CN109963591A - B7H3 antibody-drug conjugates and its medical usage - Google Patents

B7H3 antibody-drug conjugates and its medical usage Download PDF

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Publication number
CN109963591A
CN109963591A CN201880004425.6A CN201880004425A CN109963591A CN 109963591 A CN109963591 A CN 109963591A CN 201880004425 A CN201880004425 A CN 201880004425A CN 109963591 A CN109963591 A CN 109963591A
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antibody
seq
sequence
chain variable
drug conjugates
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CN109963591B (en
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顾津明
叶鑫
杨柳青
梁金栋
蒋贵阳
陶维康
张连山
应华
张玲
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Changzhou Hengbang Pharmaceutical Co ltd
Hansen Shanghai Health Technology Co ltd
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Jiangsu Hengrui Medicine Co Ltd
Shanghai Hengrui Pharmaceutical Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Abstract

A kind of B7H3 antibody-drug conjugates and its medical usage.Specifically, B7H3 antibody-cell drug toxicity conjugate or its pharmaceutically acceptable salt or solvated compounds, and the purposes in the drug of pharmaceutical composition and its preparation for treating disease or illness that B7H3 is mediated comprising aforementioned conjugate or its pharmaceutically acceptable salt or solvated compounds；Especially preparing the purposes in anticancer drug.

Description

B7H3 antibody-drug conjugates and its medical usage Technical field
Application the present invention relates to B7H3 antibody-drug conjugates and its in medicine, further, the present invention relates to B7H3 antibody-cell drug toxicity conjugate or its pharmaceutically acceptable salts or solvated compounds, and the purposes in the drug of pharmaceutical composition and its preparation for treating disease or illness that B7H3 is mediated comprising aforementioned conjugate or its pharmaceutically acceptable salt or solvated compounds；Especially preparing the purposes in anticancer drug.
Background technique
The immune response that T cell mediates plays extremely important effect during body is antitumor, and the activation of T cell and proliferation not only need the antigen signals of TCR identification, it is also necessary to the second signal that costimulatory molecules provide.B7 family molecule belongs to costimulatory molecules immunoglobulin superfamily, more and more researches show that, which has played important adjustment effect under body normal immunological function and pathological state.
B7H3 is one of the member of B7 family, belong to I type transmembrane protein, a signal peptide comprising aminoterminal, cytoplasmic tail (the Tissue Antigens.2007 Aug of an extracellular immunoglobulin-like variable region (IgV) and constant region (IgC), a transmembrane region and one containing 45 amino acid；70(2):96-104).Currently, B7H3 is primarily present 2 kinds of spliced bodies, B7H3a and B7H3b.B7H3a extracellular fragment is made of 2 immunoglobulin domains of IgV-IgC, also known as 2IgB7H3, and B7H3b extracellular fragment is made of 4 immunoglobulin domains of IgV-IgC-IgV-IgC, also known as 4IgB7H3.
B7H3 protein is not expressed in normal tissue, cell or extremely low expression, high to be expressed in kinds of tumors tissue and closely related with the progress of tumour, the existence of patient and prognosis.Clinically it is reported that B7H3 is overexpressed (Lung Cancer.2009 Nov in many cancer types, particularly in non-small cell lung cancer, kidney, urothelium cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, oophoroma and cancer of pancreas；66(2):245-249；Clin Cancer Res.2008 Aug 15；14(16):5150-5157).Furthermore, also there is document report, in prostate cancer, the expression intensity and clinicopathologia of B7H3 pernicious (invade outside such as gross tumor volume, prostate or Gleason scores) is positively correlated, and also (Cancer Res.2007 Aug 15 related to cancer progression；67(16):7893-7900).Similarly, in glioblastoma multiforme, the expression of B7H3 and the survival of no event are negatively correlated, and in cancer of pancreas, the expression of B7H3 is related to lymphatic metastasis and pathological progress.Therefore, B7H3 is considered as a kind of new tumor markers and potential therapy target
Currently, having the therapeutic strategy for B7H3 target spot for preclinical study, the antibody of such as targeting mouse B7H3 can enhance the T cell of the CD8- positive of the wellability in tumor and inhibit tumour growth (Mod Pathol.2010 Aug；23(8):1104-1112).In addition, 2008/066691 patent of WO is shown, the antibody of identification B7H3 variant B7H3a can show Anticancer effect in vivo to gland cancer.In clinical studies, the B7H3 antibody Yu radioactivity I of a kind of source of mouse 131Coupling drug can significantly inhibit patient into the growth [J Neufooocol 97 (3): 409-l 8 (2010] of neuroblastoma.But it is all at present source of mouse antibody through humanization modified humanized antibody in the project ground, and humanized antibody has that immunogenicity is relatively high when immune, is a unfavorable factor in human body application.
Display technique of bacteriophage (phage display technology) is by exogenous proteins or polypeptide and bacteriophage coat protein amalgamation and expression, thus by exogenous protein expression on the surface of bacteriophage.Phage antibody library is an antibody library set up with complex art means for combining display technique of bacteriophage, PCR amplification, protein expression techniques.
Phage antibody library biggest advantage is to simulate three processes that internal antibody generates without vivo immunization and prepare full-length human antibody.In addition to this, phage antibody library also has the advantage that the unification for 1. realizing genotype and phenotype.In addition, experimental method is simple, quickly, it is traditional need to be after the several months by hybridoma technology antibody production method, and Antibody library only needs the time in short a few weeks.2. what is expressed is complete human antibody, and molecular weight is small, is mainly expressed in the form of active fragment Fab, scFV, is all had a clear superiority in terms of tissue penetration compared with complete antibody.3. it is big to screen capacity, hybridoma technology is the screening in thousands of clones, and Antibody library can be to million or even hundreds of millions molecular selection.The antibody type screened is more.4. it is widely used, prokaryotic expression system is used, more obvious (the Curr Opin Biotechnol.2002 Dec of advantage when large-scale production；13(6):598-602；Immunotechnology,2013,48(13)48(13):63-73).
Antibody-drug conjugates (antibody drug conjugate, ADC) monoclonal antibody or antibody fragment are connected by stable chemical linker compound with biologically active cytotoxin, the high efficiency of specificity and cytotoxic substance that antibody combines normal cell and tumor cell surface antigen is taken full advantage of, while in turn avoiding the defects of the former curative effect is relatively low and the latter's toxic side effect is excessive.This is also meaned that, compared with previous traditional chemotherapeutics, antibody-drug conjugates can more accurately combine tumour cell and reduce the influence to normal cell.
There are many ADC drugs to be used for clinical or clinical research, such as Kadcyla at present, is the ADC drug for targeting the Herceptin of Her2 and being formed with DM1.Meanwhile also having the antibody of targeting B7H3 and the patent report of ADC drug, such as WO2008100934, WO2012147713, WO2014061277, WO2015184203, WO2016044383.But it lists still without the ADC drug of B7H3 target spot or is studied for clinical treatment, therefore, the ADC drug for developing new B7H3 target spot has broad prospects.
Summary of the invention
The object of the present invention is to provide the ADC drugs of monoclonal antibody and cytotoxic substance coupling in conjunction with the amino acid sequence of the extracellular region of B7H3 or three-dimensional structure.By screening the anti-human B7H3 human antibody ADC drug of high activity and high stability, the therapeutic agent for using the antibody A DC drug as active constituent is provided.
The present invention provides antibody-drug conjugates or its pharmaceutically acceptable salt or solvated compounds shown in a kind of general formula (A),
Ab-(L 2-L 1-D) y        (A)
Wherein:
D is cytotoxic drug；
L 1, L 2It is connector unit；
Y is the number selected from 1-8, is preferably selected from the number of 2-4；Y can be decimal or integer；
Ab is B7H3 antibody or its antigen-binding fragment, and in conjunction with people B7H3, the B7H3 antibody or its antigen-binding fragment are the monoclonal antibody or its antigen-binding fragment any into (c) selected from following (a):
(a) monoclonal antibody has the amino acid sequence of at least 95% sequence identity it includes one or more CDR region sequences selected from the following or with it:
Antibody heavy chain variable region HCDR region sequence: as shown in the amino acid sequence of SEQ ID NO:10,11 and 12；With antibody's light chain variable region LCDR region sequence: as shown in SEQ ID NO:13,14 and fifteen amino acid sequence；
(b) monoclonal antibody has the amino acid sequence of at least 95% sequence identity it includes one or more CDR region sequences selected from the following or with it:
Antibody heavy chain variable region HCDR region sequence: as shown in the amino acid sequence of SEQ ID NO:16,17 and 18；With antibody's light chain variable region LCDR region sequence: as shown in the amino acid sequence of SEQ ID NO:19,20 and 21；
(c) monoclonal antibody has the amino acid sequence of at least 95% sequence identity it includes one or more CDR region sequences selected from the following or with it:
Antibody heavy chain variable region HCDR region sequence: as shown in the amino acid sequence of SEQ ID NO:30,31 and 32；With antibody's light chain variable region LCDR region sequence: as shown in the amino acid sequence of SEQ ID NO:33,34 and 35.
In a preferred embodiment, the antibody-drug conjugates as shown in general formula (A) or its pharmaceutically acceptable salt or solvated compounds, wherein the B7H3 antibody or its antigen-binding fragment are the monoclonal antibody or its antigen-binding fragment any into (c) selected from following (a):
(a) monoclonal antibody, it includes the HCDR1, HCDR2, HCDR3 of the antibody heavy chain variable region as shown in the amino acid sequence of SEQ ID NO:10,11 and 12 respectively；With the LCDR1, LCDR2, LCDR3 of the antibody's light chain variable region as shown in SEQ ID NO:13,14 and fifteen amino acid sequence；
(b) monoclonal antibody, it includes the HCDR1, HCDR2, HCDR3 of the antibody heavy chain variable region as shown in the amino acid sequence of SEQ ID NO:16,17 and 18 respectively；With the LCDR1, LCDR2, LCDR3 of the antibody's light chain variable region as shown in the amino acid sequence of SEQ ID NO:19,20 and 21；
(c) monoclonal antibody, it includes the HCDR1, HCDR2, HCDR3 of the antibody heavy chain variable region as shown in the amino acid sequence of SEQ ID NO:30,31 and 32 respectively；With the LCDR1, LCDR2, LCDR3 of the antibody's light chain variable region as shown in the amino acid sequence of SEQ ID NO:33,34 and 35.
In a preferred embodiment, the antibody-drug conjugates as shown in general formula (A) or its pharmaceutically acceptable salt or solvated compounds,
Ab-(L 2-L 1-D) y        (A)
Wherein:
D is cytotoxic drug；
L 1, L 2It is connector unit；
Y is the number selected from 1-8, is preferably selected from the number of 2-4；
Ab is the monoclonal antibody or its antigen-binding fragment with B7H3 antibody as defined above or its antigen-binding fragment competitive binding people B7H3.
In a preferred embodiment, the antibody-drug conjugates as shown in general formula (A), wherein the Ab is recombinant antibodies.
In a preferred embodiment, the antibody-drug conjugates as shown in general formula (A), wherein the Ab is the recombinant antibodies or its antigen-binding fragment of source of people.
In a preferred embodiment, the antibody-drug conjugates as shown in general formula (A), wherein the light chain and heavy chain FR region sequence on the light chain and heavy chain variable region of the Ab are respectively derived from people's germline light chain and sequence of heavy chain or its mutant nucleotide sequence.
In a preferred embodiment, the antibody-drug conjugates as shown in general formula (A), wherein the constant region of the Ab includes the heavy chain constant region from humanized IgG 1, IgG2, IgG3 or IgG4 or its variant, preferably 1 heavy chain constant region of humanized IgG；With the constant region of light chain from source of people κ, λ chain or its variant, preferably source of people κ chain constant region of light chain.
In a preferred embodiment, the antibody-drug conjugates as shown in general formula (A), wherein the Ab contains SEQ ID NO:6, SEQ ID NO:8, heavy chain variable region or its variant shown in SEQ ID NO:36 or SEQ ID NO:37；The variant is the sequence on the weight chain variabl area sequence shown in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:36 or SEQ ID NO:37 with 1-10 amino acid substitution.
In a preferred embodiment, the antibody-drug conjugates as shown in general formula (A), wherein the Ab contains SEQ ID NO:7, SEQ ID NO:9, light chain variable region or its variant shown in SEQ ID NO:38 or SEQ ID NO:39；The variant is the sequence on the light-chain variable sequence shown in SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:38 or SEQ ID NO:39 with 1-10 amino acid substitution.
In a preferred embodiment, the antibody-drug conjugates as shown in general formula (A), wherein the Ab contains selected from following (1) monoclonal antibody any into (7) or its antigen-binding fragment:
(1) monoclonal antibody, it includes antibody heavy chain variable regions shown in SEQ ID NO:6；With antibody's light chain variable region shown in SEQ ID NO:7；
(2) monoclonal antibody, it includes antibody heavy chain variable regions shown in SEQ ID NO:8；With antibody's light chain variable region shown in SEQ ID NO:9；
(3) monoclonal antibody, it includes antibody heavy chain variable regions shown in SEQ ID NO:28；With antibody's light chain variable region shown in SEQ ID NO:29；
(4) monoclonal antibody, it includes antibody heavy chain variable regions shown in SEQ ID NO:36；With antibody's light chain variable region shown in SEQ ID NO:38；
(5) monoclonal antibody, it includes antibody heavy chain variable regions shown in SEQ ID NO:36；With antibody's light chain variable region shown in SEQ ID NO:39；
(6) monoclonal antibody, it includes antibody heavy chain variable regions shown in SEQ ID NO:37；With antibody's light chain variable region shown in SEQ ID NO:38；
(7) monoclonal antibody, it includes antibody heavy chain variable regions shown in SEQ ID NO:37；With antibody's light chain variable region shown in SEQ ID NO:39.
In a preferred embodiment, the antibody-drug conjugates as shown in general formula (A), wherein the Ab is full length antibody, it further comprise human antibody constant region；Wherein the full length antibody is selected from:
The full length antibody that sequence of light chain shown in h1702 antibody, the sequence of heavy chain shown in SEQ ID NO:22 and SEQ ID NO:23 forms,
The full length antibody that sequence of light chain shown in h1703 antibody, the sequence of heavy chain shown in SEQ ID NO:24 and SEQ ID NO:25 forms,
The full length antibody that sequence of light chain shown in h1702-DS antibody, the sequence of heavy chain shown in SEQ ID NO:22 and SEQ ID NO:26 forms,
Or h1704-3 antibody, the full length antibody that sequence of light chain shown in the sequence of heavy chain shown in SEQ ID NO:40 and SEQ ID NO:41 forms.
In a preferred embodiment, the antibody-drug conjugates as shown in general formula (A), wherein the antigen-binding fragment is selected from the antigen-binding fragment of Fab, Fab', F (ab') 2, single-chain antibody (scFv), the area V (double antibody) of dimerization, the disulfide-stabilized area V (dsFv) and the peptide comprising CDR.
In a preferred embodiment, the antibody-drug conjugates as shown in general formula (A), wherein cytotoxic drug is selected from toxin, chemotherapeutics, antibiotic, radioactive isotope and core lyase.
In a preferred embodiment, the antibody-drug conjugates as shown in general formula (A), wherein cytotoxic drug is selected from DM1, DM3, DM4, MMAF and MMAE.
In a preferred embodiment, the antibody-drug conjugates as shown in general formula (A), wherein cytotoxic drug is selected from:
In a preferred embodiment, the antibody-drug conjugates as shown in general formula (A) are compound or its pharmaceutically acceptable salt or solvated compounds shown in Formulas I,
Wherein:
L 1, L 2It is connector unit；
Y is the number selected from 1-8, is preferably selected from the number of 2-4；
Ab is B7H3 antibody as defined above or its antigen-binding fragment or monoclonal antibody or its antigen-binding fragment with B7H3 antibody as defined above or its antigen-binding fragment competitive binding people B7H3.
In a preferred embodiment, the antibody-drug conjugates as shown in general formula (A), wherein L 2Such as following general formula L 2It is shown:
Wherein
X 1Selected from hydrogen atom, halogen, hydroxyl, cyano, alkyl, alkoxy and naphthenic base；
X 2Selected from alkyl, naphthenic base and heterocycle；
M is the integer selected from 0-5, preferably 1,2 or 3；S is sulphur atom.
In a preferred embodiment, the antibody-drug conjugates as shown in general formula (A), wherein L 1Such as following general formula (L 1) shown in:
Wherein
X 3For alkyl, the optionally alkyl is further taken by the substituent group selected from halogen, hydroxyl and cyano
Generation；
N is the integer selected from 0-5, preferably 1,2 or 3.
In a preferred embodiment, the antibody-drug conjugates as shown in general formula (A), wherein cytotoxic drug obtains compound after connecting with attachment L1:
In a preferred embodiment, the antibody-drug conjugates as shown in general formula (A), to lead to antibody-drug conjugates shown in formula (II):
Wherein, L 2It is connector unit；
Y is the number selected from 1-8, is preferably selected from the number of 2-4；
Ab is B7H3 antibody as defined above or its antigen-binding fragment or monoclonal antibody or its antigen-binding fragment with B7H3 antibody as defined above or its antigen-binding fragment competitive binding people B7H3.
In a preferred embodiment, the antibody-drug conjugates as shown in general formula (A), to lead to antibody-drug conjugates shown in formula (III):
Wherein, L 1It is connector unit；
Y is the number selected from 1-8, is preferably selected from the number of 2-4；
Ab is B7H3 antibody as defined above or its antigen-binding fragment or monoclonal antibody or its antigen-binding fragment with B7H3 antibody as defined above or its antigen-binding fragment competitive binding people B7H3.
In a preferred embodiment, the antibody-drug conjugates as shown in general formula (A) or its officinal salt, including but not limited to:
The invention further relates to a kind of pharmaceutical compositions, and it includes the antibody-drug conjugates as shown in general formula of the present invention (A) or its pharmaceutically acceptable salts or solvated compounds and one or more pharmaceutical excipient, diluent or carrier.
The invention further relates to antibody-drug conjugates shown in general formula (A), or its pharmaceutically acceptable salt or solvated compounds, or the pharmaceutical composition comprising it, the purposes in drug of the preparation for treating disease relevant to people's B7H3 positive cell；Preferably, wherein the purposes, is to prepare the purposes in the drug for treating B7H3 high expression cancer.
The invention further relates to antibody-drug conjugates shown in general formula (A), or its pharmaceutically acceptable salt or solvated compounds, or the purposes in drug for treating disease is being prepared comprising its pharmaceutical composition, the disease is selected from treatment glioma (non-limiting embodiment is human brain astrocytoblast tumor), people's pharynx cancer, adrenal tumor, AIDS- associated cancer, alveolar soft part sarcoma, astrocytoma, bladder cancer, osteocarcinoma, brain and spinal cord cancer, metastatic brain tumor, breast cancer, carotid body tumor, cervical carcinoma, chondrosarcoma, chordoma, kidney chromophobe cell tumor, clear cell carcinoma, colon cancer, colorectal cancer, desmoplastic small round cell tumor, ependymocytoma, Juventus tumour, Extraskeletal myxoid chondrosarcoma, fibrogenesis imperfecta ossium, fibrous dysplasia of bone, Gall-bladder or cholangiocarcinoma, gastric cancer, gestational trophoblast disease, gonioma, head and neck cancer, hepatocellular carcinoma, islet-cell tumour, Kaposi sarcoma, kidney, leukaemia, embryonal-cell lipoma/pernicious lipoma tumour, liver cancer, lymthoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, Huppert's disease, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, oophoroma, cancer of pancreas, papillary thyroid carcinoma, parathyroid adenoma, paediatric cancer, Peripheral Nerve Sheath Tumors, pheochromocytoma, pituitary tumor, prostate cancer, uveal afterwards, kidney metastatic carcinoma, rhabdoid tumor, rhabdomyosarcoma, sarcoma, cutaneum carcinoma, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, carcinoma of testis, thymic carcinoma, thymoma, Thyroid metastasis cancer and son Palace cancer.
The invention further relates to a kind of methods for treating disease, this method include to need its patient apply treatment effective dose the general formula (A) shown in antibody-drug conjugates, or its pharmaceutically acceptable salt or solvated compounds, or the pharmaceutical composition comprising it, the disease is selected from human brain astrocytes' blastoma, people's pharynx cancer, adrenal tumor, AIDS- associated cancer, alveolar soft part sarcoma, astrocytoma, bladder cancer, osteocarcinoma, brain and spinal cord cancer, metastatic brain tumor, breast cancer, carotid body tumor, cervical carcinoma, chondrosarcoma, chordoma, kidney chromophobe cell tumor, clear cell carcinoma, colon cancer, colorectal cancer, desmoplastic small round cell tumor, ependymocytoma, Juventus tumour, Extraskeletal myxoid chondrosarcoma, fibrogenesis imperfecta ossium, bone fibres sexual development is not It is good, gall-bladder or cholangiocarcinoma, gastric cancer, gestational trophoblast disease, gonioma, head and neck cancer, hepatocellular carcinoma, islet-cell tumour, Kaposi sarcoma, kidney, leukaemia, embryonal-cell lipoma/pernicious lipoma tumour, liver cancer, lymthoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, Huppert's disease, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, oophoroma, cancer of pancreas, papillary thyroid carcinoma, parathyroid adenoma, paediatric cancer, Peripheral Nerve Sheath Tumors, pheochromocytoma, pituitary tumor, prostate cancer, uveal afterwards, kidney metastatic carcinoma, rhabdoid tumor, rhabdomyosarcoma, sarcoma, cutaneum carcinoma, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, carcinoma of testis, thymic carcinoma, thymoma, Thyroid metastasis cancer And uterine cancer.
Detailed description of the invention
Fig. 1: the binding force of antibody and U87MG cell；
Fig. 2: endocytosis effect of the different antibodies on U87MG cell；
Endocytosis effect of Fig. 3: the present invention difference ADC h1702-3024, h1703-3024 on U87MG cell；
Fig. 4: present invention difference ADC h1702-cys-3024, h1702DS-cys-3024 and
The endocytosis effect of h1704-3-cys-3024 and its corresponding antibodies on U87MG cell；
Fig. 5: h1702-3024 to the curative effect of nude mice U87MG transplantable tumor；
Fig. 6: the h1702-3024 influence to U87MG nude mice weight.
The stability result of Fig. 7: h1702DS-cys-3024 and h1704-3-cys-3024
Specific embodiment
One, term
In order to be easier to understand the present invention, certain technical and scientific terms are defined in detail below.Unless separately explicitly defining herein, all other technical and scientific term used herein all has the normally understood meaning of those skilled in the art of the art.
Amino acid three-letter codes used in the present invention and single letter code such as J.biol.chem, described in 243, p3558 (1968).
" antibody " of the present invention refers to immunoglobulin, is four peptide chain structures being formed by connecting by two identical heavy chains and two identical light chains by interchain disulfide bond.The amino acid of immunoglobulin heavy chain constant region forms and the difference that puts in order, therefore its antigenicity is also different.Accordingly, immunoglobulin can be divided into five classes, or be the isotype of immunoglobulin, i.e. IgM, IgD, IgG, IgA and IgE, corresponding heavy chain is respectively μ chain, δ chain, γ chain, α chain and ε chain.Same class Ig is formed according to its hinge region amino acid and the difference of the number and location of heavy chain disulfide bond, and can be divided into different subclass, as IgG can be divided into IgG1, IgG2, IgG3, IgG4.Light chain is divided into κ chain or λ chain by the difference of constant region.Every class Ig can have κ chain or λ chain in five class Ig.
Heavy chain of antibody and light chain are very big close to the sequence variation of about 110 amino acid of N-terminal, are variable region (area Fv)；Remaining amino acid sequence close to C-terminal is relatively stable, is constant region.The variable region skeleton area (FR) relatively conservative including 3 hypervariable regions (HVR) and 4 sequences.3 hypervariable regions determine the specificity of antibody, also known as complementarity-determining region (CDR).Every light chain variable region (LCVR) and heavy chain variable region (HCVR) are by 3 CDR regions, 4 FR district's groups at the sequence being arranged successively from aminoterminal to c-terminus are as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.3 CDR regions of light chain refer to LCDR1, LCDR2 and LCDR3；3 CDR regions of heavy chain refer to HCDR1, HCDR2 and HCDR3.The area LCVR of antibody or antigen-binding fragment of the present invention and the cdr amino acid residue in the area HCVR meet known Kabat coding rule (LCDR1-3 in quantity and position, HCDR2-3), or meet the coding rule (HCDR1) of kabat and chothia.
Antibody of the invention includes source of mouse antibody, chimeric antibody, humanized antibody, preferably humanized antibody.
Term " source of mouse antibody " is the monoclonal antibody to people B7H3 prepared according to this field knowledge and skills in the present invention.With B7H3 antigen injection subjects when preparation, then separation expression has the hybridoma of the antibody of required sequence or functional characteristic.In a preferred embodiment of the present invention, the source of mouse B7H3 antibody or its antigen-binding fragment, it can further include source of mouse κ, λ chain or the constant region of light chain of its variant, or further include the heavy chain constant region of mouse IgG 1, IgG2, IgG3 or its variant.
Term " recombinant antibodies " includes " chimeric antibody ", " humanized antibody " and " complete human antibody ".
Term " chimeric antibody (chimeric antibody) " is that antibody made of the constant domain of the variable region of murine antibody and human antibody can be mitigated the immune response of murine antibody induction.Establish chimeric antibody, first to establish the hybridoma of secretion mouse specific monoclonal antibody, then variable region gene is cloned from mouse hybridoma, further according to the constant region gene for needing to clone human antibody, it is inserted into expression vector after mouse variable region gene and human constant region gene are connected into mosaic gene, finally the chimeric antibody expression molecule in eukaryotic system or prokaryotic system.In a preferred embodiment of the present invention, the antibody light chain of the B7H3 chimeric antibody further includes source of people κ, λ chain or the constant region of light chain of its variant.The heavy chain of antibody of the B7H3 chimeric antibody further includes the heavy chain constant region of humanized IgG 1, IgG2, IgG3, IgG4 or its variant, preferably comprise humanized IgG 1, IgG2 or IgG4 heavy chain constant region, or IgG1, IgG2 or IgG4 variant using amino acid mutation (such as YTE mutation or back mutation).
Term " humanized antibody (humanized antibody) ", also referred to as CDR grafted antibody (CDR-grafted antibody), refer to the antibody variable region frame that the CDR sequence of mouse is transplanted to people, i.e., the antibody generated in different types of human germline antibody's Frame sequence.Chimeric antibody can be overcome due to carrying a large amount of mouse protein ingredients, thus the heterologous reaction of induction.Such frame sequence can be obtained from the public DNA database or disclosed bibliography for including germline antibody gene sequences.As people's heavy chain and the germline DNA sequence dna of light-chain variable region gene can be in " VBase " human germ line sequences' databases (in internet www.mrccpe.com.ac.uk/vbaseCan get), and in Kabat, E.A. et al., 1991Sequences of Proteins of Immunological Interest is found in the 5th edition.While to avoid immunogenicity from declining, caused activity decline can carry out minimum inverse transition or back mutation to the human antibody variable region framework sequence, to keep activity.Humanized antibody of the invention also includes further carrying out the humanized antibody after affinity maturation to CDR by phage display.In a preferred embodiment of the present invention, the CDR sequence of mouse is selected from SEQ ID NO:8-13 or 14-19 in the B7H3 humanized antibody；The antibody variable region frame of people passes through design alternative, wherein the heavy chain FR region sequence in the antibody heavy chain variable region, from the composite sequence of human germline heavy IGHV1-18*01 and hjh4.1 or the composite sequence of IGHV3-7*01 and hjh4.1；The wherein light chain FR region sequence in the antibody's light chain variable region, from the composite sequence of people's germline light chain IGKV1-33*01 and hjk4.1 or the composite sequence of IGKV1-39*01 and hjk2.1.While to avoid immunogenicity from declining, caused activity decline can carry out minimum inverse transition to the human antibody variable region, to keep activity.
The transplanting of CDR can cause the B7H3 antibody generated or its antigen-binding fragment to weaken the affinity of antigen due to the Framework residues with antigen contact.Such interact can be the result of somatic hypermutation.Accordingly, it is possible to which there is still a need for the frameworks that such donor framework amino acid is migrated to humanized antibody.The amino acid residue of participation antigen binding from inhuman B7H3 antibody or its antigen-binding fragment can be identified by checking mouse monoclonal antibody variable region sequences and structure.Each residue different to germline can be considered as relevant in CDR donor framework.It, can be by sequence with hypotype consensus sequence or the consensus sequence of the mouse sequence with high percentage similarity compares if not can determine that immediate germline.Rare Framework residues are deemed likely to be somatic hypermutation as a result, to play an important role in combination.
Term " complete human antibody " or " human antibody ", also referred to as " full human monoclonal antibody ", the variable region of antibody and constant region are all source of people, remove immunogenicity and toxic side effect.The development experience of monoclonal antibody four-stage, is respectively as follows: mouse monoclonal antibody, chimeric monoclonal antibodies, Humanized monoclonal antibodies and full human monoclonal antibody.The relevant technologies of human antibody preparation mainly have: people's hybridoma technology, EBV conversion bone-marrow-derived lymphocyte technology, phage display technology (phage display), transgenic mice antibody production techniques (transgenic mouse) and single B cell antibody technology of preparing etc.." fully human antibodies " in the present invention obtain antibody variable region using phage display technology, can further recombinate with antibody constant region and obtain complete antibody.
" antigen-binding fragment " or " function fragment " of term antibody refers to one or more segments of the ability of the holding molecule of the antigen binding (for example, B7H3) of antibody.The antigen binding function that antibody can be carried out using the segment of full length antibody has been displayed.The example for the binding fragment for including in " antigen-binding fragment " of term antibody includes (i) Fab segment, the monovalent fragment being made of VL, VH, CL and CH1 structural domain；(ii)F(ab') 2Segment, comprising the bivalent fragment of the two Fab segments connected by the disulphide bridges on hinge area, the Fd segment that (iii) is made of VH and CH1 structural domain；(iv) the Fv segment being made of VH the and VL structural domain of the single armed of antibody；(v) single domain or dAb segment (Ward et al., (1989) Nature341:544-546), are made of VH structural domain；The complementary determining region (CDR) or (vii) of (vi) separation optionally pass through the combination of the CDR of two or more separation of the connector connection of synthesis.Furthermore, although two structural domain VL and VH of Fv segment are encoded by the gene separated, but recombination method can be used, them are connected by the connector of synthesis, so that it, which can be produced as the wherein area VL and VH, matches single protein chain (the referred to as scFv (scFv) to form monovalent molecule；See, e.g., Bird et al. (1988) Science242:423-426；With Huston et al. (1988) Proc.Natl.Acad.Sci USA85:5879-5883).Such single-chain antibody is also intended to including in " antigen-binding fragment " of term antibody.Obtain such antibody fragment using routine techniques well known by persons skilled in the art, and by with for complete antibody in a manner of identical mode segment is screened with regard to instrumentality.Antigen-binding portion thereof can be generated by recombinant DNA technology or by enzymatic or chemical disruption intact immunoglobulins.Antibody can be the antibody of different isotypes, for example, IgG (for example, IgG1, IgG2, IgG3 or IgG4 hypotype), IgA1, IgA2, IgD, IgE or IgM antibody.
Antigen-binding fragment of the invention includes Fab, F (ab') 2, Fab', single-chain antibody (scFv), the area V (double antibody) of dimerization, the disulfide-stabilized area V (dsFv), peptide comprising CDR etc..
Fab is by having about 50 in protease papain enzyme (224 amino acid residues of cutting H chain) processing IgG antibody molecule segment obtained, 000 molecular weight and the antibody fragment with antigen-binding activity, wherein about half of H chain N-terminal side is together with entire L chain is by disulfide-bonded.
Fab of the invention can be by being produced with Papain enzymatic treatment specific recognition people B7H3 of the invention and the monoclonal antibody in conjunction with the amino acid sequence of extracellular region or its three-dimensional structure.Furthermore, it is possible to by the way that the DNA of the Fab of encoding said antibody is inserted into prokaryotic expression carrier or eukaryotic expression vector and the Fab will be produced in vector introduction to prokaryotes or eucaryote to express Fab.
F (ab') 2 be by in enzyme pepsin digestion IgG hinge area the section below of two disulfide bond and the molecular weight that obtains is about 100,000 and with antigen-binding activity and is included in the antibody fragment in the area Liang Ge Fab that hinge position is connected.
F (ab') 2 of the invention can be by being produced with pepsin specific recognition people B7H3 of the invention and the monoclonal antibody in conjunction with the amino acid sequence of extracellular region or its three-dimensional structure.Furthermore, it is possible to produce the F (ab') 2 by connecting Fab' described below with thioether bond or disulfide bond.
Fab' is that the molecular weight obtained by the disulfide bond for cutting the hinge area of above-mentioned F (ab') 2 is about 50,000 and the antibody fragment with antigen-binding activity.Fab' of the invention can be produced by handling specific recognition B7H3 of the invention and F (ab') 2 in conjunction with the amino acid sequence of extracellular region or its three-dimensional structure with reducing agent such as dithiothreitol (DTT).
Furthermore, it is possible to by the way that the DNA of the Fab' segment of encoding antibody is inserted into prokaryotic expression carrier or eukaryotic expression vector and the Fab' will be produced in vector introduction to prokaryotes or eucaryote to express Fab'.
Term " single-chain antibody ", " scFv " or " scFv " means heavy chain of antibody variable domains (or the region comprising connecting by connector；) and antibody light chain variable domains (or region VH；VL molecule).Such scFv molecule can have general structure: NH 2- VL- connector-VH-COOH or NH 2- VH- connector-VL-COOH.Suitable prior art connector is made of duplicate GGGGS amino acid sequence or its variant, such as uses 1-4 duplicate variants (Holliger et al. (1993), Proc.Natl.Acad.Sci.USA90:6444-6448).Other connectors for use in the present invention are by Alfthan et al. (1995), Protein Eng.8:725-731, Choi et al. (2001), Eur.J.Immuno is l.31:94-106, Hu et al. (1996), Cancer Res.56:3055-3061, Kipriyanov et al. (1999), J.Mol.Biol.293:41-56 and Roovers et al. (2001), Cancer Immunol. description.
ScFv of the invention can be produced by following steps: obtain the code cDNA of specific recognition people B7H3 of the invention and the VH and VL of the monoclonal antibody in conjunction with the amino acid sequence of extracellular region or its three-dimensional structure, the DNA of building coding scFv, the DNA is inserted into prokaryotic expression carrier or eukaryotic expression vector, then the expression vector is imported into prokaryotes or eucaryote to express scFv.
Double antibody is that wherein scFv is to combine active antibody fragment with bivalent antigen by Dimerized antibody fragment.It is combined in activity in bivalent antigen, two antigens can be identical or different.
Double antibody of the invention can be produced by following steps: obtain the code cDNA of specific recognition people B7H3 of the invention and the VH and VL of the monoclonal antibody in conjunction with the amino acid sequence of extracellular region or its three-dimensional structure, building encodes the DNA of scFv so that the length amino acid sequence of peptide linker is for 8 residues or less, the DNA is inserted into prokaryotic expression carrier or eukaryotic expression vector, then the expression vector is imported into prokaryotes or eucaryote to express double antibody.
DsFv is that the polypeptide by being replaced an amino acid residue in wherein each VH and VL by cysteine residues is connected via the disulfide bond between cysteine residues and obtains.It (Protein Engineering, 7,697 (1994)) can be predicted based on the three-dimensional structure of antibody to select the amino acid residue replaced by cysteine residues according to known methods.
DsFv of the invention can be produced by following steps: obtain the code cDNA of specific recognition people B7H3 of the invention and the VH and VL of the monoclonal antibody in conjunction with the amino acid sequence of extracellular region or its three-dimensional structure, the DNA of building coding dsFv, the DNA is inserted into prokaryotic expression carrier or eukaryotic expression vector, then the expression vector is imported into prokaryotes or eucaryote to express dsFv.
Peptide comprising CDR is one or more regions in the CDR by the inclusion of VH or VL and constitutes.Peptide comprising multiple CDR can be connected directly or is connected via suitable peptide linker.
Peptide comprising CDR of the invention can be produced by following steps: construct the coding DNA of specific recognition people B7H3 of the invention and the CDR of the VH and VL of the monoclonal antibody in conjunction with the amino acid sequence of extracellular region or its three-dimensional structure, the DNA is inserted into prokaryotic expression carrier or eukaryotic expression vector, then the expression vector is imported into prokaryotes or eucaryote to express the peptide.The peptide comprising CDR can also be produced by chemical synthesis process such as Fmoc method or tBoc method.
Term " CDR " refers to one of 6 hypervariable regions for mainly facilitating antigen binding in the variable domains of antibody.One of most common definition of 6 CDR is by Kabat E.A. et al., (1991) Sequences of proteins of immunological interest.NIH Publication91-3242) it provides.As used in this article, the Kabat of CDR, which is defined, is only applied to LCDR1, LCDR2 and LCDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of light variable domains and the HCDR2 and HCDR3 (CDR H2, CDR H3 or H2, H3) of heavy-chain variable domains.
Term " antibody framework " used herein, refers to a part of variable domains VL or VH, is used as the bracket of the antigen binding loops (CDR) of the variable domains.It in essence, is the variable domains without CDR.
Term " epitope " or " antigenic determinant " refer to the position (for example, privileged site on B7H3 molecule) that immunoglobulin or antibody specificity combine on antigen.Epitope includes usually at least 3,4,5,6,7,8,9,10,11,12,13,14 or 15 amino acid continuously or discontinuously with unique space conformation.See, e.g., Epitope Mapping Protocols in Methods in Molecular B iology, volume 66, G.E.Morris, Ed. (1996).
Term " specific binding ", " selective binding ", " selectively combining " and " specifically combining " refers to combination of the antibody to the epitope on predetermined antigen.In general, antibody is approximately to be less than 10 -7M, such as approximately less than 10 -8M、10 -9M or 10 -10M or smaller affinity (KD) combine.
Term " KD " or " Kd " refer to specific antibodies-antigen interactions Dissociation equilibrium constant.In general, antibody of the invention is to be less than about 10 -7M is, for example, less than about 10 -8M、10 -9M or 10 -10M or smaller Dissociation equilibrium constant (KD) combine B7H3, for example, as measured in BIACORE instrument using surface plasma body resonant vibration (SPR) technology.
Term " monoclonal antibody of people B7H3 in conjunction with B7H3 antibody competition " refers to and a part of same epitope (also referred to as antigenic determinant) or same epitope on the extracellular region of monoclonal antibody of the invention competition identification people B7H3 and the antibody in conjunction with the epitope.The antibody of same epitope, which refers to, in conjunction with B7H3 antibody of the invention identifies and is incorporated into the antibody that B7H3 antibody of the invention knows the amino acid sequence of others B7H3.
Term " nucleic acid molecules " used herein refers to DNA molecular and RNA molecule.Nucleic acid molecules can be single-stranded or double-stranded, but preferably double-stranded DNA.When nucleic acid and another nucleic acid sequence to be placed in functional relationship, nucleic acid is " effectively connecting ".For example, promoter or enhancer are operatively connected to the coded sequence if promoter or enhancer influence the transcription of coded sequence.
Term " carrier " is the nucleic acid molecules for referring to transport another nucleic acid connected to it.In one embodiment, carrier is " plasmid ", and other DNA section can be connected on circular double stranded DNA ring therein by referring to.In another embodiment, carrier is viral vectors, wherein other DNA section can be connected in viral genome.Carrier disclosed herein can in the host cell for having been introduced into them independently duplication (such as, have the bacteria carrier and episomal mammalian vectors of germy replication orgin) or can be integrated into the genome of host cell after introducing host cell, to replicate (for example, non-add type mammalian vector) with host genome.
The method of known production and antibody purification and antigen-binding fragment in the prior art, such as the antibody assay technical manual of Cold SpringHarbor, 5-8 chapter and 15 chapters.For example, mouse can be immune with employment B7H3 or its segment, obtained antibody can be by renaturation, purifying, and can carry out amino acid sequencing with conventional method.Antigen-binding fragment can equally be prepared with conventional method.The CDR region that the antibody or antigen-binding fragment gene engineering method are invented in non-source of people adds the one or more area source of people FR.People FR Germline sequences can be by comparing the human antibody variable domains IMGT germ line genes database and MOE software, it is obtained from the website http://imgt.cines.fr of ImMunoGeneTics (IMGT), or it is obtained from immunoglobulin magazine, 2001ISBN012441351.
Term " host cell " refers to the cell for introducing expression vector thereto.Host cell may include bacterium, microorganism, plant or zooblast.The bacterium for being easy to convert includes the member of enterobacteriaceae (enterobacteriaceae), such as the bacterial strain of Escherichia coli (Escherichia coli) or salmonella (Salmonella)；Bacillaceae (Bacillaceae) such as bacillus subtilis (Bacillus subtilis)；Pneumococcus (Pneumococcus)；Streptococcus (Streptococcus) and Hemophilus influenzae (Haemophilus influenzae).Microorganism appropriate includes saccharomyces cerevisiae (Saccharomyces cerevisiae) and Pichia pastoris (Pichia pastoris).Animal host cell system appropriate includes CHO (Chinese hamster ovary line) and NS0 cell.
The antibody or antigen-binding fragment that the present invention is engineered can be prepared and purified with conventional method.For example, the cDNA sequence of encoding heavy chain and light chain, can clone and recombinate to GS expression vector.The immunoglobulin expression carrier of recombination can steadily transfection CHO cell.As the prior art that one kind is more recommended, mammal expression system will lead to the glycosylation of antibody, the especially highly conserved N-terminal site in the area Fc.Stable clone is obtained with the people B7H3 antibody specifically bound by expressing.Positive being cloned in the serum free medium of bioreactor expands culture to produce antibody.The culture solution for having secreted antibody can be purified with routine techniques.For example, being purified with A the or G Sepharose FF column containing adjusted buffer.Wash away the component of non-specific binding.The antibody for using PH gradient method elution of bound again detects antibody fragment with SDS-PAGE, collects.Antibody can be filtered concentration with conventional method.Soluble mixture and polymer can also be removed with conventional method, such as molecular sieve, ion exchange.Obtained product need to freeze immediately, and such as -70 DEG C, or freeze-drying.
" giving " and " processing " refers to the contact of exogenous drugs, therapeutic agent, diagnosticum or composition with animal, people, subject, cell, tissue, organ or biofluid when being applied to animal, people, experimental subjects, cell, tissue, organ or biofluid." giving " and " processing " can refer to such as treatment, pharmacokinetics, diagnosis, research and experimental method.The processing of cell includes contact and reagent contact with fluid of the reagent with cell, wherein the fluid is contacted with cell." giving " and " processing " still means that through reagent, diagnosis, combining compositions or passes through another cells in vitro and ex vivo treatment such as cell." processing " refers to treatment processing, prevention or preventive measure, research and diagnostic application when being applied to people, animal medicine or study subject.
" treatment " means to give the interior or topical therapeutic agent of patient, such as the composition comprising any binding compounds of the invention, and the patient has one or more disease symptoms, and the known therapeutic agent has therapeutic effect to these symptoms.In general, therapeutic agent is given in subject or group with the amount that one or more disease symptoms are effectively relieved, to induce this kind of symptom to degenerate or inhibit this kind of symptom development to the degree of any right measurement of clinic.The amount (also referred to as " therapeutically effective amount ") that the therapeutic agent of any disease specific symptom is effectively relieved can change according to many factors, such as morbid state, age and the weight and drug of patient generate the ability for needing curative effect in patient.It is commonly evaluated for the seriousness of the symptom or any clinical testing procedure of development situation by doctor or other professional health care personages, whether evaluable disease symptoms have been mitigated.Although embodiment of the present invention (such as treatment method or product) may be invalid in terms of alleviating each target disease symptom, but examine (H inspection), Jonckheere-Terpstra inspection and Wilcoxon to examine and determine according to any statistical test method known in the art such as Student t inspection, Chi-square Test, according to the U inspection of Mann and Whitney, Kruskal-Wallis, target disease symptom should be mitigated in the patient of statistically significant number.
" conservative modification " or " conservative substitution or substitution " refer to the amino acid in other amino acid replacement albumen with similar characteristics (such as charge, side chain size, hydrophobicity/hydrophily, Conformation of the main chain and rigidity etc.), so that can biological activity of the frequent progress change without changing albumen.As known to those skilled in the art, in general, single amino acids displacement in the non-essential region of polypeptide not substantially changes biological activity (see, for example, Watson etc. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub.Co., page 224, (the 4th edition)).In addition, the unlikely broken ring biological activity of displacement of the similar amino acid of structure or function.
" effective quantity " includes to be enough to improve or the amount of the symptom of preventive medicine disease or illness.Effective quantity still means that the amount for being enough to allow or promoting diagnosis.It can change according to following factor for the effective quantity of particular patient or veterinary science subject: for example, illness to be treated, the general health of patient, the method and approach of administration and dosage and side effect seriousness.Effective quantity can be the maximum dose or dosage regimen for avoiding significant side effect or toxic effect.
" exogenous " refers to the substance according to circumstances generated outside biology, cell or human body." endogenous " refers to the substance according to circumstances generated in cell, biology or human body.
" homology " refers to the sequence similarity between two polynucleotide sequences or between two polypeptides.When the position in two comparison sequences is occupied by identical base or amino acid monomer subunit, for example, if when each position of two DNA moleculars is occupied by adenine, then the molecule is homologous in the position.Percent homology between two sequences is the function of the shared matching of two sequences or homologous position number divided by positional number × 100 compared.For example, if there are 6 matchings or homologous in 10 positions in two sequences, two sequences are 60% homologous in sequence optimal comparison；If there are 95 matchings or homologous in 100 positions in two sequences, two sequences are 95% homologous.In general, being compared when comparing two sequences and obtain maximum Percent homology.
Statement " cell ", " cell line " and " cell culture " used herein is used interchangeably, and all such titles all include offspring.Therefore, word " transformant " and " transformed cells " include primary subject cell and culture as derived from it, shift number without considering.It is to be further understood that all offsprings can not be accurate identical in terms of DNA content due to mutation deliberately or unintentionally.Including having the Mutant progeny with the identical function or biological activity screened in initial transformed cells.It is clearly visible by context in the case where meaning different names.
" polymerase chain reaction " used herein or " PCR " refer to nucleic acid, RNA and/or the DNA program or technology such as the amplification described in such as U.S. Patent number 4,683,195 of wherein micro specific part.It is generally desirable to obtain from target area end or except sequence information, allow to design Oligonucleolide primers；These primers are same or similar with the corresponding chain of template to be amplified in terms of sequence.5 ' terminal nucleotides of 2 primers can be consistent with the end of material to be amplified.PCR can be used for expanding specific RNA sequence, the specific dna sequence from total genomic dna and by the cDNA of total cell rna transcription, bacteriophage or plasmid sequence etc..Referring generally to Mullis etc. (1987) Cold Spring Harbor Symp.Ouant.Biol.51:263；Erlich is edited, (1989) PCR TECHNOLOGY (Stockton Press, N.Y.).PCR used herein is considered as an example of the nucleic acid polymerase reaction method for amplification of nucleic acid test sample, but is not unique example, and the method includes the known nucleic acids and nucleic acid polymerase used as primer, to expand or generate the specific part of nucleic acid.
" optional " or " optionally " mean ground described later event or environment can with but need not occur, which includes the event or the occasion that environment occurs or do not occur.For example, " optionally include 1-3 antibody heavy chain variable region " mean particular sequence antibody heavy chain variable region can with but necessarily exist.
" pharmaceutical composition " indicates the mixture containing one or more compounds described herein or its physiologically/pharmaceutical salt or pro-drug and other chemical constituents, the other components such as physiology/pharmaceutical carrier and excipient.The purpose of pharmaceutical composition is the administration promoted to organism, plays bioactivity in turn conducive to the absorption of active constituent.
Furthermore, diagnosticum the present invention relates to the method for the cell of B7H3 is expressed for immune detection or the method for measurement B7H3, for the reagent of immune detection or measurement B7H3, for immune detection or measurement and for diagnosing disease relevant to B7H3 positive cell, the monoclonal antibody or antibody fragment it includes specific recognition people B7H3 of the invention and in conjunction with the amino acid sequence of extracellular region or its three-dimensional structure are as active constituent.
In the present invention, the method for detecting or measuring the amount of B7H3 can be any known method.For example, it includes immune detection or measuring method.
Immune detection or measuring method are the methods using the antigen of label or antibody test or measurement amount of antibody or amount of antigen.The example of immune detection or measuring method includes immune antiboidy method (RIA), enzyme immunoassay (EIA or ELISA), fluorescence immunoassay (FIA), luminescence immunoassay, protein immunoblotting method, the physico-chemical process etc. that radioactive substance marks.
Above-mentioned disease relevant to B7H3 positive cell can be diagnosed by detecting or measuring the cell of expression B7H3 with monoclonal antibody of the invention or antibody fragment.
In order to detect the cell of expression polypeptide, known immunologic detection method can be used, and it is preferable to use immuno-precipitation, fluorecyte decoration method, immuning tissue's decoration methods etc..In addition it is possible to use utilizing the fluoresent antibody staining etc. of FMAT8100HTS system (Applied Biosystem).
In the present invention, the biopsy samples for detecting or measuring B7H3 are not particularly limited, as long as it includes such as histocyte, blood, blood plasma, serum, pancreatic juice, urine, excrement, tissue fluid or culture solution a possibility that expressing the cell of B7H3 that it, which has,.
According to required diagnostic method, the diagnosticum containing monoclonal antibody of the invention or its antibody fragment can also contain the reagent for executing antigen-antibody reaction or the reagent for detecting reaction.Reagent for executing antigen-antibody reaction includes buffer, salt etc..Reagent for detection includes the reagent commonly used in immune detection or measuring method, such as identifies the secondary antibody and substrate corresponding with the label etc. of the label of the monoclonal antibody, its antibody fragment or its conjugate.
Term " cytotoxic drug " refers to has the chemical molecular for destroying its normal growth more by force in tumour cell.Cytotoxic drug can kill tumour cell under sufficiently high concentration in principle, but due to lacking specificity, while killing tumor cell, also result in the apoptosis of normal cell, lead to serious side effect.The term is intended to include radioactive isotope (such as At 211、I 131、I 125、Y 90、Re 186、Re 188、Sm 153、Bi 212、P 32With the radioactive isotope of Lu), chemotherapeutics, the small molecule toxins or enzyme activity toxin of toxin such as bacterium, fungi, plant or animal origin, including its segment and/or variant.
Term " toxin " refers to from bacterium, fungi, the small molecule toxins and its derivative of plant or animal, including maytansinoid and its derivative (CN101573384) such as DM1, DM3, DM4, auristatin F (AF) and its derivative, such as MMAF, MMAE, 3024 (2016/127790 A1 of WO, compound 7), diphtheria toxin, exotoxin, ricin (ricin) A chain, abrin (abrin) A chain, modeccin, α-broom aspergillin (sarcin), tung oil tree (Aleutites fordii) toxalbumin, carnation (dianthin) toxalbumin, dyers' grapes (Phytolaca a Mericana) toxalbumin (PAPI, PAPII and PAP-S), balsam pear (Momordica charantia) mortifier, curcin (curcin), crotin (crotin), Saponaria officinalis (sapaonaria officinalis) mortifier, gelonin (gelonin), morphine (mitogellin) Restrictocin (restrictocin), phenomycin (phenomycin), enomycin (enomycin) and trichothecin (trichothecenes).
MMAF, MMAE are the auspicious statin derivative of Australia, and referring to US2005/0238649 and Doronina etc. (2006) Bioconjugate Chem.17:114-124, structural formula is as follows:
Specifically, the method recorded in US2005-0238649A1 and Doronina etc. (2006) Bioconjugate Chem.17:114-124 can be used to prepare in the auspicious statin of Australia/dolastatin drug moiety MMAF and its derivative.The method recorded in Doronina etc. (2003) Nat.Biotech.21:778-784 can be used to prepare in the auspicious statin of Australia/dolastatin drug moiety MMAE and its derivative.It can be readily synthesized agent-linker module MC-MMAF, MC-MMAE, MC-vc-PAB-MMAF and MC-vc-PAB-MMAE by conventional method, documented by (2003) Nat.Biotech.21:778-784 and U.S. Patent Application Publication No. US2005/0238649A1 such as such as Doronina, they are then coupled to interested antibody.
Heretofore described drug 2852, referring to 2016/127790 A1 of WO (COMPOUNDS EXAMPLE 7), structural formula is as follows:
The method that can be estimated by method described in the invention (COMPOUNDS EXAMPLE 8) or fields personnel, after synthetic drug-joint module (heretofore described 3024), structural formula is as follows:
Term " chemotherapeutics " is the chemical compound used in oncotherapy.Chemotherapeutics example includes alkylating agent, such as thio-tepa (thiotepa)；Cyclophosphamide (cyclosphamide) (CYTOXAN TM)；Alkyl sulfonic acid rouge such as busulfan (busulfan), Improsulfan (improsulfan) and piposulfan (piposulfan)；Aziridine (aziridine) such as benzo DOPA (benaodopa), carboquone (carboquone), Meturedepa (meturedopa) and uredepa (uredopa)；Aziridine and methylamelamine include hemel (altretamine), tretamine (triethylenemelamine), triethylenephosphoramide, triethylene thiophosphoramide and tri methylol melamine (trimethylolomelamine)；Mustargen (nitrogen mustards) such as Chlorambucil, Chlornaphazine, cholophosphamide (cholophosphamide), estramustine (estramustine), ifosfamide (ifosfamide), mustargen (mechlorethamine), mustron；Alkeran (melphalan), novoembichin (novembichin), phenesterine, prednimustine (prednimustine), Trofosfamide (trofosfamide), uracil mastard；Nitroso ureas (nitrosureas) such as Carmustine (carmustine), chlorozotocin (chlorozotocin), Fotemustine (fotemustine), lomustine (lomustine), Nimustine (nimustine), Ranimustine (ranimustine)；Antibiotic such as aclacinomycin, D actinomycin D, authramycin, azaserine, bleomycin, act-C (cactinomycin), Calicheamicin (calicheamicin), carabicin, carminomycin (chromomycin), carzinophillin (carzinophilin), chromomycin, actinomycin D, daunorubicin (daunorubicin), Detorubicin (detorubicin), 6- diazonium -5- oxygen-L- nor-leucine, adriamycin (doxorubicin), Epi-ADM (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin) It sends out wave mycin (marcellomycin), mitomycin, mycophenolic acid, nogalamycin (nogalamycin), olivomycin (olivomycin), Peplomycin (peplomycin), potfiromycin, puromycin, triferricdoxorubicin (quelamycin), rodorubicin (rodorubicin), streptonigrin；Streptozotocin (streptozocin), tubercidin, ubenimex (ubenimex), Zinostatin (zinostatin), zorubicin (zorubicin)；Antimetabolite such as methotrexate, 5 FU 5 fluorouracil (5-FU)；Folacin such as denopterin (denopterin), amethopterin, pteropterin, Trimetrexate (trimetrexate)；It talks endlessly and chants analog fludarabine (f1udarabine), 6- sulfydryl pterin, sulphur miaow pterin, sulphur guanopterin；Pyrimidine analogue such as ancitabine (ancitabine), azacitidine (azacitidine), 6- azauridine, Carmofur (carmofur), cytarabine, dideoxyuridine, remove fluorine oxygen uridine (doxitluridine), enocitabine (enocitabine), floxuridine, 5-FU；Androgens such as clausterone (calusterone), dromostanolone propionate (dromostanolong propionate), epithioandrostanol (epitiostanol), mepitiostane (mepitiostane), Testolactone (testolactone)；Anti- adrenal gland class such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), Trilostane (trilostane)；Folic acid supplement such as frolinic acid；Vinegar Portugal lactones；Aldophosphamideglycoside (aldophosphamideglycoside)；Amino-laevulic acid (aminolevulinic acid)；Amsacrine (amsacrine)；bestrabucil；Bisantrene (biasntrene)；Edatrexate (edatraxate)；defofamine；Demecolcine；Diaziquone (diaziquone)；elfomithine；Elliptinium Acetate (elliptinium acetate)；Ethoglucid (etoglucid)；Gallium nitrate；Hydroxycarbamide；Lentinan (lentinan)；Lonidamine (lonidamine)；Mitoguazone (mitoguazone)；Mitoxantrone (mitoxantrone)；Mopidamol (mopidamol)；Nitrafudan (nitracrine)；Pentostatin (pintostatin)；phenamet；Pirarubicin (pirarubicin)；Podophyllum emodi var chinense tree is sour (podophyllinic acid)；2- ethylhydrazide；Procarbazine (procarbazine)； Razoxane (razoxane)；Sizofiran (sizofiran)；Spirogermanium (spirogermanium)；Tenuazonic acid；Triethyleneiminobenzoquinone；2,2', 2 "-trichlorine diethylamine (trichlorrotriethylamine)；Urethane (urethan)；Vinblastine amide；Dacarbazine (dacarbazine)；Mannomustin；Dibromannitol (mitobronitol)；Mitolactol；Piperazine bromine alkane cheats (pipobroman)；gacytosine；Arabinoside (" Ara-C ")；Cyclophosphamide；Tespamin (thiotepa)；Taxane, as taxol ( Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel ( Rhone-Poulenc Rorer,Antony,France)；Chlorambucil；Gemcitabine (gemcitabine)；6-thioguanine；Purinethol；Amethopterin；Platinum analogs such as cis-platinum and carboplatin；Vinblastine；Platinum；Etoposide (etoposide) (VP-16)；Different ring phosphorus boat glue；Mitomycin C；Mitoxantrone；Vincristine；Vinorelbine (vinorelbine)；Neomycin (navelbine)；novantrone；Teniposide (teniposide)；Daunorubicin；Aminopterin；xeloda；She visits phosphate (ibandronate)；CPT-11；Topoisomerase enzyme inhibitor RFS2000；Difluoromethylornithine (DMFO)；Vitamin A acid esperamicins；capecitabine；And the officinal salt of above-mentioned any substance, acid or derivative.This definition further includes being adjustable or inhibitory hormone is to the anti-hormonal agents of the effect of tumour, if anti-estrogens include tamoxifen (tamoxifen), Raloxifene (raloxifene), aromatase inhibitor 4 (5)-imidazoles, 4-hydroxytamoxifen, Trioxifene (trioxifene), keoxifene, LY117018, onapristone and Toremifene (Fareston)；With his ammonia (flutamide) of antiandrogen preparation such as fluorine, Nilutamide (nilutamide), bicalutamide, Leuprorelin (leuprolide) and Goserelin (goserelin)；With the officinal salt of above-mentioned any substance, acid or derivative.
Term " connector unit " is L1 and L2 in the present invention, refers to the covalent linkage of one end and antibody and chemical structure segment or key that the other end is connected with cytotoxic drug.
Term " drugloading rate " refers to the cytotoxic drug par loaded on each ligand in molecule, it can also be expressed as the ratio of medication amount and amount of antibody, the range of drug carrying capacity can be each ligand (Pc) and connect 1-8 cytotoxic drug, in embodiments of the present invention, drugloading rate is expressed as y, conventional method such as UV/ Visible Spectroscopy, mass spectrum, the equal quantity of drug product of each ADC molecule after ELISA test and HPLC characterization coupling reaction can be used.
In the present invention, y may be limited by connection site quantity.In an embodiment of the invention, cytotoxic drug is coupled on the N-terminal amino of ligand and/or the epsilon-amino of lysine residue by connector unit, generally, can will be less than theoretic maximum value with the drug molecule number of antibody coupling in coupling reaction.
The carrying capacity of antibody cytotoxicity drug conjugates can be controlled with following non-limiting method, comprising:
(1) molar ratio of control connection reagent and monoclonal antibody,
(2) reaction time and temperature are controlled,
(3) different reaction reagents is selected.
Term " carrier " is used for drug of the invention, and drug can be changed by, which referring to, enters the mode of human body and the rate of release of distribution, control drug in vivo and the system for conducting drugs to target organs.Pharmaceutical carrier release and targeted system can reduce drug degradation and loss, reduce side effect, improve bioavilability.The high molecular surfactant of carrier such as be can be used as due to its unique amphipathic structure, self assembly can be carried out, form various forms of aggregations, preferred example such as micella, microemulsion, gel, liquid crystal, vesica etc..These aggregations have the ability for containing drug molecule, while having good permeability to film again, can be used as excellent pharmaceutical carrier.
Term " excipient " is the additives in pharmaceutical preparation in addition to main ingredient, alternatively referred to as auxiliary material.Such as binder, filler, disintegrating agent, the lubricant in tablet；Base portion in semi-solid preparation ointment, creme；Preservative, antioxidant, corrigent, aromatic, cosolvent, emulsifier, solubilizer, osmotic pressure regulator, colorant in liquid preparation etc. can be described as excipient.
Term " diluent " is also known as filler, is mainly used for increasing the weight and volume of tablet.The addition of diluent not only guarantees certain volume size, but also reduces the dose deviations of main component, improves the compact property etc. of drug.When the drug of tablet contains oily components, absorbent need to be added and absorb oiliness object, make to keep " drying " state, in favor of tablet is made.
Pharmaceutical composition can be sterile injectable aqueous form.Can there are water, ringer's solution and isotonic sodium chlorrde solution in the acceptable solvent and solvent used.Aseptic injection preparation can be the aseptic injection oil-in-water microemulsion that wherein active constituent is dissolved in oily phase.Can be by a large amount of injections in part, it will be in injection or the blood flow of micro emulsion injection patient.Alternatively, preferably giving solution and micro emulsion in the way of it can keep the compounds of this invention constant circulating concentration.To keep this constant density, continuous intravenous delivery device can be used.The example of this device is Deltec CADD-PLUS.TM.5400 type Iv pump.
Pharmaceutical composition can be for intramuscular and the aseptic injection water of subcutaneous administration or the form of oil suspension.Can be by known technology, the dispersing agent or wetting agent and suspending agent for being suitable for those described above prepare the suspension.Aseptic injection preparation is also possible to the aseptic injectable solution or suspension prepared in the acceptable diluent of nontoxic parenteral or solvent.Furthermore, it is convenient to use sterile fixed oil as solvent or suspension media.
The invention further relates to the methods of the relevant disease for the treatment of people B7H3 positive cell, the purposes especially in treating cancer and inflammation.
Two, embodiments and test case
The present invention is further described with reference to embodiments, but these embodiments and test case not limit the scope of the present invention.Test method without specific conditions in the embodiment of the present invention or test case, usually according to normal condition, such as the antibody technique laboratory manual of Cold SpringHarbor, molecular cloning handbook；Or according to condition proposed by raw material or commodity manufacturer.The reagent in specific source is not specified, for the conventional reagent of market purchase.
Embodiment 1.B7H3 antigen and the detection preparation of albumen
Using the leted others have a look at B7H3 of SEQ ID NO:1 as the template of B7H3 of the present invention, the amino acid sequence of antigen of the present invention and detection albumen is designed.The following non-specified otherwise of B7H3 antigen refers both to people B7H3.
1.1 people's B7H3 full length amino acid sequences: B7H3 (SEQ ID NO:1):
Annotation:
Double horizontal line parts are signal peptide (Signal peptide:1-28)；
Drawing horizontal line part is B7H3 extracellular region (Extracellular domain:29-466), and wherein 29-139 is 1 structural domain of Ig- sample v-shaped, and 145-238 be 1 structural domain of Ig- sample C2- type；243-357 is 2 structural domain of Ig- sample v-shaped, and 363-456 be 2 structural domain of Ig- sample C2- type；
Chain-dotted line part is transmembrane region part (Transmembrane domain:467-487)；
Italicized item is intracellular region (Cytoplasmic domain:488-534).
1.2 mouse B7H3 full length amino acid sequences (SEQ ID NO:2)
Annotation:
Double horizontal line parts are signal peptide (Signal peptide:1-28)；
Drawing horizontal line part is B7H3 extracellular region (Extracellular domain:29-248), wherein 29-139 be Ig- sample v-shaped structural domain, 145-238 be Ig- sample C2- type structural domain；
Chain-dotted line part is transmembrane region part (Transmembrane domain:249-269)；
Italicized item is intracellular region (Cytoplasmic domain:270-316).
1.3 screenings and detection employment B7H3 antigen (SEQ ID NO:3)
For commercially produced product (R&D cat#1949-B3-050/CF, abbreviation 2Ig-B7H3), sequence is as follows:
Annotation: drawing horizontal line part is B7H3 extracellular region；Italicized item is His-tag label.
1.4 detections employment B7H3 antigen (SEQ ID NO:4)
For commercially produced product (SinoBiological cat#11188-H08H, abbreviation 4Ig-B7H3), sequence is as follows:
Annotation: drawing horizontal line part is B7H3 extracellular region；Italicized item is His-tag label.
Mouse B7H3 antigen (SEQ ID NO:5) is used in 1.5 screenings and detection
For commercially produced product (R&D cat#1397-B3-050/CF), sequence is as follows:
Annotation: drawing horizontal line part is B7H3 extracellular region；Italicized item is His-tag label.
The preparation of the complete human antibody of embodiment 2.
The screening of 2.1 positive sequences
B cell is separated, and extracts RNA using human PBMC, spleen, lymph node tissue, constructs natural single-chain phage antibody library (storage capacity 3.2 × 10 10).By the natural single stranded phage library of building after packaging forms phage particle, carry out washing in a pan sieve using liquid phase method, bacteriophage is separated in conjunction with biotinylated B7H3 liquid phase, then using Streptavidin MagneSphere.The positive sequence combined can be intersected with people B7H3 (R&D cat#1949-B3-050/CF) and mouse B7H3 (R&D cat#1397-B3-050/CF) respectively to obtain, biotinylated people B7H3 and biotinylated mouse B7H3 is respectively adopted to carry out alternately washing in a pan sieve, the first run carries out washing in a pan sieve using the biotinylated people B7H3 of 2 μ g/ml, second wheel carries out washing in a pan sieve using the biotinylated mouse B7H3 of 2 μ g/ml, third round carries out washing in a pan sieve using the biotinylated people B7H3 of 0.5 μ g/ml, after three-wheel washes in a pan sieve, 500 monoclonals of picking are packaged into bacteriophage, it is tested for Phage-ELISA.Monoclonal phage and the combination activity of people B7H3 (R&D cat#1949-B3-050/CF) and mouse B7H3 (R&D cat#1397-B3-050/CF) are tested respectively: being coated with 1 μ g/ml people B7H3 or mouse B7H3 and 1%BSA on elisa plate respectively, the diluted bacteriophage supernatant of 1:1 Block buffer is added, is finally detected with anti-M13 HRP；The OD450 value that ELISA is tested is greater than 0.5, and the ELISA OD450 value of people and mouse B7H3 is combined to be sequenced divided by the clone that two ratios of the ELISA OD450 value for combining 1%BSA are all larger than 2.0, obtains 9 specific sequences.
The building of 2.2 intact monoclonal antibodies
It determines that wherein 2 antigen binding capacities are strong by ELISA Binding experiment after 9 specific sequences building complete antibody that phage library screens, is h1702 and h1703 respectively.It is as follows to the process of its intact monoclonal antibodies building:
According to the single chain antibody sequence that sequencing obtains, design primer PCR builds the VH/VK/VL genetic fragment of each single chain antibody sequence.Obtain the heavy light chain variable region of h1702 and h1703.
> h1702 weight chain variabl area sequence
SEQ ID NO:6
> h1702 light-chain variable sequence
SEQ ID NO:7
> h1703 weight chain variabl area sequence
SEQ ID NO:8
> h1703 light-chain variable sequence
SEQ ID NO:9
Note: being sequentially FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and italic is FR sequence in sequence, and underscore is CDR sequence.
Wherein CDR sequence is as shown in table 1 in each antibody light and weight chain.
Each heavy chain of table 1 and light chain CDR region sequence
Antibody variable region carries out homologous recombination with constant region gene (CH1-FC/CL) segment again, constructs complete antibody VH-CH1-FC/VK-CL/VL-CL.
Intact full length antibody h1702, h1703 sequence of building is as follows:
H1702:
H1702 heavy chain (IgG1) amino acid sequence: (SEQ ID NO:22)
H1702 light-chain amino acid sequence: Lamada (SEQ ID NO:23)
H1703:
H1703 heavy chain (IgG1) amino acid sequence: (SEQ ID NO:24)
H1703 light-chain amino acid sequence: Kappa (SEQ ID NO:25)
For the stability for further increasing antibody, amino acid mutation is carried out to the sequence of light chain of h1702, specifically sporting first amino acid residue Q of light chain (SEQ ID NO:23) N-terminal is replaced by D, first amino acid residue S of deletion mutation C-terminal, to obtain more stable and uniform monoclonal antibody h1702-DS.
The sequence of heavy chain of h1702-DS after mutation modification is SEQ ID NO:22, and light-chain amino acid sequence is as follows: (SEQ ID NO:26).
The expression and purification of 2.3 human antibodies
The plasmid of antibody light and weight chain being expressed respectively, HEK293E cell being transfected with the ratio of 1.5:1, expression supernatant is collected after 6 days, high speed centrifugation removes impurity, purified with Protein A column.Pillar is rinsed with PBS, until A280 reading is down to baseline.Destination protein is eluted with the acidic effluent liquid of pH3.0-pH3.5, is neutralized with 1M Tris-HCl, pH8.0-9.0.After elution samples are suitably concentrated, the gel chromatography Superdex200 (GE) balanced using PBS is further purified, and to remove aggressiveness, is collected monomer peak, is dispensed spare.
B7H3 antibody-drug conjugates prepare embodiment
The preparation of embodiment 3, source of mouse antibody
3.1 the preparation of antigen
Human B 7-H 3 (h-B7H3-Fc) sequence of coding-belt huFc label synthesizes (the above B7-H3 recombinant protein is by stencil plate sequence of the present invention) by Integrated DNA Technology (IDT) company, is cloned on pTT5 carrier (Biovector) respectively.The B7-H3 albumen of recombination is purified after the expression of 293T cell by the routine techniques of fields.The albumen of purifying can be used for mouse immune and obtain antibody.
The sequence of h-B7H3-Fc:
The preparation of 3.2 antibody
Anti-human B7H3 monoclonal antibody is generated by immune mouse.The experiment white mouse of Swiss Webster, female, 6 week old (Charles River company).Feeding environment: SPF grades.After mouse is bought, laboratory environment is raised 1 week, and light dark cycles are adjusted within 12/12 hour, and 20-25 DEG C of temperature；Humidity 40-60%.Immunizing antigen is the people B7H3 recombinant protein (huB7H3-Fc) with Fc label.It is adjuvant with Titermax (sigma Lot Num:T2684).Antigen and adjuvant (titermax) ratio are 1:1, are inoculated with after antigen emulsification, and the time is the 0th, 21,35,49,63 day.Antigen after the emulsification of 15 μ g+ palmulas (footpad) 25/ of (IP) injection in 0th day peritonaeum.21,35,49, antigen after the emulsification of 15 μ g+ palmulas (footpad) 15/ of (IP) injection in 63 days peritonaeums, preceding 3 days booster immunizations are merged carrying out splenocyte, (IP) injects the antigenic solution of the normal saline of 15 μ g+ palmulas (footpad) 15/ in peritonaeum.In progress blood examination in the 42nd, 56,70 day, mice serum is detected with ELISA and FACS method, determines the antibody titer in mice serum.After the 5th is immune, selects Serum Antibody titre high and titre tend to the mouse of platform and carries out splenocyte fusion, using optimization electro' asion step by splenic lymphocytes and myeloma cell Sp2/0 cell ( CRL-8287 TM) merged to obtain hybridoma.
Fused Hybridoma Cell Culture is after 7-14 days, take culture medium supernatant, use B7-H3 recombinant protein, ELISA experiment carries out antibody screening to hybridoma supematant, obtained positive antibody strain further uses the steady CHO-S cell for turning expression B7-H3, blank CHO-S cell is compared to exclude non-specific binding antibody hybridoma strain, is screened with flow-sorting methods, so that selected two plants combine recombinant protein and also in relation with the hybridoma of cell expression antigen.Logarithmic growth phase hybridoma is collected, extracts RNA and reverse transcription (PrimeScript with Trizol (Invitrogen, 15596-018) TMReverse Transcriptase, Takara#2680A).It is sequenced after the cDNA that reverse transcription obtains is carried out PCR amplification using mouse Ig-Primer Set (Novagen, TB326Rev.B 0503), finally obtains the sequence of source of mouse antibody m1704.
The heavy chain and light-chain variable sequence of mouse monoclonal antibody m1704 is as follows:
M1704 heavy chain
SEQ ID NO:28
M1704 light chain
SEQ ID NO:29
In order to improve the stability of antibody, the CDR sequence resisted to above-mentioned mouse is optimized.CDR region after optimization has following sequence:
Title Sequence Number
1704-HCDR1 RYGMS SEQ ID NO:30
1704-HCDR2 ISSGGGSIYYPDTVKG SEQ ID NO:31
1704-HCDR3 TRHYLLFEMDY SEQ ID NO:32
1704-LCDR1 KASQNVNTAVA SEQ ID NO:33
1704-LCDR2 SASNRYT SEQ ID NO:34
1704-LCDR3 QQYSSSLT SEQ ID NO:35
Heavy chain and light chain variable region that mouse resists are cloned to the pTT vector plasmid (Biovector) into heavy chain constant region containing human IgG1 and κ constant region of light chain respectively, then wink is transfected into HEK293 cell, the chimeric antibody of anti-B7-H3 is obtained, routinely technical method is spare after purification.
The humanization of 3.3 mouse antibodies
The method of many document publicities in source of mouse anti human B 7-H 3 monoclonal antibody humanization such as this field carries out.In short, user's constant domain substitutes parent (source of mouse antibody) constant domain, ethnic group antibody sequence is selected according to the homology of source of mouse antibody and human antibody, antibody m1704 is carried out humanization by the present invention.
On the basis of source of mouse antibody VH/VL CDR typical structure obtained, by weight, light-chain variable sequence compared with human antibody germline database, the high ethnic group system template of homology is obtained.Wherein human germline light chain framework region comes from human kappa light chain gene people germline light chain template IGkV1-33, and human germline heavy chain framework regions come from people's heavy chain template IGHV3-23
The CDR region of source of mouse antibody m1704 is transplanted in selected corresponding humanization template, replaces humanization variable region, then recombinate with IgG constant region (preferably heavy chain is IgG1, light chain κ).Then, based on the three-dimensional structure of source of mouse antibody, there is the residue of direct interaction to embedding residue, with CDR region, and back mutation is carried out to the residue that the conformation of VL and VH has a major impact, and CDR region chemically unstable amino acid residue is optimized, it designs and has detected the antibody being composed of following humanization weight chain variable region sequence.
H1704VH1 (SEQ ID NO:36):
H1704VH2 (SEQ ID NO:37):
H1704VL1 (SEQ ID NO:38):
H1704VL2 (SEQ ID NO:39):
  h1704VL1 h1704VL2
h1704VH1 h1704-1 h1704-3
h1704VH2 h1704-2 h1704-4
Through expression test and back mutation quantitative comparison, final humanization h1704-3 antibody molecule (using VH1 heavy chain variable region and VL2 light chain variable region) is selected, heavy chain and sequence of light chain are as shown in SEQ ID NO:40 and 41.
H1704-3 heavy chain of antibody (IgG1) sequence:
SEQ ID NO:40
H1704-3 antibody light chain (Kappa) sequence:
SEQ ID NO:41
CDNA segment is synthesized according to the gene order of above each humanized antibody light chain and heavy chain, is inserted into pcDNA3.1 expression vector (Life Technologies Cat.No.V790-20).By expression vector and transfection reagent PEI (Polysciences, Inc.Cat.No.23966) with the ratio transfected HEK 293 (Life Technologies Cat.No.11625019) of 1:2, it is placed in CO 2It is incubated for 4-5 days in incubator.After the antibody of expression is recovered by centrifugation, antibody purification is carried out, humanized antibody albumen of the invention is obtained.
B7H3 antibody-drug conjugates prepare embodiment
Following example 4 to embodiment 11 is the preparation process of correlation ADC of the invention.Wherein the antibody (h1702, h1703) into embodiment 7 of embodiment 4 is reacted by being coupled the drug (MMAF or 3024) with connection unit on its lysine, prepares ADC；Embodiment 8, by the sulfydryl on antibody (h1702, h1704-3, h1702DS) cysteine, preparation ADC is reacted with the drug (3024) with connection unit to embodiment 11.
The preparation of embodiment 4:B7H3-h1702-L2-MC-MMAF (h1702-MMAF)
By thioacetic acid S- (3- carbonyl propyl) ester (0.10mg, 0.75 μm of ol), it is dissolved in 0.2mL acetonitrile solution, it is spare；To antibody B7H3-h1702, the acetic acid of PH=4.3/sodium acetate buffer (2.5mg/ml, 2.0mL, the acetonitrile solution of above-mentioned prefabricated thioacetic acid S- (3- carbonyl propyl) ester 0.034umol) is added, then sodium cyanoborohydride (the 3.4mg of 0.1mL is added dropwise, 53 μm of ol) aqueous solution, oscillating reactions 2 hours at 25 DEG C.By reaction solution Sephadex G25 gel column desalting and purifying (elution phase: the PBS buffered aqueous solution for the 0.05M that pH is 6.5, EDTA containing 0.001M) it is purified, remove unreacted thioacetic acid S- (3- carbonyl propyl) ester and sodium cyanoborohydride, the PBS buffer solution (about 5.5mL) of title product 1b is obtained, ultrafiltration centrifugal concentrating to about 2.5ml carries out next step reaction.
Second step
The 2.0M hydroxylamine hydrochloride solution of 0.07mL is added into the PBS buffer solution (2.5mL) of 1b, finishes, is placed in water bath chader, oscillating reactions 30 minutes at 25 DEG C stop reaction.By reaction solution Sephadex G25 gel column desalting and purifying (elution phase: the PBS buffered aqueous solution for the 0.05M that pH is 6.5, EDTA containing 0.001M) it is purified, obtain title product B7H3-h1702 monoclonal antibody-propanethiol 1c PBS buffer solution (concentration 0.84mg/ml, 5.0mL).
Third step
By compound (S) -2- ((2R, 3R) -3- ((1S, 3S, 5S) -2- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (6- (2, 5- dicarbapentaborane -2, 5- dihydro -1H- pyrroles -1- base)-N- methyl hexanoyl amine) -3- methylbutyryl amine)-N, 3- amide dimethyl butyrate) -3- methoxyl group -5- methylheptanoyl base) -2- azabicyclo [3.1.0] hexane -3- base) -3- methoxyl group -2- methyl propanamide) -3- benzenpropanoic acid MC-MMAF (0.32mg, 0.34 μm of ol) it is dissolved in 0.5mL acetonitrile, B7H3-h1702 monoclonal antibody-propanethiol PBS buffer solution 1c (0.84mg/mL is added, 5.0 ML it in), is placed in water bath chader, stops reaction after oscillating reactions 4 hours at 25 DEG C.
By reaction solution Sephadex G25 gel column desalting and purifying (elution phase: the PBS buffered aqueous solution for the 0.05M that pH is 6.5, EDTA containing 0.001M), the PBS buffer solution (0.21mg/mL, 14.5mL) of title product h1702-MMAF is obtained, in 4 DEG C of stored frozens.
Q-TOF LC/MS calculates average value: y=1.76.
The preparation of embodiment 5:B7H3-h1703-L2-MC-MMAF (h1703-MMAF)
By thioacetic acid S- (3- carbonyl propyl) ester (0.11mg, 0.82 μm of ol), it is dissolved in 0.2mL acetonitrile solution, it is spare；To antibody B7H3-h1703, the acetic acid of PH=4.3/sodium acetate buffer (2.5mg/ml, 2.0mL, the acetonitrile solution of above-mentioned prefabricated thioacetic acid S- (3- carbonyl propyl) ester 0.034umol) is added, then sodium cyanoborohydride (the 3.4mg of 0.1mL is added dropwise, 53 μm of ol) aqueous solution, oscillating reactions 2 hours at 25 DEG C.By reaction solution Sephadex G25 gel column desalting and purifying (elution phase: the PBS buffered aqueous solution for the 0.05M that pH is 6.5, EDTA containing 0.001M) it is purified, remove unreacted thioacetic acid S- (3- carbonyl propyl) ester and sodium cyanoborohydride, the PBS buffer solution (about 5.5mL) of title product 2b is obtained, ultrafiltration centrifugal concentrating to about 2.5ml carries out next step reaction.
Second step
The 2.0M hydroxylamine hydrochloride solution of 0.07mL is added into the PBS buffer solution (2.5mL) of 2b, finishes, is placed in water bath chader, oscillating reactions 30 minutes at 25 DEG C stop reaction.By reaction solution Sephadex G25 gel column desalting and purifying (elution phase: the PBS buffered aqueous solution for the 0.05M that pH is 6.5, EDTA containing 0.001M) it is purified, obtain title product B7H3-h1703 monoclonal antibody-propanethiol 2c PBS buffer solution (concentration 0.82mg/ml, 5.0mL).
Third step
By compound (S) -2- ((2R, 3R) -3- ((1S, 3S, 5S) -2- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (6- (2, 5- dicarbapentaborane -2, 5- dihydro -1H- pyrroles -1- base)-N- methyl hexanoyl amine) -3- methylbutyryl amine)-N, 3- amide dimethyl butyrate) -3- methoxyl group -5- methylheptanoyl base) -2- azabicyclo [3.1.0] hexane -3- base) -3- methoxyl group -2- methyl propanamide) -3- benzenpropanoic acid MC-MMAF (0.32mg, 0.34 μm of ol) it is dissolved in 0.5mL acetonitrile, B7H3-h1703 monoclonal antibody-propanethiol PBS buffer solution 2c (0.82mg/mL is added, 5.0 ML it in), is placed in water bath chader, stops reaction after oscillating reactions 4 hours at 25 DEG C.
By reaction solution Sephadex G25 gel column desalting and purifying (elution phase: the PBS buffered aqueous solution for the 0.05M that pH is 6.5, EDTA containing 0.001M), the PBS buffer solution (0.20mg/mL, 13.0mL) of title product h1703-MMAF is obtained, in 4 DEG C of stored frozens.
Q-TOF LC/MS calculates average value: y=2.29.
The preparation of embodiment 6:B7H3-h1702-L2-3024 (h1702-3024)
By thioacetic acid S- (3- carbonyl propyl) ester (0.45mg, 3.38 μm of ol), it is dissolved in 0.8mL acetonitrile solution, it is spare；To antibody B7H3-h1702, the acetic acid of pH=4.3/sodium acetate buffer (5.0mg/ml, 8.0mL, 0.270 μm of ol) acetonitrile solution of above-mentioned prefabricated thioacetic acid S- (3- carbonyl propyl) ester is added, then sodium cyanoborohydride (the 12.8mg of 0.1mL is added dropwise, aqueous solution 0.2mmol), oscillating reactions 2 hours at 25 DEG C.By reaction solution Sephadex G25 gel column desalting and purifying (elution phase: the PBS buffered aqueous solution for the 0.05M that pH is 6.5, EDTA containing 0.001M) it is purified, remove unreacted thioacetic acid S- (3- carbonyl propyl) ester and sodium cyanoborohydride, the PBS buffer solution (about 14.5mL) of title product 3b is obtained, ultrafiltration centrifugal concentrating to about 7.5ml carries out next step reaction.
Second step
The 2.0M hydroxylamine hydrochloride solution of 0.20mL is added into the PBS buffer solution (7.5mL) of 3b, finishes, is placed in water bath chader, oscillating reactions 30 minutes at 25 DEG C stop reaction.By reaction solution Sephadex G25 gel column desalting and purifying (elution phase: the PBS buffered aqueous solution for the 0.05M that pH is 6.5, EDTA containing 0.001M) it is purified, obtain title product B7H3-h1702 monoclonal antibody-propanethiol 3c PBS buffer solution (concentration 2.84mg/ml, 14.0mL).
Third step
By compound (S) -2- ((2R, 3R) -3- ((1S, 3S, 5S) -2- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (6- (2, 5- dicarbapentaborane -2, 5- dihydro -1H- pyrroles -1- base)-N- methyl hexanoyl amine) -3- methylbutyryl amine)-N, 3- amide dimethyl butyrate) -3- methoxyl group -5- methylheptanoyl base) -2- azabicyclo [3.1.0] hexane -3- base) -3- methoxyl group -2- methyl propanamide) 3024 (2.6mg of -3- (2- fluorophenyl) phenylpropionic acid compound, 2.7 μm of ol) it is dissolved in 1.4mL acetonitrile, B7H3-h1702 monoclonal antibody-propanethiol PBS buffer solution 3c (2.84mg/mL is added , 14.0mL) in, it is placed in water bath chader, stops reaction after oscillating reactions 4 hours at 25 DEG C.
By reaction solution Sephadex G25 gel column desalting and purifying (elution phase: the PBS buffered aqueous solution for the 0.05M that pH is 6.5, EDTA containing 0.001M), the PBS buffer solution (1.35mg/mL, 27.5mL) of title product h1702-3024 is obtained, in 4 DEG C of stored frozens.
Q-TOF LC/MS calculates average value: y=2.05.
The preparation of embodiment 7:B7H3-h1703-L2-3024 (h1703-3024)
By thioacetic acid S- (3- carbonyl propyl) ester (0.50mg, 3.78 μm of ol), it is dissolved in 0.8mL acetonitrile solution, it is spare；To antibody B7H3-h1703, the acetic acid of pH=4.3/sodium acetate buffer (5.0mg/ml, 8.0mL, the acetonitrile solution of above-mentioned prefabricated thioacetic acid S- (3- carbonyl propyl) ester 0.270umol) is added, then sodium cyanoborohydride (the 12.8mg of 0.1mL is added dropwise, aqueous solution 0.2mmol), oscillating reactions 2 hours at 25 DEG C.By reaction solution Sephadex G25 gel column desalting and purifying (elution phase: the PBS buffered aqueous solution for the 0.05M that pH is 6.5, EDTA containing 0.001M) it is purified, remove unreacted thioacetic acid S- (3- carbonyl propyl) ester and sodium cyanoborohydride, the PBS buffer solution (about 14.0mL) of title product 4b is obtained, ultrafiltration centrifugal concentrating to about 7.5ml carries out next step reaction.
Second step
The 2.0M hydroxylamine hydrochloride solution of 0.20mL is added into the PBS buffer solution (7.5mL) of 4b, finishes, is placed in water bath chader, oscillating reactions 30 minutes at 25 DEG C stop reaction.By reaction solution Sephadex G25 gel column desalting and purifying (elution phase: the PBS buffered aqueous solution for the 0.05M that pH is 6.5, EDTA containing 0.001M) it is purified, obtain title product B7H3-h1703 monoclonal antibody-propanethiol 4c PBS buffer solution (concentration 2.64mg/ml, 14.5mL).
Third step
By compound (S) -2- ((2R, 3R) -3- ((1S, 3S, 5S) -2- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (6- (2, 5- dicarbapentaborane -2, 5- dihydro -1H- pyrroles -1- base)-N- methyl hexanoyl amine) -3- methylbutyryl amine)-N, 3- amide dimethyl butyrate) -3- methoxyl group -5- methylheptanoyl base) -2- azabicyclo [3.1.0] hexane -3- base) -3- methoxyl group -2- methyl propanamide) 3024 (2.6mg of -3- (2- fluorophenyl) phenylpropionic acid compound, 2.7 μm of ol) it is dissolved in 1.45mL acetonitrile, B7H3-h1703 monoclonal antibody-propanethiol PBS buffer solution 6c (2.64mg/m is added L, 14.5mL) in, it is placed in water bath chader, stops reaction after oscillating reactions 4 hours at 25 DEG C.
By reaction solution Sephadex G25 gel column desalting and purifying (elution phase: the PBS buffered aqueous solution for the 0.05M that pH is 6.5, EDTA containing 0.001M), the PBS buffer solution (1.20mg/mL, 28.0mL) of title product h1703-3024 is obtained, in 4 DEG C of stored frozens.
Q-TOF LC/MS calculates average value: y=1.62.
The preparation of embodiment 8.B7H3-h1702-cys-3024 (h1702-cys-3024)
Under the conditions of 37 DEG C, to antibody h1702, PBS buffered aqueous solution (the 10.0mg/ml of the 0.05M of pH=6.5,5.0mL, the 10mM aqueous solution 0.07mL (0.7umol) of three (2- carboxyethyl) phosphines (TCEP) of configured 0.01mM 0.333umol) is added, it is placed in water bath chader, oscillating reactions 3 hours at 37 DEG C stop reaction；
Reaction solution is cooled to 25 DEG C with water-bath, again by compound (S) -2- ((2R, 3R) -3- ((1S, 3S, 5S) -2- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (6- (2, 5- dicarbapentaborane -2, 5- dihydro -1H- pyrroles -1- base)-N- methyl hexanoyl amine) -3- methylbutyryl amine)-N, 3- amide dimethyl butyrate) -3- methoxyl group -5- methylheptanoyl base) -2- azabicyclo [3.1.0] hexane -3- base) -3- methoxyl group -2- methyl propanamide) -3- (2- fluorophenyl) propionic acid SHR3024 (2.6mg, 2.7 μm of ol) it is dissolved in 0.5mL acetonitrile, the antibody h1702 for being cooled to 25 DEG C is added to (containing TC EP in), it is placed in water bath chader, oscillating reactions 3 hours at 25 DEG C stop reaction；
By reaction solution Sephadex G25 gel column desalting and purifying (elution phase: the PBS buffered aqueous solution for the 0.05M that pH is 6.5, EDTA containing 0.001M), the PBS buffer solution (6.85mg/mL, 7.5mL) of title product is obtained, in 4 DEG C of stored frozens.
HIC-HPLC calculates average value: y=3.5.
The preparation of embodiment 9.B7H3-h1704-3-cys-3024 (h1704-3-cys-3024)
Under the conditions of 37 DEG C, to antibody B7H3-h1704-3, PBS buffered aqueous solution (the 10.0mg/ml of the 0.05M of PH=6.5,5.0mL, the 10mM aqueous solution 0.075mL (0.75umol) of three (2- carboxyethyl) phosphines (TCEP) of configured 0.01mM 0.333umol) is added, it is placed in water bath chader, oscillating reactions 3 hours at 37 DEG C stop reaction；
Reaction solution is cooled to 25 DEG C with water-bath, again by compound (S) -2- ((2R, 3R) -3- ((1S, 3S, 5S) -2- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (6- (2, 5- dicarbapentaborane -2, 5- dihydro -1H- pyrroles -1- base)-N- methyl hexanoyl amine) -3- methylbutyryl amine)-N, 3- amide dimethyl butyrate) -3- methoxyl group -5- methylheptanoyl base) -2- azabicyclo [3.1.0] hexane -3- base) -3- methoxyl group -2- methyl propanamide) 3024 (2.6mg of -3- (2- fluorophenyl) phenylpropionic acid compound, 2.7 μm of ol) it is dissolved in 0.5mL acetonitrile, it is added to and is cooled to 25 DEG C of antibody B7H3-h1704 In -3 (containing TCEP), it is placed in water bath chader, oscillating reactions 3 hours at 25 DEG C stop reaction；
By reaction solution Sephadex G25 gel column desalting and purifying (elution phase: the PBS buffered aqueous solution for the 0.05M that pH is 6.5, EDTA containing 0.001M), the PBS buffer solution (6.75mg/mL, 7.8mL) of title product is obtained, in 4 DEG C of stored frozens.
HIC-HPLC calculates average value: y=3.4.
The preparation of embodiment 10.B7H3-h1702DS-cys-3024 (h1702DS-cys-3024)
Under the conditions of 37 DEG C, to antibody H1702-DS, PBS buffered aqueous solution (the 10.0mg/ml of the 0.05M of pH=6.5,2.0mL, the 10mM aqueous solution 0.028mL (0.28umol) of three (2- carboxyethyl) phosphines (TCEP) of configured 0.01mM 0.133umol) is added, it is placed in water bath chader, oscillating reactions 3 hours at 37 DEG C stop reaction；
Reaction solution is cooled to 25 DEG C with water-bath, again by compound (S) -2- ((2R, 3R) -3- ((1S, 3S, 5S) -2- ((3R, 4S, 5S) -4- ((S) -2- ((S) -2- (6- (2, 5- dicarbapentaborane -2, 5- dihydro -1H- pyrroles -1- base)-N- methyl hexanoyl amine) -3- methylbutyryl amine)-N, 3- amide dimethyl butyrate) -3- methoxyl group -5- methylheptanoyl base) -2- azabicyclo [3.1.0] hexane -3- base) -3- methoxyl group -2- methyl propanamide) 3024 (1.02mg of -3- (2- fluorophenyl) phenylpropionic acid compound, 1.1 μm of ol) it is dissolved in 0.2mL acetonitrile, be added to be cooled to 25 DEG C antibody h1702-DS ( Contain TCEP) in, it is placed in water bath chader, oscillating reactions 3 hours at 25 DEG C stop reaction；
By reaction solution Sephadex G25 gel column desalting and purifying (elution phase: the PBS buffered aqueous solution for the 0.05M that pH is 6.5, EDTA containing 0.001M), the PBS buffer solution (6.65mg/mL, 2.7mL) of title product is obtained, in 4 DEG C of stored frozens.
HIC-HPLC calculates average value: y=3.55.
Antibody performance and beneficial effect of the present invention are verified with test method below.
Test case 1.ELISA Binding experiment
To detect the B7H3 antibody screened, and corresponding difference B7H3-ADC is for the external binding ability of people's different form B7H3, people 2Ig-B7H3 (Cat.#1949-B3-050/CF, R&D) and people 4Ig-B7H3 (Cat.#11188-H08H, Sino Biological) be used to carry out to combine detection in vitro.
With the PBS (Sigma of pH7.4, P4417-100TAB) people B7H3 albumen (2Ig/4Ig) is diluted to 1 μ g/ml concentration by buffer, 96 hole elisa Plates (Corning are added with the volume in 100 holes μ l/, CLS3590-100EA it in), is stood overnight in 4 DEG C 16-20 hours.It after discarding liquid, is added and uses PBST buffer (PH7.4PBS contains 0.05%Tween-20) diluted 5% skim milk (bright skimmed milk power) confining liquid, 120 hole μ l/, 37 DEG C of incubators, which are incubated for 2 hours, to be closed.After closing, discard confining liquid, and with the corresponding B7H3 antibody (or B7H3-ADC) that 100 μ l/ hole initial concentrations are 1 μM after PBST buffer board-washing 4 times, is added, with 8 gradients of PBST buffer doubling dilution, it is placed in 37 DEG C of incubators and is incubated for 1 hour.After incubation, discard the reaction solution in ELISA Plate, with PBST board-washing 4 times, secondary antibody (the Jackson Immuno Research of goat anti-human igg (Goat anti-Human IgG) Fc γ fragments specific of the HRP label of 100 μ l/ hole PBST dilution (1:4000) is added, 109-005-008), it is incubated for 1 hour for 37 DEG C.After PBST board-washing 4 times, 100 hole μ l/ TMB chromogenic substrates (KPL, 52-00-03) are added, in incubation at room temperature 3-5min, 100 hole μ l/ 1M H are added 2SO 4Reaction is terminated, reads absorption value at 450nm with NOVOStar microplate reader, calculating antibody the results are shown in Table 2 to the combination EC50 value of antigen.
The binding force of 2. different antibodies of table and antibody A DC and people's 2Ig-B7H3 and 4Ig-B7H3 antigen
EC50 People 2Ig-B7H3 (nM) People 4Ig-B7H3 (nM)
h1702 0.11 0.16
h1703 10.28 2.10
h1702-3024 0.17 0.26
h1703-3024 15.07 4.97
h1702-MMAF 0.16 0.18
h1703-MMAF 2.36 3.08
h1702-cys-3024   0.29
h1702DS-cys-3024   0.30
h1704-3-cys-3024   0.09
Conclusion: for people 2Ig-B7H3 and people 4Ig-B7H3, ADC with it is naked resist experimentally there is similar binding force in ELISA, i.e. ADC does not reduce binding force after marking.
Test case 2. intersects Binding experiment with different genera B7H3's
To detect the B7H3 antibody screened, and corresponding difference B7H3-ADC, for the external binding ability of the B7H3 in different genera source, mouse B7H3 (Cat.#1397-B3-050/CF, R&D) be used to carry out to combine detection in vitro.
With the PBS (Sigma of pH7.4, P4417-100TAB) different genera B7H3 albumen (mouse B7H3) is diluted to 1 μ g/ml concentration by buffer, it is added in 96 hole elisa Plates, is stood overnight in 4 DEG C 16-20 hours with the volume in 100 holes μ l/.It after discarding liquid, is added and uses PBST buffer (PH7.4PBS contains 0.05%Tween-20) diluted 5% skim milk (bright skimmed milk power) confining liquid, 120 hole μ l/, 37 DEG C of incubators, which are incubated for 2 hours, to be closed.After closing, discard confining liquid, and with the corresponding B7H3 antibody (or B7H3-ADC) that 100 μ l/ hole initial concentrations are 1 μM after PBST buffer board-washing 4 times, is added, with 8 gradients of PBST buffer doubling dilution, it is placed in 37 DEG C of incubators and is incubated for 1 hour.After incubation, discard the reaction solution in ELISA Plate, with PBST board-washing 4 times, the Goat anti-Human IgG of the HRP label of 100 μ l/ hole PBST dilution (1:4000) is added, secondary antibody (the Jackson Immuno Research of Fc γ fragments specific, 109-005-008), it is incubated for 1 hour for 37 DEG C.After PBST board-washing 4 times, 100 hole μ l/ TMB chromogenic substrates (KPL, 52-00-03) are added, in incubation at room temperature 3-5min, 100 hole μ l/ 1M H are added 2SO 4Reaction is terminated, reads absorption value, combination EC50 value (the results are shown in Table 3) of the calculating antibody to antigen at 450nm with NOVOStar microplate reader.
The binding force of 3. different antibodies of table and mouse B7H3 antigen
EC50 Mouse B7H3 (nM)
h1702 18.12
h1703 86.68
h1702-3024 130.8
h1703-3024 115.3
h1702-MMAF 58.95
h1703-MMAF 115.6
The results show that h1702 and h1703, and difference ADC and mouse B7H3 binding force it is relatively weak, show that two monoclonal antibodies have good people B7H3 binding specificity.
The experiment of test case 3.Biacore affinity of antibody
With Biacore, the anti-B7H3 antibody of GE Instrument measuring and anti-B7H3-ADC and people's 2Ig-B7H3 antigen, the affinity reaction between the various antigens of people's 4Ig-B7H3 antigen.
With bio-sensing chip Protein A (Cat.#29127556, GE) a certain amount of test antibodies of affinity capture/ADC to be measured, then a series of people's 2Ig-B7H3 antigen (Cat.#1949-B3-050/CF under concentration gradients is flowed through in chip surface, R&D), people 4Ig-B7H3 antigen (Cat.#11188-H08H, Sino Biological), using Biacore instrument (Biacore T200, GE) real-time detection reaction signal to obtaining association and dissociation curve.After the completion of the dissociation of each circulation, biochip is cleaned into regeneration with glycine-HCI actified solution (pH 1.5) (Cat.#BR-1003-54, GE).The buffer used in experiment is HBS-EP buffer solution (pH 7.4) (Cat.#BR-1001-88, GE).
Obtained data BIAevaluation version 4.1 is tested, GE software is fitted with (1:1) Langmuir model, to obtain affinity numerical value.Experimental result is shown in Table 4.
Affinity reaction (unit: M) between 4. different antibodies of table/ADC and various antigens
Antibody People 2Ig-B7H3 People 4Ig-B7H3
h1702 7.97E-7 8.55E-9
h1703 4.48E-7 -
h1702-MMAF 4.52E-7  
h1702-3024 5.80E-7 1.05E-8
h1703-MMAF 1.93E-7  
h1703-3024 2.19E-7 In conjunction with extremely weak
h1702-cys-3024   1.12E-8
h1702DS-cys-3024   1.06E-8
h1704-3-cys-3024   7.78E-10
Conclusion: show that ADC is similar to naked anti-affinity in the affinity test of Biacore experimentally for people 2Ig-B7H3, people 4Ig-B7H3.
4. cell in vitro Binding experiment of test case
This experiment evaluates the combination of antibody according to fluorescence signal power by the fluorescence signal of detection cell surface antibodies.By 10 μ g primary antibodies and 2 × 10 5A U87MG cell washes off extra antibody after being incubated on ice 30 minutes.By cell and APC anti-human igg Fc (Biolegend, 409306) in incubation at room temperature 30 minutes, the fluorescence signal (the result is shown in Figure 1) for using BD Verse to read cell surface after Excess antibody is washed off.
The result shows that: the B7H3 antigen of h1702 and cell surface has very strong binding ability.
The experiment of 5. cell in vitro endocytosis of test case
This experiment evaluates the endocytosis effect of antibody according to fluorescence signal power by detection intrabody or the fluorescence signal of difference ADC.B7H3 antibody/ADC and APC anti-human igg Fc (Biolegend, 409306) is mixed with the molar ratio of 1:2 and is incubated for 15 minutes on ice.By antibody mixture and 2 × 10 5A U87MG cell washes off extra antibody after being incubated on ice 30 minutes, is then transferred to cell in 37 DEG C of pre-temperature of culture medium, is incubated for respectively at 37 DEG C 0,15,30,60 and 120 minute.Centrifuge cell and cell is resuspended in antibody elution liquid is incubated at room temperature 7 minutes, washes off antibody elution liquid, is read intracellular Fluorescence signal using BD Verse, is seen Fig. 2, Fig. 3, Fig. 4.As a result endocytosis effect of the visible ADC on U87MG cell resists quite with naked.
Test case 6.SD rat T1/2 evaluation
SD rat 4, half male and half female, light dark is adjusted within 12/12 hour, 24 ± 3 DEG C of constant temperature of temperature, humidity 50-60%, ad lib drinking-water.Purchased from Jie Sijie experimental animal Co., Ltd.Experimental day distinguishes tail vein injection test medicine B7H3 antibody/ADC, dosage 3mg/kg, volume injected 5ml/kg to SD rat.
Take blood time point are as follows: 5min, 8h, for 24 hours (the 2nd day) after administration in the 1st day the 3rd day, the 5th day, the 8th day, the 11st day, the 15th day, take blood in rat eyeground vein, every time 200 μ L (be equivalent to and take 100 μ L of serum)；The blood sample of collection puts half an hour to being aggregated at room temperature, and then 10000 × g is centrifuged 10 minutes at 4 DEG C.Supernatant is collected, places -80 DEG C of storages immediately.
With the B7H3 antibody concentration in ELISA detection serum, PK analysis is carried out, the results are shown in Table 5.
T of the table 5.B7H3 antibody in SD rat 1/2
Test medicine Administration mode T 1/2(average value ± SD, h)
h1702 IV(3mg/kg) 185±17
The result shows that h1702 of the present invention is about (7.7 days) 185h in rat intracorporal half-life period.
With the concentration of the B7H3 antibody A DC in ELISA detection serum, PK analysis is carried out, the results are shown in Table 6
T1/2 of the table 6.B7H3 antibody coupling matter in SD rat
Test medicine Analyte Administration mode T 1/2(average value ± SD, h)
h1702-3024 Total ADC (total ADC) IV(3mg/kg) 97±15
h1702DS-cys-3024 Total ADC (total ADC) IV(3mg/kg) 98.20±10.16
h1704-3-cys-3024 Total ADC (total ADC) IV(3mg/kg) 65.20±4.18
The result shows that, rat vein gives 3mg/kg Subject antibodies conjugate h1702-3024, h1702DS-cys-3024, after h1704-3-cys-3024, total ADC (total ADC) is about (4.04 days) 97h in rat intracorporal half-life period, 98h (4.08 days), 65h (2.71 days).
The physical stability of test case 7.B7H3 antibody
Using the thermal stability of DSC detection different antibodies, the thermal stability situation under different buffer system condition of different pH is compared, different pH corresponding exemplary buffer system such as 10mM PB (pH7), 10mM Acetate (pH5.2).By sample displacement into corresponding buffer, sample concentration is controlled in 1mg/ml or so, is detected using MicroCal*VP-Capillary DSC (Malvern).Before detection, by each sample and plain buffer 1~2min of vacuum degasifer degassing.400 μ l samples or plain buffer is added in each hole of sample panel (instrument applied sample amount is 300 μ l).Last two pairs of orifice plates are separately added into 14%Decon 90 and ddH 2O after sample panel is loaded, puts on the soft cover board of plastics in case cleaning is used.Scanning temperature terminates since 25 DEG C to 100 DEG C, 60 DEG C/h of sweep speed.Concrete outcome is as shown in table 7, and h1702, h1703 are demonstrated by preferable thermal stability in several test systems.
The DSC experimental result of 7. different antibodies of table
Sample purity, which is monitored, by SEC-HPLC investigates a certain concentration condition periodical stability, illustrative condition such as controls sample concentration compares the steadiness that different antibodies save one month in such as -80 DEG C of multigelations and 4 DEG C, 30 DEG C, 40 DEG C in about 40-50mg/ml in PBS (pH7.4) system and pH5.2 acetic acid/sucrose system.Antibody purity is detected using Xbridge protein BEH SEC 200A (Waters) HPLC column, by investigation in one month, h1702, h1703 were demonstrated by good stability, and the results are shown in Table 8.
The stability of period result of 8. different antibodies of table
Sample 4 DEG C/month/purity 30 DEG C/month/purity 40 DEG C/month/purity
H1702/ acetic acid 99.25% 98.68% 97.85%
h1702/PBS 99.21% 98.07% 96.34%
H1703/ acetic acid 99.31% 99.04% 98.38%
h1703/PBS 99.18% 98.56% 96.99%
The results show that h1702 and h1703 all shows good stability of period in acetic acid and PBS buffer solution.
The chemical stability of test case 8.B7H3 antibody
Chemical modification is to lead to one of FAQs of product stability after Antibody preparation, and the especially partial amino-acid height deamidation in CDR region domain, oxidation or isomerization modification is typically chosen and avoids or be mutated reduction as far as possible.500 μ g test antibodies are taken to be dissolved in the PBS of 500 μ l pH 7.4,40 DEG C of water-baths；It was sampled respectively at 0,10,20 day, for digesting experiment.The sample that 100 μ g different time points sample is dissolved in 100 μ l 0.2M His-HCl, 8M G μ a-HCl, in 6.0 solution of pH, add 3 μ l 0.1g/mL DTT, 50 DEG C water-bath 1 hour, afterwards use 0.02M His-HCl, the solution ultrafiltration of pH 6.0 is twice, the trypsase (trypsin) of 3 μ L 0.25mg/mL is added, 37 DEG C of water enzyme digestions are stayed overnight.Agilent 6530Q-TOF carries out LC-MS detection and analysis, potential decorating site is analyzed by mass spectrometry and (the results are shown in Table 9), h1702, h1703 involved in the present invention aggravate trend without apparent deamidation, oxidation or isomerization as the result is shown, prompt the good physical and chemical stability of molecule.
The chemical stability of 9. different antibodies of table
Test case 9: cell proliferation experiment
This experiment evaluates B7H3-ADC to the inhibitory effect of U87MG cell and ZR-75-1 cell Proliferation by detection intracellular ATP content, according to IC50 size.
1, to the inhibitory effect of U87MG cell Proliferation
U87MG cell (Chinese Academy of Sciences's cell bank, Catalog#TCHu138) is cultivated in the EMEM culture medium containing 10%FBS, is passed on 2~3 times within one week, and passage is than column 1:2 or 1:5.When passage, culture medium is sopped up, cellular layer is rinsed with the pancreatin of 5ml 0.25%, then sops up pancreatin, cell is put and is digested 3~5 minutes in the incubator, fresh culture is added, cell is resuspended.The cell suspension of 90 μ L is added in 96 porocyte culture plates, density is 4 × 10 4Cell/ml, culture medium are the DMEM of 10%FBS, and the DMEM culture medium of 100 μ l 10%FBS is only added in 96 orifice plates periphery.By culture plate incubator culture 24 hours (37 DEG C, 5%CO 2)。
Sample to be tested is diluted to 50mM with PBS or DMSO, and is successively diluted to 10 concentration with 3 times, is arranged to the hole of blank and control.The compound solution for taking 10 μ l to be configured to gradient concentration is added in 90 μ l fresh cultures.The culture medium solution of the 10 above-mentioned drug containing of μ l is added into culture plate again.By culture plate incubator be incubated for 3 days (37 DEG C, 5%CO 2).In 96 porocyte culture plates, 100 μ l CellTiter-Glo reagents are added in every hole, and room temperature avoid light place 5-10min reads chemiluminescence signal value in Victor3, and data are handled using GraphPad software.The IC50 value measured is shown in Table 10.
Proliferation experiment result of the 10. difference ADC of table on U87MG cell
Compound IC50(nM) Maximum suppression (%)
IgG >500 -0.7
h1702 >500 3.9
h1703 >500 2.4
2852 298.4 50.9
h1702-3024 14.63 49.4
h1703-3024 22.58 54.4
MMAF 1477 55.9
h1702-MMAF 20.09 46.4
h1703-MMAF 37.29 55.8
Conclusion: the ADC that naked anti-coupling toxin obtains has good lethal effect on U87MG cell.
2, to the inhibitory effect of ZR-75-1 cell Proliferation
ZR-75-1 cell (ATCC, Catalog#CRL-1500) is cultivated in the RPMI-1640 culture medium containing 10%FBS, is passed on 2~3 times within one week, and passage is than column 1:4 or 1:6.When passage, culture medium is sopped up, cellular layer is rinsed with the pancreatin of 5mL0.25%, then sops up pancreatin, cell is put and is digested 3~5 minutes in the incubator, fresh culture is added, cell is resuspended.The cell suspension of 90 μ L is added in 96 porocyte culture plates, density is 2.8 × 10 4The RPMI-1640 culture medium of 100 μ L 10%FBS is only added in cell/mL, 96 orifice plates periphery.By culture plate incubator culture 24 hours (37 DEG C, 5%CO 2)。
Sample to be tested is diluted to 50mM with PBS or DMSO, and is successively diluted to 10 concentration with 3 times, is arranged to the hole of blank and control.The compound solution for taking 10 μ l to be configured to gradient concentration is added in 90 μ l fresh cultures.The culture medium solution of the 10 above-mentioned drug containing of μ l is added into culture plate again.By culture plate incubator be incubated for 6 days (37 DEG C, 5%CO 2).In 96 porocyte culture plates, 100 μ L CellTiter-Glo reagents are added in every hole, and room temperature avoid light place 5-10min reads chemiluminescence signal value in Victor3, and data are handled using GraphPad software.The IC50 value measured is shown in Table 11.
Proliferation experiment result of the 11. difference ADC of table on ZR-75-1 cell
Conclusion: the ADC that naked anti-coupling toxin obtains has good lethal effect on ZR-75-1 cell.
3, to the inhibitory effect of 562 cell Proliferation of Detroit
562 cell of Detroit (ATCC, Catalog#CCL-138) is cultivated in the EMEM culture medium containing 10%FBS, is passed on 2~3 times within one week, and passage is than column 1:4 or 1:6.When passage, culture medium is sopped up, cellular layer is rinsed with the pancreatin of 5mL 0.25%, then sops up pancreatin, cell is put and is digested 3~5 minutes in the incubator, fresh culture is added, cell is resuspended.The cell suspension of 90 μ L is added in 96 porocyte culture plates, density is 2.2 × 10 4The EMEM culture medium of 100 μ L 10%FBS is only added in cell/mL, 96 orifice plates periphery.By culture plate incubator culture 24 hours (37 DEG C, 5%CO 2)。
Sample to be tested is diluted to 50mM with PBS or DMSO, and is successively diluted to 10 concentration with 3 times, is arranged to the hole of blank and control.The compound solution for taking 10 μ l to be configured to gradient concentration is added in 90 μ l fresh cultures.The culture medium solution of the 10 above-mentioned drug containing of μ l is added into culture plate again.By culture plate incubator be incubated for 6 days (37 DEG C, 5%CO 2).In 96 porocyte culture plates, 100 μ L CellTiter-Glo reagents are added in every hole, and room temperature avoid light place 5-10min reads chemiluminescence signal value in Victor3, and data are handled using GraphPad software.The IC50 value measured is shown in Table 12.
Proliferation experiment result of the 12. difference ADC of table on 562 cell of Detroit
Compound IC50(nM) Maximum suppression (%)
IgG >500 6.4
h1702DS >500 4.55
2852 133.8 87.99
h1702DS-3024 20.78 85.92
Conclusion: the ADC that naked anti-coupling toxin obtains has good lethal effect on 562 cell of Detroit.
4, in the above way to h1702DS, h1704-3 antibody and its corresponding ADC:h1702-cys-3024, h1702DS-cys-3024, h1704-3-cys-3024 tests the proliferation inhibiting effect to 562 cell of U87MG, ZR-75-1 and Detroit, as a result such as the following table 13:
Conclusion: the ADC that naked anti-coupling toxin obtains has good lethal effect on U87MG, ZR-75-1 and Detroit562 cell.
Activity in vivo biological assessment
Therapeutic evaluation of the test case 10:ADC to human brain astrocytes' blastoma U87MG Nude Mice
One, test method
BALB/cA-nude nude mouse is used in experiment, and female 6-7 weeks, is purchased from the western Poole Bi Kai experimental animal Co., Ltd in Shanghai (quality certification number: SCXK (Shanghai) 2008-0016).Feeding environment: SPF grades.Nude mouse inoculates human brain astrocytes' blastoma U87MG cell (Chinese Academy of Sciences, article No. TCHu138), the tenth day (tumor average volume 122mm after inoculating cell 3), animal is grouped (D0) at random, every group 8, starts 1 times/week of intraperitoneal injection, is administered altogether 3 times, 2-3 knurl product and weight is surveyed weekly, records data.Gross tumor volume (V) calculation formula are as follows:
V=1/2 × a × b2
Wherein a, b respectively indicate length and width.
Relative volume (RTV)=VT/V0
Tumour inhibiting rate (%)=(CRTV-TRTV)/CRTV (%)
Wherein V0, VT are respectively the gross tumor volume tested when starting and at the end of experiment.CRTV, TRTV are respectively the relative tumour volume of the control group (blank) and experimental group at the end of testing.
Two, test object
h1702-3024 ADC(1mpk,3mpk,10mpk)；
h1702DS-cys-3024 ADC(1mpk,3mpk)；
h1704-3-cys-3024 ADC(1mpk,3mpk)；
Blank group (blank): the PBS buffer solution of PH7.4
Three, the tumor killing effect of antibody A DC
1) when the 26th day (D26) after observation to administration starting, the tumour inhibiting rate of Subject antibodies h1702-3024 ADC (1mpk, 3mpk, 10mpk) is respectively 33.68%, 99.13%, 100%；See Fig. 5, there were significant differences compared with the control group.When being administered the 21st day, for control group since tumour is excessive, individual mouse weight loss are obvious, and when being administered the 26th day, control group has two mouse because of the too big death of tumour.The equal Normal-weight of other each groups, is shown in Fig. 6, table 14, does not occur dead mouse in administration process, and each dosage of h1702-3024 is prompted not have obvious toxic-side effects.
There is certain dose-dependence between each dosage of h1702-3024,95% or more tumour inhibiting rate is had reached when 3mpk, 10mpk reaches 100% tumour inhibiting rate.
Curative effect of the 14. administered antibody h1702-3024 of table to tumor bearing nude mice U87MG transplantable tumor
Vs blank: p < 0.001 * p < 0.05, * * p < 0.01, * * *
2) 15 are shown in Table to Subject antibodies h1702DS-cys-3024 ADC, h1704-3-cys-3024 ADC tumor killing effect experimental result.The results show that 1 times/week of intraperitoneal injection, it is administered 3 times altogether, when observation is to D26, the inhibitory rate of tested ADC h1702DS-cys-3024 (1mpk) to 22.99%；The inhibitory rate of h1702DS-cys-3024 (3mpk) is to 98.16%；The inhibitory rate of h1704-3-cys-3024 (1mpk) is to 31.05%；The inhibitory rate of h1704-3-cys-3024 (3mpk) is to 94.83%；For 3 Subject antibodies groups of 1mpk compared to the blank group all without significant difference (P > 0.05), 3 Subject antibodies groups of 3mpk have extremely significant difference (P < 0.001) compared to the blank group.
Groups of animals Normal-weight in administration process prompts ADC without obvious toxic-side effects.
Blank group and 1mpk each group gross tumor volume at 26 days are larger, therefore put to death blank and 1mpk each group when D26, and when remaining group observation is to D36, tumour growth is still relatively slow after h1702DS-cys-3024 (3mpk) group is discontinued, and tumor killing effect is best.The tumour inhibiting rate comparison among groups no significant difference (P > 0.05) of h1702DS-cys-3024 (3mpk) and h1704-3-cys-3024 (3mpk) in D26 and D36.
Curative effect (D26) of 15. administered antibody of table to tumor bearing nude mice U87MG transplantable tumor
Vs blank: p < 0.001 * p < 0.05, * * p < 0.01, * * *
Therapeutic evaluation of the test case 11:ADC to 562 Nude Mice of people's pharynx cancer hydrothorax metastatic cells Detroit
One, test method
BALB/cA-nude nude mouse is used in experiment, and female 6-7 weeks, inoculates 562 cell of people's pharynx cancer hydrothorax metastatic cells Detroit.The tenth day after inoculating cell, animal is grouped (D0) at random, every group 8, starts 1 times/week of intraperitoneal injection, be administered altogether 3 times, surveyed 2-3 knurl product and weight weekly, record data.Gross tumor volume (V) calculation formula are as follows:
V=1/2 × a × b2
Wherein a, b respectively indicate length and width.
Relative volume (RTV)=VT/V0
Tumour inhibiting rate (%)=(CRTV-TRTV)/CRTV (%)
Wherein V0, VT are respectively the gross tumor volume tested when starting and at the end of experiment.CRTV, TRTV are respectively the relative tumour volume of the control group (blank) and experimental group at the end of testing.
Two, test object
h1702DS-cys-3024 ADC(1mpk,3mpk)；
Blank group (blank): the PBS buffer solution of PH7.4
Three, the tumor killing effect of antibody A DC
When observation is to D35, Subject antibodies ADC tumour inhibiting rate is respectively: the inhibitory rate of h1702DS-cys-3024 ADC (1mpk) to 39.22% (P < 0.05)；The inhibitory rate of h1702DS-cys-3024 ADC (3mpk) is to 70.50% (P < 0.001) (being shown in Table 16).Groups of animals Normal-weight during medicine.
Curative effect of 16. administered antibody of table to 562 transplantable tumor of tumor bearing nude mice Detroit
Vs blank: p < 0.001 * p < 0.05, * * *
Estimation of stability
The physical stability of test case 12:B7H3 ADC
Using the thermal stability of DSC detection different antibodies ADC, the thermal stability situation under different buffer system condition of different pH is compared, different pH corresponding exemplary buffer system such as 10mM PBS (pH7.4), 10mM Acetate (pH5.5).By sample displacement into corresponding buffer, sample concentration is controlled in 1mg/ml or so, is detected using MicroCal*VP-Capillary DSC (Malvern).Before detection, by each sample and plain buffer 1~2min of vacuum degasifer degassing.400 μ l samples or plain buffer is added in each hole of sample panel (instrument applied sample amount is 300 μ l).Last two pairs of orifice plates are separately added into 14%Decon 90 and ddH 2O after sample panel is loaded, puts on the soft cover board of plastics in case cleaning is used.Scanning temperature terminates since 25 DEG C to 100 DEG C, 60 DEG C/h of sweep speed.Concrete outcome is as shown in table 17, and h1702-3024, h1703-3024 are demonstrated by preferable thermal stability in several test systems.
The DSC experimental result of 17. difference ADC of table
The stability of test case 13:ADC
Sample (h1702DS-cys-3024 ADC is monitored by SEC-HPLC, h1704-3-cys-3024 ADC) purity investigates a certain concentration condition stability inferior, illustrative condition such as controls sample concentration in about 50mg/ml, and relatively different antibodies save 28 days steadiness at 40 DEG C in PBS (pH7.4) system and pH5.5 acetic acid/sucrose system (generation claims 559 systems).Antibody purity is detected using Xbridge protein BEH SEC 200A (Waters) HPLC column, by investigation in 28 days, h1702DS-cys-3024 ADC was demonstrated by good stability, as a result as shown in Figure 7.
The results show that h1702DS-cys-3024 all shows good stability in acetic acid and PBS buffer solution.
Although foregoing invention is described in detail by means of attached drawing and example in order to be clearly understood from, description and example are not construed as limiting the scope of the invention.The disclosure of all patents referred to herein and scientific literature is completely clearly combined by reference.

Claims (22)

  1. Antibody-drug conjugates shown in a kind of general formula (A),
    Ab-(L 2-L 1-D) y         (A)
    Wherein:
    D is cytotoxic drug；
    L 1, L 2It is connector unit；
    Y is the number selected from 1-8, is preferably selected from the number of 2-4；
    Ab is B7H3 antibody or its antigen-binding fragment, and in conjunction with people B7H3, the B7H3 antibody or its antigen-binding fragment are the monoclonal antibody or its antigen-binding fragment any into (c) selected from following (a):
    (a) monoclonal antibody has the amino acid sequence of at least 95% sequence identity it includes one or more CDR region sequences selected from the following or with it:
    Antibody heavy chain variable region HCDR region sequence: as shown in the amino acid sequence of SEQ ID NO:10,11 and 12；With antibody's light chain variable region LCDR region sequence: as shown in SEQ ID NO:13,14 and fifteen amino acid sequence；
    (b) monoclonal antibody has the amino acid sequence of at least 95% sequence identity it includes one or more CDR region sequences selected from the following or with it:
    Antibody heavy chain variable region HCDR region sequence: as shown in the amino acid sequence of SEQ ID NO:16,17 and 18；With antibody's light chain variable region LCDR region sequence: as shown in the amino acid sequence of SEQ ID NO:19,20 and 21；
    (c) monoclonal antibody has the amino acid sequence of at least 95% sequence identity it includes one or more CDR region sequences selected from the following or with it:
    Antibody heavy chain variable region HCDR region sequence: as shown in the amino acid sequence of SEQ ID NO:30,31 and 32；With antibody's light chain variable region LCDR region sequence: as shown in the amino acid sequence of SEQ ID NO:33,34 and 35.
  2. Antibody-drug conjugates shown in a kind of general formula (A),
    Ab-(L 2-L 1-D) y         (A)
    Wherein:
    D is cytotoxic drug；
    L1, L2 are connector units；
    Y is the number selected from 1-8, is preferably selected from the number of 2-4；
    Ab is the monoclonal antibody or its antigen-binding fragment with B7H3 antibody or its antigen-binding fragment competitive binding people B7H3 described in claim 1.
  3. Antibody-drug conjugates as claimed in claim 1 or 2, wherein the Ab is recombinant antibodies.
  4. Antibody-drug conjugates as claimed in claim 3, wherein the constant region of the Ab and/or framework region are derived from the recombinant antibodies or its antigen-binding fragment of people.
  5. Antibody-drug conjugates as claimed in claim 4, wherein the area light chain FR and heavy chain FR region sequence on the light chain and heavy chain variable region of the Ab are respectively derived from people's germline light chain and sequence of heavy chain or its mutant nucleotide sequence；Wherein the constant region includes the heavy chain constant region from humanized IgG 1, IgG2, IgG3 or IgG4 or its variant, preferably 1 heavy chain constant region of humanized IgG；With the constant region of light chain from source of people κ, λ chain or its variant.
  6. Antibody-drug conjugates as claimed in claim 5, wherein the Ab contains SEQ ID NO:6, SEQ ID NO:8, heavy chain variable region or its variant shown in SEQ ID NO:36 or SEQ ID NO:37；The variant is the sequence on the weight chain variabl area sequence shown in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:36 or SEQ ID NO:37 with 1-10 amino acid substitution；
    With contain SEQ ID NO:7, SEQ ID NO:9, light chain variable region or its variant shown in SEQ ID NO:38 or SEQ ID NO:39；The variant is the sequence on the light-chain variable sequence shown in SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:38 or SEQ ID NO:39 with 1-10 amino acid substitution.
  7. Antibody-drug conjugates as claimed in claim 5, wherein the Ab contains selected from following (1) monoclonal antibody any into (7) or its antigen-binding fragment:
    (1) monoclonal antibody, it includes antibody heavy chain variable regions shown in SEQ ID NO:6；With antibody's light chain variable region shown in SEQ ID NO:7；
    (2) monoclonal antibody, it includes antibody heavy chain variable regions shown in SEQ ID NO:8；With antibody's light chain variable region shown in SEQ ID NO:9；
    (3) monoclonal antibody, it includes antibody heavy chain variable regions shown in SEQ ID NO:28；With antibody's light chain variable region shown in SEQ ID NO:29；
    (4) monoclonal antibody, it includes antibody heavy chain variable regions shown in SEQ ID NO:36；With antibody's light chain variable region shown in SEQ ID NO:38；
    (5) monoclonal antibody, it includes antibody heavy chain variable regions shown in SEQ ID NO:36；With antibody's light chain variable region shown in SEQ ID NO:39；
    (6) monoclonal antibody, it includes antibody heavy chain variable regions shown in SEQ ID NO:37；With antibody's light chain variable region shown in SEQ ID NO:38；
    (7) monoclonal antibody, it includes antibody heavy chain variable regions shown in SEQ ID NO:37；With antibody's light chain variable region shown in SEQ ID NO:39.
  8. Such as antibody-drug conjugates of any of claims 1-7, wherein the Ab is full length antibody, it further comprise human antibody constant region；Wherein the full length antibody is selected from:
    The full length antibody that sequence of light chain shown in h1702 antibody, the sequence of heavy chain shown in SEQ ID NO:22 and SEQ ID NO:23 forms,
    The full length antibody that sequence of light chain shown in h1703 antibody, the sequence of heavy chain shown in SEQ ID NO:24 and SEQ ID NO:25 forms,
    The full length antibody that sequence of light chain shown in h1702-DS antibody, the sequence of heavy chain shown in SEQ ID NO:22 and SEQ ID NO:26 forms, and
    The full length antibody that sequence of light chain shown in h1704-3 antibody, the sequence of heavy chain shown in SEQ ID NO:40 and SEQ ID NO:41 forms.
  9. Such as antibody-drug conjugates of any of claims 1-7, wherein the antigen-binding fragment is selected from the antigen-binding fragment of Fab, Fab', F (ab') 2, single-chain antibody (scFv), the area V (double antibody) of dimerization, the disulfide-stabilized area V (dsFv) and the peptide comprising CDR.
  10. Such as the described in any item antibody-drug conjugates of claim 1-9, wherein the cytotoxic drug is selected from toxin, chemotherapeutics, antibiotic, radioactive isotope and core lyase.
  11. Antibody-drug conjugates as claimed in claim 10, wherein the cytotoxic drug is selected from DM1, DM3, DM4, MMAF and MMAE.
  12. Antibody-drug conjugates as claimed in claim 10, wherein the cytotoxic drug is selected from:
  13. Antibody-drug conjugates as claimed in claim 10 are compound or its pharmaceutically acceptable salt or solvated compounds shown in Formulas I,
    Wherein:
    L 1, L 2It is connector unit；
    Y is the number selected from 1-8, is preferably selected from the number of 2-4；
    Ab is B7H3 antibody of any of claims 1-9 or its antigen-binding fragment.
  14. Antibody-drug conjugates as claimed in claim 13, wherein L 2Such as following general formula L 2It is shown:
    Wherein
    X 1Selected from hydrogen atom, halogen, hydroxyl, cyano, alkyl, alkoxy and naphthenic base；
    X 2Selected from alkyl, naphthenic base and heterocycle；
    M is the integer selected from 0-5, preferably 1,2 or 3；S is sulphur atom.
  15. Antibody-drug conjugates as claimed in claim 14, wherein L 1Such as following general formula (L 1) shown in:
    Wherein
    X 3For alkyl, the optionally alkyl is further selected from replaced the substituent group of halogen, hydroxyl and cyano；
    N is the integer selected from 0-5, preferably 1,2 or 3.
  16. Antibody-drug conjugates as claimed in claim 13, to lead to antibody-drug conjugates shown in formula (II):
  17. Antibody-drug conjugates as claimed in claim 13, to lead to antibody-drug conjugates shown in formula (III):
  18. Antibody-drug conjugates as claimed in claim 1 or 2 are selected from following compound:
  19. A kind of pharmaceutical composition, it includes such as described in any item antibody-drug conjugates of claim 1-18 or the antibody-drug conjugates pharmaceutically acceptable salt or solvated compounds and one or more pharmaceutical excipient, diluent or carrier.
  20. Such as the purposes of the described in any item antibody-drug conjugates of claim 1-18 or pharmaceutical composition as claimed in claim 19 in the drug that preparation is used to treat disease relevant to people B7H3.
  21. Purposes as claimed in claim 20 is to prepare the purposes in the drug for treating B7H3 high expression cancer.
  22. As the described in any item antibody-drug conjugates of claim 1-18 or pharmaceutical composition as claimed in claim 19 are preparing the purposes in the drug for treating disease, the disease is selected from human brain astrocytes' blastoma, people's pharynx cancer, adrenal tumor, AIDS- associated cancer, alveolar soft part sarcoma, astrocytoma, bladder cancer, osteocarcinoma, brain and spinal cord cancer, metastatic brain tumor, breast cancer, carotid body tumor, cervical carcinoma, chondrosarcoma, chordoma, kidney chromophobe cell tumor, clear cell carcinoma, colon cancer, colorectal cancer, desmoplastic small round cell tumor, ependymocytoma, Juventus tumour, Extraskeletal myxoid chondrosarcoma, fibrogenesis imperfecta ossium, fibrous dysplasia of bone, gall-bladder or cholangiocarcinoma, gastric cancer, gestational trophoblast disease, gonioma, head and neck cancer, Hepatocellular carcinoma, islet-cell tumour, Kaposi sarcoma, kidney, leukaemia, embryonal-cell lipoma/pernicious lipoma tumour, liver cancer, lymthoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, Huppert's disease, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, oophoroma, cancer of pancreas, papillary thyroid carcinoma, parathyroid adenoma, paediatric cancer, Peripheral Nerve Sheath Tumors, pheochromocytoma, pituitary tumor, prostate cancer, uveal afterwards, kidney metastatic carcinoma, rhabdoid tumor, rhabdomyosarcoma, sarcoma, cutaneum carcinoma, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, carcinoma of testis, thymic carcinoma, thymoma, Thyroid metastasis cancer and uterine cancer.
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