CN110305213B - anti-B7-H3 antibody, preparation method thereof, conjugate thereof and application thereof - Google Patents
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Abstract
The invention discloses an anti-B7-H3 antibody, a preparation method thereof, a conjugate thereof and application thereof. The anti-B7-H3 antibody comprises a complementarity determining region, and the sequence of the complementarity determining region is described in the invention. The anti-B7-H3 antibody is a fully humanized antibody screened by a phage library, and has a unique antigen binding epitope; the B7-H3 antigen can be specifically combined with a B7-H3 antigen on a tumor cell, can be quickly internalized into the cell after being combined with the tumor cell, can be used for ADC drug development, and is expected to obtain better anti-tumor activity and efficacy so as to achieve the purpose of treating cancer.
Description
Technical Field
The invention belongs to the field of antibodies, and particularly relates to an antibody specifically binding to B7-H3 of mammals, particularly humans, and a preparation method, a conjugate and an application thereof; in particular to a fully human antibody and a fully human antibody conjugate for treating cancers.
Background
B7-H3, also known as CD276, was first reported in 2001 (Chapoval AI et al, nat Immunol 2001,2 (3): 269-274), the protein of which was not considered to belong to the lactotrophic protein and myelin oligodendrocyte glycoprotein due to its lack of a header structure and B30.2 domain, and identified as belonging to the B7 family as a member of the immunoglobulin superfamily (Chapoval AI et al, nat Immunol 2001,2 (3): 269-274), unlike other members of the family such as PD-L1, B7-H4, CD80, CD86, etc.: B7-H3 exists in two different variant forms in humans, namely 2IgB7-H3 and 4IgB7-H3, where 4IgB7-H3 is an exon duplication of 2IgB7-H3, and mainly in humans in The form of 4Ig B7-H3 (Sun M et al, the Journal of Immunology 2002,168 (12): 6294-6297, ling V et al, genomics 2003,82 (3): 365-377 Steinberger P et al, J IMMUNOL 2004,172 (4): 2352-2359), while in mice only The 2IgB7-H3 structure (Sun M et al, the Journal of Immunology 2002,168 (12): 6294-6297). The results of the study showed that 2IgB7-H3 from native mice showed similar functions and no functional differences to 4IgB7-H3 from humans (Ling V et al, genomics 2003,82 (3): 365-377 Hofmeyer KA et al, proc Natl Acad Sci U S2008, 105 (30): 10277-10278.) and that the crystal Structure indicated that FG loop in the IgV region of the protein is an important epitope for B7-H3 function (Vigdorovich V et al, structure 2013,21 (5): 707-717).
Although B7-H3 has a wide mRNA level, such as B7-H3 with high level detected in various organs including heart, liver, placenta, prostate, testis, uterus, pancreas, small intestine and colon, the protein expression level is relatively limited to non-immune cells such as resting fibroblasts, endothelial cells, osteoblasts, amniotic fluid stem cells, etc., and induced antigen presenting cells, NK cell surface (Hofmeyer KA et al, proc Natl Acad Sci U S A2008, 105 (30): 10277-10278 Yi K et al, immunol Rev 2009,229 (1): 145-151 Picarda E et al, CLIN CANCER RES, 2016,22 (14): 3425-3431. B7-H3 protein levels are low in expression in normal healthy tissues, for example, B7-H3 can be detected at low protein levels in normal human liver, LUNG, bladder, testis, prostate, breast, placenta, and lymph organs, but B7-H3 protein is overexpressed in a large number of malignant tumors and is a marker antigen of tumor cells, and studies have shown that B7-H3 can be highly expressed in many CANCERs such as prostate CANCER, ovarian CANCER, colorectal CANCER, renal cell carcinoma, non-small cell LUNG CANCER, pancreatic CANCER, melanoma, gastric CANCER, bladder CANCER, malignant glioma, and osteosarcoma, and particularly in many CANCERs such as head and neck CANCER, renal CANCER, brain glioma, and thyroid CANCER (Roth TJ et al, cancel RES 2007,67 (16): 7893-7900, zang X et al, MODERN PATHOL 2010,23 (8): 1104-1112, ingebrigtsen VA et al, INT J CANCER 2012,131 (11), 2528-2536, sun J et al, CANCER Immunology, immunotherapy 2010,59 (8), 1163-1171, crispen PL et al, CLIN CANCER RES 2008,14 (16), 5150-5157, zhang G et al, LUNG CANCER 2009,66 (2), 245-249 Yamato I et al, br J CANCER 2009,101 (10), 9-1716 TekC et al, INT CANCER 2012,130 (10), 2282-2290, katayama A et al, INT J ONCOL 2011, 201538 (5), 1219-1226 Wu CP et al, world J Straterol 2006,12 (3. Wu et al, ONCOL 3, 3H-459, 3H-3H, 3H-1424, the protein is also highly expressed on tumor neovascular endothelial cells and is a very broad-spectrum tumor marker antigen. High expression of the B7-H3 protein can promote cancer progression, associated with poor prognosis and poor survival benefit in patients.
Although early research results show that B7-H3 can stimulate the function of activated T cells, promote the proliferation of CD4 and CD8 cells and the secretion of IFN-gamma, with the intensive research, the B7-H3 is shown as an immune check point, mainly plays a role in inhibiting the function of the T cells, regulates the activity of the T cells and is a negative regulatory molecule of the T cells. Both Woong-Kyung Suh and Durbaka V.R.Prasad studies showed that murine B7-H3 protein can significantly inhibit CD4, CD8 cell proliferation dose-dependently (Suh W et al, NAT IMMUNOL 2003,4 (9): 899-906, prasad DVR et al, the Journal of Immunology 2004,173 (4): 2500-2506). Judith Leitner et al also showed that human 4Ig-B7-H3Ig and 2Ig-B7-H3Ig inhibited T cell proliferation in vitro and also inhibited secretion of related cytokines (IFN-. Gamma., IL-2, IL-10, IL-13) by CD4, CD8 cells (Leitner J et al, EUR J IMMUNOL2009,39 (7): 1754-1764), further analysis indicated that B7-H3 mediated inhibition of T cell proliferation primarily by inhibition of IL-2 production. While targeting antibodies that neutralize B7-H3 in mice can significantly promote The progression of Experimental Autoimmune Encephalomyelitis (EAE) and promote The proliferation of CD4 cells, objectively suggesting that B7-H3 inhibits T cell function in vivo (Prasad DVR et al, the Journal of Immunology 2004,173 (4): 2500-2506). B7-H3 deficient mice also showed earlier development of experimental autoimmune encephalomyelitis (caused by Th1 cells) under immune EAE conditions than wild type mice in the Woong-Kyung Suh study, suggesting that B7-H3 primarily inhibits Th1 cells (Suh W et al, NAT IMMUNOL 2003,4 (9): 899-906). As mentioned above, the function of B7-H3 on T cells is controversial, but the promotion of T cell function by B7-H3 is only currently found in mouse studies, while the promotion of T cell function by human B7-H3 is not reported for a moment, and although the receptor of B7-H3 is not determined, the main idea of B7-H3 is that B7-H3 is a negative regulatory molecule of T cells.
Based on that B7-H3 can inhibit T cell activity to mediate escape immune surveillance of tumor cells, it is effective to block the binding of B7-H3 and unknown receptors to mediate T cell activation and inhibit tumor cell activity, for example, the existing clinical results of Enoblituzumab (US 2018134790A 1) show that the T cell has different degrees of remission on different tumors and has better curative effect, but a plurality of patients still have disease progression, so that the single development of monoclonal antibodies aiming at B7-H3 still has larger clinical unsatisfied; the antibody is screened through hybridoma and then subjected to humanization transformation, the hybridoma screening still contains a mouse-derived sequence despite the humanization transformation, the potential immunogenicity risk is realized, and the existing clinical result shows that the anti-tumor effect of the antibody needs to be further improved.
Due to the fact that B7-H3 is highly expressed in various tumors and has high antigen kurtosis in different tumors, the B7-H3 is a target suitable for developing antibody conjugate drugs.
Disclosure of Invention
The invention aims to solve the technical problems of the prior art that the number of anti-B7-H3 antibodies is insufficient and most antibodies containing murine sequences are insufficient, and provides an anti-B7-H3 antibody, a preparation method thereof, a conjugate thereof and application thereof. The invention adopts phage whole-human library screening, and the obtained antibody sequences are all whole-human sequences, so that the potential immunogenicity risk is lower, the antibody has certain clinical application safety and internalization function, and can be used for ADC drug development to hopefully obtain better anti-tumor activity and efficacy so as to achieve the purpose of treating cancer.
The present invention mainly solves the above-mentioned problems by the following technical means.
The present invention provides an anti-B7-H3 antibody comprising complementarity determining regions: one or more of a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and/or one or more of a light chain CDR1, a light chain CDR2, and a light chain CDR3; the amino acid sequence of the heavy chain CDR1 is shown as SEQ ID NO.7, 16, 25 or 34 in the sequence table; the amino acid sequence of the heavy chain CDR2 is shown as SEQ ID NO.8, 17, 26 or 35 in the sequence table; the amino acid sequence of the heavy chain CDR3 is shown as SEQ ID NO.9, 18, 27, 36, 43 or 49 in the sequence table; the amino acid sequence of the light chain CDR1 is shown as SEQ ID NO.11, 20, 29, 38, 45 or 51 in the sequence table; the amino acid sequence of the light chain CDR2 is shown as SEQ ID NO.12, 21, 30, 39 or 52 in the sequence table; the amino acid sequence of the light chain CDR3 is shown as SEQ ID NO.13, 22, 31, 40, 46 or 53 in the sequence table.
Preferably, in the above B7-H3 antibody:
the amino acid sequence of the heavy chain CDR1 is shown as SEQ ID NO.7, the amino acid sequence of the heavy chain CDR2 is shown as SEQ ID NO.8, and the amino acid sequence of the heavy chain CDR3 is shown as SEQ ID NO. 9;
or, the amino acid sequence of the heavy chain CDR1 is shown as SEQ ID NO.16, the amino acid sequence of the heavy chain CDR2 is shown as SEQ ID NO.17, and the amino acid sequence of the heavy chain CDR3 is shown as SEQ ID NO. 18;
or, the amino acid sequence of the heavy chain CDR1 is shown as SEQ ID NO.25, the amino acid sequence of the heavy chain CDR2 is shown as SEQ ID NO.26, and the amino acid sequence of the heavy chain CDR3 is shown as SEQ ID NO. 27;
or the amino acid sequence of the heavy chain CDR1 is shown as SEQ ID NO.34, the amino acid sequence of the heavy chain CDR2 is shown as SEQ ID NO.35, and the amino acid sequence of the heavy chain CDR3 is shown as SEQ ID NO. 36;
or the amino acid sequence of the heavy chain CDR1 is shown as SEQ ID NO.7, the amino acid sequence of the heavy chain CDR2 is shown as SEQ ID NO.8, and the amino acid sequence of the heavy chain CDR3 is shown as SEQ ID NO. 43;
or, the amino acid sequence of the heavy chain CDR1 is shown as SEQ ID NO.7, the amino acid sequence of the heavy chain CDR2 is shown as SEQ ID NO.8, and the amino acid sequence of the heavy chain CDR3 is shown as SEQ ID NO. 49.
The amino acid sequence of the light chain CDR1 is shown as SEQ ID NO.11, the amino acid sequence of the light chain CDR2 is shown as SEQ ID NO.12, and the amino acid sequence of the light chain CDR3 is shown as SEQ ID NO. 13;
or, the amino acid sequence of the light chain CDR1 is shown as SEQ ID NO.20, the amino acid sequence of the light chain CDR2 is shown as SEQ ID NO.21, and the amino acid sequence of the light chain CDR3 is shown as SEQ ID NO. 22;
or, the amino acid sequence of the light chain CDR1 is shown as SEQ ID NO.29, the amino acid sequence of the light chain CDR2 is shown as SEQ ID NO.30, and the amino acid sequence of the light chain CDR3 is shown as SEQ ID NO. 31;
or, the amino acid sequence of the light chain CDR1 is shown as SEQ ID NO.38, the amino acid sequence of the light chain CDR2 is shown as SEQ ID NO.39, and the amino acid sequence of the light chain CDR3 is shown as SEQ ID NO. 40;
or the amino acid sequence of the light chain CDR1 is shown as SEQ ID NO.45, the amino acid sequence of the light chain CDR2 is shown as SEQ ID NO.12, and the amino acid sequence of the light chain CDR3 is shown as SEQ ID NO. 46;
or, the amino acid sequence of the light chain CDR1 is shown as SEQ ID NO.51, the amino acid sequence of the light chain CDR2 is shown as SEQ ID NO.52, and the amino acid sequence of the light chain CDR3 is shown as SEQ ID NO. 53.
More preferably, the anti-B7-H3 antibody comprises a heavy chain variable region (also referred to as VH domain) and/or a light chain variable region (also referred to as VL domain), or equivalents with one or more conservative amino acid substitutions and their homologues (homologs); the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.6, 15, 24, 33, 42 or 48 in the sequence table, or has at least 90% homology with the amino acid sequence shown as SEQ ID NO.6, 15, 24, 33, 42 or 48 in the sequence table; the amino acid sequence of the light chain variable region is shown as SEQ ID NO.10, 19, 28, 37, 44 or 50 in the sequence table, or has at least 90% homology with the amino acid sequence shown as SEQ ID NO.10, 19, 28, 37, 44 or 50 in the sequence table. The amino acid sequence "having at least 90% homology" in the present invention is obtained by insertion, deletion or substitution of the amino acid sequence shown in the aforementioned sequence listing, and the substitution may be: for example, computer structure modeling analysis of sequences, analysis of posttranscriptional modification (PTMs) sites that may be present, particularly CDR regions, including analysis and substitution of antibody aggregation, deamidation-sensitive (NG, NS, NH, etc.), aspartic acid isomerization (DG, DP) sensitive sites, N-glycosylation (N- { P } S/T) sensitive sites, and oxidation-sensitive sites.
The anti-B7-H3 antibody comprises or consists of a polypeptide having the amino acid sequence from any one of the VH domains shown in SEQ ID nos. 6, 15, 24, 33, 42 and 48 and the amino acid sequence from any one of the VL domains shown in SEQ ID nos. 10, 19, 28, 37, 44 and 50 to provide a VH/VL pair representing the antigen binding site of the antibody.
More preferably, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.6 in the sequence table, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.10 in the sequence table;
or the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.15 in the sequence table, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.19 in the sequence table;
or the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.24 in the sequence table, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.28 in the sequence table;
or the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.33 in the sequence table, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.37 in the sequence table;
or the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.42 in the sequence table, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.44 in the sequence table;
or the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.48 in the sequence table, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.50 in the sequence table.
Still more preferably, the anti-B7-H3 antibody further comprises an antibody heavy chain constant region and/or an antibody light chain constant region; the antibody heavy chain constant region is preferably a human or mouse antibody heavy chain constant region; the antibody light chain constant region is preferably a human or mouse antibody light chain constant region.
In a preferred embodiment of the invention, the amino acid sequence of the heavy chain of the anti-B7-H3 antibody is shown as SEQ ID No.58, 60, 62, 64, 66 or SEQ ID No.68 of the sequence Listing, or has at least 90% homology with the amino acid sequence shown as SEQ ID No.58, 60, 62, 64, 66 or SEQ ID No.68 of the sequence Listing; the amino acid sequence of the light chain of the anti-B7-H3 antibody is shown as SEQ ID NO.59, 61, 63, 65, 67 or SEQ ID NO.69 in the sequence table, or has at least 90% homology with the amino acid sequence shown as SEQ ID NO.59, 61, 63, 65, 67 or SEQ ID NO.69 in the sequence table; preferably, the amino acid sequence of the heavy chain of the anti-B7-H3 antibody is shown as SEQ ID NO.58 in the sequence table, and the amino acid sequence of the light chain of the anti-B7-H3 antibody is shown as SEQ ID NO.59 in the sequence table; or the amino acid sequence of the heavy chain of the anti-B7-H3 antibody is shown as SEQ ID NO.60 in the sequence table, and the amino acid sequence of the light chain of the anti-B7-H3 antibody is shown as SEQ ID NO.61 in the sequence table; or the amino acid sequence of the heavy chain of the anti-B7-H3 antibody is shown as SEQ ID NO.62 in the sequence table, and the amino acid sequence of the light chain of the anti-B7-H3 antibody is shown as SEQ ID NO.63 in the sequence table; or the amino acid sequence of the heavy chain of the anti-B7-H3 antibody is shown as SEQ ID NO.64 in the sequence table, and the amino acid sequence of the light chain of the anti-B7-H3 antibody is shown as SEQ ID NO.65 in the sequence table; or the amino acid sequence of the heavy chain of the anti-B7-H3 antibody is shown as SEQ ID NO.66 in the sequence table, and the amino acid sequence of the light chain of the anti-B7-H3 antibody is shown as SEQ ID NO.67 in the sequence table; or the amino acid sequence of the heavy chain of the anti-B7-H3 antibody is shown as SEQ ID NO.68 in the sequence table, and the amino acid sequence of the light chain of the anti-B7-H3 antibody is shown as SEQ ID NO.69 in the sequence table.
The anti-B7-H3 antibody of the present invention may be in the form of any one of the following antibodies:
(a) An intact immunoglobulin molecule;
(b) A scFv; the amino acid sequence of the scFv is shown as SEQ ID NO.5, 14, 23, 32, 41 or 47 in the sequence table;
(c) A fusion protein comprising a scFv;
(d) A Fab fragment;
(e) A Fab' fragment;
(f) F (ab) 2 。
The anti-B7-H3 antibody of the invention can be a monoclonal antibody or a polyclonal antibody, and the monoclonal antibody is preferably a fully human monoclonal antibody.
The B7-H3 antibody of the present invention may be a super-humanized antibody or a diabody.
The B7-H3 involved in the anti-B7-H3 antibody described in the present invention may be B7-H3 conventional in the art, such as soluble B7-H3, membrane form B7-H3, etc., and the soluble form of B7-H3 is, for example, human B7-H3 variant 1 having the sequence shown in SEQ ID No.1, human B7-H3 variant 2 having the sequence shown in SEQ ID No.2, B7-H3 expressed on human monocytes, murine B7-H3 having the sequence shown in SEQ ID No.3, B7-H3 expressed on murine monocytes, monkey B7-H3 having the sequence shown in SEQ ID No.4, B7-H3 expressed on monkey monocytes, B7-H3 having the sequence shown in SEQ ID No.54, B7-H3 having the sequence shown in SEQ ID No.55, B7-H3 having the sequence shown in SEQ ID No.56, C7-H3 having the sequence shown in SEQ ID No.56, or IgH 3 domain having the sequence shown in SEQ ID No.57, V domain shown in SEQ ID No.57, etc. Preferably, the B7-H3 in the present invention is endogenously expressed B7-H3 on the surface of cancer cells; more preferably, the anti-B7-H3 antibody is internalized upon binding to B7-H3 expressed on the surface of a cancer cell.
The dissociation constant (KD) of the anti-B7-H3 antibody described in the present invention is preferably 10 -9 M is less than or equal to, more preferably 10 -11 M is less than or equal to M.
The invention also provides a panel of antibodies (including molecules comprising or consisting of antibody fragments or variants), wherein the panel members correspond to one, two, three, four, five, or more different antibodies of the invention [ e.g., complete antibody, fab, F (ab) 2 Fragments and scFv and the like]。
The anti-B7-H3 antibody of the invention is a B7-H3 antagonist, in particular a human B7-H3 antagonist. Protein-specific antagonists of B7-H3 (or referred to herein as "B7-H3-specific antagonists") are B7-H3 protein-specific binding molecules or proteins effective in inhibiting B7-H3 function, e.g., can be groups (sets) containing the disclosed CDR domains or heavy and/or light chain CDR domains and their equivalents characterized by having one or more conservative amino acid substitutions.
They are of great importance in the treatment of conditions associated with B7-H3 function or expressing B7-H3, including but not limited to prostate cancer, ovarian cancer, colorectal cancer, renal cell carcinoma, non-small cell lung cancer, pancreatic cancer, melanoma, gastric cancer, bladder cancer, malignant glioma, and osteosarcoma, among other relevant B7-H3 expressing tumors. The B7-H3 specific antagonists are characterized by selective recognition and binding of B7-H3. B7-H3 specific antagonists do not show significant binding to substances other than B7-H3, except in those special cases: the antagonist is supplemented with an additional specificity different from that of the B7-H3 specific binding moiety. In particular embodiments, the B7-H3-specific antagonist binds human B7-H3 with a KD of 1.2X 10 -6 Or less. In a specific embodiment, the B7-H3-specific antagonist binds to human B7-H3 with a KD of 1X 10 -7 Or less. In other embodiments, the B7-H3-specific antagonist binds human B7-H3 with a KD of 1X 10 -8 Or less. In other embodiments, the B7-H3-specific antagonist binds human B7-H3 with a KD of 5X 10 -9 Or less, or 1X 10 -9 Or less. In other embodiments, the B7-H3-specific antagonist binds human B7-H3 with a KD of 1X 10 -10 Or less, 1X 10 -11 Or less, or 1X 10 -12 Or less. In particular embodiments, the B7-H3 specific antagonist does not bind other proteins at the levels described above.
A B7-H3-specific antagonist can be efficiently internalized into a cell upon specific binding to B7-H3. It has been frequently demonstrated that the binding of B7-H3-specific antagonists can bring their conjugated poison molecules into the cell by internalization, leading to apoptosis, and that the specific activity leading to apoptosis appears dose-dependent. Therefore, B7-H3 specific antagonists are important for killing tumor cells. The B7-H3 specific antagonists of the invention may also be used for various diagnostic purposes in the detection and quantification of B7-H3.
Those skilled in the art will appreciate that fragments of B7-H3-specific antagonists that retain the ability to antagonize B7-H3 can be inserted into various frameworks (frames), see, for example, U.S. patent No.6,818,418 and references therein, which discuss various scaffolds (scaffolds) that can be used to display antibody rings that were previously selected based on antigen binding.
The B7-H3 specific antagonists and fragments can be in the form of various non-antibody based scaffolds, including but not limited to high affinity multimers (avimers) (Avidia); DARPins (molecular partners); adnectins (Adnexus), anticalins (Pieris) and Affinibands (Affinibody). Many evaluations have been made in the scientific literature on the use of alternative scaffolds for protein binding, see for example Binz & Pl u ckthun,2005curr. 1-11. Thus, a non-antibody based scaffold or antagonist molecule that specifically binds to B7-H3 with specificity selectivity for B7-H3, or that internalizes upon binding, constitutes an important embodiment of the present invention.
The present invention also provides a nucleic acid encoding the above-described anti-B7-H3 antibody.
The invention also provides a recombinant expression vector comprising the nucleic acid.
The present invention also provides a transformant comprising the above recombinant expression vector.
The invention also provides a preparation method of the anti-B7-H3 antibody, which comprises the following steps: the above-mentioned transformant is cultured, and the anti-B7-H3 antibody is obtained from the culture.
The invention also provides an immunoconjugate comprising the anti-B7-H3 antibody described above; preferably, the immunoconjugate is an Antibody Drug Conjugate (ADC) or a chimeric antigen receptor T cell (CAR-T); more preferably, in the antibody drug conjugate, the anti-B7-H3 antibody and the cytotoxic agent are linked by a linker, the linker is SMCC, the cytotoxic agent is maytansine, and the antibody drug ratio (DAR) of the anti-B7-H3 antibody to the maytansine is 3.2 to 3.5.
The invention also provides a pharmaceutical composition, which comprises the immunoconjugate and a pharmaceutically acceptable carrier.
The invention also provides application of the anti-B7-H3 antibody, the immunoconjugate or the pharmaceutical composition in preparing medicines for diseases related to abnormal B7-H3 expression, wherein the diseases related to abnormal B7-H3 expression are preferably tumors, the tumors are preferably cancers, and the cancers are preferably lung cancer, breast cancer, prostate cancer, pancreatic cancer, colorectal cancer, melanoma, liver cancer, ovarian cancer, bladder cancer, gastric cancer, esophageal cancer or kidney cancer.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
the anti-B7-H3 antibody is a fully humanized antibody screened by a phage library, and has a unique antigen binding epitope; the B7-H3 antigen can be specifically combined with a B7-H3 antigen on a tumor cell, can be quickly internalized into the cell after being combined with the tumor cell, can be used for ADC drug development, and is expected to obtain better anti-tumor activity and efficacy so as to achieve the purpose of treating cancer.
Drawings
FIG. 1 shows the binding of different antibodies to the proteins of B7-H3.
FIG. 2 shows the binding of antibodies P1G2, P1D1, P3B9 and P2E3 to Hs-700T cells.
FIG. 3 shows that P2E5 and P1E11 bind to B7-H3 transfected CHO cells.
FIG. 4A is P1G2, P2E5, P1E11 competing for biotin-P1E11 binding to the B7H3/4Ig antigen; FIG. 4B shows P1G2, P2E5 competing for biotin-P2E5 binding to the B7H3/4Ig antigen.
FIG. 5 shows that different anti-B7-H3 antibodies have internalizing ability after binding to tumor cell lines.
FIG. 6 shows the killing activity of anti-B7-H3 antibody DM1 ADC.
Detailed Description
The term "B7-H3" as used herein is used in the same sense as the B7-H3 protein and also denotes B7-H3 variant 1 and/or B7-H3 variant 2.
A B7-H3-specific antagonist as defined herein selectively recognizes and specifically binds B7-H3.
The term "selectivity" or "specificity" as used herein refers to the fact that: the disclosed antagonists do not show significant binding to substances other than B7-H3, except in those special cases: wherein the antagonist is supplemented such that it has an additional, different specificity than the B7-H3-specific binding moiety (e.g., a bispecific or bifunctional molecule, wherein the molecule is designed to bind or perform both functions, at least one of which is specifically binding to B7-H3).
KD refers to the dissociation constant obtained from the ratio of KD (off-rate of specific binding molecule-target protein interaction) to Ka (on-rate of specific binding molecule-target protein interaction) (or KD/Ka, expressed as molar concentration (M)). KD values can be determined using methods well established in the art. A preferred method of determining the KD of a binding molecule is by using surface plasmon resonance, for example biosensor systems such as the Biacore (TM) (GE Healthcare Life Sciences) system.
An "antibody molecule" or "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. Thus, the term antibody encompasses not only intact antibody molecules, but also fragments of said antibodies and variants (including derivatives) of said antibodies and antibody fragments. The term antibody molecule as used in this specification includes, for example, but is not limited to, single chain Fv (scFv), fab fragments, fab 'fragments, F (ab') 2, disulfide linked Fv (sdFv), fv, and whole or full length antibodies. The term "single chain Fv" or "scFv" refers to a polypeptide comprising the VL domain of an antibody linked to the VH domain of an antibody. Antibodies that immunospecifically bind to B7-H3 can cross-react with other antigens. Preferably, antibodies that immunospecifically bind to B7-H3 do not cross-react with other antigens. Antibodies that immunospecifically bind to B7-H3 can be identified, for example, by immunoassay or other methods known to those skilled in the art. A "complete" antibody or a "full-length" antibody refers to a protein comprising two heavy chains (H) and two light chains (L) which are interconnected by disulfide bonds, said protein comprising: (1) For the heavy chain, a variable region (abbreviated herein as "VH") and a heavy chain constant region containing three domains CH1, CH2, CH 3; and (2) for the light chain, a light chain variable region (abbreviated herein as "VL") and a light chain constant region comprising one domain CL. Antibodies of the invention include, but are not limited to, monoclonal, multispecific, human or chimeric antibodies, single chain antibodies, fab fragments, F (ab') fragments, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above. The immunoglobulin molecules of the invention may be of any type (e.g., igG, igE, igM, igD, igA and IgY), class (e.g., igG1, igG2, igG3, igG4, igA1 and IgA 2) or subclass of immunoglobulin. Preferably, the antibody of the invention comprises or consists of a VH domain, a VH CDR, a VL domain, or a VL CDR having any one of the amino acid sequences set forth in table 1, or a fragment or variant thereof.
An antibody of the invention that binds to soluble form B7-H3 "is one that binds to 435 amino acid soluble form of human B7-H3 protein (SEQ ID NO: 54), i.e., the 4Ig B7-H3 protein, and/or to 217 amino acid soluble form of human B7-H3 (SEQ ID NO: 55), i.e., the 2Ig B7-H3 protein, and/or to the IgC domain of 102 amino acid soluble form of human B7-H3 (SEQ ID NO: 56), and/or to the IgV domain of 101 amino acid soluble form of human B7-H3 (SEQ ID NO: 57). In a specific embodiment of the invention, the B7-H3-specific antagonist of the invention binds to the 435 amino acid soluble form of the human B7-H3 protein, preferably to an antibody which simultaneously binds to the 216 amino acid soluble form of human B7-H3.
An antibody of the present invention "binding to membrane form B7-H3" is an antibody that binds to membrane B7-H3 protein. In a specific embodiment of the invention, an antibody of the invention "binding to membrane form B7-H3" does not bind to the soluble form of B7-H3. Binding to B7-H3 transfected CHO cells (as described herein) in an ELISA was a test for specificity of antibody binding to membrane form B7-H3. Assays that can be used as antibodies specific for membrane form B7-H3 include, but are not limited to, expression of B7-H3 in binding membranes as described in example 5. An antibody of the invention that "binds to both soluble and membrane forms B7-H3" is an antibody that binds both membrane form B7-H3 and soluble form B7-H3.
The term "variant" refers to a polypeptide that has a similar or identical function to a B7-H3 polypeptide, a B7-H3 fragment, an anti-B7-H3 antibody or antibody fragment thereof, but does not necessarily comprise the amino acid sequence of a similar or identical B7-H3 polypeptide, a B7-H3 fragment, an anti-B7-H3 antibody or fragment thereof, or a structure having a similar or identical B7-H3 polypeptide, a B7-H3 fragment, an anti-B7-H3 antibody or fragment thereof. A variant having a similar amino acid sequence is referred to as a polypeptide, which corresponds to at least one of the following polypeptides: (a) A polypeptide comprising or consisting of an amino acid sequence that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least 99% identical to the amino acid sequence of a B7-H3 polypeptide, a B7-H3 fragment, an anti-B7-H3 antibody or fragment thereof (including a VH domain, a VHCDR, a VL domain, or a VLCDR having any one of the amino acid sequences set forth in table 1); (b) A polypeptide comprising at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 125, or at least 150 amino acid residues encoded by a nucleotide sequence the complement of which hybridizes under stringent hybridization conditions to a nucleotide sequence encoding a B7-H3 polypeptide, a B7-H3 fragment, an anti-B7-H3 antibody or fragment thereof (including a VH domain, a VHCDR, a VL domain, or a VLCDR having any one of the amino acid sequences set forth in table 1); (c) A polypeptide encoded by a nucleotide sequence that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least 99% identical to a nucleotide sequence encoding a B7-H3 polypeptide, a B7-H3 fragment, an anti-B7-H3 antibody or fragment thereof (including a VH domain, a VHCDR, a VL domain, or a VLCDR having any one of the amino acid sequences set forth in table 1). A polypeptide having a structure similar to that of a B7-H3 polypeptide, a B7-H3 fragment, an anti-B7-H3 antibody or fragment thereof refers to a polypeptide having a secondary, tertiary or quaternary structure similar to that of a B7-H3 polypeptide, a B7-H3 fragment, an anti-B7-H3 antibody or fragment thereof. The structure of a polypeptide can be determined by methods known to those skilled in the art, including, but not limited to, X-ray crystallization, nuclear magnetic resonance, and crystallization electron microscopy.
As used herein, the percent homology between two amino acid sequences is equal to the percent identity (identity) between the two sequences. The percent sequence identity between two sequences is a function of the number of identical positions shared by the sequences (i.e.,% homology = number of identical positions/total number of positions X100), where the number of gaps (gaps) and the length of each gap are taken into account and need to be introduced for optimal alignment of the two sequences. Sequence comparisons and determination of percent identity between sequences can be performed by methods generally known in the art, and such sequence comparisons and determination of percent identity can be accomplished using mathematical algorithms. For example, meyers and Miller,1988comput.appl.biosci.4:11-17 (incorporated into the ALIGN program (version 2.0)) to determine the percent identity between amino acid sequences and/or between nucleotide sequences. In addition, the GAP program (using its default parameters) in the GCG software package, available online from Accelrys, can be used to determine percent identity between amino acid sequences or between nucleotide sequences. In one embodiment, the two sequences are of equal length.
The term "epitope" refers to a portion of B7-H3 that has antigenic or immunogenic activity in the body of an animal, preferably a mammal. The epitope with immunogenic activity is part of B7-H3, which elicits an antibody response in the animal. The epitope having antigenic activity is part of B7-H3 to which an antibody immunospecifically binds, which can be determined by methods known in the art, such as the immunoassay methods described herein. The antigenic epitope is not necessarily immunogenic.
In a particular aspect, the invention provides isolated B7-H3 specific antagonists that antagonize B7-H3. In particular embodiments, the B7-H3 specific antagonist specifically binds to B7-H3 and blocks binding to its unknown receptor. By repeated experiments, the B7-H3-specific antagonists of the invention (e.g., antibody molecules P1G2 or P2E 5) bind to B7-H3 dose-dependently. Thus, in particular embodiments, the invention encompasses B7-H3 specific antagonists, and in more particular embodiments, the invention encompasses antibody molecules comprising heavy and/or light chain variable regions, and their equivalents (characterized by having one or more conservative amino acid substitutions) or homologues. The term "domain" or "region" as used herein simply refers to portions of an antibody molecule in which a (resuride) or currently controversial (at issue) sequence or fragment will be present.
One skilled in the art will appreciate that a conservative amino acid substitution is one that replaces an amino acid residue with one that confers similar or better (for the intended purpose) functional and/or chemical properties. For example, conservative amino acid substitutions are often those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art.
These families include amino acids with the following side chains: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β -branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). Such modifications are not intended to significantly reduce or alter the binding or functional characteristics of B7-H3 specific antagonists, although they may improve these properties. The purpose of making the substitution is not critical and can include, but is in no way limited to, replacing a residue with one that better maintains or enhances the structure of the molecule, the charge or hydrophobicity of the molecule, or the size of the molecule. For example, it may be desirable to replace a less desirable residue with a residue having the same polarity or charge. Such modifications can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. One particular way in which one skilled in the art can achieve conservative amino acid substitutions is alanine scanning mutagenesis, described, for example, in MacLennan et al, 1998Acta physiol.Scand.suppl.643:55-67, and Sasaki et al, 1998Adv.Biophys.35: 1-24. The altered antagonists are then tested for retention or better function using functional assays available in the art or described herein. A B7-H3 specific antagonist having the following characteristics is referred to herein as a "functional equivalent" of the antagonist disclosed herein and constitutes a particular embodiment of the invention: having one or more of the above-described conservative amino acid substitutions, retains the ability to selectively bind to human B7-H3 and antagonizes B7-H3 function at the same or better level relative to molecules without the above-described amino acid changes.
B7-H3 specific antagonists having an amino acid sequence homologous to the amino acid sequence of the antagonists described herein are typically prepared to improve one or more properties of the antagonist without altering its specificity for B7-H3. One way to obtain such sequences, not the only way available to those skilled in the art, is to mutate sequences encoding B7-H3 specific antagonists or their specificity determining regions, express antagonists comprising these mutated sequences, and test whether these encoded antagonists retain function using available functional assays, including those described herein. Mutations can be made by site-directed mutagenesis or by random mutagenesis. However, one skilled in the art will appreciate that other mutagenesis methods can readily achieve the same effect. For example, in some approaches, the mutant spectrum (spectrum) is limited by non-random targeted conservative substitutions based on the chemical or structural properties of the amino acid or by structural considerations of the protein. In affinity maturation experiments, several such mutations can be found in a single selected molecule (selected randomly or non-randomly). For affinity maturation, there are various structure-based methods, described, for example, in U.S. Pat. No.7,117,096, PCT publication No.: WO 02/084277 and WO03/099999.
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Example 1 preparation of human B7-H3 protein
Selecting a nucleic acid sequence of 29-245 amino acids of human 2Ig B7-H3, wherein a purified 10His tag is added at the N end and a Myc tag is added at the C end, and the nucleic acid sequence is named as H2M; selecting a nucleic acid sequence of 27-461 amino acids of human 4Ig B7-H3, wherein a purification tag 10His is added at the N end and a detection Myc tag is added at the C end, and the nucleic acid sequence is named as H4M; the nucleic acid sequence of amino acids 27-461 of human 4Ig B7-H3 is selected, wherein a myc tag is added at the N end and a purified 10His tag is added at the C end, and the nucleic acid sequence is named as M4H. Respectively synthesizing gene plasmids H2M-pUC57, M4H-pUC57 and H4M-pUC57 of the three B7-H3 antigens, synthesizing a pv81 expression vector plasmid, respectively performing enzyme digestion by EcoRI and SmaI, then connecting and transforming an escherichia coli competent cell Trans-T1, screening and amplifying correct clones by PCR, and performing mass extraction on the plasmids. The extracted and prepared expression vectors H2M, H4M and M4H are transiently expressed in Expi293 for 7 days respectively, and the expression supernatant is purified and prepared after ELISA detection.
Biotinylated antigen preparation, prepared H4M antigen (concentration 3.36 mg/mL) was incubated with PBS and 10uM biotin at 37 ℃ at an antigen to biotin mass ratio of 1.
Example 2 library preparation
1ml of each library was read with a Lambda size of 2.91 x10 9 And the size of the library Kappa is 3.72 x10 9 The bacterial liquid is added into 2.0L of fresh culture medium 2YT +100ug/ml Amp +2% glucose, the initial OD600 of the bacterial liquid of the library is less than 0.1, and the bacterial liquid is cultured under the conditions of 37 ℃ and 200rpm of rotation speed. When the OD600 was 0.5-0.6, 365ul of M13K07 was added with a titer of 9.6X 10 12 The addition amount of the additive is 10 times of the bacterial amount, and the bacterial amount = OD600 × 8.0 × 10 8 Volume of shake bacteria/ml, to aid phagemid infestation. Adding auxiliary phage, culturing at 37 deg.C for 30min, culturing at 200rpm for 30min, centrifuging at 4000rpm for 10min, resuspending with 2.0L of 2YT +100ug/mlAmp +50ug/ml Kana, and culturing at 30 deg.C for 16h for expression. After the expression culture is finished, taking the bacterial liquid, centrifuging for 30 minutes at 8000rpm under the condition of 4 ℃, and removing the supernatant. Centrifuging at high speed, removing thallus, adding 1/5 volume of PEG/NACL into supernatant, precipitating Phage in supernatant, dissolving Phage in PBS after centrifuging, and measuring Phage titer to be about 2.1 × 10 11 。
Example 3 anti-B7-H3 antibody phage library screening
Liquid phase elutriation: 150uL of magnetic bead Dynabeads M-280 was blocked with 1% casein at room temperature for 1h, then 100uL of the prepared Phage library Phage was added, and the mixture was gently shaken and blocked at room temperature for 1h. And after the sealing is finished, 30ug of biotinylated B7-H3 antigen H4M is added, the incubation is carried out for 1H at room temperature, after the incubation is finished, the complex of the antigen H4M and the phage antibody is incubated with the sealed magnetic beads for 15min, so that the complex is combined on the magnetic beads, and the PBST and the PBS are respectively used for washing for 15 times. The phage bound to the antigen was then eluted by adding 1ml of trypsin (10 ug/ml) in a volume of 1ml, which was infected with TG1 in the log phase of growth, the titer was measured, and the amplified phage was used for the next round of panning. A total of 3 rounds of panning were performed, followed by 2 rounds of panning in the same manner as the first round, but with the addition of biotinylated antigen H4M gradually reduced to 10ug and 5ug or 7.5ug and 2.5ug, respectively.
Phage ELISA screening: dissolving streptavidin with concentration of 1mg/ml at room temperature, mixing uniformly, gradually diluting to 5ug/ml with coating solution, adding into 96-well plate with each well at 100 uL/well, setting 3 wells, adding only 100uL coating solution as blank control, and standing at 2-8 deg.C overnight. Collecting biotinThe digested B7-H3 antigen H4M was diluted to 50ng/ml with 0.5% BSA-PBST diluent, added to streptavidin-coated plates at a concentration of 20ng/ml and 100uL, placed in a microplate shaker, shaken at 37 ℃ and 600rpm for 1 hour, discarded, washed 3 times with washing solution, and then blotted dry. And (4) taking the positive phage obtained in the second round or the third round of panning, dissolving and uniformly mixing at room temperature, and diluting by using a diluent by 500 times. Adding diluted phage into the ELISA plate according to the amount of 100 uL/hole, placing into a microplate oscillator, oscillating and incubating for 1 hour at 37 deg.C and 600rpm, discarding the solution, and washing the plate with washing solution for 3 times. Then 100uL of diluted enzyme-linked antibody of HRP/Anti-M137 is added into each hole, the mixture is put into a microplate oscillator, the solution is discarded and the plate is washed for 3 times after the mixture is oscillated at 37 ℃ and 600rpm for 1 hour, TMB color developing solution is added in the amount of 100 uL/hole for developing for 15 minutes, and then 1mol/L H color developing solution is added in the amount of 100 uL/hole for developing 2 SO 4 The reaction is stopped by the stop solution, and the absorbance is measured at the wavelength of 450nm by using an enzyme-linked immunosorbent assay with the wavelength of 650nm as the reference wavelength.
scFv protein ElISA screening: dissolving B7-H3 antigen H4M at room temperature, diluting to 2ng/ml with coating solution, adding 100 uL/well of the coating solution into a hydrophobic enzyme label plate strip, arranging 3 wells of the coating solution, adding 100uL of the coating solution as a blank control, and standing overnight at 2-8 ℃. 100uL of scfv protein sample was added to each well, placed in a microplate shaker, shaken at 37 deg.C and 600rpm for 1 hour, discarded, and the plate was washed 3 times with wash solution. Then adding diluted enzyme-linked antibody in an amount of 100 μ L/well, placing into a microplate oscillator, oscillating at 37 deg.C and 600rpm for 1 hr, discarding the solution, washing the plate for 3 times, adding TMB color developing solution in an amount of 100 uL/well for developing for 15min, and adding 1mol/L H in an amount of 100 uL/well 2 SO 4 The reaction was terminated with the stop solution, and absorbance was measured at a wavelength of 450nm using a microplate reader with a wavelength of 650nm as a reference wavelength.
As described above, a total of 60 scFv antibodies against B7-H3 with unique sequences were screened, wherein the sequence composition of the exemplified scFv antibodies P1D1, P1G2, P2E5, P1E11, P2E3, P3B9 is shown in table 1 (wherein the determination of the CDRs is according to Kabat rules).
TABLE 1
The binding signal values of scFv antibodies P1D1, P1G2, P2E5, P1E11, P2E3, P3B9 and the like in phage ELISA and scFv protein ELISA to B7-H3 antigen H4M are shown in Table 2 below. The result shows that the selected scFv antibodies have better binding signal value on the B7-H3 protein.
TABLE 2 Phage scFv and protein scFv bind H4M ELISA Signal values
| Antibodies | Sequence of | Phage scFv ELISA Signal values | 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 |
Example 4 anti-B7-H3 full-Length antibody protein ELISA binding
anti-B7-H3 full length antibody protein ELISA binding
IgG1 antibody preparation: the scFv is converted to the IgG1 format, and the VH and VL domains of the scFv that we wish to convert to an IgG molecule are cloned into vectors containing nucleotide sequences encoding the appropriate heavy (human IgG 1) or light (human kappa or human lambda) constant regions so that the complete heavy or light chain molecule can be expressed from these vectors when transfected into an appropriate host cell. Alternatively, when cloned heavy and light chains are expressed simultaneously in a cell line (from one vector or two vectors), they can assemble into a complete functional antibody molecule, which is secreted into the cell culture medium. Conversion of scFv to conventional antibody molecules is a well-known technique in the art (assembly of VH and VL of scFv sequences directly onto the heavy and light chain constant regions, respectively, of a full-length antibody). The light and heavy chain primer sequences of 6 pairs of the above exemplified antibodies, such as P1G2, P1E11, P2E3, P2E5, P3B9, and P1D1, were designed, respectively, except that the light chain of P1E11 was constructed on pfu-CLIg-hk (invitrogen) vector by EcoRI + BsiWI double digestion, the remaining antibody light chain was constructed on pfu-CLIg-hl2 (invitrogen) vector by EcoRI + ArvII double digestion, and the heavy chain was constructed on pfu-CLIg-Hg1 (invitrogen) vector by EcoRI + NheI double digestion H chain (IgG 1), and the correct clones were amplified by PCR screening for mass extraction of plasmids (see J.Sambuk. Molecular cloning, experimental guidelines, 2 edition [ M ]. Scientific Press, 1992.). The extracted expression vectors are transiently expressed in CHO-S cells for 7 days respectively, and the expression supernatant is purified and prepared after ELISA detection.
Protein binding: antigen B7-H3 (R) coated with 100uL 20ng/ml&D) On a microplate, overnight at 2-8 ℃. Then 100uL of the prepared full-length IgG1 antibody with the concentration starting from 2000ng/ml and 3-fold gradient dilution are added respectively, the incubation is carried out for 1 hour at 37 ℃ and 600rpm, the washing is carried out for three times, the Goat anti human IgG (Fc) -HRP enzyme-linked antibody is added, the oscillation is carried out for 1 hour at 37 ℃ and 600rpm, the washing is carried out for 4 times, the TMB developing solution is added, the developing is carried out for 10 minutes, and 100uL of 1mol/L H is added 2 SO 4 The reaction was terminated and the absorbance was measured. Results except that P1E11 and P2E3 did not bind to the coated B7-H3 protein, the remaining antibodies specifically bound to the B7-H3 antigen, as shown in FIG. 1 for antibodies P1D1, P3B9, P1G2, and P2E5, which bind to the B7-H3 protein level.
Example 5 anti-B7-H3 antibody Cell-Base ELISA binding evaluation
Culturing pancreatic cancer cell strain Hs-700T, adding FBS/DMEM culture medium, plating with 20000 cells/well, setting plate hole without inoculated cells and cell complete culture medium as blank control, at 37 deg.C, 5% 2 Culturing for 20-24 hours under the condition of (1). PBST washing plate 1 times, adding formaldehyde 200 ul/hole, room temperature fixation for 0.5 hours, washing 3 times, adding 10% skimmed milk powder, sealing for 1 hour, washing again for 3 times, and lightly tapping for use. After mixing IgG1 antibodies prepared as described above, such as P2E3, P1D1 and P3B9, respectively, each antibody was diluted to 10000ng/ml with 0.5% BSA-PBST diluent, and then diluted in multiples, totaling 8 concentration points, added to Hs-700T-coated well plates at 100 ul/well, incubated at room temperature for 1 hour, and then washed 3 times. Adding TMB for color development, stopping reaction and detecting. The results are shown in FIG. 2 for the antibodies P1D1,P1G2, P3B9 and P2E3 bind to Hs-700T cells.
Binding of the IgG1 antibody prepared to B7-H3-transfected CHO cells was also confirmed using the same method as above, and the results are shown in FIG. 3 for P2E5 and P1E11 to B7-H3-transfected CHO cells.
The other antibody P1G2 specifically binds to both protein and Hs-700T cells as well as CHO cells, and the binding level of the antibody is listed in the map, and the binding activity at the cellular level is only relatively weak, but the ADC activity is best in the subsequent ADC killing evaluation.
Example 6 anti-B7-H3 antibodies bind to the B7-H3 domain
In the same manner as in example 4, igG1 antibodies P1G2, P2E5, and P1E11 were examined which domains of B7-H3, such as domain 2IgB7-H3, igC domain, and IgV domain, were bound by the protein ELISA method. As a result, as shown in Table 3, it was confirmed that P1G2 binds to both IgC and IgV domains of B7-H3, P2E5 binds to the IgC domain of B7-H3 and weakly binds to the IgV domain, and P1E11 binds to the IgC domain of B7-H3 and does not bind to the IgV domain. These results demonstrate that the P1G2 antibody binds to the IgC domain (SEQ ID NO: 56) and the IgV domain (SEQ ID NO: 57) of B7-H3. In the same way, it was also demonstrated that P2E5 binds to the IgC domain weakly and the IgV domain, and P1E11 only binds to the IgC domain.
TABLE 3 binding of P1G2, P2E5 and P1E11 to different domains of B7-H3
Note: +, weak bonding; + + +, moderate binding level; + + + +, strong bonding; x, do not bind
The 4Ig-B7H3 antigen was immobilized on an elisa plate, bound to a biotin-labeled P1E11 antibody (biotin-labeled DAR-2.5), and the IgG1 antibodies P1G2 and P2E5 series concentrations were competitively bound to P1E11, enzyme-linked secondary antibody was Streptavidin-HRP (1. The blank response value range is less than or equal to 0.1 as the basic requirement by using commercial developing solution reagent and proper developing time. The results are shown in fig. 4A, and both P1G2 and P2E5 have no competitive binding relationship with P1E11, indicating that P1G2 and P2E5 bind to different epitopes with P1E 11. In the same manner, the series of concentrations of P1G2 and P2E5 competed for binding with the biotinylated P2E5 antibody (biotinylated DAR-2.0), and it was also confirmed in FIG. 4B that the prepared antibody P1G2 did not compete for binding to B7-H3 with P2E5, indicating that P1G2 and P2E5 bind to different epitopes of B7-H3, respectively. Thus prepared P1G2 and P2E5 and P1E11 bind to different epitopes of B7-H3, respectively.
Example 7 binding of anti-B7-H3 antibodies to B7-H3 internalization
The ability of the anti-B7-H3 antibodies of the invention to be internalized upon binding to cancer cells was investigated. Pancreatic cell carcinoma Hs700T (purchased from ATCC) was seeded in 96-well cell culture plates at a cell density of 80000 cells/50. Mu.l per well for 20-24hr; secondly, 8 concentration gradients are prepared by diluting the anti-B7-H3 Antibody of the invention, such as P1G2, P2E5 and P1E11 to 12.5, 6.3, 3.1, 1.6, 0.8, 0.4, 0.2nM and 0nM with the cell-corresponding medium containing 15% FBS, respectively, transferring the diluted anti-B7-H3 Antibody of the invention to a culture plate inoculated with 200nM (30. Mu.g/ml) PA-Goat anti-Human IgG Fc Antibody (DAR-10.6) after 1; then, excitation wavelength of a microplate reader: 532nm, emission wavelength: 560nm, reading by selecting bottom reading mode. The results are shown in FIG. 5, which demonstrates the ability of the anti-B7-H3 antibodies of the invention to be internalized within 24hr after binding to cancer cells.
Example 8 evaluation of killing by ADC
Coupling preparation of DM1 ADC samples. Appropriate amounts of antibody samples P1G12, P2E5 and P1E11 were each prepared, and replaced with 50mM potassium phosphate, 50mM NaCl,1mM HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) and pH7.4 buffer solutions, and the antibody samples were placed in a reaction vessel, stirred, and then added with SMCC (molar ratio of antibody to SMCC: 1: 7.5), and reacted at room temperature for 2 hours. The prepared sample is replaced in a buffer solution system of 50mM sodium phosphate, 100mM NaCl,60mM sodium caprylate and pH7.5, placed in a reaction kettle, added with DM1 drug dissolved by DMA (the molar ratio of the antibody to the DM1 is 1. After preparation, the DAR values of the prepared ADC samples were analyzed by mass spectrometry.
The lung cancer cell strain NCI-H322 (purchased from Beibai Biotechnology Co., ltd., beijing, south China) and anti-B7-H3 antibody conjugate drugs P1G2-DM1, P2E5-DM1 and P1E11-DM1 prepared by MCC-DM1 coupling are incubated together. The internalizing killing activity of the DM1 ADC (DAR 3.2-3.5) of the anti-B7-H3 antibodies of the invention against tumor cells was evaluated by measuring the viability of the cells.
The results are shown in FIG. 6: the same shows that the anti-B7-H3 antibody coupling drugs P1G2-DM1, P2E5-DM1 and P1E11-DM1 all present better ADC internalization killing activity to tumor cells.
Example 9 evaluation of the kinetics of antibody interaction with B7-H3 by surface plasmon resonance ("SPR")
SPR detection was performed using the BIAcore T200 (GE) system. Sensor Chip CM5 (Series Sensor Chip CM 5) and amine coupling kit for immobilization were purchased from GE.
Respectively diluting the test samples of the antibodies P1G2, P2E5 and P1E11 to 10 mu G/ml by NaAc buffer solution with pH 5.5, setting the flow rate to be 10 mu L/min, setting the chip activation time of EDC and NHS mixed solution to be a default value of 420s, respectively fixing the F0006 test sample to 200RU level by adopting a preset coupling amount mode, and blocking the activated groups which are not combined with the test samples by using ethanolamine.
The B7-H3 antigen was diluted with HBS-EP buffer at a given ratio to give concentrations of 0nM, 0.15625nM, 0.3125nM, 0.625nM, 1.25nM (two replicates), 2.5nM, 5nM, 10nM and 20nM, respectively, and the flow rate was set at 30. Mu.L/min, the binding time was 120s, and the dissociation time was 1800s during the sample analysis. Then, regeneration was carried out using Gly-HCl buffer (pH 1.5) as a regeneration buffer, and the flow rate was set at 30. Mu.L/min for 30 seconds. The experiment adopts multi-cycle operation, the response signal of the experiment takes the analysis time as an abscissa, and the response value is an ordinate. The obtained data are fitted by BIAcore T200 analysis software, the adopted fitting model is a 1.
TABLE 4 binding kinetics parameters of anti-B7-H3 antibodies
| Name of antibody | Antigens | 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 Compound Dangjiang biomedical corporation
<120> anti-B7-H3 antibody, preparation method, conjugate and application thereof
<130> P180115881C
<160> 69
<170> PatentIn version 3.5
<210> 1
<211> 534
<212> PRT
<213> Intelligent
<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
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Met Leu Arg Arg Arg Gly Ser Pro Gly Met Gly Val His Val Gly Ala
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Ala Leu Gly Ala Leu Trp Phe Cys Leu Thr Gly Ala Leu Glu Val Gln
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Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr Leu
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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
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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
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Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys
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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
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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
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Asp Gly Glu Gly Glu Gly Ser Lys Thr Ala Leu Gln Pro Leu Lys His
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Ser Asp Ser Lys Glu Asp Asp Gly Gln Glu Ile Ala
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Met Leu Arg Gly Trp Gly Gly Pro Ser Val Gly Val Cys Val Arg Thr
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Ala Leu Gly Val Leu Cys Leu Cys Leu Thr Gly Ala Val Glu Val Gln
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Val Ser Glu Asp Pro Val Val Ala Leu Val Asp Thr Asp Ala Thr Leu
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Arg Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn
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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
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Ser Glu Asn Lys Glu Asp Asp Gly Gln Glu Ile Ala
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Met Leu His Arg Arg Gly Ser Pro Gly Met Gly Val His Val Gly Ala
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Ala Leu Gly Ala Leu Trp Phe Cys Leu Thr Gly Ala Leu Glu Val Gln
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Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr Leu
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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
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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
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Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr
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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
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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
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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
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Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr
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Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
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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
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Gly Tyr Ser Phe Thr Ser Tyr Trp
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Ile Tyr Pro Gly Asp Ser Asp Thr
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Ala Arg Arg Ser Val Ala Gly Leu Asp Tyr
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Lys Thr Val Val Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
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Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Lys
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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
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Ser Ser Asn Ile Gly Ser Lys Tyr
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Arg Asn Asn
1
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Ala Ser Trp Asp Asp Arg Leu Lys Gly Tyr Ala
1 5 10
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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
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Gln Val Thr Leu Lys Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
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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
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Gly Phe Thr Phe Ser Asn Ser Tyr
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Met Lys Pro Asp Gly Ser Val Lys
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Ser Ser Tyr Asp Thr Arg Trp Gly Trp Phe Asp Pro
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Gln Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly
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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
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Ser Gly Ser Val Ser Thr Ser Tyr Phe
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Asn Thr Asn
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Leu Leu Tyr Met Asp Ser Gly Pro His Trp Val
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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
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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 sequences
<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>
<223> 435 amino acid B7-H3Ig protein
<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>
<223> 217 amino acid B7-H3Ig protein
<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>
<223> 102 amino acid B7-H3 IgC domain
<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>
<223> 101 amino acid B7-H3 IgV domian
<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 full-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 chain
<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 full-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 chain
<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 full-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 chain
<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 full-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 chain
<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 full-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 chain
<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 full-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 chain
<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 (18)
1. An anti-B7-H3 antibody comprising the complementarity determining regions: a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, and, a light chain CDR1, a light chain CDR2, and a light chain CDR3; the amino acid sequence of the heavy chain CDR1 is shown as SEQ ID NO.34, the amino acid sequence of the heavy chain CDR2 is shown as SEQ ID NO.35, the amino acid sequence of the heavy chain CDR3 is shown as SEQ ID NO.36, the amino acid sequence of the light chain CDR1 is shown as SEQ ID NO.38, the amino acid sequence of the light chain CDR2 is shown as SEQ ID NO.39, and the amino acid sequence of the light chain CDR3 is shown as SEQ ID NO. 40.
2. The anti-B7-H3 antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region has at least 90% homology with the amino acid sequence shown as SEQ ID No.33 of the sequence listing, and the amino acid sequence of the light chain variable region has at least 90% homology with the amino acid sequence shown as SEQ ID No.37 of the sequence listing.
3. The anti-B7-H3 antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region is represented by SEQ ID No.33 of the sequence Listing, and the amino acid sequence of the light chain variable region is represented by SEQ ID No.37 of the sequence Listing.
4. The anti-B7-H3 antibody of claim 1, wherein said anti-B7-H3 antibody further comprises an antibody heavy chain constant region and an antibody light chain constant region.
5. The anti-B7-H3 antibody of claim 4, wherein said antibody heavy chain constant region is a human or mouse antibody heavy chain constant region; the antibody light chain constant region is a human or mouse antibody light chain constant region.
6. The anti-B7-H3 antibody of claim 5, wherein the amino acid sequence of the heavy chain of said anti-B7-H3 antibody is at least 90% homologous to the amino acid sequence set forth in SEQ ID No.58 of the sequence listing, and the amino acid sequence of the light chain of said anti-B7-H3 antibody is at least 90% homologous to the amino acid sequence set forth in SEQ ID No.59 of the sequence listing.
7. The anti-B7-H3 antibody of claim 5, wherein the amino acid sequence of the heavy chain of said anti-B7-H3 antibody is represented by SEQ ID No.58 of the sequence Listing, and the amino acid sequence of the light chain of said anti-B7-H3 antibody is represented by SEQ ID No.59 of the sequence Listing.
8. The anti-B7-H3 antibody of any one of claims 1 to 7, in the form of any one of the following antibodies:
(a) An intact immunoglobulin molecule;
(b) A scFv; the amino acid sequence of the scFv is shown as SEQ ID NO. 32 in the sequence table;
(c) A fusion protein comprising a scFv; the amino acid sequence of the scFv is shown as SEQ ID NO. 32 in the sequence table;
(d) A Fab fragment;
(e) A Fab' fragment;
(f) F (ab) 2 ；
Or, the antibody is a monoclonal antibody or a polyclonal antibody;
alternatively, the antibody is a super-humanized antibody or a diabody.
9. The anti-B7-H3 antibody of claim 8, wherein said monoclonal antibody is a fully human monoclonal antibody.
10. A nucleic acid encoding the anti-B7-H3 antibody of any one of claims 1 to 9.
11. A recombinant expression vector comprising the nucleic acid of claim 10.
12. A transformant comprising the recombinant expression vector of claim 11.
13. A method for preparing an anti-B7-H3 antibody, which is characterized by comprising the following steps: culturing the transformant according to claim 12, and obtaining the anti-B7-H3 antibody from the culture.
14. An immunoconjugate comprising an anti-B7-H3 antibody according to any one of claims 1 to 9.
15. The immunoconjugate of claim 14, wherein the immunoconjugate is an antibody drug conjugate or a chimeric antigen receptor T cell.
16. The immunoconjugate of claim 14 or claim 15, wherein the anti-B7-H3 antibody and cytotoxic agent are linked by a linker, the linker is SMCC, the cytotoxic agent is maytansine, and the antibody-drug ratio of the anti-B7-H3 antibody to the maytansine is 3.2 to 3.5.
17. A pharmaceutical composition comprising an immunoconjugate according to any one of claims 14 to 16 and a pharmaceutically acceptable carrier.
18. Use of an anti-B7-H3 antibody according to any one of claims 1 to 9, or an immunoconjugate according to any one of claims 14 to 16, or a pharmaceutical composition according to claim 17, for the manufacture of a medicament for treating a disease associated with abnormal expression of B7-H3; the diseases related to abnormal B7-H3 expression are lung cancer, breast cancer, prostatic cancer, pancreatic cancer, colorectal cancer, melanoma, liver cancer, ovarian cancer, bladder cancer, gastric cancer, esophageal cancer or renal cancer.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811333430.XA CN110305213B (en) | 2018-11-09 | 2018-11-09 | anti-B7-H3 antibody, preparation method thereof, conjugate thereof and application thereof |
| US17/292,488 US20220017620A1 (en) | 2018-11-09 | 2019-11-08 | Anti-b7-h3 antibody, preparation method therefor, conjugate and application thereof |
| PCT/CN2019/116590 WO2020094120A1 (en) | 2018-11-09 | 2019-11-08 | Anti-b7-h3 antibody, preparation method therefor, conjugate and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811333430.XA CN110305213B (en) | 2018-11-09 | 2018-11-09 | anti-B7-H3 antibody, preparation method thereof, conjugate thereof and application thereof |
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| CN110305213B true CN110305213B (en) | 2023-03-10 |
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Families Citing this family (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW201909926A (en) * | 2017-08-04 | 2019-03-16 | 大陸商江蘇恆瑞醫藥股份有限公司 | B7H3 antibody-drug conjugate and its medical use |
| US20220017620A1 (en) * | 2018-11-09 | 2022-01-20 | Shanghai Fudan-Zhangjiang Bio-Pharmaceutical Co., Ltd. | Anti-b7-h3 antibody, preparation method therefor, conjugate and application thereof |
| CN110642948B (en) * | 2019-10-09 | 2021-06-29 | 达石药业(广东)有限公司 | B7-H3 nano antibody, preparation method and application thereof |
| CN113754766A (en) * | 2020-06-02 | 2021-12-07 | 明慧医药（上海）有限公司 | anti-B7-H3 antibody and preparation and application thereof |
| JP2023550533A (en) * | 2020-12-18 | 2023-12-01 | シャンハイ フダン－チャンジャン バイオ－ファーマシューティカル カンパニー リミテッド | Antibody-drug conjugate targeting B7-H3, its production method and use |
| CN114763381A (en) * | 2021-01-13 | 2022-07-19 | 博生吉医药科技(苏州)有限公司 | B7-H3 chimeric antigen receptor modified T cell and application thereof |
| CN114763388A (en) * | 2021-01-13 | 2022-07-19 | 博生吉医药科技(苏州)有限公司 | B7-H3-targeted CAR-T cells and application thereof in treatment of acute myeloid leukemia |
| CN114763382A (en) * | 2021-01-13 | 2022-07-19 | 博生吉医药科技(苏州)有限公司 | Monoclonal antibody targeting human CD276 and application thereof |
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