CN107973854A - PDL1 monoclonal antibodies and its application - Google Patents

PDL1 monoclonal antibodies and its application Download PDF

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Publication number
CN107973854A
CN107973854A CN201711303454.6A CN201711303454A CN107973854A CN 107973854 A CN107973854 A CN 107973854A CN 201711303454 A CN201711303454 A CN 201711303454A CN 107973854 A CN107973854 A CN 107973854A
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seq
heavy chain
variable region
light chain
chain variable
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CN107973854B (en
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周宏林
蔡斌
刘杰
陈罡
董欣
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Suzhou Yinhe Biological Medicine Co Ltd
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Suzhou Yinhe Biological Medicine Co Ltd
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Priority to PCT/CN2018/092330 priority patent/WO2019114235A1/en
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    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Abstract

The present invention relates to PDL1 monoclonal antibodies and its application, belong to immunological technique field.The present invention provides a kind of separated human specific binding molecule, includes a）And b）；a）Three light chain CDR：Light chain CDR1, light chain CDR2 and light chain CDR3；b）Three heavy chain CDR：Heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3；The separated people PDL1 specific binding molecules are separated antibody or antigen-binding fragment.PDL1 monoclonal antibodies provided by the invention can effectively suppress Local tumor growth；Block PD1/PDL1 signals to promote the propagation of specific for tumour antigen T cell, play the effect of killing tumor cell；Correlation PDL1 signals on tumour cell are blocked to raise infiltration CD8+The secretion of T cell IFN γ.

Description

PDL1 monoclonal antibodies and its application
Technical field
The invention belongs to oncotherapy and molecular immunology field, be related to a variety of anti-PDL1 antibody, its pharmaceutical composition and Purposes.Specifically, the monoclonal antibody the present invention relates to a variety of anti-PDL1.
Background technology
The cellular immunity of T cell mediation is identifying and is playing an important role in killing tumor cell that T cell is thin by T Born of the same parents' acceptor（T cell receptor, TCR）It is compound with the ajor histocompatibility with specific antigen of tumor cell surface Body（major histocompatibility complex, MHC）With reference to so that tumor cell.TCR and MHC molecule Interaction is controlled be subject to a series of immunologic test points, wherein having costimulatory signal and coinhibitory signals, can swash T cell Living or suppression.Wherein PD1 and its ligand PDL1 paths are inhibition immunologic test points, they, which are combined, passes on co-suppression signal, It can be suppressed the immunocompetence of T cell, play a significant role in immune tolerance, while be also that tumour cell is immunized and escapes The major reason of ease.
Programmed death acceptor 1 (programmed death 1, PD1), is a kind of important immunosuppression molecule.For The I type transmembrane proteins of CD28 superfamily members, it is initially to clone to come from the mouse Tcell hybridoma 2B4.11 of apoptosis.With PD1 has antitumor, anti-infective, anti-autoimmune disease and organ transplant survival etc. for the immunological regulation of target spot important Meaning.PD1 has two kinds of binding partners, PDL1 (B7-H1) and PDL2 (B7-DC), both expression are different, PDL2 expression ratios Compared with limitation, main expression is in the macrophage of activation, dendritic cells and a small number of tumours；Then the T cell in activation, B are thin by PDL1 Born of the same parents, macrophage, dendritic cells and tumour cell wide expression, at the same in body some immune masked segment such as placentas, eye and The tissue expressions such as its epithelium, muscle, liver and blood vessel endothelium, therefore the effects of PDL1 in vivo will be considerably beyond PDL2.
Apoptosis ligand 1（1 ligand 1, PDL1 of Programmed cell death）Also referred to as surface resists Original differentiation cluster 274（Cluster of differentiation 274, CD274）Or B7 autoploids（B7 homolog 1, B7- H1）, it is a kind of protein in mankind's body, by CD274 gene codes.
PDL1 has IgV and IgC samples area, transmembrane region and cytoplasmic tail, wherein cytoplasmic tail and intracellular signal Transduction is related, and IgV and IgC then participate in intercellular signal transduction.Research finds, TNF, IFN-γ, IL-4, granulocyte stimulate because The cytokine profiles such as son and IL-10 can raise expression of the PDL1 in different cells.
PDL1 interacts with the acceptor PD1 in its T cell, and important work is played in terms of the negativity regulation and control of immune response With；The molecule has extensive distribution expression pattern, and higher expression is fastened in some tumour cells, and many researchs show it It is related to the Immune escaping mechanism of tumour.The microenvironment of tumor section can induce the expression of the PDL1 on tumour cell, and express Extensively, the PDL1 of expression is conducive to the generation and growth of tumour, the apoptosis of inducing antitumor T cell.
After PD1 is combined with PDL1, inhibition signal is transmitted, the propagation and activity, suppression CD4 of lymphocyte can be suppressed+ T For cell to Th1 and Th17 cell differentiations, the release of suppression inflammatory cytokine, these all play the role of immune negative regulation. Under normal circumstances, the combination of PDL1 and PD1 can maintain periphery lymphocyte to exempt from autoantigen by above-mentioned effect Epidemic disease is resistant to, so as to prevent the generation of autoimmune disease.But in the occurrence and development of tumour, the PDL1 of tumor cells expression with PD1 but can be by acting on the inhibition of lymphocyte, so as to promote the immunologic escape of tumour after combining.
The mechanism of tumor immune escape includes：Under the expression of tumor cell membrane surface compatability compound (MHC) quasi-molecule Adjust, lack co-stimulators, the secretory immune inhibitory cells factor, expresses death ligand or expression inhibiting ligand etc.. The high expression PDL1 molecules of many tumor cell lines and tumour cell, after it is combined with the PD1 molecules of lymphocytic cell surface, weaken The anti-tumor immune response of body, so as to cause the generation of tumor immune escape.In addition, tumor microenvironment is by tumour cell, blood Pipe, tissue fluid, interstitial cell and the lymphocyte composition infiltrated on a small quantity.Tumor microenvironment also there are some inflammatory cytokines, Such as IFN-α and IFN-γ, these cell factors can promote the expression of tumor cell surface PDL1, in the immunologic escape mistake of tumour Important function has been played in journey.
Although at present regulatory mechanisms of PDL1 and the PD1 in tumor immune escape not yet illustrate completely, for PDL1 with The blocking antibody of PD1 achieves preferable therapeutic effect in clinical test.With deepening continuously for research, PDL1 and PD1 Effect and regulatory mechanism in tumor immune escape can definitely, and PDL1 and PD1 will also control as effective antitumour Treat target spot.
The content of the invention
The present invention gives expression to the PDL1 of restructuring as mice immunized with antigen by the use of mammalian cell expression system, through mouse Spleen cell is merged with myeloma cell obtains hybridoma.The present invention is obtained by being screened to substantial amounts of sample Several hybridoma cell strains, can secrete the monoclonal antibody specific of generation and PDL1 specific bindings, and the monoclonal Antibody has further obtained chimeric antibody by means such as molecular clonings, and the chimeric antibody being prepared can combine people's T cells The PDL1 on surface（Fig. 1, Table III）, there is high-affinity（Table II）.And a series of characterizations and functional experiment research, table are carried out These bright chimeric antibodies can be with the T cell and DC cell combinations of activation（Fig. 2,3）, and can highly desirable block PDL1 with Combination between PD1 or CD80（Fig. 4, Table IV）.Cross-species reaction experiment shows that chimeric antibody does not have with machin and mouse Or there is smaller cross reaction（Fig. 5, Table V）.External function test the results show that these chimeric antibodies can promote T cell lines or The immune function of primary T cells（Fig. 6,7）；Results of animal shows that these druggability antibody can effectively suppress tumour and exist Humanization PDL1 knocks in the growth in Mice Body（Fig. 8）.Finally, these antibody have been carried out with humanization with reduce immunogenicity and Obtain therapeutic antibodies.Thus provide following inventions：
The present invention provides the antibody of specific binding people PDL1, the antibody producing is from hybridoma cell line.It is provided by the invention Hybridoma cell line derives from mouse immune, and cell fusion, by sequence of operations such as screenings, can produce specific inhibition The monoclonal antibody of the combination of PDL1 and PD1/CD80.（Referring to embodiment 1）
Present invention also offers the chimeric antibody of specific binding people PDL1, wherein by the way that the variable region of mouse source PDL1 antibody is connected IgG1 the and κ constant regions of people are connected to, construct the heavy chain and light chain of chimeric antibody.（Referring to embodiment 2）
Chimeric antibody provided by the invention has high-affinity, wherein equilibrium association constant K in kinetic measurementDFor≤ 3.89x10-10, one aspect of the present invention provide kinetic parameter use bio-molecular interaction system Octet-96（Pall Life Sciences, S‐000959）Measure.（Referring to embodiment 3）
The chimeric antibody that one aspect of the present invention provides, is combined, EC50 is in 62.6 ng/mL and 437.7 ng/ with cell surface antigen In the range of mL；Wherein, term EC50 refers to half-maximal effect concentration (50% of maximal of concentration for effect).（Referring to embodiment 4）
The chimeric antibody that one aspect of the present invention provides, T cell and DC cell combinations with activation.（Referring to embodiment 5）
The chimeric antibody that another aspect of the present invention provides, blocks the combination of PDL1 and PD1 or CD80, IC50 is respectively 119.8 In the range of ng/mL and 178.5 ng/mL and in the range of 608.5 ng/mL and 1867 ng/mL.Wherein, IC50 (half Maximal inhibitory concentration) refer to the 503nhibiting concentration of measured antagonist.（Referring to embodiment 6）
In the present invention, if not otherwise specified, the CD80 is B7-1；Its specific protein sequence is sequence known in the state of the art Row, may be referred to the sequence disclosed in existing literature or GenBank.For example, B7-1 (CD80, NCBI Gene ID:941).
The chimeric antibody that one aspect of the present invention provides, with mouse without species cross reaction, has the cross-species anti-with machin Should.（Referring to embodiment 7）
Chimeric antibody provided by the invention can strengthen the immune function of Jurkat cell.（Referring to embodiment 8）
Chimeric antibody provided by the invention has stronger In vitro biological activity.In allosome mix lymphocyte reaction （Mixed Lymphocyte Reaction, MLR）, antibody of the present invention can strengthen the secretion of IFN-γ（Referring to embodiment 9）.
Druggability antibody provided by the invention has stronger internal pharmacological activity.In animal model for tumour, this hair Bright antibody can effectively suppress tumour growth and reduce gross tumor volume.（Referring to embodiment 10）
One aspect of the present invention provides separated human specific binding molecule, includes a）And b）, a）Three light chain CDR：Light chain CDR1, light chain CDR2 and light chain CDR3,
b）Three heavy chain CDR：Heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3；
The separated people PDL1 specific binding molecules are separated antibody or antigen-binding fragment；
(i) heavy chain CDR1 is selected from：SEQ ID NO: 37、SEQ ID NO: 43、SEQ ID NO: 49、SEQ ID NO: 55、 SEQ ID NO: 61、SEQ ID NO: 67、SEQ ID NO: 73、SEQ ID NO:79 or SEQ ID NO: 85；
(ii) heavy chain CDR2 is selected from：SEQ ID NO: 38、SEQ ID NO: 44、SEQ ID NO: 50、SEQ ID NO: 56、 SEQ ID NO: 62、SEQ ID NO: 68、SEQ ID NO: 74、SEQ ID NO:80 or SEQ ID NO: 86；
(iii) heavy chain CDR3 is selected from：SEQ ID NO: 39、SEQ ID NO: 45、SEQ ID NO: 51、SEQ ID NO: 57、SEQ ID NO: 63、SEQ ID NO: 69、SEQ ID NO: 75、SEQ ID NO:81 or SEQ ID NO: 87；
(iv) light chain CDR1 is selected from：SEQ ID NO: 40、SEQ ID NO: 46、SEQ ID NO: 52、SEQ ID NO: 58、 SEQ ID NO: 64、SEQ ID NO: 70、SEQ ID NO: 76、SEQ ID NO:82 or SEQ ID NO: 88；
(v) light chain CDR2 is selected from：SEQ ID NO: 41、SEQ ID NO: 47、SEQ ID NO: 53、SEQ ID NO: 59、 SEQ ID NO: 65、SEQ ID NO: 71、SEQ ID NO: 77、SEQ ID NO: 83、SEQ ID NO:9 or SEQ ID NO: 89；With
(vi) light chain CDR3 is selected from：SEQ ID NO: 42、SEQ ID NO: 48、SEQ ID NO: 54、SEQ ID NO: 60、 SEQ ID NO: 66、SEQ ID NO: 72、SEQ ID NO: 78、SEQ ID NO:84 or SEQ ID NO: 90.
The separated human specific binding molecule that another aspect of the present invention provides, the specific binding molecules include heavy chain Variable region and light chain variable region, wherein：
(i) heavy chain variable region includes such as SEQ ID NO：Heavy chain CDR1, such as SEQ ID NO shown in 37：Heavy chain shown in 38 CDR2 and such as SEQ ID NO：Heavy chain CDR3 shown in 39；And/or light chain variable region includes such as SEQ ID NO：Shown in 40 Light chain CDR1, such as SEQ ID NO：Light chain CDR2 and such as SEQ ID NO shown in 41：Light chain CDR3 shown in 42；Or Person
(ii) heavy chain variable region includes such as SEQ ID NO：Heavy chain CDR1, such as SEQ ID NO shown in 43：Weight shown in 44 Chain CDR2 and such as SEQ ID NO：Heavy chain CDR3 shown in 45；And/or light chain variable region includes such as SEQ ID NO：46 institutes Light chain CDR1, such as SEQ ID NO shown：Light chain CDR2 and such as SEQ ID NO shown in 47：Light chain CDR3 shown in 48； Or
(iii) heavy chain variable region includes such as SEQ ID NO：Heavy chain CDR1, such as SEQ ID NO shown in 49：Weight shown in 50 Chain CDR2 and such as SEQ ID NO：Heavy chain CDR3 shown in 51；And/or light chain variable region includes such as SEQ ID NO：52 institutes Light chain CDR1, such as SEQ ID NO shown：Light chain CDR2 and such as SEQ ID NO shown in 53：Light chain CDR3 shown in 54； Or
(iv) heavy chain variable region includes such as SEQ ID NO：Heavy chain CDR1, such as SEQ ID NO shown in 55：Weight shown in 56 Chain CDR2 and such as SEQ ID NO：Heavy chain CDR3 shown in 57；And/or light chain variable region includes such as SEQ ID NO：58 institutes Light chain CDR1, such as SEQ ID NO shown：Light chain CDR2 and such as SEQ ID NO shown in 593：Light chain shown in 60 CDR3；Or
(v) heavy chain variable region includes such as SEQ ID NO：Heavy chain CDR1, such as SEQ ID NO shown in 61：Heavy chain shown in 62 CDR2 and such as SEQ ID NO：Heavy chain CDR3 shown in 63；And/or light chain variable region includes such as SEQ ID NO：Shown in 64 Light chain CDR1, such as SEQ ID NO：Light chain CDR2 and such as SEQ ID NO shown in 65：Light chain CDR3 shown in 66；Or Person
(vi) heavy chain variable region includes such as SEQ ID NO：Heavy chain CDR1, such as SEQ ID NO shown in 67：Weight shown in 68 Chain CDR2 and such as SEQ ID NO：Heavy chain CDR3 shown in 69；And/or light chain variable region includes such as SEQ ID NO：70 institutes Light chain CDR1, such as SEQ ID NO shown：Light chain CDR2 and such as SEQ ID NO shown in 71：Light chain CDR3 shown in 72； Or
(vii) heavy chain variable region includes such as SEQ ID NO：Heavy chain CDR1, such as SEQ ID NO shown in 73：Weight shown in 74 Chain CDR2 and such as SEQ ID NO：Heavy chain CDR3 shown in 75；And/or light chain variable region includes such as SEQ ID NO：76 institutes Light chain CDR1, such as SEQ ID NO shown：Light chain CDR2 and such as SEQ ID NO shown in 77：Light chain CDR3 shown in 78； Or
(viii) heavy chain variable region includes such as SEQ ID NO：Heavy chain CDR1, such as SEQ ID NO shown in 79：Shown in 80 Heavy chain CDR2 and such as SEQ ID NO：Heavy chain CDR3 shown in 81；And/or light chain variable region includes such as SEQ ID NO：82 Shown light chain CDR1, such as SEQ ID NO：Light chain CDR2 and such as SEQ ID NO shown in 83：Light chain shown in 84 CDR3；Or
(ix) heavy chain variable region includes such as SEQ ID NO：Heavy chain CDR1, such as SEQ ID NO shown in 85：Weight shown in 86 Chain CDR2 and such as SEQ ID NO：Heavy chain CDR3 shown in 87；And/or light chain variable region includes such as SEQ ID NO：88 institutes Light chain CDR1, such as SEQ ID NO shown：Light chain CDR2 and such as SEQ ID NO shown in 89：Light chain CDR3 shown in 90.
In some specific embodiments of the present invention, the antibody is full length antibody.
In some specific embodiments of the present invention, the antigen-binding fragment is Fab or F (ab) '2Or scFv.
One aspect of the present invention provides separated antibody or its antigen-binding fragment, can it includes light chain variable region and heavy chain Become area, wherein：
(i) heavy chain variable region is selected from：SEQ ID NO: 19、SEQ ID NO: 21、SEQ ID NO: 23、SEQ ID NO: 25；SEQ ID NO: 27、SEQ ID NO: 29、SEQ ID NO: 31、SEQ ID NO:33 and SEQ ID NO: 35；
(ii) light chain variable region is selected from：SEQ ID NO: 20、SEQ ID NO: 22、SEQ ID NO: 24、SEQ ID NO: 26；SEQ ID NO: 28、SEQ ID NO: 30、SEQ ID NO: 32、SEQ ID NO:34 and SEQ ID NO: 36；
(iii) sequence of sequence at least 80% homology and described in (i), (ii).
One aspect of the present invention provides separated antibody or its antigen-binding fragment, can it includes light chain variable region and heavy chain Become area, wherein：
(i) heavy chain variable region is SEQ ID NO:19 and the light chain variable region be SEQ ID NO: 20；
(ii) heavy chain variable region is SEQ ID NO:21 and the light chain variable region be SEQ ID NO: 22；
(iii) heavy chain variable region is SEQ ID NO:23 and the light chain variable region be SEQ ID NO: 24；
(iv) heavy chain variable region is SEQ ID NO:25 and the light chain variable region be SEQ ID NO: 26；
(v) heavy chain variable region is SEQ ID NO:27 and the light chain variable region be SEQ ID NO: 28；
(vi) heavy chain variable region is SEQ ID NO:29 and the light chain variable region be SEQ ID NO: 30；
(vii) heavy chain variable region is SEQ ID NO:31 and the light chain variable region be SEQ ID NO: 32；
(viii) heavy chain variable region is SEQ ID NO:33 and the light chain variable region be SEQ ID NO: 34；With
(ix) heavy chain variable region is SEQ ID NO:35 and the light chain variable region be SEQ ID NO: 36.
In some specific embodiments of the present invention, the antibody or its antigen-binding fragment, specifically with reference to people PDL1 and show at least one following property：
(i) combination of PDL1 and PD1 or CD80 is blocked；
(ii) PDL1 on human T-cell surface is combined；
(iii) with 3.89 × 10-10 M or lower KDCombined with people PDL1；
(iv) IFN-γ is improved in mixed lymphocyte reaction (MLP) (MLR) experiment to produce.
In certain embodiments, the monoclonal antibody or its antigen-binding portion thereof are being prepared for suppressing patient Purposes in the medicine of inner tumour cell growth.
In certain embodiments, the monoclonal antibody or its antigen-binding portion thereof are being prepared for treating infectious disease Purposes in medicine.
One aspect of the present invention provides separated nucleic acid, in its encoding antibody light variable region and heavy chain of antibody variable region One or both, wherein
(i) fragment of encoding said antibody heavy chain variable region is selected from：SEQ ID NO: 1、SEQ ID NO: 3、SEQ ID NO: 5、SEQ ID NO: 7、SEQ ID NO: 9、SEQ ID NO: 11、SEQ ID NO: 13、SEQ ID NO:15 and SEQ ID NO: 17；
(ii) fragment of encoding said antibody light chain variable region is selected from：SEQ ID NO: 2、SEQ ID NO: 4、SEQ ID NO: 6、SEQ ID NO: 8、SEQ ID NO: 10、SEQ ID NO: 12、SEQ ID NO: 14、SEQ ID NO:16 and SEQ ID NO: 18；
(iii) sequence of sequence at least 80% homology and described in (i), (ii).
Present invention also offers a kind of carrier, it contains the nucleic acid molecules.
Present invention also offers a kind of host cell, it contains the nucleic acid molecules or carrier.
Present invention also offers a kind of conjugate, and it includes be covalently attached with isotope, immunotoxin and/or chemicals Anti-human PDL1 monoclonal antibodies；The anti-human PDL1 monoclonal antibodies are the separated people PDL1 specific binding molecules.
Present invention also offers a kind of conjugate, it is by the separated people PDL1 specific binding molecules and/or institute The conjugate stated is coupled to be formed with solid dielectric or semi-solid medium.
The invention further relates to the anti-PDL1 monoclonal antibodies and/or conjugate and/or conjugate to prepare treatment disease Application in the medicine of disease；
The disease for breast cancer, lung cancer, stomach cancer, intestinal cancer, the cancer of the esophagus, oophoroma, cervical carcinoma, kidney, carcinoma of urinary bladder, cancer of pancreas, Glioma or melanoma.
The present invention provides a kind of composition, its contain anti-human PDL1 Humanized monoclonal antibodies or its antigen-binding portion thereof, Nucleic acid molecules, carrier, host cell, conjugate or conjugate, and optional pharmaceutically acceptable carrier or figuration Agent, and optional other bioactive substances.
The invention further relates to kit, and it includes the separated antibody or its antigen-binding fragment and one group to be used for Detection is incorporated into the reagent of the antibody of people PDL1 or the compound of the antigen-binding fragment.
The present invention is described further below：In the present invention, unless otherwise stated, science used herein There is the normally understood implication of those skilled in the art institute with technical term.Also, protein used herein and nucleination , molecular biology, cell and tissue culture, microbiology, immunology relational language and laboratory operation step are corresponding Widely used term and conventional steps in field.Meanwhile for a better understanding of the present invention, determining for relational language is provided below Justice and explanation.
Term " antibody " mentioned herein includes complete antibody and its any antigen-binding fragment (i.e. " antigen-binding portion Point ") or it is single-stranded." antibody " refers to comprising at least two weight (H) chains and two light (L) being mutually connected to each other by disulfide bond The glycoprotein of chain, or its antigen-binding portion thereof.Each heavy chain is by heavy chain variable region (being abbreviated as VH herein) and light chain constant district's groups Into.Heavy chain constant region is made of three domain Cs H1, CH2 and CH3.Every light chain by light chain variable region (being abbreviated as VL) herein Formed with constant region of light chain.Constant region of light chain is made of a domain C L.VH and VL areas can be further subdivided into hypervariable region, claim For complementary determining region (CDR), CDR is dispersed in the more conservative region for being referred to as framework region (FR).Each VH and VL by Three CDR and four FR compositions, they are arranged in the following order from aminoterminal to c-terminus：FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.The variable region of heavy chain and light chain is contained can be with the binding structural domain of antigen interactions.The constant region of antibody can be with The combination of mediated immunity globulin and host tissue or the factor, the host tissue or the factor include the various cells of immune system (such as effector cell) and the first composition (C1q) of classical complement system.
" antigen-binding portion thereof " (or being referred to as " antibody moiety ") of term antibody used herein refers to retain and antigen One or more fragments of the antibody of the ability of (such as PDL1) specific binding.The antigen binding function of being proved antibody can be by The fragment of full length antibody is exercised.The example of included binding fragment includes in " antigen-binding portion thereof " of term antibody：(i) Fab fragments, i.e., the monovalent fragment being made of VL, VH, CL and CH1 domain；(ii)F(ab’)2Fragment, i.e., included in hinge area The divalent fragments thereof for two Fab fragments that place is connected by disulfide bond；(iii) the Fd fragments being made of VH and CH1 domains；(iv) The Fv fragments being made of VL the and VH domains of antibody single armed；(v) the dAb fragments being made of VH domains；(vi) is separated Complementary determining region (CDR).In addition, although two domain VL and VH of Fv fragments by single gene code, but they can To be linked together using recombination method by synthesizing connector, which allows them to that a protein chain is made, its Middle VL and VH areas pairing forms monovalent molecule and is known as scFv (scFv).This single-chain antibody is also included within the " anti-of term antibody In former bound fraction ".These antibody fragments to be obtained with well known to a person skilled in the art routine techniques, and with complete antibody phase Same method screens the practicality of these fragments.
" separated antibody " used herein refers to the antibody for being substantially free of other antibody with different antigentic specificities (for example, with PDL1 specific binding separated antibody be substantially free of combined with the antigentic specificity in addition to PDL1 resist Body).But can with the separated antibody of PDL1 specific bindings and other antigens such as PDL1 molecules from other species There can be cross reactivity.Moreover, separated antibody can be substantially free of other cell materials and/or chemical substance.
Terms used herein " monoclonal antibody " or " monoclonal antibody combination " refer to the antibody of single molecular composition The preparation of molecule.Monoclonal antibody combination shows the single binding specificity and compatibility to defined epitope.
Terms used herein " human antibody " include with following variable region antibody, in the variable region, framework region and CDR region is derived from human germline immunoglobulin's sequence.If moreover, the antibody contains constant region, constant region also originates from ethnic group It is immunoglobulin sequences.The human antibody of the present invention can include not residual by the amino acid of human germline immunoglobulin's sequential coding Base (for example, passing through external random or site-specific mutagenesis or the mutation introduced by internal somatic mutation).But herein The CDR sequence that the term " human antibody " used does not include the wherein germline from another mammal kind such as mouse has been transplanted to Antibody on people's frame sequence.
Term " human monoclonal antibodies " refers to the antibody for showing single binding specificity, it has wherein framework region and CDR Area is derived from the variable region of human germline immunoglobulin's sequence.In one embodiment, human monoclonal antibodies are produced by hybridoma Raw, which includes the B cell merged with immortalized cell, and the B cell is from heavy chain transgene containing people and light chain Obtained in the transgenic nonhuman animal (such as transgenic mice) of the genome of transgenosis.
Terms used herein " recombinant human antibody " includes preparing by recombination method, expresses, generation or separated all Human antibody, such as：(a) from the transgenosis for human immunoglobulin gene or trans-chromosome animal (such as mouse) or by its system Separated antibody in standby hybridoma (being discussed further below), the host cell such as transfection of (b) from inverted expression human antibody Separated antibody in knurl, (c) separated antibody from restructuring combination human antibody library, and (d) pass through including by people's immune globulin White gene order montage is prepared, expressed for any other method of other DNA sequence dnas, producing or separated antibody.These restructuring Human antibody is derived from the variable region of human germline immunoglobulin's sequence with wherein framework region and CDR region.But in some implementations In scheme, these recombinant human antibodies can undergo mutagenesis in vitro and (alternatively, when the transgenic animals of user's Ig sequences, undergo Internal somatic mutagenesis), therefore the amino acid sequence in the VH and VL areas of recombinant antibodies is despite from people's germline VH and VL sequence And associated sequence, but may not be the repertoire for being naturally occurring in human antibody germline in vivo (repertoire) in.
Term " humanized antibody " refers to the CDR sequence for wherein deriving from the germline of another mammal kind such as mouse The antibody being transplanted on people's frame sequence.Other framework region modifications can also be carried out in people's frame sequence.
Term " chimeric antibody " refer to wherein variable region sequences from a species and constant-region sequences from another The antibody of a species, such as wherein variable region sequences derive from the antibody of human antibody from mouse antibodies and constant-region sequences.
Terms used herein " Kassoc" or " Ka" refer to the association rates of specific antibodies-antigen interactions, and herein Term " the K useddis" or " Kd" refer to the dissociation rates of specific antibodies-antigen interactions.Terms used herein " KD" be Refer to dissociation constant, it is by KdWith KaRatio obtain (i.e. Kd/Ka), and it is expressed as molar concentration (M).The K of antibodyDValue can The method that can be established with this area measures.Measure antibody KDA kind of method for optimizing be to use surface plasmon resonance method, it is excellent Choosing uses bio-sensor system.
" homology " refers to the sequence between two polynucleotide sequences or between two polypeptide sequences when most preferably comparing Row similitude.
" separated nucleic acid molecules " mean not with all or part of polynucleotides association genome DNA or RNA, MRNA, cDNA or its synthesis source or some combinations, wherein separated polynucleotides are visible in nature or are connected to certainly The polynucleotides being not connected with right boundary.
PDL1 monoclonal antibodies provided by the invention can effectively suppress Local tumor growth；Block PD1/PDL1 signals can be with Promote the propagation of specific for tumour antigen T cell, play the effect of killing tumor cell；Block correlation PDL1 letters on tumour cell It number can raise infiltration CD8+The secretion of T cell IFN-γ, shows the blockings of PD1/PDL1 signal paths to induce immune response For the purpose of tumor immune response in play a role；Select anti-PDL1 monoclonal antibodies to coordinate tumor vaccine to carry out tumour immunity to control The immune activation of tumor vaccine can effectively be strengthened by treating.At present, anti-PD1/PDL1 treatment with its it is good the effect of and security walk The forefront of immunization therapy, becomes the popular target spot in nearly 2 years lung cancer therapy fields.
Brief description of the drawings
Fig. 1：FACS detects the combination of PDL1 chimeric antibodies and B16-hPDL1 cells；
Fig. 2：FACS detects PDL1 chimeric antibodies and activates the combination of T cell；
Fig. 3：FACS detects the combination of PDL1 chimeric antibodies and DC cells；
Fig. 4 a：ELISA detection PDL1 chimeric antibodies block the interaction between PD1 and PDL1；
Fig. 4 b：ELISA detection PDL1 chimeric antibodies block the interaction between CD80 and PDL1；
Fig. 5 a：ELISA detects PDL1 chimeric antibodies and is reacted with machin PDL1 cross-species；
Fig. 5 b：FACS detects PDL1 chimeric antibodies and is reacted with mouse PDL1 cross-species；
Fig. 6：Luciferase Assay Reagent box detects influence of the PDL1 chimeric antibodies to Jurkat cell immune response；
Fig. 7：The secretion of PDL1 chimeric antibodies enhancing IFN-γ in ELISA detection MLR experiments；
Fig. 8：Growth of the PDL1 druggabilities antibody inhibiting tumor in PDL1 Transgenic mouse bodies.
Fig. 9 a- Fig. 9 c：Table one, the sequence number table of the anti-PDL1 antibody of the present invention；
Figure 10：Table two, people's mouse PDL1 chimeric antibody dynamic analysis tables；
Figure 11：Table three, the cell combination medium effective concentration table of people's mouse PDL1 chimeric antibodies；
Figure 12：Table four, the ligand blocking experiment table of people's mouse PDL1 chimeric antibodies；
Figure 13：Table five, the cross reaction table of people's mouse PDL1 chimeric antibodies.
Embodiment
The invention discloses monoclonal antibody and its application, those skilled in the art can use for reference present disclosure, suitably change Realized into technological parameter.In particular, all similar substitutions and modifications are for a person skilled in the art aobvious And be clear to, they are considered as being included in the present invention.The method of the present invention and application are carried out by preferred embodiment Description, related personnel substantially can not depart from present invention, in the range of to method described herein and application be modified or Suitably change with combining, to realize and using the technology of the present invention.
Raw materials used and reagent can be bought by market in monoclonal antibody provided by the invention and its application.
With reference to embodiment, the present invention is further explained：
Embodiment 1：The screening of animal immune and anti-PDL1 mouse antibody
The Balb/c mouse of Suitable Age are selected, carry out inoculation.By the use of hPDL1-mFc fusion proteins as antigen and completely not Family name's adjuvant（Sigma-Aldrich）After mixing, by being subcutaneously injected in immune Mice Body, corresponding bone-marrow-derived lymphocyte is stimulated to clone. Then, every about the 1 of two to three 100 μ g of all intraperitoneal injections:1 is emulsifiable in incomplete Freund's adjuvant（Sigma-Aldrich）In HPDL1-mFc, so as to carry out booster immunization to immune mouse.Mouse spleen lymphocyte is taken out by sterile working, with Ready SP2/0 myeloma cell is by a certain percentage（Splenocyte 1x108, myeloma cell 2x107）Mixing, and add poly- second Glycol（Sigma, P7181）Carry out cell fusion.
After fusion, fused cell is added in 96 orifice plates, 0.1 mL HAT culture mediums are added per hole, is put into carbon dioxide training Support in case, 37 DEG C of cultures；4th day, often 0.1 mL HT culture mediums are added in hole；At the 7th day into, culture medium is changed to HT trainings completely Support base.Screened at the 8-12 days, positive cell is subcloned by limiting dilution assay, expands culture, and pass through ELISA Tested with FACS, specific method is as follows:
The hPDL1-His of 2 μ g/mL is coated on 96 orifice plates（Corning, catalog #9018）, 4 DEG C are incubated overnight.With PBST（PBS contains 0.05% Tween-20）Board-washing three times, uses 5% skimmed milk power（Oxoid, catalog #LP0031B）Envelope Close.With PBST board-washings three times after, add hybridoma supematant, 37 DEG C be incubated 2 it is small when.After board-washing, 1/5000 diluted HRP marks are added The sheep anti-mouse igg secondary antibody of note（BioLegend, catalog #405306）, when 37 DEG C of incubations 1 are small.After board-washing, TMB is added （Tiangen, catalog # PA107-02）Colour developing.
Antibody and B16-hPDL1 cells（Endogenous mouse PDL1 gene knockouts）Combination be detected using FACS. 96 orifice plates（Corning, catalog #3799）In pancreatin postdigestive 2.5x10 is added per hole5B16-hPDL1 cells, with Hybridoma supematant is incubated 30 minutes at 4 DEG C.Use buffer solution（PBS contains 1% BSA and 5 mM EDTA）After washing cell twice, add Enter the sheep anti-mouse igg secondary antibody of APC marks（BD, catalog #550826）, 4 DEG C are incubated 30 minutes.The buffered liquid washing of cell Afterwards, in Attune Nxt flow cytometers（Thermo Fisher）Upper analysis.
PDL1 antibody can block the combination of PDL1 and PD1, therefore further screen hybridoma using ligand blocking experiment Supernatant.The hPD1-hFc of 1.5 μ g/mL is coated with 96 orifice plates, 4 DEG C overnight.After board-washing 3 times, using 5% skimmed milk power in 37 DEG C of envelopes Close 2 it is small when.Board-washing three times after, add the hybridoma supematant being pre-mixed and the biotinylated hPDL1-hFc of 200 ng/mL, room When the lower incubation 1 of temperature is small.After board-washing, the Streptavidin of HRP marks is added, when incubation at room temperature 1 is small.After board-washing, add TMB and show Color.
PDL1 antibody can block the combination of PDL1 and CD80, therefore further screen hybridoma using ligand blocking experiment Supernatant.The hCD80-hFc of 2 μ g/mL is coated with 96 orifice plates, 4 DEG C overnight.After board-washing 3 times, using 5% skimmed milk power in 37 DEG C of envelopes Close 2 it is small when.Board-washing three times after, add the hybridoma supematant being pre-mixed and the biotinylated hPDL1-hFc of 1000 ng/mL, room When the lower incubation 1 of temperature is small.After board-washing, the Streptavidin of HRP marks is added, when incubation at room temperature 1 is small.After board-washing, add TMB and show Color.
The cross reaction of PDL1 antibody and machin PDL1 is examined using ELISA method.By the anti-mouse of 2 μ g/mL IgG Fc antibody（Sigma, catalog #M4280）It is coated with onto 96 orifice plates, 4 DEG C overnight.Using 5% skimmed milk power at 37 DEG C Close 2 it is small when.After PBST board-washings, hybridoma supematant is added, is incubated at room temperature 30 minutes.PBST board-washings three times after, by biotin The machin PDL1 of mark（SinoBiological, catalog #90251-C08H）Add in plate, when incubation at room temperature 1 is small. With PBST board-washings three times, the Streptavidin of 1/1000 diluted HRP marks is added, when incubation at room temperature 1 is small.After board-washing, add TMB develops the color.
Embodiment 2：The clone of mouse source antibody cDNA and the structure of chimeric antibody
With degenerate primer PCR method, the variable region gene sequence of acquisition hybridoma antibody heavy chain and light chain.Hybridoma monoclonal is thin Born of the same parents use Trizol（Invitrogen, catalog #15596-018）Isolated total serum IgE after cracking, and use SuperScript III First-Strand Synthesis System（Invitrogen, catalog #18080- 051）, reverse transcription is carried out using RNA as template, obtains cDNA storehouses.Using obtained cDNA storehouses as template, using degenerate primer into Row PCR（Zhou H, et al., Nucleic Acids Research 22: 888-889 (1994), Chardes T et al., FEBS Letters 452: 386-394 (1999)）.PCR product is detected through agarose gel electrophoresis, heavy chain and The estimated size of the pcr amplification product of light chain variable region is 400 base-pairs.PCR product is cloned into pClone007 carriers （Tsingke, catalog #TSV-007BS）, and convert to 5 α of E. coli competent Trans（Transgen, catalog# CD201-02）, and select 3-6 Escherichia coli clones on a lbmc agar plate and carry out gene sequencing.In some situations Under, PCR product also can be directly used for gene sequencing.By above method, the heavy chain of antibody and the variable region of light chain are finally obtained Full-length gene order.Through NCBI Ig-BLAST（https://www.ncbi.nlm.nih.gov/projects/igblast/） Further the complementary determining region of heavy chain of antibody and light chain is obtained after analysis（Complementarity Determining Region）With skeleton area（Framework Region）Sequencing and analyzing（Kabat E.A., et al., 1991, Sequences of proteins of immunological interest, in: NIH Publication No. 91- 3242, US Department of Health and Human Services, Bethesda, MD）.Specifying information is shown in anti- Body sequence table and Fig. 9 a- Fig. 9 c（Table I）.
By the IgG1 that the heavy chain of mouse source PDL1 antibody and light chain variable region are respectively connected to people（Or IgG1 mutant N297A）With κ constant regions, the heavy chain and light chain of chimeric antibody are constructed.By PCR method, introduced in heavy chain and light chain variable region Suitable restriction enzyme site, is cloned into corresponding chimeric antibody expression vector respectively.By lipofection, by chimeric antibody The plasmid of heavy chain and light chain imports 293F cells, and continues culture 6 days.Antibody Protein A columns in cells and supernatant （GE Healthcare, catalog #17549112）Purified.Protein A columns are washed with 1 mM PBS, then with 50 The PBS of mM（pH3.0）Antibody is eluted from column.Eluent adjusts pH to neutrality with the sodium hydroxide solution of 0.5M, and uses 0.22 μm of membrane filtration.Antibody-solutions are through Ultra-15 centrifugal concentrators（Millipore, catalog #ACS500024）After concentration, quantitative antibody concentration is detected with Nanodrop spectrophotometers.In antibody purification solution Endotoxin content use Gel Clot TAL kit（Xiamen Bioendo Technology, catalog #010250） Detection, standard are less than 1 EU/mL for content.
Embodiment 3：People's mouse PDL1 chimeric antibody kinetic measurements
Use bio-molecular interaction system Octet-96（Pall Life Sciences, S-000959）, measurement antibody with The kinetic constant combined between antigen（k assoc And k dissoc ）, and further calculated equilibrium binding constant K D .By hPDL1-mFc Antigen protein is coupled to AMC sensors（Pall Life Sciences, PN18-5099）Surface, and add various concentrations and resist Body, measures the association and dissociation between PDL1 chimeric antibodies and sensor surface PDL1-mFc albumen.Specifically, AMC is sensed Device is in buffer solution（PBS containing 0.02% Tween-20 and 0.1% BSA）Middle pre-wetted 10 minutes, then hPDL1-mFc's Balanced 5 minutes in sample buffer so that PDL1-mFc is protein coupled to sensor surface.The AMC sensings of PDL1-mFc couplings Device balances 2 minutes first in buffer solution, is subsequently placed in containing various concentrations antibody（3-200 nM）Buffer solution in be incubated 5 altogether Minute, to measure the combination of antibody and PDL1-mFc albumen；Finally, the sensor for combining antigen and antibody is replaced in sample Buffer solution is savored, and is waited 10 minutes, to measure the dissociation of antibody and hPDL1-mFc albumen.Measure obtained data and use 1：1 mould Type carries out overall fit, to be combined and dissociation kinetics, and is further balanced binding constant on this basis K D .Experimental result is shown in Figure 10（Table II）.
Embodiment 4：The combination of people's mouse PDL1 chimeric antibodies and B16-hPDL1 cells
Human mouse chimeric antibody further detects the combination of itself and cell surface hPDL1 using FACS methods.B16-hPDL1 cells （Endogenous mouse PDL1 gene knockouts）Use FACS buffer solution（PBS contains 1% BSA）It is resuspended to 4x106Cell/mL, 50 μ L/ holes Add 96 hole round bottom plates（Corning, catalog #3795）.By antibody purification to be measured according in a certain concentration adding hole （50 μ L/ holes）, when 4 DEG C of incubations 1 are small.After the buffered liquid washing three times of cell, the fluorescence secondary antibody of PE marks, 4 DEG C of incubations are added 0.5 it is small when.Cell is washed three times with FACS buffer solution, is resuspended to 200 μ L/ holes, is used Attune Nxt flow cytometers （Thermo Fisher）Detect fluorescence signal.The results are shown in Figure 1, sees Figure 11 with reference to medium effective concentration（Table III）.
Embodiment 5：The combination of people's mouse PDL1 chimeric antibodies and activation T cell and DC cells
Human mouse chimeric antibody further detects itself and activation T cell and the knot of surface of dendritic cells hPDL1 using FACS methods Close.Use density-gradient centrifugation method（Ficoll-Paque Premium, GE Healthcare, catalog #17-5442- 02）, the separating periphery blood monocytic cell from human peripheral（PBMC）.People CD3+ T cell uses Pan T Cell Isolation Kit（Miltenyi, catalog #130-096-535）It is isolated from PBMC, and add the CD3/CD28 of equivalent Dynabeads（Invitrogen 11132D）Activate 48 it is small when.The T cell FACS buffer solution of activation（PBS contains 1% BSA）Weight Hang to 4x106Cell/mL, 50 μ L/ holes add 96 hole round bottom plates（Corning, catalog #3795）.By purifying to be measured Antibody is according in a certain concentration adding hole（50 μ L/ holes）, when 4 DEG C of incubations 1 are small.After the buffered liquid washing three times of cell, PE is added The fluorescence secondary antibody of mark, when 4 DEG C of incubations 0.5 are small.Cell is washed three times with FACS buffer solution, is resuspended to 200 μ L/ holes, is used Attune Nxt flow cytometers（Thermo Fisher）Detect fluorescence signal.The results are shown in Figure 2.
Use CD14 Cell Isolation Kit（Miltenyi, catalog #130-050-201）From PBMC into One step isolates and purifies to obtain monocyte（monocyte）.By adding 1000 U/mL GM-CSF of cell factor in the medium （Prospec, catalog #CYT-221）With 1000 U/mL IL-4（Prospec, catalog #CYT211）, with induction Monocyte is divided into dendritic cells（Dendritic cells）.Cell factor per 2-3 days, once, after 5-6 days received by supplement Obtain immature dendritic cells.Dendritic cells FACS buffer solution（PBS contains 1% BSA）It is resuspended to 4x106Cell/mL, 50 μ L/ Hole adds 96 hole round bottom plates（Corning, catalog #3795）.By antibody purification to be measured according in a certain concentration adding hole （50 μ L/ holes）, when 4 DEG C of incubations 1 are small.After the buffered liquid washing three times of cell, the fluorescence secondary antibody of PE marks, 4 DEG C of incubations are added 0.5 it is small when.Cell is washed three times with FACS buffer solution, is resuspended to 200 μ L/ holes, is used Attune Nxt flow cytometers （Thermo Fisher）Detect fluorescence signal.The results are shown in Figure 3.
Embodiment 6：People mouse PDL1 chimeric antibodies block the interaction between PD1 or CD80 and PDL1
The combination between hPD1 or CD80 and hPDL1 can be blocked using ELISA method detection antibody.Use Biotin Labeling Kit-NH2（Dojindo, catalog#LK03）Kit carries out biotin labeling to hPDL1-hFc albumen.Will HPD1-hFc or hCD80-hFc is coated with onto 96 orifice plates, and 4 DEG C overnight.Using 5% skimmed milk power when 37 DEG C of closings 2 are small.Board-washing After three times, the biotinylated hPDL1-hFc and PDL1 chimeric antibodies being pre-mixed are added, when incubation at room temperature 1 is small.Washed with PBST Plate, adds the Streptavidin of HRP marks, when incubation at room temperature 1 is small.After board-washing, TMB colour developings are added.As a result such as Fig. 4 a and 4b, knot Fruit, which is summarized, sees Figure 12（Table IV）.
Embodiment 7：The cross-species reaction of people's mouse PDL1 chimeric antibodies
The cross-species that people's mouse PDL1 chimeric antibodies and machin are detected using ELISA method are reacted.Will quantitative antibody purification bag By on 96 orifice plates, 4 DEG C overnight.Using 5% skimmed milk power when 37 DEG C of closings 2 are small.By the machin PDL1 of biotin labeling （SinoBiological, catalog # 90251-C08H-200）Add in plate, when incubation at room temperature 1 is small.With PBST board-washings three It is secondary, the Streptavidin of 1/1000 diluted HRP marks is added, when incubation at room temperature 1 is small.After board-washing, TMB colour developings are added.As a result Such as Fig. 5 a, and Figure 13（Table V）Shown in.
The cross-species that people's mouse PDL1 chimeric antibodies and mouse are detected using FACS methods are reacted.By wild type B16 cells FACS buffer solution is used after being digested with pancreatin（PBS contains 1% BSA）It is resuspended to 1.25x106Cell/mL.Take 80 μ L cell suspensions with The antibody mixing of 80 μ L gradient dilutions, room temperature are placed 30 minutes.After PBST washings three times, fluorescent marker secondary antibody is added （BioLegend, catalog #409304）, when 4 DEG C of incubations 1 are small.After PBST washings, Attune Nxt flow cytometers are used （Thermo Fisher）Detect fluorescence signal.As a result such as Fig. 5 b, and Figure 13（Table V）Shown in.
Embodiment 8：People mouse PDL1 chimeric antibodies strengthen the immune function of Jurkat cell
The cell biological of people's mouse PDL1 chimeric antibodies is detected using the Jurkat-PD1 cells containing luciferase-reporter Activity.In this cell experiment, Raji/hPDL1 cells are as antigen presenting cell, Jurkat/hPD1/NF κ B- Luciferase cells are as effector cell.Add the interaction that people's mouse PDL1 chimeric antibodies have blocked iuntercellular PD1/PDL1 It has activated Jurkat/hPD1/NF κ B-luciferase cells expression Luciferase reporter genes.Specifically, pass through The hPD1 of exogenous expression and the Luciferase reporter genes containing NF κ B promoters are imported in Jurkat cell；And The hPDL1 of exogenous expression is imported in Raji cells.Raji/hPDL1 cells and Jurkat/hPD1/NF κ B-luciferase are thin After born of the same parents' mixing, add the test antibodies of φt cell receptor activator and gradient dilution, when 6-16 is small after detected using luciferase Kit（Promega,G7940）Detection.The results show that people's mouse PDL1 chimeric antibodies can strengthen fluorescence signal, and agent is presented Amount relies on effect, and the result is shown in Fig. 6.
Embodiment 9：The allosome mixed lymphocytes experiment of people's mouse PDL1 chimeric antibodies（Mixed Lymphocyte Reaction, MLR）
Use the In vitro biological activity of mixed lymphocyte reaction (MLP) detection people's mouse PDL1 chimeric antibodies.Use density gradient centrifugation Method（Ficoll-Paque Premium, GE Healthcare, catalog #17-5442-02）, separated from human peripheral Peripheral blood mononuclear cells（PBMC）.Use CD14 Cell Isolation Kit（Miltenyi, catalog #130-050- 201）Further isolate and purify to obtain monocyte from PBMC（monocyte）.By adding cell factor in the medium 1000 U/mL GM-CSF （Prospec, catalog #CYT-221）With 1000 U/mL IL-4（Prospec, catalog #CYT211）, dendritic cells are divided into induced monocyte（Dendritic cells）.Cell factor supplement one per 2-3 days It is secondary, dendritic cells were harvested after 5-6 days and are used for follow-up experiment.
People CD3+T cell uses Pan T Cell Isolation Kit（Miltenyi, catalog #130-096- 535）It is isolated from PBMC.In 96 hole round bottom plates（Corning, catalog #3799）Middle addition CD3+T cell and not Ripe dendritic cells, and the PDL1 antibody of various concentrations.After cell culture 5-6 days, harvest supernatant and determined with ELISA method Amount detection cytokine content.As a result as Fig. 7 shows that people's mouse PDL1 chimeric antibodies can strengthen the secretion of IFN-γ in MLR.
Embodiment 10：People mouse PDL1 chimeric antibodies suppress tumour growth
Heretofore described animal model for tumour, is to pass through genetic modification by being inoculated with PDL1 Transgenic mouses Mouse colonic cell MC38/mPDL1KO/ hPDL1, and build in mouse internal test druggability PDL1 antibody bodies The specific tumor animal model of interior drug effect.
To PDL1 Transgenic mouses（7-11 week old）The 5 × 10 of 0.1 mL volumes are subcutaneously injected in bodyside face5It is a MC38/mPDL1KO/ hPDL1 tumour cells.After inoculated tumour cell 7 days, with 10 mg/kg dosage（10 mL/ of dose volume kg）The PDL1 antibody, reference antibody or control PBS are injected twice a week, carry out 6 administrations altogether.In experimentation, weekly Vernier caliper measurement subcutaneous tumor volumes are used twice.As shown in figure 8, after 6 administrations, test antibodies substantially suppress tumour Growth, Partial tumors almost disappear after drug-treated.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should It is considered as protection scope of the present invention.
Sequence table
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aggaactaac 1800
tgggacggga 1860
ggtacttcga 1920
tgtctggggc 1980
gcagggacca 2040
cggtcaccgt 2100
ctcctcag 394
<210> 6
<211> 339
<212> DNA
<213>Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.339)
<223>Light chain variable region DNA sequence dna
<400> 6
gacattgtga 60
tgtcacagtc 120
tccatcctcc 180
ctggctgtgt 240
cagcaggaga 300
gaaggtcact 360
atgagctgca 420
aatccagtca 480
gagtctgttc 540
aacagtggaa 600
cccgaaagaa 660
ctacttggct 720
tggtacctgc 780
agaaaccagg 840
gcagtctcct 900
aaactgctga 960
tctactgggc 1020
atccactagg 1080
gaatctgggg 1140
tccctgatcg 1200
cttcacaggc 1260
agtggatctg 1320
ggacagactt 1380
cactctcacc 1440
atcagcagtg 1500
tgcaggctga 1560
agacctggca 1620
gtttattact 1680
gcaagcaatc 1740
ttataatctc 1800
atgttcacgt 1860
tcggaggggg 1920
gaccaagctg 1980
gaaataaaa 373
<210> 7
<211> 355
<212> DNA
<213>Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.355)
<223>Heavy chain variable region DNA sequence dna
<400> 7
gaggtgcagc 60
ttcaggagtc 120
aggacctagc 180
ctcgtgaaac 240
cttctcagac 300
tctgtccctc 360
acctgttctg 420
tcactggcga 480
ctccatcacc 540
agtggttact 600
ggaactggat 660
ccggaaattc 720
ccagggaata 780
aacttgagta 840
catggggttc 900
ataagctaca 960
ctggtagcac 1020
ttactacaat 1080
ccatctctca 1140
aaagtcgaat 1200
ctccatcact 1260
cgagacacat 1320
ccaagaacca 1380
gtactacctg 1440
cagttgaatt 1500
ctgtgactac 1560
tgaggacaca 1620
gccacatatt 1680
actgtgcaag 1740
catggcggga 1800
tggttagcct 1860
ggtttgctta 1920
ctggggccaa 1980
gggactctgg 2040
tcactgtctc 2100
tgcag 391
<210> 8
<211> 339
<212> DNA
<213>Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.339)
<223>Light chain variable region DNA sequence dna
<400> 8
gacattgtga 60
tgtcacagtc 120
tccatcctcc 180
ctagctgtgt 240
cagttggaga 300
gaaggttact 360
atgagctgca 420
agtccagtca 480
gagcctttta 540
tatagtagca 600
atcaaaagaa 660
ctccttggcc 720
tggtaccagc 780
agaaaccagg 840
gcagtctcct 900
aaactgctga 960
tttactgggc 1020
atccactagg 1080
gaatctgggg 1140
tccctgatcg 1200
cttcacaggc 1260
agtggatctg 1320
ggacagattt 1380
cactctcacc 1440
atcagcagtg 1500
tgaaggctga 1560
agacctggca 1620
gtttattact 1680
gtcagcaata 1740
ttatagctat 1800
ccgtggacgt 1860
tcggtggagg 1920
caccaagctg 1980
gaaatcaaa 373
<210> 9
<211> 355
<212> DNA
<213>Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.355)
<223>Heavy chain variable region DNA sequence dna
<400> 9
gaggtgcagc 60
ttcaggagtc 120
aggacctagc 180
ctcgtgaaac 240
cttctcagac 300
tctgtccctc 360
acctgttctg 420
tcactggcga 480
ctccatcacc 540
agtggttact 600
ggcactggat 660
ccggaaattc 720
cccgggaata 780
aacttgagta 840
catggggtac 900
ataagctaca 960
gtgggagcac 1020
ttacttcatt 1080
ccatctctca 1140
aaagtcgaat 1200
ctccatcact 1260
cgagacacat 1320
ccaagaacca 1380
gtactacctg 1440
cagttgaatt 1500
ctgtgactac 1560
tgaggacaca 1620
gccacatatt 1680
actgtgcaag 1740
atcaggggga 1800
tggttactgc 1860
attttgctta 1920
ctggggccaa 1980
gggactctgg 2040
tcactgtctc 2100
tgcag 391
<210> 10
<211> 339
<212> DNA
<213>Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.339)
<223>Light chain variable region DNA sequence dna
<400> 10
gacgttgtga 60
tgtcacagtc 120
tccatcctcc 180
ctagctgtgt 240
cagttggaga 300
gaaggttact 360
atgagctgca 420
agtccagtca 480
gagcctttta 540
tatagtagca 600
atcaaaagaa 660
ctccttggcc 720
tggtaccagc 780
agaaaccagg 840
gcagtctcct 900
aaactgctga 960
tttactgggc 1020
atccactagg 1080
gaatctgggg 1140
tccctgatcg 1200
cttcacaggc 1260
agtggatctg 1320
ggacagattt 1380
cactctcacc 1440
atcagcaatg 1500
tgaagactga 1560
agacctggca 1620
gtttattact 1680
gtcagcaata 1740
ttatggctat 1800
ccttacacgt 1860
tcggaggggg 1920
gaccaagctg 1980
gaaataaaa 373
<210> 11
<211> 349
<212> DNA
<213>Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.349)
<223>Heavy chain variable region DNA sequence dna
<400> 11
gaagtgatgc 60
tggtggagtc 120
tgggggcggc 180
ttagtgaagc 240
ctggagggtc 300
cctgaaactc 360
tcctgtgcag 420
tctctggatt 480
cactttcagt 540
agctatgcca 600
tgtcttgggt 660
tcgccagact 720
ccggaaaaga 780
ggctggagtg 840
ggtcgcaacc 900
attactagtg 960
atggtcatta 1020
cacctactat 1080
ccagacaatg 1140
tgaaggggcg 1200
attcaccatc 1260
tccagagaca 1320
atgccaagaa 1380
caccctctac 1440
ctgcaaatga 1500
gcagtctgag 1560
gtctgaggac 1620
acggccatgt 1680
attactgtac 1740
aagacgtacg 1800
aactactttg 1860
actactgggg 1920
ccaaggcacc 1980
actctcacag 2040
tctcctcag 384
<210> 12
<211> 315
<212> DNA
<213>Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.315)
<223>Light chain variable region DNA sequence dna
<400> 12
caaattgttc 60
tcacccagtc 120
tccagcactc 180
atgtctgcat 240
atccagggga 300
gaaggtcacc 360
atgacctgca 420
gtgccaactc 480
aagtgttact 540
tacatgtatt 600
ggtaccagca 660
gaagccaaga 720
tcctccccca 780
aaccctggat 840
ttttctcaca 900
tccaacctgg 960
cttctggagt 1020
ccctgctcgc 1080
ttcagtggca 1140
gtgggtctgg 1200
gacctcttac 1260
tctctcacaa 1320
tcagcagcat 1380
ggaggctgaa 1440
gatgctgcca 1500
cttattactg 1560
ccagcagtgg 1620
agaagtaacc 1680
cgacgttcgg 1740
tggaggcacc 1800
aagctggaaa 1860
tcaaa 347
<210> 13
<211> 358
<212> DNA
<213>Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.358)
<223>Heavy chain variable region DNA sequence dna
<400> 13
gaggtgcagc 60
ttcaggagtc 120
aggacctagc 180
ctcgtgaaac 240
cttctcagac 300
tctgtccctc 360
acctgctctg 420
tcactggcga 480
ctccatcacc 540
agtggttact 600
ggaactggat 660
ccggaaattc 720
ccagggaata 780
aacttgagta 840
catggggtac 900
ataagctaca 960
ctggtagcac 1020
ttactacaat 1080
ccatctctca 1140
aaagtcgaat 1200
ctccatcact 1260
cgagacacat 1320
ccaagaacca 1380
gtactacctg 1440
cagttgaatt 1500
ctgtgactac 1560
tgaggacaca 1620
gccacatatt 1680
actgtgcaag 1740
atatagagac 1800
tgggtcgtcg 1860
gctactttga 1920
ctactggggc 1980
caaggcacca 2040
ctctcacagt 2100
ctcctcag 394
<210> 14
<211> 321
<212> DNA
<213>Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.321)
<223>Light chain variable region DNA sequence dna
<400> 14
gactttgtga 60
tgacccagtc 120
tcaaaaattc 180
atgtccacat 240
cagtgggaga 300
cagggtcagc 360
gtcacctgca 420
aggccagtca 480
gaatgtgggt 540
actaatgtag 600
cctggtatca 660
acagaaacca 720
ggacaatctc 780
ctaaagcact 840
gatttactcg 900
gcatcctacc 960
ggtacagtgg 1020
agtccctgat 1080
cgcttcacag 1140
gcagtggatc 1200
tgggacagat 1260
ttcactctca 1320
ccatcagcaa 1380
tgtgcagtct 1440
gaagacttgg 1500
cagaatattt 1560
ctgtcagcaa 1620
tataacagct 1680
atcctctcac 1740
gttcggctcg 1800
gggacaaagt 1860
tggaaataaa 1920
a 354
<210> 15
<211> 355
<212> DNA
<213>Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.355)
<223>Heavy chain variable region DNA sequence dna
<400> 15
gaggtgcagc 60
ttcaggagtc 120
aggacctagc 180
ctcgtgaaac 240
cttctcagac 300
tctgtccctc 360
acctgttctg 420
tcactggcga 480
ctccatcacc 540
agtggttact 600
ggaactggat 660
ccggaaattc 720
ccagggaata 780
aacttgagta 840
catggggtac 900
ataagctaca 960
ctggtagtac 1020
ttactacaat 1080
ccatctctca 1140
aaagtcgaat 1200
ctccatcact 1260
cgagacactt 1320
ccaagaacca 1380
gtactacctg 1440
cagttgaatt 1500
ctgtgactgc 1560
tgaggacaca 1620
gccacatatt 1680
actgtgcaag 1740
aaggggggga 1800
tggttactgc 1860
cttttgacta 1920
ctggggccaa 1980
ggcaccactc 2040
tcacagtctc 2100
ctcag 391
<210> 16
<211> 339
<212> DNA
<213>Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.339)
<223>Light chain variable region DNA sequence dna
<400> 16
gacattgtga 60
tgacacagtc 120
tccatcctcc 180
ctgactgtga 240
cagcaggaga 300
gaaggtcact 360
atgagctgca 420
agtccagtca 480
gagtctgtta 540
aacagtggaa 600
atcaaaagaa 660
ctgcttgacc 720
tggtaccagc 780
agaaaccagg 840
gcagcctcct 900
aaactgttga 960
tctcctgggc 1020
ttccactagg 1080
gaatctgggg 1140
tccctgatcg 1200
cttcacaggc 1260
agtggatctg 1320
gaacagattt 1380
cactctcacc 1440
atcagcagtg 1500
tgcaggctga 1560
agacctggca 1620
gtttattact 1680
gtcagaatga 1740
ttatggttat 1800
ccgctcacgt 1860
tcggtgctgg 1920
gaccaagctg 1980
gagctgaaa 373
<210> 17
<211> 367
<212> DNA
<213>Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.367)
<223>Heavy chain variable region DNA sequence dna
<400> 17
gaggttcagc 60
tgcagcagtc 120
tggggcagag 180
cttgtgaagc 240
caggggcctc 300
agtcaaattg 360
tcctgcacag 420
cttctggctt 480
caacattaaa 540
gacacctata 600
tgtactgggt 660
gaagcagagg 720
cctgaacagg 780
gcctggagtg 840
gattggaagg 900
attgatcctg 960
cgaatggtaa 1020
tactaaatat 1080
gacccgaagt 1140
tccagggcaa 1200
ggccactata 1260
acagcagaca 1320
catcctccaa 1380
cacagccttc 1440
ttgcagctca 1500
gcagcctgac 1560
atctgaggac 1620
actgccgtct 1680
attactgtgc 1740
taggagggga 1800
ttatttttta 1860
ctacggtaac 1920
agctattgac 1980
tactggggcc 2040
aaggcaccac 2100
tctcacagtc 2160
tcctcag 404
<210> 18
<211> 321
<212> DNA
<213>Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.321)
<223>Light chain variable region DNA sequence dna
<400> 18
gacattgtga 60
tgacccagtc 120
tcacaaattc 180
atgtccacat 240
cagtaggaga 300
cagggtcagc 360
attacctgca 420
aggccagtca 480
ggatgtgagt 540
actgctgtag 600
cctggtatca 660
acaaaaacca 720
gggcaatctc 780
ctaaactact 840
gatttactgg 900
gcatccaccc 960
ggcacactgg 1020
agtccctgat 1080
cgcttcacag 1140
gcagtggatc 1200
tgggacagat 1260
tattctctca 1320
ccatcagcag 1380
tgtgcagtct 1440
gaagacctga 1500
cactttatta 1560
ctgtcagcaa 1620
cattatgaca 1680
ctccgtggac 1740
gttcggtgga 1800
ggcaccaagc 1860
tggaaatcaa 1920
a 354
<210> 19
<211> 115
<212> PRT
<213>Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.115)
<223>Heavy chain variable amino acid sequence
<400> 19
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Leu Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr
20 25 30
Trp Leu Glu Trp Leu Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Leu Pro Gly Ser Gly Asn Pro Asn Tyr Asn Glu Asn Phe
50 55 60
Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Val Tyr
65 70 75 80
Met Gln Leu Ile Ser Leu Ile Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Arg Ala Met Asp Tyr Trp Gly His Gly Thr Ser Val Thr
100 105 110
Val Ser Ser
115
<210> 20
<211> 107
<212> PRT
<213>Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.107)
<223>Chain variable region amino acid sequence
<400> 20
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 21
<211> 119
<212> PRT
<213>Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.119)
<223>Heavy chain variable amino acid sequence
<400> 21
Glu Val Lys Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Arg Tyr
20 25 30
Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Gln Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Thr Pro Ser Leu
50 55 60
Lys Asp Tyr Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Thr Lys Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Ser Pro Asp Tyr Tyr Gly Ser Ser Leu Pro Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala
115
<210> 22
<211> 112
<212> PRT
<213>Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.112)
<223>Chain variable region amino acid sequence
<400> 22
Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Thr Ile Val His Ser
20 25 30
Asn Ala Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Phe Lys Val Ser Asn Arg Phe Ala Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly
85 90 95
Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 23
<211> 119
<212> PRT
<213>Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.119)
<223>Heavy chain variable amino acid sequence
<400> 23
Glu Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Thr Ser Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Asp Tyr Met
35 40 45
Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu
65 70 75 80
Gln Leu Asn Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Tyr Cys Ala
85 90 95
Arg Gly Thr Asn Trp Asp Gly Arg Tyr Phe Asp Val Trp Gly Ala Gly
100 105 110
Thr Thr Val Thr Val Ser Ser
115
<210> 24
<211> 113
<212> PRT
<213>Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.113)
<223>Chain variable region amino acid sequence
<400> 24
Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser
20 25 30
Gly Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Leu Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln
85 90 95
Ser Tyr Asn Leu Met Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 25
<211> 118
<212> PRT
<213>Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.118)
<223>Heavy chain variable amino acid sequence
<400> 25
Glu Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Thr Ser Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Lys Leu Glu Tyr Met
35 40 45
Gly Phe Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu
65 70 75 80
Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala
85 90 95
Ser Met Ala Gly Trp Leu Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ala
115
<210> 26
<211> 113
<212> PRT
<213>Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.113)
<223>Chain variable region amino acid sequence
<400> 26
Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30
Ser Asn Gln Lys Asn Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Ser Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 27
<211> 118
<212> PRT
<213>Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.118)
<223>Heavy chain variable amino acid sequence
<400> 27
Glu Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Thr Ser Gly
20 25 30
Tyr Trp His Trp Ile Arg Lys Phe Pro Gly Asn Lys Leu Glu Tyr Met
35 40 45
Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Tyr Phe Ile Pro Ser Leu Lys
50 55 60
Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu
65 70 75 80
Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala
85 90 95
Arg Ser Gly Gly Trp Leu Leu His Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ala
115
<210> 28
<211> 113
<212> PRT
<213>Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.113)
<223>Chain variable region amino acid sequence
<400> 28
Asp Val Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30
Ser Asn Gln Lys Asn Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Asn Val Lys Thr Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Gly Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 29
<211> 116
<212> PRT
<213>Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.116)
<223>Heavy chain variable amino acid sequence
<400> 29
Glu Val Met Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val
35 40 45
Ala Thr Ile Thr Ser Asp Gly His Tyr Thr Tyr Tyr Pro Asp Asn Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Thr Arg Arg Thr Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu
100 105 110
Thr Val Ser Ser
115
<210> 30
<211> 105
<212> PRT
<213>Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.105)
<223>Chain variable region amino acid sequence
<400> 30
Gln Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Tyr Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser Ala Asn Ser Ser Val Thr Tyr Met
20 25 30
Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Phe
35 40 45
Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Arg Ser Asn Pro Thr Phe
85 90 95
Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 31
<211> 119
<212> PRT
<213>Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.119)
<223>Heavy chain variable amino acid sequence
<400> 31
Glu Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Thr Ser Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Lys Leu Glu Tyr Met
35 40 45
Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu
65 70 75 80
Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala
85 90 95
Arg Tyr Arg Asp Trp Val Val Gly Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Leu Thr Val Ser Ser
115
<210> 32
<211> 113
<212> PRT
<213>Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.107)
<223>Chain variable region amino acid sequence
<400> 32
Asp Phe Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser
65 70 75 80
Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu
85 90 95
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 33
<211> 118
<212> PRT
<213>Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.118)
<223>Heavy chain variable amino acid sequence
<400> 33
Glu Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Thr Ser Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Lys Leu Glu Tyr Met
35 40 45
Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu
65 70 75 80
Gln Leu Asn Ser Val Thr Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala
85 90 95
Arg Arg Gly Gly Trp Leu Leu Pro Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Thr Leu Thr Val Ser Ser
115
<210> 34
<211> 164
<212> PRT
<213>Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.164)
<223>Chain variable region amino acid sequence
<400> 34
Glu Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln
1 5 10 15
Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Thr Ser Gly
20 25 30
Tyr Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Lys Leu Glu Tyr Met
35 40 45
Gly Tyr Ile Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val
50 55 60
Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu
65 70 75 80
Leu Asn Ser Gly Asn Gln Lys Asn Cys Leu Thr Trp Tyr Gln Gln Lys
85 90 95
Pro Gly Gln Pro Pro Lys Leu Leu Ile Ser Trp Ala Ser Thr Arg Glu
100 105 110
Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe
115 120 125
Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr
130 135 140
Cys Gln Asn Asp Tyr Gly Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys
145 150 155 160
Leu Glu Leu Lys
<210> 35
<211> 122
<212> PRT
<213>Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.122)
<223>Heavy chain variable amino acid sequence
<400> 35
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Met Tyr Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Phe
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Gly Leu Phe Phe Thr Thr Val Thr Ala Ile Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
115 120
<210> 36
<211> 107
<212> PRT
<213>Artificial sequence ()
<220>
<221> V_region
<222> (1)..(.107)
<223>Chain variable region amino acid sequence
<400> 36
Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Ser Val Gln Ser
65 70 75 80
Glu Asp Leu Thr Leu Tyr Tyr Cys Gln Gln His Tyr Asp Thr Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 37
<211> 10
<212> PRT
<213>Artificial sequence ()
<220>
<221> misc_feature

Claims (21)

1. separated people PDL1 specific binding molecules, include a）And b）,
a）Three light chain CDR：Light chain CDR1, light chain CDR2 and light chain CDR3,
b）Three heavy chain CDR：Heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3；
The separated people PDL1 specific binding molecules are separated antibody or antigen-binding fragment；
It is characterized in that：
Heavy chain CDR1 is selected from：SEQ ID NO: 37、SEQ ID NO: 43、SEQ ID NO: 49、SEQ ID NO: 55、SEQ ID NO: 61、SEQ ID NO: 67、SEQ ID NO: 73、SEQ ID NO:79 or SEQ ID NO:One kind in 85；
Heavy chain CDR2 is selected from：SEQ ID NO: 38、SEQ ID NO: 44、SEQ ID NO: 50、SEQ ID NO: 56、SEQ ID NO: 62、SEQ ID NO: 68、SEQ ID NO: 74、SEQ ID NO:80 or SEQ ID NO:One kind in 86；
Heavy chain CDR3 is selected from：SEQ ID NO: 39、SEQ ID NO: 45、SEQ ID NO: 51、SEQ ID NO: 57、SEQ ID NO: 63、SEQ ID NO: 69、SEQ ID NO: 75、SEQ ID NO:81 or SEQ ID NO:One kind in 87；
Light chain CDR1 is selected from：SEQ ID NO: 40、SEQ ID NO: 46、SEQ ID NO: 52、SEQ ID NO: 58、SEQ ID NO: 64、SEQ ID NO: 70、SEQ ID NO: 76、SEQ ID NO:82 or SEQ ID NO:One kind in 88；
Light chain CDR2 is selected from：SEQ ID NO: 41、SEQ ID NO: 47、SEQ ID NO: 53、SEQ ID NO: 59、SEQ ID NO: 65、SEQ ID NO: 71、SEQ ID NO: 77、SEQ ID NO: 83、SEQ ID NO:9 or SEQ ID NO: One kind in 89；
Light chain CDR3 is selected from：SEQ ID NO: 42、SEQ ID NO: 48、SEQ ID NO: 54、SEQ ID NO: 60、SEQ ID NO: 66、SEQ ID NO: 72、SEQ ID NO: 78、SEQ ID NO:84 or SEQ ID NO:One kind in 90.
2. separated people PDL1 specific binding molecules according to claim 1, it is characterised in that：The specificity knot Close molecule and include heavy chain variable region and light chain variable region, wherein：
Heavy chain variable region includes such as SEQ ID NO：Heavy chain CDR1, such as SEQ ID NO shown in 37：Heavy chain shown in 38 CDR2 and such as SEQ ID NO：Heavy chain CDR3 shown in 39；And/or light chain variable region includes such as SEQ ID NO：Shown in 40 Light chain CDR1, such as SEQ ID NO：Light chain CDR2 and such as SEQ ID NO shown in 41：Light chain CDR3 shown in 42；Or Person
Heavy chain variable region includes such as SEQ ID NO：Heavy chain CDR1, such as SEQ ID NO shown in 43：Heavy chain shown in 44 CDR2 and such as SEQ ID NO：Heavy chain CDR3 shown in 45；And/or light chain variable region includes such as SEQ ID NO：Shown in 46 Light chain CDR1, such as SEQ ID NO：Light chain CDR2 and such as SEQ ID NO shown in 47：Light chain CDR3 shown in 48；Or Person
Heavy chain variable region includes such as SEQ ID NO：Heavy chain CDR1, such as SEQ ID NO shown in 49：Heavy chain shown in 50 CDR2 and such as SEQ ID NO：Heavy chain CDR3 shown in 51；And/or light chain variable region includes such as SEQ ID NO：Shown in 52 Light chain CDR1, such as SEQ ID NO：Light chain CDR2 and such as SEQ ID NO shown in 53：Light chain CDR3 shown in 54；Or Person
Heavy chain variable region includes such as SEQ ID NO：Heavy chain CDR1, such as SEQ ID NO shown in 55：Heavy chain shown in 56 CDR2 and such as SEQ ID NO：Heavy chain CDR3 shown in 57；And/or light chain variable region includes such as SEQ ID NO：Shown in 58 Light chain CDR1, such as SEQ ID NO：Light chain CDR2 and such as SEQ ID NO shown in 593：Light chain CDR3 shown in 60；Or Person
Heavy chain variable region includes such as SEQ ID NO：Heavy chain CDR1, such as SEQ ID NO shown in 61：Heavy chain shown in 62 CDR2 and such as SEQ ID NO：Heavy chain CDR3 shown in 63；And/or light chain variable region includes such as SEQ ID NO：Shown in 64 Light chain CDR1, such as SEQ ID NO：Light chain CDR2 and such as SEQ ID NO shown in 65：Light chain CDR3 shown in 66；Or Person
Heavy chain variable region includes such as SEQ ID NO：Heavy chain CDR1, such as SEQ ID NO shown in 67：Heavy chain shown in 68 CDR2 and such as SEQ ID NO：Heavy chain CDR3 shown in 69；And/or light chain variable region includes such as SEQ ID NO：Shown in 70 Light chain CDR1, such as SEQ ID NO：Light chain CDR2 and such as SEQ ID NO shown in 71：Light chain CDR3 shown in 72；Or Person
Heavy chain variable region includes such as SEQ ID NO：Heavy chain CDR1, such as SEQ ID NO shown in 73：Heavy chain shown in 74 CDR2 and such as SEQ ID NO：Heavy chain CDR3 shown in 75；And/or light chain variable region includes such as SEQ ID NO：Shown in 76 Light chain CDR1, such as SEQ ID NO：Light chain CDR2 and such as SEQ ID NO shown in 77：Light chain CDR3 shown in 78；Or Person
Heavy chain variable region includes such as SEQ ID NO：Heavy chain CDR1, such as SEQ ID NO shown in 79：Heavy chain shown in 80 CDR2 and such as SEQ ID NO：Heavy chain CDR3 shown in 81；And/or light chain variable region includes such as SEQ ID NO：Shown in 82 Light chain CDR1, such as SEQ ID NO：Light chain CDR2 and such as SEQ ID NO shown in 83：Light chain CDR3 shown in 84；Or Person
Heavy chain variable region includes such as SEQ ID NO：Heavy chain CDR1, such as SEQ ID NO shown in 85：Heavy chain shown in 86 CDR2 and such as SEQ ID NO：Heavy chain CDR3 shown in 87；And/or light chain variable region includes such as SEQ ID NO：Shown in 88 Light chain CDR1, such as SEQ ID NO：Light chain CDR2 and such as SEQ ID NO shown in 89：Light chain CDR3 shown in 90.
3. separated people PDL1 specific binding molecules according to claim 2, it is characterised in that：The antibody is total length Antibody.
4. separated people PDL1 specific binding molecules according to claim 2, it is characterised in that：The antigen binding fragment Section is Fab or F (ab) '2Or scFv.
5. separated people PDL1 specific binding molecules according to claim 1, it is characterised in that：The specificity knot Close molecule and include light chain variable region and heavy chain variable region, wherein：
The heavy chain variable region is selected from：SEQ ID NO: 19、SEQ ID NO: 21、SEQ ID NO: 23、SEQ ID NO: 25；SEQ ID NO: 27、SEQ ID NO: 29、SEQ ID NO: 31、SEQ ID NO:33 or SEQ ID NO:In 35 It is a kind of；
The light chain variable region is selected from：SEQ ID NO: 20、SEQ ID NO: 22、SEQ ID NO: 24、SEQ ID NO: 26；SEQ ID NO: 28、SEQ ID NO: 30、SEQ ID NO: 32、SEQ ID NO:34 or SEQ ID NO:In 36 It is a kind of.
6. separated people PDL1 specific binding molecules according to claim 1, it is characterised in that：The specificity knot Close molecule and include light chain variable region and heavy chain variable region, wherein the sequence of light chain variable region is with sequence（X）At least 80% is homologous Property；The sequence of heavy chain variable region is with sequence（Y）At least 80% homology；The sequence（Y）It is selected from：SEQ ID NO: 19、SEQ ID NO: 21、SEQ ID NO: 23、SEQ ID NO: 25；SEQ ID NO: 27、SEQ ID NO: 29、SEQ ID NO: 31、SEQ ID NO:33 or SEQ ID NO:One kind in 35；The sequence（X）It is selected from：SEQ ID NO: 20、SEQ ID NO: 22、SEQ ID NO: 24、SEQ ID NO: 26；SEQ ID NO: 28、SEQ ID NO: 30、SEQ ID NO: 32、 SEQ ID NO:34 or SEQ ID NO:One kind in 36.
7. separated people PDL1 specific binding molecules according to claim 1, it is characterised in that：The specificity knot Close molecule and include light chain variable region and heavy chain variable region, wherein：
The heavy chain variable region is SEQ ID NO:19 and the light chain variable region be SEQ ID NO: 20；Or
The heavy chain variable region is SEQ ID NO:21 and the light chain variable region be SEQ ID NO: 22；Or
The heavy chain variable region is SEQ ID NO:23 and the light chain variable region be SEQ ID NO: 24；Or
The heavy chain variable region is SEQ ID NO:25 and the light chain variable region be SEQ ID NO: 26；Or
The heavy chain variable region is SEQ ID NO:27 and the light chain variable region be SEQ ID NO: 28；Or
The heavy chain variable region is SEQ ID NO:29 and the light chain variable region be SEQ ID NO: 30；Or
The heavy chain variable region is SEQ ID NO:31 and the light chain variable region be SEQ ID NO: 32；Or
The heavy chain variable region is SEQ ID NO:33 and the light chain variable region be SEQ ID NO: 34；Or
The heavy chain variable region is SEQ ID NO:35 and the light chain variable region be SEQ ID NO: 36.
8. separated people PDL1 specific binding molecules according to claim 1, it is characterised in that：The specific binding Molecule at least shows a kind of following property：
Block the combination of PDL1 and PD1 or CD80；
With reference to the PDL1 on human T-cell surface；
With 3.89 × 10-10 M or lower KDCombined with people PDL1；
IFN-γ is improved in mixed lymphocyte reaction (MLP) (MLR) experiment to produce.
9. prepared according to the separated people PDL1 specific binding molecules of claim 1-7 any one of them for suppressing sufferer Purposes in the medicine of tumor cell growth in vivo.
10. prepared according to the separated people PDL1 specific binding molecules of claim 1-7 any one of them for treating biography The purposes caught an illness in medicine.
11. separated nucleic acid, one or both of encoding antibody light variable region and heavy chain of antibody variable region, its feature exist In：
The fragment of encoding said antibody heavy chain variable region is selected from：SEQ ID NO: 1、SEQ ID NO: 3、SEQ ID NO: 5、 SEQ ID NO: 7、SEQ ID NO: 9、SEQ ID NO: 11、SEQ ID NO: 13、SEQ ID NO:15 or SEQ ID NO: 17；
The fragment of encoding said antibody light chain variable region is selected from：SEQ ID NO: 2、SEQ ID NO: 4、SEQ ID NO: 6、 SEQ ID NO: 8、SEQ ID NO: 10、SEQ ID NO: 12、SEQ ID NO: 14、SEQ ID NO:16 or SEQ ID NO: 18。
12. separated nucleic acid, one or both of encoding antibody light variable region and heavy chain of antibody variable region, its feature exist In：The nucleic acid is the fragment with encoding said antibody heavy chain variable region（X）Shown sequence and encoding said antibody weight chain variable The fragment in area（Y）The sequence of shown sequence at least 80% homology；The fragment（X）For：SEQ ID NO: 1、SEQ ID NO: 3、SEQ ID NO: 5、SEQ ID NO: 7、SEQ ID NO: 9、SEQ ID NO: 11、SEQ ID NO: 13、SEQ ID NO:15 or SEQ ID NO: 17；The fragment（Y）For：SEQ ID NO: 2、SEQ ID NO: 4、SEQ ID NO: 6、SEQ ID NO: 8、SEQ ID NO: 10、SEQ ID NO: 12、SEQ ID NO: 14、SEQ ID NO:16 or SEQ ID NO: 18。
A kind of 13. carrier, it is characterised in that：The carrier contains such as 11 or 12 any one of them nucleic acid molecules of claim.
A kind of 14. host cell, it is characterised in that：The host cell contains such as 11 or 12 any one of them core of claim Acid molecule, or
Contain carrier as claimed in claim 13.
A kind of 15. conjugate, it is characterised in that：The conjugate includes covalent with isotope, immunotoxin and/or chemicals The anti-human PDL1 monoclonal antibodies of connection；The anti-human PDL1 monoclonal antibodies are such as claim 1-7 any one of them point From people's PDL1 specific binding molecules.
A kind of 16. conjugate, it is characterised in that：It is resisted by anti-human PDL1 monoclonals according to any one of claims 1 to 7 Conjugate described in body and/or claim 15 is coupled to be formed with solid dielectric or semi-solid medium.
17. preparing treatment disease according to the separated people PDL1 specific binding molecules of claim 1-7 any one of them Application in medicine；The separated people PDL1 specific binding molecules are anti-PDL1 monoclonal antibodies；The disease is breast Gland cancer, lung cancer, stomach cancer, intestinal cancer, the cancer of the esophagus, oophoroma, cervical carcinoma, kidney, carcinoma of urinary bladder, cancer of pancreas, glioma or melanocyte Knurl.
18. application of the conjugate according to claim 15 in the medicine for preparing treatment disease；The disease is mammary gland Cancer, lung cancer, stomach cancer, intestinal cancer, the cancer of the esophagus, oophoroma, cervical carcinoma, kidney, carcinoma of urinary bladder, cancer of pancreas, glioma or melanoma.
19. application of the conjugate according to claim 16 in the medicine for preparing treatment disease；The disease is mammary gland Cancer, lung cancer, stomach cancer, intestinal cancer, the cancer of the esophagus, oophoroma, cervical carcinoma, kidney, carcinoma of urinary bladder, cancer of pancreas, glioma or melanoma.
A kind of 20. composition, it is characterised in that：The composition contains main matter（M）And auxiliary substance（N）；It is described main Material（M）Selected from the separated people PDL1 specific binding molecules of claim 1-7 any one of them, 11 or 12 institute of claim Carrier described in the separated nucleic acid stated, claim 13, the host cell described in claim 14, described in claim 15 The one or more in conjugate described in conjugate or claim 16；The auxiliary substance（N）Selected from pharmaceutically acceptable Carrier or excipient, and optional other bioactive substances.
A kind of 21. kit, it is characterised in that：The kit includes such as the separated people of claim 1-7 any one of them PDL1 specific binding molecules and for detect combined with the molecular specificity after the reagent of immune complex that is formed.
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