence table, and the amino acid of the light chain of the anti-B7-H3 antibody Sequence is as shown in SEQ ID NO.69 in sequence table.

Heretofore described anti-B7-H3 antibody can be following any antibody formation:

(a) a kind of complete immunoglobulin molecules；

(b) a kind of scFv；In the amino acid sequence of the scFv such as sequence table SEQ ID NO.5,14,23,32,41 or Shown in person 47；

(c) a kind of fusion protein comprising scFv；

(d) a kind of Fab segment；

(e) a kind of Fab ' segment；

(f) a kind of F (ab)₂。

Heretofore described anti-B7-H3 antibody can be a kind of monoclonal antibody or polyclonal antibody, the Dan Ke The grand preferred human monoclonal antibodies of antibody.

Heretofore described B7-H3 antibody can be super humanized antibodies or double antibody.

B7-H3 involved in heretofore described anti-B7-H3 antibody can be the B7-H3 of this field routine, such as solvable Property B7-H3, form membrane B7-H3 etc., the B7-H3 of the soluble form is, such as sequence people B7- as shown in SEQ ID NO.1 H3 variant 1, sequence human B 7-H 3 variant 2 as shown in SEQ ID NO.2, the B7-H3 expressed on person monocytic cell, sequence is such as Mouse B7-H3 shown in SEQ ID NO.3, the B7-H3 expressed on mouse monocyte, sequence monkey as shown in SEQ ID NO.4 B7-H3, the B7-H3 expressed on monkey monocyte, sequence B7-H3 as shown in SEQ ID NO.54, sequence such as SEQ ID B7-H3 shown in NO.55, sequence is as shown in SEQ ID NO.56, the IgC structural domain of B7-H3 or sequence such as SEQ ID Shown in NO.57, the IgV structural domain of B7-H3 etc..Preferably, the B7-H3 in the present invention is endogenous on cancer cell surfaces The B7-H3 of expression；More preferably, it is internalized by after the B7-H3 that the anti-B7-H3 antibody is expressed on combining cancer cell surfaces.

The dissociation constant (KD) of heretofore described anti-B7-H3 antibody is preferably 10^-9M is hereinafter, be more preferably 10^-11M Below.

The present invention also provides antibody group (including comprising or by the molecule that forms of antibody fragment or variant), wherein group membership Corresponding to of the invention one, two, three, four, five, or more different antibody [such as complete antibody, Fab, F (ab)₂Segment and scFv etc.].

The anti-B7-H3 antibody in the present invention is a kind of B7-H3 antagonist, especially human B 7-H 3 antagonist.B7-H3 Protein-specific antagonist (or herein referred as " B7-H3 specific antagonists ") be effective B7-H3 egg for inhibiting B7-H3 function White specific binding molecules or albumen may be, for example, containing disclosed CDR structural domain or heavy chain and/or light chain CDR structural domain Group (sets) and their equivalent characterized by there are one or more conservative amino acids to replace.

They are extremely important in treating illness that is related to B7-H3 function or expressing B7-H3, and the illness includes but not It is limited to prostate cancer, oophoroma, colorectal cancer, clear-cell carcinoma, non-small cell lung cancer, cancer of pancreas, melanoma, gastric cancer, bladder The tumour of the correlated expressions such as cancer, glioblastoma and osteosarcoma B7-H3.B7-H3 specific antagonists are characterized in that, Selective recognition and combination to B7-H3.B7-H3 specific antagonists do not show the significant knot to the substance in addition to B7-H3 Close, in addition to those in special circumstances: make antagonist supplement with other, different from B7-H3 specific binding member spies It is anisotropic.In a specific embodiment, KD of the B7-H3 specific antagonists in conjunction with human B 7-H 3 is 1.2 × 10^-6Or it is less.Having In body embodiment, KD of the B7-H3 specific antagonists in conjunction with human B 7-H 3 is 1 × 10^-7Or it is less.In other embodiment party In formula, KD of the B7-H3 specific antagonists in conjunction with human B 7-H 3 is 1 × 10^-8Or it is less.In other embodiments, B7-H3 KD of the specific antagonists in conjunction with human B 7-H 3 is 5 × 10^-9Or it is less or 1 × 10^-9Or it is less.In other embodiments, KD of the B7-H3 specific antagonists in conjunction with human B 7-H 3 is 1 × 10^-10Or it is less, 1 × 10^-11Or it is less or 1 × 10^-12Or more It is few.In a specific embodiment, B7-H3 specific antagonists are not with the other albumen of above-mentioned horizontal integration.

It can effectively be internalized by after B7-H3 specific antagonists specific binding B7-H3 into cell.It repeatedly proves, B7- H3 specific antagonists can bring cell by being internalized by the poisonous substance molecule that can be coupled after combining, and cause apoptosis, and cause thin Dose dependent is presented in the concrete activity of born of the same parents' apoptosis.Therefore, B7-H3 specific antagonists are extremely important to killing tumor cell. B7-H3 specific antagonists of the present invention can also detect and quantitatively be used for various diagnostic purposes in B7-H3.

It will be understood by those skilled in the art that the B7-H3 specific antagonists segment for remaining antagonism B7-H3 ability can be inserted Enter in various frameworks (frameworks), see, for example, United States Patent (USP) 6,818,418 and bibliography therein, they are discussed It can be used for showing the various brackets (scaffolds) of antibody ring, be based on antigen binding before the antibody ring to select.

B7-H3 specific antagonists and segment can be the form of the various brackets for being not based on antibody, including but not limited to Avimers (avimers) (Avidia)；DARPins(MolecularPartners)；Adnectins (Adnexus), Anticalins (Pieris) and Affibodies (Affibody).To for protein bound in scientific literature The use of optional bracket has carried out many evaluations, see, for example, Binz&Pl ü ckthun, 2005Curr.Opin.Biotech.16: 1-11.Therefore, with B7-H3 specific selective, B7-H3 can be specifically bound, or can be internalized by after combining, be not based on antibody Bracket or antagonist molecules constitute important embodiment of the invention.

The present invention also provides a kind of nucleic acid for encoding above-mentioned anti-B7-H3 antibody.

The present invention also provides a kind of recombinant expression carriers comprising above-mentioned nucleic acid.

The present invention also provides a kind of transformant comprising above-mentioned recombinant expression carrier.

The present invention also provides a kind of preparation methods of anti-B7-H3 antibody comprising and following steps: cultivating above-mentioned transformant, The anti-B7-H3 antibody is obtained from culture.

The present invention also provides a kind of immune conjugates comprising above-mentioned anti-B7-H3 antibody；Preferably, the immune idol Connection object is antibody drug conjugates (ADC) or Chimeric antigen receptor T cell (CAR-T)；More preferably, the antibody drug is even Join in object, connected the anti-B7-H3 antibody with cytotoxic agent by connexon, the connexon is SMCC, described Cytotoxic agent is maytansine, the antibody drug ratio (DAR) of the anti-B7-H3 antibody and the maytansine is 3.2~ 3.5。

The present invention also provides a kind of pharmaceutical compositions comprising above-mentioned immune conjugate and pharmaceutically acceptable carrier.

The present invention also provides a kind of above-mentioned anti-B7-H3 antibody or above-mentioned immune conjugate or aforementioned pharmaceutical compositions Application in the drug for preparing B7-H3 abnormal expression related disease, the B7-H3 abnormal expression related disease are preferably swollen Tumor, the preferred cancer of the tumour, the preferred lung cancer of the cancer, breast cancer, prostate cancer, cancer of pancreas, colorectal cancer, melanocyte Tumor, liver cancer, oophoroma, bladder cancer, gastric cancer, the cancer of the esophagus or kidney.

On the basis of common knowledge of the art, above-mentioned each optimum condition, can any combination to get each preferable reality of the present invention Example.

The reagents and materials used in the present invention are commercially available.

The positive effect of the present invention is that:

Anti- B7-H3 antibody of the invention is the human antibody of phage library screening, has unique antigen binding table Position；The B7-H3 antigen on tumour cell can be specifically bound, and in conjunction with tumour cell after can rapid internalization into cell, can use It is expected to obtain more preferably anti-tumor activity and effect in ADC drug development, to reach the purposes for the treatment of cancer.

Detailed description of the invention

Fig. 1 is protein binding of the different antibodies with B7-H3.

Fig. 2 is antibody P1G2, P1D1, P3B9 and P2E3 with Hs-700T cell combination.

Fig. 3 is that P2E5, P1E11 are combined with the Chinese hamster ovary celI that B7-H3 is transfected.

Fig. 4 A is that P1G2, P2E5, P1E11 compete biotin-P1E11 combination B7H3/4Ig antigen；Fig. 4 B is P1G2, P2E5 Compete biotin-P2E5 combination B7H3/4Ig antigen.

Fig. 5 is after different anti-B7-H3 antibody combination tumor cell lines with internalization capability.

Fig. 6 is anti-B7-H3 antibody DM1 class ADC killing activity.

Specific embodiment

Term " B7-H3 " used herein is used with meaning identical with B7-H3 albumen, and is also represented by B7-H3 change Body 1 and/or B7-H3 variant 2.

B7-H3 specific antagonists Selective recognition defined herein simultaneously specifically binds B7-H3.

Terms used herein " selectivity " or " specificity " refer to the fact that the disclosed antagonist is not shown to removing The significant combination of substance except B7-H3, in addition to those in special circumstances: wherein supplement antagonist make it have it is other, The specificity different from B7-H3 specific binding member (for example, bispecific or bifunctional molecule, wherein the MOLECULE DESIGN For combining or exercising two kinds of functions, wherein at least one is specific binding B7-H3).

KD refers to obtained from Kd (specific binding molecule-target protein interaction dissociation rate) and Ka (specific binding molecule- Target protein interaction association rate) the ratio between (or Kd/Ka, indicated with molar concentration (M)) dissociation constant.Ability can be used The method that domain is sufficiently established measures KD value.Measure binding molecule KD preferred method be by using surface plasma resonance, Such as bio-sensor system, such as Biacore TM (GE Healthcare Life Sciences) system.

" antibody molecule " or " antibody " refers to exempting from for immunoglobulin molecules and immunoglobulin molecules as described herein Epidemic disease active part, i.e., the molecule of the antigen binding site containing immunologic specificity combination antigen.Therefore, term antibody is not only covered Complete antibody molecule further includes the segment of the antibody and the variant (including derivative) of the antibody and antibody fragment.? Term antibody molecule described in this specification for example including but be not limited to scFv (scFv), Fab segment, Fab ' segment, F (ab ') 2, the Fv (sdFv) of disulfide bond connection, Fv and complete antibody or full length antibody.Term " scFv " or " scFv " refer to A kind of polypeptide, it includes the VL structural domains for the antibody connecting with antibody VH domain.The antibody of immunologic specificity combination B7-H3 can Cross reaction occurs with other antigens.Preferably, the antibody of immunologic specificity combination B7-H3 does not intersect with other antigens Reaction.The antibody of immunologic specificity combination B7-H3 can be for example by immunoassays or other well known by persons skilled in the art Method identifies." complete " antibody or " overall length " antibody refer to the albumen comprising two heavy chains (H) and two light chains (L), the heavy chain It is connected with each other with light chain by disulfide bond, the albumen includes: (1) for heavy chain, (being abbreviated as herein comprising variable region " VH ") and containing there are three the heavy chain constant region of domain C H1, CH2, CH3；It (2) include light chain variable region for light chain (being abbreviated as " VL " herein) and constant region of light chain containing a domain C L.Antibody of the invention includes, but are not limited to Dan Ke It is grand, polyspecific, people or chimeric antibody, single-chain antibody, Fab segment, F (ab ') segment, antiidiotype (anti-Id) antibody (including Such as the anti-Id antibody of antibody of the present invention) and above-mentioned any antibody epitope binding fragments.Immunoglobulin of the invention point Son can be any type (such as IgG, IgE, IgM, IgD, IgA and IgY) of immunoglobulin, classification (such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.Preferably, antibody of the invention includes or as with any ammonia described in table 1 The VH structural domain of base acid sequence or its segment or variant, VH CDR, VL structural domain or VL CDR composition.

Antibody of the present invention in conjunction with soluble form B7-H3 " is such a antibody, in conjunction with 435 amino acid can The human B 7-H 3 albumen (SEQ ID NO:54) namely 4Ig B7-H3 albumen of molten form, and/or combine the solvable of 217 amino acid The people of the soluble form of the human B 7-H 3 (SEQ ID NO:55) namely 2Ig B7-H3 albumen, and/or 102 amino acid of form The IgC structural domain (SEQ ID NO:56) of B7-H3, and/or 101 amino acid soluble form human B 7-H 3 IgV structural domain (SEQ ID NO:57).In specific embodiments of the invention, B7-H3 specific antagonists of the invention combine 435 ammonia The human B 7-H 3 albumen of the soluble form of base acid, the preferably antibody in combination with the human B 7-H 3 of the soluble form of 216 amino acid.

The antibody of the present invention of " in conjunction with form membrane B7-H3 " is such a antibody, in conjunction with film B7-H3 albumen.At this In the specific embodiments of invention, the antibody of the present invention of " in conjunction with form membrane B7-H3 " is not in conjunction with the B7-H3 of soluble form.? The combination of the Chinese hamster ovary celI (as described herein) transfected in ELISA and through B7-H3 is antibody combination form membrane B7-H3 specificity It examines.Can be used as antibody there is the inspection of specificity to include, but are not limited to combine film as described in Example 5 form membrane B7-H3 Express B7-H3.The antibody of the present invention of " in conjunction with soluble form and form membrane B7-H3 " is to combine again in conjunction with the B7-H3 of form membrane The antibody of soluble form B7-H3.

Term " variant " refers to a kind of polypeptide, with B7-H3 polypeptide, B7-H3 segment, anti-B7-H3 antibody or its antibody piece Section have the function of it is similar or identical, but it is nonessential include similar or identical B7-H3 polypeptide, B7-H3 segment, anti-B7-H3 resist The amino acid sequence of body or its segment, or with similar or identical B7-H3 polypeptide, B7-H3 segment, anti-B7-H3 antibody or its The structure of segment.A kind of variant with similar amino acid sequence is known as a kind of polypeptide, at least conforms to a kind of following polypeptide: (a) include or by a kind of polypeptide that amino acid sequence forms, the amino acid sequence and B7-H3 polypeptide, B7-H3 segment, anti-B7- H3 antibody or its segment (including the VH structural domain with any amino acid sequence shown in table 1, VHCDR, VL structural domain, or VLCDR amino acid sequence) have at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, the 95% or at least 99% phase same sex；(b) by it is a kind of it is nucleotide sequence coded containing at least 5,10,15, 20,25,30,40,50,60,70,80,90,100,125 or at least 150 amino acid residues polypeptide, the nucleotide sequence Complementary series under stringent hybridization conditions with coding B7-H3 polypeptide, B7-H3 segment, anti-B7-H3 antibody or its segment (including VH structural domain with any amino acid sequence shown in table 1, VHCDR, VL structural domain or VLCDR) nucleotide sequence it is miscellaneous It hands over；(c) resisted by a kind of nucleotide sequence coded polypeptide, the nucleotide sequence and coding B7-H3 polypeptide, B7-H3 segment B7-H3 antibody or its segment (including the VH structural domain with any amino acid sequence shown in table 1, VHCDR, VL structural domain, or VLCDR nucleotide sequence) have at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, the 95% or at least 99% phase same sex.With B7-H3 polypeptide, B7-H3 segment, anti-B7-H3 antibody or its segment Polypeptide with similar structure refers to such polypeptide, and with B7-H3 polypeptide, B7-H3 segment resists and B7-H3 antibody or its piece Section has similar second level, three or four structure.The structure of polypeptide can determine by methods known to those skilled in the art, Include, but are not limited to X-ray crystallisation, nuclear magnetic resonance, and crystallization electron microscopy.

As used herein, the percentage that the Percent homology between two amino acid sequences is equal between two sequences is same One property (identity).Percent sequence identity between two sequences is the function of the number of the shared same position of sequence (that is, % homology=same position number/total number of positions mesh X100), wherein consider notch (gap) number and it is each lack The length of mouth, needs to be introduced into the optimal comparison for two sequences.Sequence can be carried out with the generally known method in this field Compare and determine the percentage identity between sequence, can realize that this sequence compares with percentage identity really with mathematical algorithm It is fixed.For example, Meyers and Miller can be used, the algorithm of 1988Comput.Appl.Biosci.4:11-17 (has been integrated into ALIGN Program (version 2 .0)) determine the percentage identity between amino acid sequence and/or between nucleotide sequence.In addition, available It is determined between amino acid sequence from the GAP program (using its default parameter) in the GCG software package that Accelrys is obtained online Or the percentage identity between nucleotide sequence.In one embodiment, described two sequences are isometric.

Term " epitope " refers to the B7-H3 preferably in the mammalian body in animal body with antigenicity or immunogenic activity A part.Epitope with immunogenic activity is a part of B7-H3, excites antibody response in animal body.Have The epitope of antigenic activity is a part of B7-H3, and for antibody in conjunction with its immunologic specificity, this can be by known in the art Method measurement, such as method of immunity as described herein.Nonessential antigenic epitopes are immunogenicities.

In a specific aspect, the present invention provides the isolated B7-H3 specific antagonists of antagonism B7-H3.Specific implementation In mode, the B7-H3 specific antagonists specifically bind the combination of B7-H3 and blocking and its unknown receptor.Pass through repetition Experiment, B7-H3 specific antagonists of the invention (such as antibody molecule P1G2 or P2E5) dose-dependently combine B7-H3. Therefore, in a specific embodiment, the present invention includes B7-H3 specific antagonists, of the invention in a more specific embodiment, Including antibody molecule, (it is characterized in having comprising heavy chain and/or light chain variable region and their equivalent in the antibody molecule There are one or more conservative amino acids to replace) or homologue.Terms used herein " structural domain " or " region " simply refer to anti- Each section of body molecule, wherein (reside) will be present or there is currently the sequences or segment that have arguement (at issue).

It will be understood by those skilled in the art that conservative amino acid substitution be can be brought with one it is similar or better (to pre- For the purpose of phase) amino acid residue of functional characteristic and/or chemical characteristic carrys out the displacement of substituted amino acid residue.For example, protecting Keeping acidic amino acid substitution, often wherein amino acid residue is replaced by the amino acid residue with similar side chain.Determine in this field Amino acid residue families of the justice with similar side chain.

These families include the amino acid with following side chain: basic side chain (for example, lysine, arginine, histidine), Acid side-chain (for example, aspartic acid, glutamic acid), uncharged polar side chain are (for example, glycine, asparagine, glutamy Amine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar sidechain is (for example, alanine, valine, bright ammonia Acid, isoleucine, proline, phenylalanine, methionine), β-branched building block (for example, threonine, valine, isoleucine) With aromatic side chains (for example, tyrosine, phenylalanine, tryptophan, histidine).Such modification is not intended to significantly reduce or change The combination of B7-H3 specific antagonists or functional character, although they may improve these characteristics.The purpose replaced does not weigh It wants, it may include but be not limited to, with can preferably maintain or enhance molecular structure, the charge of molecule or hydrophobicity or molecule The residue of size substitute a certain residue.Such as, it may be desirable to replace one kind not with a kind of residue with same polarity or charge Too ideal residue.It can be such to introduce with standard technique known in the art (such as mutagenesis of site-directed mutagenesis and PCR mediation) Modification.A kind of concrete mode that those skilled in the art realize that conservative amino acid replaces is alanine scanning mutagenesis, is described in For example, MacLennan etc., 1998Acta Physiol.Scand.Suppl.643:55-67 and Sasaki etc., In 1998Adv.Biophys.35:1-24.Then available or functional trial as described herein test is through changing with this field Whether the antagonist of change remains function or has better function.It is herein that the B7-H3 specificity with following characteristics is short of money Anti-agent is known as " functionally equivalent " of antagonist disclosed herein and constitutes a specific embodiment of the invention: have one or Multiple above-mentioned conservative amino acids replace, and remain selective binding in the ability of human B 7-H 3, and above-mentioned relative to not having The level of the molecule of amino acid variation, antagonism B7-H3 function is identical or more preferably.

Usually preparation has special with the B7-H3 of the amino acid sequence of the amino acid sequence homologous of antagonist as described herein Property antagonist improves one or more characteristics of the antagonist without the specificity that changes it to B7-H3.Obtain such sequence A kind of method (the not available unique method of those skilled in the art) of column is to make to encode B7-H3 specific antagonists or its spy The series jump of anisotropic determining area, expression include the antagonist of these mutant nucleotide sequences, use available functional trial (packet Include those described herein test) come test these coding antagonist whether remain function.Can by site-directed mutagenesis or Random mutagenesis is mutated.It will be understood by those skilled in the art, however, that other method of mutagenesis can easily realize identical effect Fruit.For example, in certain methods, with the nonrandom targeting conservative substitution of chemical characteristic or architectural characteristic based on amino acid or (spectrum) is composed with the considerations of protein structure aspect come restriction mutant.It is single seleced in affinity maturation experiment Several such mutation can be found in molecule (through random or nonrandom selection).For affinity maturation, structure is based on there are also various Method, be described in such as United States Patent (USP) 7,117,096, PCT Publication: WO 02/084277 and WO03/099999.

The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient The selection of product specification.

The preparation of 1 human B 7-H 3 albumen of embodiment

The nucleic acid sequence of the 29-245 amino acids of people 2Ig B7-H3 is chosen, wherein N-terminal addition purifying 10His label and C End addition detection Myc label, is named as H2M；The nucleic acid sequence of the 27-461 amino acids of people 4Ig B7-H3 is chosen, wherein N-terminal Purification tag 10His and C-terminal addition detection Myc label are added, H4M is named as；Choose the 27-461 bit amino of people 4Ig B7-H3 The nucleic acid sequence of acid, wherein N-terminal addition detection myc label and C-terminal addition purifying 10His label, are named as M4H.It is respectively synthesized Gene plasmid H2M-pUC57, M4H-pUC57 and H4M-pUC57 of above-mentioned three kinds of B7-H3 antigen, and synthesize pv81 expression vector Plasmid screens through PCR and expands respectively by connection conversion competent escherichia coli cell Trans-T1 after EcoRI and SmalI digestion Increase a large amount of extractings that correctly clone carries out plasmid.By expression vector H2M, H4M and M4H for preparing of extracting respectively at Turn expression 7 days in Expi293 wink, expression supernatant purifies preparation after ELISA is detected.

Biotinylated antigen preparation, by the biotin of the H4M antigen (concentration 3.36mg/mL) prepared and PBS and 10uM It is incubated under the conditions of 37 DEG C, the amount ratio of antigen and biotin substance is 1:10, then 0.1mL 3M is added in 30min to be incubated for Tris is reacted with terminating, biotinylated antigen concentration displacement buffer to 20mM PB, with removal in 150mM NaCl, pH7.4 Free biotin is computed, and H4M antigen its final labelled amount after biotin labeling is about 2.5.

The preparation of 2 library of embodiment

The library 1ml Lambda size 2.91*10 is taken respectively⁹It is 3.72*10 with library Kappa size⁹Bacterium solution be added 2.0L In fresh culture 2YT+100ug/ml Amp+2% glucose, library bacterium solution originates OD600 < 0.1, in 37 DEG C, revolving speed It is cultivated under conditions of 200rpm.When OD600 is 0.5-0.6, the M13K07 of 365ul, titre 9.6*10 is added¹²/ ml, adds Enter 10 times that amount is bacterium amount, bacterium amount=OD600*8.0*10⁸/ ml* shakes thallus product, and helper phage grain infects.Helper phage is added In 37 DEG C of condition static gas wave refrigerator 30min after body, then under the conditions of 200rpm 30min cultivate, finally under the conditions of 4000rpm from Heart 10min is resuspended, 30 DEG C of culture 16h, for expressing with 2.0L isometric 2YT+100ug/mlAmp+50ug/ml Kana. After the completion of culture to be expressed, take bacterium solution under the conditions of 4 DEG C, 8000rpm is centrifuged 30 minutes, removes supernatant.High speed centrifugation removes bacterium The PEG/NACL of 1/5 volume is added in supernatant for body, precipitates the Phage in supernatant, Phage is dissolved in PBS after centrifugation, surveys Phage titre is about 2.1*10¹¹。

The screening of the anti-B7-H3 antibody phage libraries of embodiment 3

Liquid phase elutriation: 150uL magnetic bead Dynabeads M-280 closes 1h through 1% casein room temperature, has added 100uL The phage library Phage prepared, is jiggled, and room temperature closes 1h.The biotinylated B7-H3 of 30ug is added after the completion of closing Antigen H4M is incubated at room temperature 1h, and the compound of antigen H4M and phage antibody is incubated for closed magnetic bead again after the completion of being incubated for 15min is integrated to its compound on magnetic bead, is respectively washed 15 times with PBST and PBS.Then the trypsase of 1ml is added The bacteriophage of (10ug/ml) elution and antigen binding, elution volume 1ml are infected the TG1 for being growing logarithmic phase, survey drop Degree, amplification bacteriophage are used for next round elutriation.3 wheel elutriations, the rear 2 wheel panning process same first round, but the biology being added are carried out altogether Elementization amount of antigen H4M gradually decreases respectively 10ug and 5ug or 7.5ug and 2.5ug.

Phage-ELISA screening: take concentration be 1mg/ml Streptavidin, room-temperature dissolution, after mixing with coating buffer by Step is diluted to 5ug/ml, and the hole every hole 100uL/ in 96 orifice plates is added, and 3 holes of another setting are only added 100uL coating buffer and make blank control, 2-8 DEG C overnight.Take biotinylated B7-H3 antigen H4M, with 0.5%BSA-PBST diluted to 50ng/ml, then with The concentration 100uL of 20ng/ml is added in the coated lath of Streptavidin, is put into microplate oscillator, 37 DEG C, 600rpm vibration After swinging 1 hour, liquid is abandoned, is patted dry with after cleaning solution board-washing 3 times.The positive bacteriophage for taking the second wheel or third round elutriation to obtain, room After temperature dissolution mixes, with 500 times of diluted.ELISA Plate is added according to the amount in the hole 100uL/ in the bacteriophage diluted In, it is put into microplate oscillator, oscillation incubation 1 hour under the conditions of 37 DEG C, 600rpm, liquid is abandoned, with cleaning solution board-washing 3 times. Then the enzyme-linked antibody of 100uL diluted HRP/Anti-M137 is added in every hole, is put into microplate oscillator, 37 DEG C, It after 600rpm vibrates 1 hour, abandons liquid board-washing 3 times, TMB developing solution is added with the amount in the hole 100uL/ and is developed the color 15 minutes, is then added 1mol/L H is added with the hole 100ul/₂SO₄Terminate liquid terminates reaction, with microplate reader using 650nm wavelength as reference wavelength, in wavelength Absorbance is measured at 450nm.

ScFv albumen ElISA screening: taking B7-H3 antigen H4M, and room-temperature dissolution is diluted to 2ng/ml with coating buffer, with The amount in the hole 100uL/ is added in hydrophobic ELIAS strip, and 3 holes of another setting are only added 100uL coating buffer and make blank control, 2-8 DEG C of mistake Night.The scfv protein sample of 100uL is added into every hole, is put into microplate oscillator, is vibrated under the conditions of 37 DEG C, 600rpm 1 hour, liquid is abandoned, with cleaning solution board-washing 3 times.Then the enzyme-linked antibody after dilution is added with the amount in 100 holes μ l/, is put into microwell plate It in oscillator, after 37 DEG C, 600rpm vibrate 1 hour, abandons liquid board-washing 3 times, the colour developing of TMB developing solution is added with the amount in the hole 100uL/ It 15 minutes, is then added and 1mol/L H is added with the hole 100ul/₂SO₄Terminate liquid terminates reaction, is with 650nm wavelength with microplate reader Reference wavelength measures absorbance at wavelength 450nm.

As described above, screening the scFv antibody of 60 anti-B7-H3 with unique sequences, wherein example scFv antibody altogether (wherein the determination basis Kabat of CDR is advised the sequence composition of P1D1, P1G2, P2E5, P1E11, P2E3, P3B9 as shown in table 1 Then).

Table 1

ScFv antibody P1D1, P1G2, P2E5, P1E11, P2E3, P3B9 etc. are in Phage-ELISA and scFv protein ELISA It is as shown in table 2 below with the binding signal value of B7-H3 antigen H4M.The result shows that the scFv antibody screened is to B7-H3 albumen Has preferable binding signal value.

2 Phage scFv of table and albumen scFv combination H4M ELISA signal value

Antibody	Sequence	Phage scFv ELISA signal value	ScFv protein ELISA signal value
				P1D1	SEQ ID NO:5	3.65	2.97
P2E5	SEQ ID NO:14	0.74	0.84
				P1E11	SEQ ID NO:23	0.92	0.20
P1G2	SEQ ID NO:32	3.55	0.54
				P3B9	SEQ ID NO:41	3.09	3.39
P2E3	SEQ ID NO:47	3.54	3.58

The anti-B7-H3 full length antibody protein ELISA of embodiment 4 combines

Anti- B7-H3 full length antibody protein ELISA combines

IgG1 Antibody preparation: changing into IgG1 form for scFv, will be it is desirable that changing into the VH structural domain of the scFv of IgG molecule It clones with VL structural domain into containing the core for encoding suitable heavy chain (human IgG1) or light chain (people kappa or people lambda) constant region In the carrier of nucleotide sequence, to have been expressed from these carriers when transfecting these carriers into suitable host cell Whole heavy chain or light chain molecule.In addition, the heavy chain and light chain as clone (carry in a cell line from a carrier or two Body) simultaneously when expressing, they can be assembled into complete functional antibody molecule, are secreted into cell culture growth medium.It will It is that the VH of scFv sequence and VL (are distinguished direct-assembling to resist in overall length by technology well known in the art that scFv, which changes into conventional antibody molecule, On the heavy chain constant region and constant region of light chain of body).Separately design above-mentioned example antibody such as P1G2, P1E11, P2E3, P2E5, P3B9 And 6 pairs of light and weight chain primer sequences such as P1D1, except the light chain of P1E11 is building up to pfu-CLIg- using EcoRI+BsiWI double digestion Outer on hk (invitrogen) carrier, remaining antibody light chain is building up to pfu-CLIg-hl2 using EcoRI+ArvII double digestion (invitrogen) on carrier, heavy chain is building up to pfu-CLIg-Hg1 using EcoRI+NheI double digestion H chain (IgG1) (invitrogen) on carrier, (J. Pehanorm cloth Shandong is detailed in through PCR screening amplification a large amount of extractings that correctly clone carries out plasmid Gram .2 editions [M] Science Presses of Molecular Cloning:A Laboratory guide, 1992.).The expression vector that extracting is prepared is respectively at CHO- Turn expression 7 days in S cell wink, expression supernatant purifies preparation after ELISA is detected.

Protein binding: the antigen B7-H3 (R&D) of coating 100uL 20ng/ml is on ELISA Plate, and 2-8 DEG C overnight.Then divide Not Jia Ru 100uL concentration from 2000ng/ml originate, 3 times of gradient dilutions, amount to 8 concentration points the above-mentioned overall length prepared IgG1 antibody is incubated for 1 hour under the conditions of 37 DEG C, 600rpm, and washing three times, adds Goat anti human IgG (Fc)-HRP Enzyme-linked antibody, 37 DEG C, 600rpm oscillation 1 hour, washs 4 times, adds TMB developing solution, develop the color 10 minutes, add the 1mol/L of 100uL H₂SO₄Reaction is terminated, absorbance is surveyed.As a result in addition to P1E11 and P2E3 is not combined with coated B7-H3 albumen, remaining antibody is equal B7-H3 antigen is specifically bound, the antibody such as P1D1, P3B9, P1G2 and P2E5 as shown in Figure 1 are combined with B7-H3 protein level.

The anti-B7-H3 antibody Cell-Base ELISA combining assessment of embodiment 5

Pancreas cancer cell strain Hs-700T after cultivation, adds FBS/DMEM culture medium, is spread with the amount in the hole 20000cells/ The plate hole of plate, setting not inoculating cell adds cell culture complete medium to make blank control, in 37 DEG C, 5%CO₂Under conditions of cultivate 20 ~24 hours.PBST is washed board-washing 1 time, and the hole formaldehyde 200ul/ is added, and room temperature fixes 0.5 hour, is washed 3 times, and it is de- to be added 10% Rouge milk powder room temperature closes cleaning solution 3 times again in 1 hour, gently pats dry stand-by.Respectively such as by the above-mentioned IgG1 antibody prepared After P2E3, P1D1 and P3B9 are mixed, each antibody 0.5%BSA-PBST diluted to 10000ng/ml, then again Than dilution, amount to 8 concentration points, be coated in the orifice plate of Hs-700T by the addition in the hole 100ul/ respectively, is incubated at room temperature 1 hour, It is washed out 3 times.Reaction, detection are terminated after adding TMB to develop the color.As a result antibody P1D1, P1G2, P3B9 and P2E3 as shown in Figure 2 With Hs-700T cell combination.

It is combined using the IgG1 antibody as prepared by upper type same susceptible of proof with the CHO transfected through B7-H3, as a result as schemed P2E5, P1E11 shown in 3 are combined with the Chinese hamster ovary celI that B7-H3 is transfected.

Another antibody P1G2 is specifically bound with albumen and Hs-700T cell and Chinese hamster ovary celI, is shown in map The combination of the antibody is horizontal, and temporarily unlisted cellular level combines, and only its cellular level combines activity relatively weak, but subsequent ADC killing evaluation in its ADC activity it is best.

The anti-B7-H3 antibody of embodiment 6 is combined with B7-H3 structural domain

In mode in the same manner as in Example 4, by protein ELISA method examine IgG1 antibody P1G2, P2E5 and Which structural domain of P1E11 combination B7-H3 such as structural domain 2IgB7-H3, IgC structural domain and IgV structural domain.As a result, such as table Shown in 3, it was confirmed that P1G2 is combined with IgC the and IgV structural domain of B7-H3, P2E5 with B7-H3 IgC structural domain in conjunction with and it is same IgV structural domain weak binding, and P1E11 is combined with the IgC structural domain of B7-H3 and is combined without same IgV structural domain.These results card The real IgC structural domain (SEQ ID NO:56) and IgV structural domain (SEQ ID NO:57) of P1G2 antibody combination B7-H3.With phase Same mode, has also demonstrated P2E5 combination IgC structural domain weak binding IgV structural domain, P1E11 only combines IgC structural domain.

Table 3 P1G2, P2E5 and P1E11 are combined with B7-H3 different structure territory

Note :+, weak binding；++, medium combination is horizontal；+++, it is strong to combine；×, it does not combine

By the fixed ELISA Plate of 4Ig-B7H3 antigen, in conjunction with P1E11 antibody (the biotin labeling DAR- through biotin labeling 2.5), by IgG1 antibody P1G2 and P2E5 series of concentrations and P1E11 competitive binding, enzyme-linked secondary antibody is Streptavidin-HRP (1:400).With commercialization developing solution reagent and suitable developing time, blank response range≤0.1 is basic demand.As a result P1G2, P2E5 as shown in Figure 4 A do not have competitive binding relationship with P1E11, show P1G2, P2E5 different tables in conjunction with P1E11 Position.The P2E5 antibody (biotin labeling DAR-2.0) of the same manner P1G2 and P2E5 series of concentrations and biotin labeling competition knot It closes, if Fig. 4 B also demonstrates that prepared antibody P1G2 and P2E5 do not have competitive binding B7-H3, shows P1G2 and P2E5 difference In conjunction with the different epitopes of B7-H3.Therefore different epitopes of the prepared P1G2 and P2E5 and P1E11 respectively in connection with B7-H3.

The anti-B7-H3 antibody of embodiment 7 is combined with B7-H3 and is internalized by

Have studied the ability that anti-B7-H3 antibody of the invention is internalized by after combining cancer cell.By pancreatic cell cancer Hs700T (being purchased from ATCC) is inoculated in 96 porocyte culture plates, every hole 80000cells/50 μ l, cultivates 20-24hr；Secondly with cell pair The culture medium containing 15%FBS answered dilute respectively anti-B7-H3 antibody such as P1G2, P2E5 and P1E11 to 12.5 of the invention, 6.3, 3.1,1.6,0.8,0.4,0.2nM and 0nM prepare 8 concentration gradients, the anti-B7-H3 antibody of the invention that will have been diluted respectively It is mixed in equal volume with PA-Goat anti-Human IgG Fc Antibody (DAR-10.6) the elder generation 1:1 of 200nM (30 μ g/ml) It is directly transferred to after reaction in the culture plate of inoculating cell, 50 holes μ l/ mix gently 1-2 minutes, train under the conditions of setting 37 DEG C It supports, internalization reacts r for 24 hours, inhales after taking-up and abandons culture solution, adds 1 × PBS (pH8.0), 100 μ l；Then microplate reader excitation wavelength: 532nm, launch wavelength: 560nm selects bottom reading mode to be read.As a result show that anti-B7-H3 of the invention is anti-as shown in Figure 5 Body has the ability being internalized by r for 24 hours after combining cancer cell.

Embodiment 8ADC killing evaluation

It is prepared by the coupling of DM1 class ADC sample.Appropriate antibody samples P1G12, P2E5 and P1E11 are taken respectively, are replaced in 50mM Potassium phosphate, 50mM NaCl, 1mM HEPES (4- (2- ethoxy) -1- piperazine ethanesulfonic acid), pH be 7.4 buffer solution system in, Above-mentioned antibody samples are placed in reaction kettle by controlled concentration in 10mg/mL or so, under stirring, be added SMCC (antibody with The molar ratio of SMCC is 1:7.5), it reacts at room temperature 2 hours.By above-mentioned obtained sample displacement in 50mM sodium phosphate, 100mM NaCl, 60mM Sodium Caprylate in the buffer solution system of pH7.5, is placed in reaction kettle, under stirring, is added and dissolves through DMA DM1 drug (molar ratio of antibody and DM1 are 1:5), reacts at room temperature 1 hour, takes out prepared DM1 class ADC after reaction Sample is replaced in 10mM citric acid, 60g/L sucrose, pH5.0 buffer solution system.It is made using mass spectral analysis after the completion of preparation The DAR value of standby ADC sample.

Lung cancer cell line NCI-H322 (this is bought from one hundred Biotechnology Co., Ltd, Nanjing section) is coupled system with through MCC-DM1 Standby anti-B7-H3 antibody coupling drug P1G2-DM1, P2E5-DM1 and P1E11-DM1 is incubated with.By the work for measuring cell Power, the DM1 class ADC (DAR 3.2-3.5) that anti-B7-H3 antibody of the invention is evaluated with this live to the internalization killing of tumour cell Power.

As a result as shown in Figure 6: again show that anti-B7-H3 antibody coupling drug P1G2-DM1, P2E5-DM1 of the invention and P1E11-DM1 is presented preferable ADC to tumour cell and is internalized by killing activity.

Embodiment 9 assesses the dynamics of antibody and B7-H3 interaction with surface plasma resonance (" SPR ")

SPR detection is carried out with BIAcore T200 (GE) system.Sensor chip CM5 (Series Sensor Chip CM5) and amine coupling kit for fixing is purchased from GE.

Antibody P1G2, P2E5, P1E11 test sample are diluted to 10 μ g/ml, flow velocity with the NaAc buffer of pH 5.5 respectively Being set as 10 μ L/min, EDC and NHS mixed liquors activation chip time is default value 420s, is distinguished using default coupling amount mode Fixed F0006 test sample closes the activated group of unbonded test sample with ethanol amine to 200RU level.

B7-H3 antigen with HBS-EP buffer be diluted by a certain percentage for concentration gradient be respectively 0nM, 0.15625nM, 0.3125nM, 0.625nM, 1.25nM (two repetitions), 2.5nM, 5nM, 10nM, 20nM, when sample analysis, set Setting flow velocity is 30 μ L/min, binding time 120s, Dissociation time 1800s.Then it is regenerated, with Gly-HCl buffer (pH 1.5) can be used as regeneration buffer, and flow velocity is set as 30 μ L/min when regeneration, regenerate 30s.Experiment is run using multi-cycle, For its response signal using analysis time as abscissa, response is ordinate.The data obtained analyzes software by BIAcore T200 It is fitted, used model of fit is 1:1Langmuir binding model, determines that its association rate constant, dissociation rate are normal The kinetic constants such as number and combination dissociation constant.

The binding kinetics parameter of the anti-B7-H3 antibody of table 4

Antibody Designation	Antigen	ka(1/Ms)	kd(1/s)	KD(M)
					P1E11	4IgB7-H3	3.133E+6	7.763E-5	2.478E-11
P1G2	4IgB7-H3	7.264E+6	1.670E-3	2.299E-10
					P2E5	4IgB7-H3	4.25E+4	5.90E-5	2.40E-9

SEQUENCE LISTING

<110>Shanghai Fudan Zhangjiang biomedical Co., Ltd

<120>a kind of anti-B7-H3 antibody and preparation method thereof, its conjugate and application

<130> P180115881C

<160> 69

<170> PatentIn version 3.5

<210> 1

<211> 534

<212> PRT

<213>homo sapiens

<400> 1

Met Leu Arg Arg Arg Gly Ser Pro Gly Met Gly Val His Val Gly Ala

1 5 10 15

Ala Leu Gly Ala Leu Trp Phe Cys Leu Thr Gly Ala Leu Glu Val Gln

20 25 30

Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr Leu

35 40 45

Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn

50 55 60

Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Ala

65 70 75 80

Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe

85 90 95

Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val

100 105 110

Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser Ile Arg Asp

115 120 125

Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys

130 135 140

Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr

145 150 155 160

Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro Glu Ala Glu Val

165 170 175

Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr Thr

180 185 190

Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp Val His Ser Ile Leu

195 200 205

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

210 215 220

Pro Val Leu Gln Gln Asp Ala His Ser Ser Val Thr Ile Thr Pro Gln

225 230 235 240

Arg Ser Pro Thr Gly Ala Val Glu Val Gln Val Pro Glu Asp Pro Val

245 250 255

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

260 265 270

Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr

275 280 285

Asp Thr Lys Gln Leu Val His Ser Phe Thr Glu Gly Arg Asp Gln Gly

290 295 300

Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe Pro Asp Leu Leu Ala Gln

305 310 315 320

Gly Asn Ala Ser Leu Arg Leu Gln Arg Val Arg Val Ala Asp Glu Gly

325 330 335

Ser Phe Thr Cys Phe Val Ser Ile Arg Asp Phe Gly Ser Ala Ala Val

340 345 350

Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu

355 360 365

Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr Val Thr Ile Thr Cys Ser

370 375 380

Ser Tyr Arg Gly Tyr Pro Glu Ala Glu Val Phe Trp Gln Asp Gly Gln

385 390 395 400

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

405 410 415

Gln Gly Leu Phe Asp Val His Ser Val Leu Arg Val Val Leu Gly Ala

420 425 430

Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn Pro Val Leu Gln Gln Asp

435 440 445

Ala His Gly Ser Val Thr Ile Thr Gly Gln Pro Met Thr Phe Pro Pro

450 455 460

Glu Ala Leu Trp Val Thr Val Gly Leu Ser Val Cys Leu Ile Ala Leu

465 470 475 480

Leu Val Ala Leu Ala Phe Val Cys Trp Arg Lys Ile Lys Gln Ser Cys

485 490 495

Glu Glu Glu Asn Ala Gly Ala Glu Asp Gln Asp Gly Glu Gly Glu Gly

500 505 510

Ser Lys Thr Ala Leu Gln Pro Leu Lys His Ser Asp Ser Lys Glu Asp

515 520 525

Asp Gly Gln Glu Ile Ala

530

<210> 2

<211> 316

<212> PRT

<213>homo sapiens

<400> 2

Met Leu Arg Arg Arg Gly Ser Pro Gly Met Gly Val His Val Gly Ala

1 5 10 15

Ala Leu Gly Ala Leu Trp Phe Cys Leu Thr Gly Ala Leu Glu Val Gln

20 25 30

Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr Leu

35 40 45

Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn

50 55 60

Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Ala

65 70 75 80

Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe

85 90 95

Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val

100 105 110

Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser Ile Arg Asp

115 120 125

Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys

130 135 140

Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr

145 150 155 160

Val Thr Ile Thr Cys Ser Ser Tyr Arg Gly Tyr Pro Glu Ala Glu Val

165 170 175

Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr Thr

180 185 190

Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp Val His Ser Val Leu

195 200 205

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

210 215 220

Pro Val Leu Gln Gln Asp Ala His Gly Ser Val Thr Ile Thr Gly Gln

225 230 235 240

Pro Met Thr Phe Pro Pro Glu Ala Leu Trp Val Thr Val Gly Leu Ser

245 250 255

Val Cys Leu Ile Ala Leu Leu Val Ala Leu Ala Phe Val Cys Trp Arg

260 265 270

Lys Ile Lys Gln Ser Cys Glu Glu Glu Asn Ala Gly Ala Glu Asp Gln

275 280 285

Asp Gly Glu Gly Glu Gly Ser Lys Thr Ala Leu Gln Pro Leu Lys His

290 295 300

Ser Asp Ser Lys Glu Asp Asp Gly Gln Glu Ile Ala

305 310 315

<210> 3

<211> 316

<212> PRT

<213>mouse

<400> 3

Met Leu Arg Gly Trp Gly Gly Pro Ser Val Gly Val Cys Val Arg Thr

1 5 10 15

Ala Leu Gly Val Leu Cys Leu Cys Leu Thr Gly Ala Val Glu Val Gln

20 25 30

Val Ser Glu Asp Pro Val Val Ala Leu Val Asp Thr Asp Ala Thr Leu

35 40 45

Arg Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn

50 55 60

Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Thr

65 70 75 80

Glu Gly Arg Asp Gln Gly Ser Ala Tyr Ser Asn Arg Thr Ala Leu Phe

85 90 95

Pro Asp Leu Leu Val Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val

100 105 110

Arg Val Thr Asp Glu Gly Ser Tyr Thr Cys Phe Val Ser Ile Gln Asp

115 120 125

Phe Asp Ser Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys

130 135 140

Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asn Met

145 150 155 160

Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro Glu Ala Glu Val

165 170 175

Phe Trp Lys Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr Thr

180 185 190

Ser Gln Met Ala Asn Glu Arg Gly Leu Phe Asp Val His Ser Val Leu

195 200 205

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

210 215 220

Pro Val Leu Gln Gln Asp Ala His Gly Ser Val Thr Ile Thr Gly Gln

225 230 235 240

Pro Leu Thr Phe Pro Pro Glu Ala Leu Trp Val Thr Val Gly Leu Ser

245 250 255

Val Cys Leu Val Val Leu Leu Val Ala Leu Ala Phe Val Cys Trp Arg

260 265 270

Lys Ile Lys Gln Ser Cys Glu Glu Glu Asn Ala Gly Ala Glu Asp Gln

275 280 285

Asp Gly Asp Gly Glu Gly Ser Lys Thr Ala Leu Arg Pro Leu Lys Pro

290 295 300

Ser Glu Asn Lys Glu Asp Asp Gly Gln Glu Ile Ala

305 310 315

<210> 4

<211> 534

<212> PRT

<213>monkey

<400> 4

Met Leu His Arg Arg Gly Ser Pro Gly Met Gly Val His Val Gly Ala

1 5 10 15

Ala Leu Gly Ala Leu Trp Phe Cys Leu Thr Gly Ala Leu Glu Val Gln

20 25 30

Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr Leu

35 40 45

Arg Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn

50 55 60

Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Thr

65 70 75 80

Glu Gly Arg Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe

85 90 95

Leu Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val

100 105 110

Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser Ile Arg Asp

115 120 125

Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys

130 135 140

Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr

145 150 155 160

Val Thr Ile Thr Cys Ser Ser Tyr Arg Gly Tyr Pro Glu Ala Glu Val

165 170 175

Phe Trp Gln Asp Gly Gln Gly Ala Pro Leu Thr Gly Asn Val Thr Thr

180 185 190

Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp Val His Ser Val Leu

195 200 205

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

210 215 220

Pro Val Leu Gln Gln Asp Ala His Gly Ser Ile Thr Ile Thr Pro Gln

225 230 235 240

Arg Ser Pro Thr Gly Ala Val Glu Val Gln Val Pro Glu Asp Pro Val

245 250 255

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

260 265 270

Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr

275 280 285

Asp Thr Lys Gln Leu Val His Ser Phe Thr Glu Gly Arg Asp Gln Gly

290 295 300

Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe Leu Asp Leu Leu Ala Gln

305 310 315 320

Gly Asn Ala Ser Leu Arg Leu Gln Arg Val Arg Val Ala Asp Glu Gly

325 330 335

Ser Phe Thr Cys Phe Val Ser Ile Arg Asp Phe Gly Ser Ala Ala Val

340 345 350

Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu

355 360 365

Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr Val Thr Ile Thr Cys Ser

370 375 380

Ser Tyr Arg Gly Tyr Pro Glu Ala Glu Val Phe Trp Gln Asp Gly Gln

385 390 395 400

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

405 410 415

Gln Gly Leu Phe Asp Val His Ser Val Leu Arg Val Val Leu Gly Ala

420 425 430

Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn Pro Val Leu Gln Gln Asp

435 440 445

Ala His Gly Ser Val Thr Ile Thr Gly Gln Pro Met Thr Phe Pro Pro

450 455 460

Glu Ala Leu Trp Val Thr Val Gly Leu Ser Val Cys Leu Val Ala Leu

465 470 475 480

Leu Val Ala Leu Ala Phe Val Cys Trp Arg Lys Ile Lys Gln Ser Cys

485 490 495

Glu Glu Glu Asn Ala Gly Ala Glu Asp Gln Asp Gly Glu Gly Glu Gly

500 505 510

Ser Lys Thr Ala Leu Gln Pro Leu Lys His Ser Asp Ser Lys Glu Asp

515 520 525

Asp Gly Gln Glu Leu Ala

530

<210> 5

<211> 253

<212> PRT

<213>artificial sequence

<220>

<223>P1D1 scfv sequence

<400> 5

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

1 5 10 15

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

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Arg Ser Val Ala Gly Leu Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

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

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys

130 135 140

Thr Val Val Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg

145 150 155 160

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

165 170 175

Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Arg Leu Leu Ile

180 185 190

Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg Ser

210 215 220

Glu Asp Glu Ser Asp Tyr Tyr Cys Ala Ser Trp Asp Asp Arg Leu Lys

225 230 235 240

Gly Tyr Ala Phe Gly Thr Gly Thr Lys Val Thr Val Leu

245 250

<210> 6

<211> 117

<212> PRT

<213>artificial sequence

<220>

<223> VH

<400> 6

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

1 5 10 15

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

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Arg Ser Val Ala Gly Leu Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 7

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223> VH CDR

<400> 7

Gly Tyr Ser Phe Thr Ser Tyr Trp

1 5

<210> 8

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223> VH CDR2

<400> 8

Ile Tyr Pro Gly Asp Ser Asp Thr

1 5

<210> 9

<211> 10

<212> PRT

<213>artificial sequence

<220>

<223> VH CDR3

<400> 9

Ala Arg Arg Ser Val Ala Gly Leu Asp Tyr

1 5 10

<210> 10

<211> 110

<212> PRT

<213>artificial sequence

<220>

<223> VL

<400> 10

Lys Thr Val Val Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Lys

20 25 30

Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ser Asp Tyr Tyr Cys Ala Ser Trp Asp Asp Arg Leu

85 90 95

Lys Gly Tyr Ala Phe Gly Thr Gly Thr Lys Val Thr Val Leu

100 105 110

<210> 11

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223> VL CDR1

<400> 11

Ser Ser Asn Ile Gly Ser Lys Tyr

1 5

<210> 12

<211> 3

<212> PRT

<213>artificial sequence

<220>

<223> VL CDR2

<400> 12

Arg Asn Asn

1

<210> 13

<211> 11

<212> PRT

<213>artificial sequence

<220>

<223> VL CDR3

<400> 13

Ala Ser Trp Asp Asp Arg Leu Lys Gly Tyr Ala

1 5 10

<210> 14

<211> 256

<212> PRT

<213>artificial sequence

<220>

<223>P2E5 scFv sequence

<400> 14

Gln Val Thr Leu Lys Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ser

20 25 30

Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Met Gly Leu Glu Trp Val

35 40 45

Ala Ser Met Lys Pro Asp Gly Ser Val Lys His Tyr Val Asp Ser Val

50 55 60

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

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Ser Tyr Asp Thr Arg Trp Gly Trp Phe Asp Pro Trp Gly Glu Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Gly Ser Ala Ser Ala Pro Thr Leu Gly

115 120 125

Gln Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Gln Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly

145 150 155 160

Gly Thr Val Thr Leu Thr Cys Gly Leu Asn Ser Gly Ser Val Ser Thr

165 170 175

Ser Tyr Phe Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg

180 185 190

Thr Leu Ile Tyr Asn Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg

195 200 205

Phe Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly

210 215 220

Ala Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Leu Leu Tyr Met Asp

225 230 235 240

Ser Gly Pro His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

245 250 255

<210> 15

<211> 119

<212> PRT

<213>artificial sequence

<220>

<223> VH

<400> 15

Gln Val Thr Leu Lys Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ser

20 25 30

Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Met Gly Leu Glu Trp Val

35 40 45

Ala Ser Met Lys Pro Asp Gly Ser Val Lys His Tyr Val Asp Ser Val

50 55 60

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

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Ser Tyr Asp Thr Arg Trp Gly Trp Phe Asp Pro Trp Gly Glu Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 16

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223> VH CDR1

<400> 16

Gly Phe Thr Phe Ser Asn Ser Tyr

1 5

<210> 17

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223> VH CDR2

<400> 17

Met Lys Pro Asp Gly Ser Val Lys

1 5

<210> 18

<211> 12

<212> PRT

<213>artificial sequence

<220>

<223> VH CDR3

<400> 18

Ser Ser Tyr Asp Thr Arg Trp Gly Trp Phe Asp Pro

1 5 10

<210> 19

<211> 111

<212> PRT

<213>artificial sequence

<220>

<223> VL

<400> 19

Gln Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Leu Asn Ser Gly Ser Val Ser Thr Ser

20 25 30

Tyr Phe Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Thr

35 40 45

Leu Ile Tyr Asn Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Leu Leu Tyr Met Asp Ser

85 90 95

Gly Pro His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 20

<211> 9

<212> PRT

<213>artificial sequence

<220>

<223> VL CDR1

<400> 20

Ser Gly Ser Val Ser Thr Ser Tyr Phe

1 5

<210> 21

<211> 3

<212> PRT

<213>artificial sequence

<220>

<223> VL CDR2

<400> 21

Asn Thr Asn

1

<210> 22

<211> 11

<212> PRT

<213>artificial sequence

<220>

<223> VL CDR3

<400> 22

Leu Leu Tyr Met Asp Ser Gly Pro His Trp Val

1 5 10

<210> 23

<211> 254

<212> PRT

<213>artificial sequence

<220>

<223>P1E11 scFv sequence

<400> 23

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 Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Arg Gly Gly Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

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

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val

130 135 140

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

145 150 155 160

Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser Asp Gly Asn

165 170 175

Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser Pro Arg Arg

180 185 190

Leu Ile Tyr Lys Val Ser Asn Arg Asp Ser Gly Val Pro Asp Arg Phe

195 200 205

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val

210 215 220

Glu Ala Glu Asp Val Gly Phe Tyr Tyr Cys Met Gln Gly Thr His Trp

225 230 235 240

Pro Pro Gly Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

245 250

<210> 24

<211> 115

<212> PRT

<213>artificial sequence

<220>

<223> VH

<400> 24

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 Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Arg Gly Gly Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 25

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223> VH CDR1

<400> 25

Gly Tyr Thr Phe Thr Gly Tyr Tyr

1 5

<210> 26

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223> VH CDR2

<400> 26

Ile Asn Pro Asn Ser Gly Gly Thr

1 5

<210> 27

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223> VH CDR3

<400> 27

Ala Arg Ala Arg Gly Gly Asp Tyr

1 5

<210> 28

<211> 113

<212> PRT

<213>artificial sequence

<220>

<223> VL

<400> 28

Glu Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asp Gly Asn Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Phe Tyr Tyr Cys Met Gln Gly

85 90 95

Thr His Trp Pro Pro Gly Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 29

<211> 11

<212> PRT

<213>artificial sequence

<220>

<223> VL CDR1

<400> 29

Gln Ser Leu Val His Ser Asp Gly Asn Thr Tyr

1 5 10

<210> 30

<211> 3

<212> PRT

<213>artificial sequence

<220>

<223> VL CDR2

<400> 30

Lys Val Ser

1

<210> 31

<211> 10

<212> PRT

<213>artificial sequence

<220>

<223> VL CDR3

<400> 31

Met Gln Gly Thr His Trp Pro Pro Gly Thr

1 5 10

<210> 32

<211> 256

<212> PRT

<213>artificial sequence

<220>

<223>P1G2 scFv sequence

<400> 32

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala 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 Val Asn Tyr Tyr Asp Ser Ser Gly Tyr Tyr Ser Asp Tyr Trp Gly

100 105 110

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

115 120 125

Leu Gly Gln Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Ser Tyr Glu Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser

145 150 155 160

Pro Gly Gln Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val

165 170 175

Gly Gly Tyr Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala

180 185 190

Pro Arg Leu Met Ile Tyr Gly Val Ser Gln Arg Pro Ser Gly Val Pro

195 200 205

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

210 215 220

Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr

225 230 235 240

Ala Asn Asn Asn Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu

245 250 255

<210> 33

<211> 121

<212> PRT

<213>artificial sequence

<220>

<223> VH

<400> 33

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala 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 Val Asn Tyr Tyr Asp Ser Ser Gly Tyr Tyr Ser Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 34

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223> VH CDR1

<400> 34

Gly Gly Thr Phe Ser Ser Tyr Ala

1 5

<210> 35

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223> VH CDR2

<400> 35

Ile Ile Pro Ile Leu Gly Ile Ala

1 5

<210> 36

<211> 14

<212> PRT

<213>artificial sequence

<220>

<223> VH CDR3

<400> 36

Ala Val Asn Tyr Tyr Asp Ser Ser Gly Tyr Tyr Ser Asp Tyr

1 5 10

<210> 37

<211> 109

<212> PRT

<213>artificial sequence

<220>

<223> VL

<400> 37

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Arg Leu

35 40 45

Met Ile Tyr Gly Val Ser Gln Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Ala Asn Asn

85 90 95

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

100 105

<210> 38

<211> 9

<212> PRT

<213>artificial sequence

<220>

<223> VL CDR1

<400> 38

Ser Ser Asp Val Gly Gly Tyr Asn Tyr

1 5

<210> 39

<211> 3

<212> PRT

<213>artificial sequence

<220>

<223> VL CDR2

<400> 39

Gly Val Ser

1

<210> 40

<211> 9

<212> PRT

<213>artificial sequence

<220>

<223> VL CDR3

<400> 40

Ser Ser Tyr Ala Asn Asn Asn Tyr Val

1 5

<210> 41

<211> 253

<212> PRT

<213>artificial sequence

<220>

<223>P3B09 scFv sequence

<400> 41

Gln Ile Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

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

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Arg Thr Pro Arg Gly Met Asp Val Trp Gly Gln Gly Thr Thr

100 105 110

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

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

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

145 150 155 160

Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr

165 170 175

Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile

180 185 190

Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg Ser

210 215 220

Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Ser

225 230 235 240

Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

245 250

<210> 42

<211> 117

<212> PRT

<213>artificial sequence

<220>

<223> VH

<400> 42

Gln Ile Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

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

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Arg Thr Pro Arg Gly Met Asp Val Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 43

<211> 10

<212> PRT

<213>artificial sequence

<220>

<223> VH CDR3

<400> 43

Ala Arg Arg Thr Pro Arg Gly Met Asp Val

1 5 10

<210> 44

<211> 110

<212> PRT

<213>artificial sequence

<220>

<223> VL

<400> 44

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

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 45

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223> VL CDR1

<400> 45

Ser Ser Asn Ile Gly Ser Asn Tyr

1 5

<210> 46

<211> 11

<212> PRT

<213>artificial sequence

<220>

<223> VL CDR3

<400> 46

Ala Ala Trp Asp Asp Ser Leu Ser Gly Trp Val

1 5 10

<210> 47

<211> 253

<212> PRT

<213>artificial sequence

<220>

<223>P2E3 scFv sequence

<400> 47

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

1 5 10 15

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

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Arg Ser Arg Glu Gly Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

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

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

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

145 150 155 160

Val Thr Met Ser Cys Ser Gly Ala Ala Ser Asn Ile Gly Lys Asn Phe

165 170 175

Val Tyr Trp Tyr Gln Gln Leu Pro Gly Arg Ala Pro Arg Leu Leu Ile

180 185 190

Ser Arg Asn Ile Gln Arg Pro Ser Glu Val Pro Asp Arg Phe Ser Gly

195 200 205

Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln Ser

210 215 220

Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Asn

225 230 235 240

Gly Phe Val Phe Gly Thr Gly Thr Ala Val Thr Val Leu

245 250

<210> 48

<211> 117

<212> PRT

<213>artificial sequence

<220>

<223> VH

<400> 48

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

1 5 10 15

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

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Arg Ser Arg Glu Gly Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 49

<211> 10

<212> PRT

<213>artificial sequence

<220>

<223> VH CDR3

<400> 49

Ala Arg Arg Ser Arg Glu Gly Phe Asp Pro

1 5 10

<210> 50

<211> 110

<212> PRT

<213>artificial sequence

<220>

<223> VL

<400> 50

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

1 5 10 15

Arg Val Thr Met Ser Cys Ser Gly Ala Ala Ser Asn Ile Gly Lys Asn

20 25 30

Phe Val Tyr Trp Tyr Gln Gln Leu Pro Gly Arg Ala Pro Arg Leu Leu

35 40 45

Ile Ser Arg Asn Ile Gln Arg Pro Ser Glu Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Asn Gly Phe Val Phe Gly Thr Gly Thr Ala Val Thr Val Leu

100 105 110

<210> 51

<211> 8

<212> PRT

<213>artificial sequence

<220>

<223> VL CDR1

<400> 51

Ala Ser Asn Ile Gly Lys Asn Phe

1 5

<210> 52

<211> 3

<212> PRT

<213>artificial sequence

<220>

<223> VL CDR2

<400> 52

Arg Asn Ile

1

<210> 53

<211> 11

<212> PRT

<213>artificial sequence

<220>

<223> VL CDR3

<400> 53

Ala Ala Trp Asp Asp Ser Leu Asn Gly Phe Val

1 5 10

<210> 54

<211> 435

<212> PRT

<213>artificial sequence

<220>

The B7-H3 4Ig albumen of<223>435 amino acid

<400> 54

Gly Ala Leu Glu Val Gln Val Pro Glu Asp Pro Val Val Ala Leu Val

1 5 10 15

Gly Thr Asp Ala Thr Leu Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe

20 25 30

Ser Leu Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln

35 40 45

Leu Val His Ser Phe Ala Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala

50 55 60

Asn Arg Thr Ala Leu Phe Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser

65 70 75 80

Leu Arg Leu Gln Arg Val Arg Val Ala Asp Glu Gly Ser Phe Thr Cys

85 90 95

Phe Val Ser Ile Arg Asp Phe Gly Ser Ala Ala Val Ser Leu Gln Val

100 105 110

Ala Ala Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu Pro Asn Lys Asp

115 120 125

Leu Arg Pro Gly Asp Thr Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly

130 135 140

Tyr Pro Glu Ala Glu Val Phe Trp Gln Asp Gly Gln Gly Val Pro Leu

145 150 155 160

Thr Gly Asn Val Thr Thr Ser Gln Met Ala Asn Glu Gln Gly Leu Phe

165 170 175

Asp Val His Ser Ile Leu Arg Val Val Leu Gly Ala Asn Gly Thr Tyr

180 185 190

Ser Cys Leu Val Arg Asn Pro Val Leu Gln Gln Asp Ala His Ser Ser

195 200 205

Val Thr Ile Thr Pro Gln Arg Ser Pro Thr Gly Ala Val Glu Val Gln

210 215 220

Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr Leu

225 230 235 240

Arg Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn

245 250 255

Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Thr

260 265 270

Glu Gly Arg Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe

275 280 285

Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val

290 295 300

Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser Ile Arg Asp

305 310 315 320

Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys

325 330 335

Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr

340 345 350

Val Thr Ile Thr Cys Ser Ser Tyr Arg Gly Tyr Pro Glu Ala Glu Val

355 360 365

Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr Thr

370 375 380

Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp Val His Ser Val Leu

385 390 395 400

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

405 410 415

Pro Val Leu Gln Gln Asp Ala His Gly Ser Val Thr Ile Thr Gly Gln

420 425 430

Pro Met Thr

435

<210> 55

<211> 217

<212> PRT

<213>artificial sequence

<220>

The B7-H3 2Ig albumen of<223>217 amino acid

<400> 55

Leu Glu Val Gln Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr

1 5 10 15

Asp Ala Thr Leu Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu

20 25 30

Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val

35 40 45

His Ser Phe Ala Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn Arg

50 55 60

Thr Ala Leu Phe Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg

65 70 75 80

Leu Gln Arg Val Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val

85 90 95

Ser Ile Arg Asp Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala

100 105 110

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

115 120 125

Pro Gly Asp Thr Val Thr Ile Thr Cys Ser Ser Tyr Arg Gly Tyr Pro

130 135 140

Glu Ala Glu Val Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr Gly

145 150 155 160

Asn Val Thr Thr Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp Val

165 170 175

His Ser Val Leu Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys

180 185 190

Leu Val Arg Asn Pro Val Leu Gln Gln Asp Ala His Gly Ser Val Thr

195 200 205

Ile Thr Gly Gln Pro Met Thr Phe Pro

210 215

<210> 56

<211> 102

<212> PRT

<213>artificial sequence

<220>

The B7-H3 IgC domain of<223>102 amino acid

<400> 56

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

1 5 10 15

Pro Gly Asp Thr Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro

20 25 30

Glu Ala Glu Val Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr Gly

35 40 45

Asn Val Thr Thr Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp Val

50 55 60

His Ser Ile Leu Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys

65 70 75 80

Leu Val Arg Asn Pro Val Leu Gln Gln Asp Ala His Ser Ser Val Thr

85 90 95

Ile Thr Pro Gln Arg Ser

100

<210> 57

<211> 101

<212> PRT

<213>artificial sequence

<220>

The B7-H3 IgV domian of<223>101 amino acid

<400> 57

Glu Val Gln Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp

1 5 10 15

Ala Thr Leu Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala

20 25 30

Gln Leu Asn Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His

35 40 45

Ser Phe Ala Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr

50 55 60

Ala Leu Phe Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg Leu

65 70 75 80

Gln Arg Val Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser

85 90 95

Ile Arg Asp Phe Gly

100

<210> 58

<211> 451

<212> PRT

<213>artificial sequence

<220>

<223>P1G2 total length heavy chain

<400> 58

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala 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 Val Asn Tyr Tyr Asp Ser Ser Gly Tyr Tyr Ser Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

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

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

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

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

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

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

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

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 59

<211> 215

<212> PRT

<213>artificial sequence

<220>

<223>P1G2 full-length light chains

<400> 59

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Ala Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Arg Leu

35 40 45

Met Ile Tyr Gly Val Ser Gln Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Ala Asn Asn

85 90 95

Asn Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gln Pro

100 105 110

Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu

115 120 125

Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro

130 135 140

Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala

145 150 155 160

Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala

165 170 175

Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg

180 185 190

Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr

195 200 205

Val Ala Pro Thr Glu Cys Ser

210 215

<210> 60

<211> 449

<212> PRT

<213>artificial sequence

<220>

<223>P2E5 total length heavy chain

<400> 60

Gln Val Thr Leu Lys Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ser

20 25 30

Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Met Gly Leu Glu Trp Val

35 40 45

Ala Ser Met Lys Pro Asp Gly Ser Val Lys His Tyr Val Asp Ser Val

50 55 60

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

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Ser Tyr Asp Thr Arg Trp Gly Trp Phe Asp Pro Trp Gly Glu Gly

100 105 110

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

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

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

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

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

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

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

305 310 315 320

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

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

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

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 61

<211> 217

<212> PRT

<213>artificial sequence

<220>

<223>P2E5 full-length light chains

<400> 61

Gln Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Leu Asn Ser Gly Ser Val Ser Thr Ser

20 25 30

Tyr Phe Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Thr

35 40 45

Leu Ile Tyr Asn Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Leu Leu Tyr Met Asp Ser

85 90 95

Gly Pro His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu

115 120 125

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

130 135 140

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val

145 150 155 160

Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys

165 170 175

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

180 185 190

His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

195 200 205

Lys Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 62

<211> 445

<212> PRT

<213>artificial sequence

<220>

<223>P1E11 total length heavy chain

<400> 62

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 Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Arg Gly Gly Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

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

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

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

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

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

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 63

<211> 220

<212> PRT

<213>artificial sequence

<220>

<223>P1E11 full-length light chains

<400> 63

Glu Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asp Gly Asn Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Phe Tyr Tyr Cys Met Gln Gly

85 90 95

Thr His Trp Pro Pro Gly Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

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

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

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

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

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

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 64

<211> 447

<212> PRT

<213>artificial sequence

<220>

<223>P1D1 total length heavy chain

<400> 64

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

1 5 10 15

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

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Arg Ser Val Ala Gly Leu Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

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

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

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

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

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

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

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

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 65

<211> 216

<212> PRT

<213>artificial sequence

<220>

<223>P1D1 full-length light chains

<400> 65

Lys Thr Val Val Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Lys

20 25 30

Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ser Asp Tyr Tyr Cys Ala Ser Trp Asp Asp Arg Leu

85 90 95

Lys Gly Tyr Ala Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 66

<211> 447

<212> PRT

<213>artificial sequence

<220>

<223>P3B9 total length heavy chain

<400> 66

Gln Ile Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

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

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Arg Thr Pro Arg Gly Met Asp Val Trp Gly Gln Gly Thr Thr

100 105 110

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

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

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

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

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

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

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

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 67

<211> 216

<212> PRT

<213>artificial sequence

<220>

<223>P3B9 full-length light chains

<400> 67

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

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 68

<211> 447

<212> PRT

<213>artificial sequence

<220>

<223>P2E3 total length heavy chain

<400> 68

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

1 5 10 15

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

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Arg Ser Arg Glu Gly Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

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

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

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

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

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

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

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

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 69

<211> 216

<212> PRT

<213>artificial sequence

<220>

<223>P2E3 full-length light chains

<400> 69

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

1 5 10 15

Arg Val Thr Met Ser Cys Ser Gly Ala Ala Ser Asn Ile Gly Lys Asn

20 25 30

Phe Val Tyr Trp Tyr Gln Gln Leu Pro Gly Arg Ala Pro Arg Leu Leu

35 40 45

Ile Ser Arg Asn Ile Gln Arg Pro Ser Glu Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Asn Gly Phe Val Phe Gly Thr Gly Thr Ala Val Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

Claims

1. a kind of anti-B7-H3 antibody, which is characterized in that it includes complementary determining regions: heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 One of or it is a variety of, and/or, one of light chain CDR1, light chain CDR2 and light chain CDR3 or a variety of；The heavy chain CDR1's Amino acid sequence is as shown in SEQ ID NO.7,16,25 or 34 in sequence table；The amino acid sequence such as sequence of the heavy chain CDR2 In table shown in SEQ ID NO.8,17,26 or 35；SEQ ID NO.9 in the amino acid sequence of the heavy chain CDR3 such as sequence table, 18, shown in 27,36,43 or 49；SEQ ID NO.11 in the amino acid sequence of the light chain CDR1 such as sequence table, 20,29,38, Shown in 45 or 51；The amino acid sequence of the light chain CDR2 is as shown in SEQ ID NO.12,21,30,39 or 52 in sequence table；Institute The amino acid sequence of light chain CDR3 is stated as shown in SEQ ID NO.13,22,31,40,46 or 53 in sequence table.

2. anti-B7-H3 antibody as described in claim 1, which is characterized in that the amino acid sequence such as SEQ of the heavy chain CDR1 Shown in ID NO.7, the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID NO.8, and the ammonia of the heavy chain CDR3 Base acid sequence is as shown in SEQ ID NO.9；The amino acid sequence of the heavy chain CDR1 is described as shown in SEQ ID NO.16 The amino acid sequence of heavy chain CDR2 is as shown in SEQ ID NO.17, and the amino acid sequence of the heavy chain CDR3 such as SEQ ID Shown in NO.18；The amino acid sequence of the heavy chain CDR1 is as shown in SEQ ID NO.25, the amino acid of the heavy chain CDR2 Sequence is as shown in SEQ ID NO.26, and the amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID NO.27；Described The amino acid sequence of heavy chain CDR1 is as shown in SEQ ID NO.34, the amino acid sequence of the heavy chain CDR2 such as SEQ ID Shown in NO.35, and the amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID NO.36；The amino of the heavy chain CDR1 For acid sequence as shown in SEQ ID NO.7, the amino acid sequence of the heavy chain CDR2 is and described as shown in SEQ ID NO.8 The amino acid sequence of heavy chain CDR3 is as shown in SEQ ID NO.43；Alternatively, the amino acid sequence such as SEQ of the heavy chain CDR1 Shown in ID NO.7, the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID NO.8, and the ammonia of the heavy chain CDR3 Base acid sequence is as shown in SEQ ID NO.49；And/or

The amino acid sequence of the light chain CDR1 is as shown in SEQ ID NO.11, and the amino acid sequence of the light chain CDR2 is such as Shown in SEQ ID NO.12, and the amino acid sequence of the light chain CDR3 is as shown in SEQ ID NO.13；The light chain The amino acid sequence of CDR1 is as shown in SEQ ID NO.20, the amino acid sequence of the light chain CDR2 such as SEQ ID NO.21 institute Show, and the amino acid sequence of the light chain CDR3 is as shown in SEQ ID NO.22；The amino acid sequence of the light chain CDR1 As shown in SEQ ID NO.29, the amino acid sequence of the light chain CDR2 is as shown in SEQ ID NO.30, and the light chain The amino acid sequence of CDR3 is as shown in SEQ ID NO.31；The amino acid sequence of the light chain CDR1 such as SEQ ID NO.38 institute Show, the amino acid sequence of the light chain CDR2 is as shown in SEQ ID NO.39, and the amino acid sequence of the light chain CDR3 As shown in SEQ ID NO.40；The amino acid sequence of the light chain CDR1 is as shown in SEQ ID NO.45, the light chain The amino acid sequence of CDR2 is as shown in SEQ ID NO.12, and the amino acid sequence of the light chain CDR3 such as SEQ ID NO.46 It is shown；Alternatively, the amino acid sequence of the light chain CDR1 is as shown in SEQ ID NO.51, the amino acid of the light chain CDR2 Sequence is as shown in SEQ ID NO.52, and the amino acid sequence of the light chain CDR3 is as shown in SEQ ID NO.53.

3. anti-B7-H3 antibody as claimed in claim 2, which is characterized in that the anti-B7-H3 antibody includes heavy chain variable region And/or light chain variable region, in the amino acid sequence of the heavy chain variable region such as sequence table SEQ ID NO.6,15,24,33,42 or Shown in person 48, either have at least with the amino acid sequence as shown in SEQ ID NO.6,15,24,33,42 or 48 in sequence table 90% homology；

The amino acid sequence of the light chain variable region as shown in SEQ ID NO.10,19,28,37,44 or 50 in sequence table, or Person with as the amino acid sequence as shown in SEQ ID NO.10,19,28,37,44 or 50 in sequence table have at least 90% it is same Source property.

4. anti-B7-H3 antibody as claimed in claim 3, which is characterized in that the amino acid sequence of the heavy chain variable region is such as In sequence table shown in SEQ ID NO.6, and SEQ ID NO.10 in the amino acid sequence of the light chain variable region such as sequence table It is shown；The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.15 in sequence table, and the light chain variable The amino acid sequence in area is as shown in SEQ ID NO.19 in sequence table；Such as sequence table of the amino acid sequence of the heavy chain variable region Shown in middle SEQ ID NO.24, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.28 in sequence table； The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.33 in sequence table, and the ammonia of the light chain variable region Base acid sequence is as shown in SEQ ID NO.37 in sequence table；SEQ in the amino acid sequence of the heavy chain variable region such as sequence table Shown in ID NO.42, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.44 in sequence table；Alternatively, institute The amino acid sequence for the heavy chain variable region stated is as shown in SEQ ID NO.48 in sequence table, and the amino of the light chain variable region Acid sequence is as shown in SEQ ID NO.50 in sequence table.

5. such as the described in any item anti-B7-H3 antibody of Claims 1 to 4, which is characterized in that the anti-B7-H3 antibody also wraps Include heavy chain constant region and/or antibody light chain constant region.

6. anti-B7-H3 antibody as claimed in claim 5, which is characterized in that the heavy chain constant region is source of people or small Source of mouse heavy chain constant region；The antibody light chain constant region is source of people or small source of mouse antibody light chain constant region；Preferably:

In the amino acid sequence of the heavy chain of the anti-B7-H3 antibody such as sequence table SEQ ID NO.58,60,62,64,66 or Shown in SEQ ID NO.68, either and as shown in SEQ ID NO.58,60,62,64,66 or SEQ ID NO.68 in sequence table Amino acid sequence have at least 90% homology；

In the amino acid sequence of the light chain of the anti-B7-H3 antibody such as sequence table SEQ ID NO.59,61,63,65,67 or Shown in SEQ ID NO.69, either and as shown in SEQ ID NO.59,61,63,65,67 or SEQ ID NO.69 in sequence table Amino acid sequence have at least 90% homology；

More preferably, the amino acid sequence of the heavy chain of the anti-B7-H3 antibody is as shown in SEQ ID NO.58 in sequence table, and institute The amino acid sequence of the light chain for the anti-B7-H3 antibody stated is as shown in SEQ ID NO.59 in sequence table；Or the anti-B7-H3 The amino acid sequence of the heavy chain of antibody is as shown in SEQ ID NO.60 in sequence table, and the light chain of the anti-B7-H3 antibody Amino acid sequence is as shown in SEQ ID NO.61 in sequence table；Or the amino acid sequence of the heavy chain of the anti-B7-H3 antibody As shown in SEQ ID NO.62 in sequence table, and SEQ in the amino acid sequence of the light chain of the anti-B7-H3 antibody such as sequence table Shown in ID NO.63；Or the amino acid sequence of the heavy chain of the anti-B7-H3 antibody such as SEQ ID NO.64 institute in sequence table Show, and the amino acid sequence of the light chain of the anti-B7-H3 antibody is as shown in SEQ ID NO.65 in sequence table；Or it is described The amino acid sequence of the heavy chain of anti-B7-H3 antibody is as shown in SEQ ID NO.66 in sequence table, and the anti-B7-H3 antibody The amino acid sequence of light chain is as shown in SEQ ID NO.67 in sequence table；Or the amino of the heavy chain of the anti-B7-H3 antibody Acid sequence is as shown in SEQ ID NO.68 in sequence table, and the amino acid sequence such as sequence of the light chain of the anti-B7-H3 antibody In table shown in SEQ ID NO.69.

7. anti-B7-H3 antibody as described in any one of claims 1 to 6, which is characterized in that the antibody is following any one Kind antibody formation:

(a) a kind of complete immunoglobulin molecules；

(b) a kind of scFv；SEQ ID NO.5,14,23,32,41 or 47 in the amino acid sequence of the scFv such as sequence table It is shown；

(c) a kind of fusion protein comprising scFv；

(d) a kind of Fab segment；

(e) a kind of Fab ' segment；

(f) a kind of F (ab)₂；

Alternatively, the antibody is monoclonal antibody or polyclonal antibody, the preferred human monoclonal of the monoclonal antibody Antibody；

Alternatively, the antibody is super humanized antibodies or double antibody.

8. anti-B7-H3 antibody as described in any one of claims 1 to 7, which is characterized in that the B7-H3 is cancer cell table The B7-H3 of endogenous expression on face；Preferably, anti-B7-H3 antibody endogenous in conjunction with the cancer cell surfaces It is internalized by after the B7-H3 of expression.

9. anti-B7-H3 antibody as described in any one of claims 1 to 8, which is characterized in that the dissociation of the anti-B7-H3 antibody Constant (KD) is 10^-9M is hereinafter, preferably 10^-11M or less.

10. a kind of nucleic acid for encoding anti-B7-H3 antibody as described in any one of claims 1 to 9.

11. a kind of recombinant expression carrier comprising nucleic acid as claimed in claim 10.

12. a kind of transformant comprising recombinant expression carrier as claimed in claim 11.

13. a kind of preparation method of anti-B7-H3 antibody, which is characterized in that it includes the following steps: to cultivate such as claim 12 institute The transformant stated obtains the anti-B7-H3 antibody from culture.

14. a kind of immune conjugate, which is characterized in that it includes anti-B7-H3 antibody as described in any one of claims 1 to 9； Preferably, the immune conjugate is antibody drug conjugates or Chimeric antigen receptor T cell；More preferably, described anti- In body drug conjugates, the anti-B7-H3 antibody is connected with cytotoxic agent by connexon, the connexon is SMCC, the cytotoxic agent are maytansine, and the antibody drug ratio of the anti-B7-H3 antibody and the maytansine is 3.2~3.5.

15. a kind of pharmaceutical composition, which is characterized in that it includes that immune conjugate and drug as claimed in claim 14 can connect The carrier received.

16. a kind of anti-B7-H3 antibody as described in any one of claims 1 to 9 or immune idol as claimed in claim 14 Join object or pharmaceutical composition as claimed in claim 15 answering in the drug for preparing B7-H3 abnormal expression related disease With, the preferred tumour of B7-H3 abnormal expression related disease, the preferred cancer of the tumour, the preferred lung cancer of the cancer, Breast cancer, prostate cancer, cancer of pancreas, colorectal cancer, melanoma, liver cancer, oophoroma, bladder cancer, gastric cancer, the cancer of the esophagus or kidney.