CN105777906A

CN105777906A - Anti-PD - L1 human antibody and application thereof

Info

Publication number: CN105777906A
Application number: CN201410798817.8A
Authority: CN
Inventors: 栾彦; 王敏; 彭建建; 马树立; 马慧; 李香; 傅士龙; 秦颂兵; 汪皛皛; 徐霆
Original assignee: Suzhou Ding Fu Target Spot Bioisystech Co Ltd
Current assignee: Suzhou Ding Fu Target Spot Bioisystech Co Ltd
Priority date: 2014-12-19
Filing date: 2014-12-19
Publication date: 2016-07-20
Anticipated expiration: 2034-12-19
Also published as: CN105777906B

Abstract

The present invention relates to anti-PD - L1 human antibody and application thereof, in particular, human antibody capable of specifically binding PD-L1 can be screened by yeast display technology, and the affinity of the antibody can be further improved by affinity maturation. The anti-PD - L1 human antibody has good specificity, high affinity and good stability, T cell activation effect can be enhanced by binding with activated T cell, and the anti-PD - L1 human antibody has significant tumor growth inhibition effect. The present invention further relates to application of the anti-PD - L1 human antibody in diagnosis and treatment of PD-L1-associated tumors.

Description

Anti-PD-L1 human antibody and application thereof

Technical field

The invention belongs to field of pharmaceutical biology, relate to anti-PD-L1 human antibody and medical usage thereof.

Background technology

T cell needs antigen presenting cell APC to provide two signals to static T lymphocyte when exogenous antigen is responded: first signal is: T cell identifies the antigenic peptides being combined with MHC molecule by TCR, transmits antigen recognition signal by TCR/CD3 complex；Secondary signal is: provided by a series of costimulatory molecules；Such T cell could activate normally, thus producing normal immunne response.Effect is produced different according to secondary signal, costimulatory molecules can be divided into positivity costimulatory molecules and negativity costimulatory molecules, and the adjustment of positivity and negativity costimulatory signal and balance between the two play important adjustment effect in the whole process of immune response.

PD-1 is a member of CD28 receptor family, and this family also includes CTLA4, CD28, ICOS and BTLA.Initial member CD28 and the ICOS of this family finds (Hutloff etc. (1999) Nature397:263-266 by increasing the function of T cell propagation after adding monoclonal antibody；Hansen etc. (1980) Immunogenics10:247-260).The part of PD-1 includes PD-L1 and PD-L2, and existing result of study shows, receptor and part can lower the activation of T cell and secretion (Freeman etc. (2000) JExpMed192:1027-34 of relevant cell factor after combining；Latchman etc. (2001) NatImmunol2:261-8；Carter etc. (2002) EurJImmunol32:634-43；Ohigashi etc. (2005) ClincancerRes11:2947-53).

PD-L1 (B7-H1) is cell surface glycoprotein, belongs to B7 family, has IgV and IgC sample district, cross-film district and cytoplasmic domain afterbody.This gene was found first in 1999 and clones (DongH etc. (1999) NatMed5:1365-1369), and it interacts with the receptor PD1 in T cell, plays an important role in the negativity regulation and control of immunne response.PD-L1 is except with the PD-1 effect of expression in T cell, and the PD-L1 expressed in T cell can interact with the CD80 on APC and conduct negative-going signal, it is suppressed that the function of T cell.PD-L1 except expressing on the cell of macrophage lineage, in the mankind organize normally, expression is relatively low, but fasten at some tumor cells and but have higher expression, such as: pulmonary carcinoma, ovarian cancer, colon cancer and melanoma (Iwai etc. (2002) PNAS99:12293-7；Ohigashi etc. (2005) ClinCancerRes11:2947-53).Existing result shows, the PD-L1 of tumor cell high expressed passes through the apoptosis increasing T cell thus playing an important role in the immunologic escape of tumor.Researcher finds, the P815 tumor cell line of transfection PD-L1 gene can resist the cracking of specific CTL in vitro, has higher oncogenicity and aggressive after in its Mice Inoculated body.These biological characteristicses all can reverse by blocking PD-L1.Knock out the mice of PD1 gene, block PD-L1/PD-1 path, then inoculated tumour cell can not form tumor (DongH etc. (2002) NatMed8:793-800).

This area remains a need for being combined and can block PD-1 and the PD-L1 anti-PD-L1 antibody combined with PD-L1 high-affinity.

Summary of the invention

The present inventor utilizes yeast display, obtains the anti-PD-L1 human antibody with good specificity, higher affinity and stability by screening with affinity maturation, this completes the present invention.

First aspect present invention relates to anti-PD-L1 antibody or its antigen-binding portion thereof, and it includes being selected from such as the CDR region of next group:

(1) sequence of heavy chain CDR1, CDR2, CDR3 is respectively as shown in SEQIDNO:1-3, the sequence of light chain CDR1, CDR2, CDR3 respectively as shown in SEQIDNO:4-6, or respectively with the homogeneity of above-mentioned sequence more than 70%, the sequence of 80%, 85%, 90%, 95%；

(2) sequence of heavy chain CDR1, CDR2, CDR3 is respectively as shown in SEQIDNO:7-9, the sequence of light chain CDR1, CDR2, CDR3 respectively as shown in SEQIDNO:10-12, or respectively with the homogeneity of above-mentioned sequence more than 70%, the sequence of 80%, 85%, 90%, 95%；

(3) sequence of heavy chain CDR1, CDR2, CDR3 is respectively as shown in SEQIDNO:13-15, the sequence of light chain CDR1, CDR2, CDR3 respectively as shown in SEQIDNO:16-18, or respectively with the homogeneity of above-mentioned sequence more than 70%, the sequence of 80%, 85%, 90%, 95%；

(4) sequence of heavy chain CDR1, CDR2, CDR3 is respectively as shown in SEQIDNO:1,2,19, the sequence of light chain CDR1, CDR2, CDR3 respectively as shown in SEQIDNO:4-6, or respectively with the homogeneity of above-mentioned sequence more than 70%, the sequence of 80%, 85%, 90%, 95%；

(5) sequence of heavy chain CDR1, CDR2, CDR3 is respectively as shown in SEQIDNO:7,20,9, the sequence of light chain CDR1, CDR2, CDR3 respectively as shown in SEQIDNO:10-12, or respectively with the homogeneity of above-mentioned sequence more than 70%, the sequence of 80%, 85%, 90%, 95%；

(6) sequence of heavy chain CDR1, CDR2, CDR3 is respectively as shown in SEQIDNO:13-15, the sequence of light chain CDR1, CDR2, CDR3 respectively as shown in SEQIDNO:21,17,18, or respectively with the homogeneity of above-mentioned sequence more than 70%, the sequence of 80%, 85%, 90%, 95%.

The anti-PD-L1 antibody of any one or its antigen-binding portion thereof according to a first aspect of the present invention, it also includes being selected from such as the framework region, variable region of heavy chain of next group:

1) sequence of FR1, FR2, FR3, FR4 is respectively as shown in SEQIDNO:22-25, or respectively with the homogeneity of above-mentioned sequence more than 70%, the sequence of 80%, 85%, 90%, 95%, 99%；

2) sequence of FR1, FR2, FR3, FR4 is respectively as shown in SEQIDNO:30-33, or respectively with the homogeneity of above-mentioned sequence more than 70%, the sequence of 80%, 85%, 90%, 95%, 99%；

3) sequence of FR1, FR2, FR3, FR4 is respectively as shown in SEQIDNO:38-41, or respectively with the homogeneity of above-mentioned sequence more than 70%, the sequence of 80%, 85%, 90%, 95%, 99%；

4) sequence of FR1, FR2, FR3, FR4 is respectively as shown in SEQIDNO:30-33, or respectively with the homogeneity of above-mentioned sequence more than 70%, the sequence of 80%, 85%, 90%, 95%, 99%.

The anti-PD-L1 antibody of any one or its antigen-binding portion thereof according to a first aspect of the present invention, it also includes being selected from such as the framework region, variable region of light chain of next group:

1) sequence of FR1, FR2, FR3, FR4 is respectively as shown in SEQIDNO:26-29, or respectively with the homogeneity of above-mentioned sequence more than 70%, the sequence of 80%, 85%, 90%, 95%, 99%；

The anti-PD-L1 antibody of any one or its antigen-binding portion thereof according to a first aspect of the present invention, it includes being selected from such as the variable region of heavy chain of next group:

Shown in its sequence such as SEQIDNO:47,49,51,53,54, or respectively with the homogeneity of above-mentioned sequence more than 70%, the sequence of 80%, 85%, 90%, 95%, 99%.

The anti-PD-L1 antibody of any one or its antigen-binding portion thereof according to a first aspect of the present invention, it includes being selected from such as the variable region of light chain of next group:

Shown in its sequence such as SEQIDNO:48,50,52,55,56, or respectively with the homogeneity of above-mentioned sequence more than 70%, the sequence of 80%, 85%, 90%, 95%, 99%.

The anti-PD-L1 antibody of any one or its antigen-binding portion thereof according to a first aspect of the present invention, it is whole antibody, bi-specific antibody, scFv, Fab, Fab', F (ab')₂Or Fv.

In one embodiment of the invention, when it is scFv, possibly together with connection peptides between heavy chain and the variable region of light chain of described anti-PD-L1 antibody or its antigen-binding portion thereof.

In specific embodiments of the present invention, the sequence of described connection peptides is such as shown in SEQIDNO:67.

The anti-PD-L1 antibody of any one or its antigen-binding portion thereof according to a first aspect of the present invention, it is whole antibody.

The anti-PD-L1 antibody of any one or its antigen-binding portion thereof according to a first aspect of the present invention, its CH is selected from IgG, IgM, IgE, IgD and IgA.

In embodiments of the invention, its CH is selected from IgG1, IgG2, IgG3 and IgG4.

In specific embodiments of the present invention, its CH is IgG1.

In specific embodiments of the present invention, the aminoacid sequence of IgG1 is such as shown in SEQIDNO:68.

The anti-PD-L1 antibody of any one or its antigen-binding portion thereof according to a first aspect of the present invention, its constant region of light chain is κ type or λ type.

In specific embodiments of the present invention, the aminoacid sequence of κ type constant region of light chain is such as shown in SEQIDNO:70.

In specific embodiments of the present invention, the aminoacid sequence of λ type constant region of light chain is such as shown in SEQIDNO:72.

Second aspect present invention relates to nucleic acid molecules, its comprise can the nucleotide sequence of encoding antibody heavy variable region, described antibody heavy chain variable region comprises the aminoacid sequence being selected from a group:

(i) SEQIDNO:1-3；

(ii) SEQIDNO:7-9；

(iii) SEQIDNO:13-15；

(iv) SEQIDNO:1,2,19；

(v) SEQIDNO:7,20,9.

The nucleic acid molecules of any one according to a second aspect of the present invention, described antibody heavy chain variable region comprises the aminoacid sequence being selected from a group:

Several amino acid whose replacements are arrived containing one in SEQIDNO:47, SEQIDNO:49, SEQIDNO:51, SEQIDNO:53, SEQIDNO:54 or above-mentioned sequence framework region.

In embodiments of the invention, described nucleic acid molecules comprises selected from the sequence as shown in SEQIDNO:57-61.

In embodiments of the invention, also comprise can the nucleotide sequence of encoding antibody heavy constant region for described nucleic acid molecules；Described CH is selected from IgG, IgM, IgE, IgD and IgA.

In specific embodiments of the present invention, its CH is IgG1.

In specific embodiments of the present invention, the nucleotide sequence of IgG1 is such as shown in SEQIDNO:69.

Third aspect present invention relates to nucleic acid molecules, its comprise can the nucleotide sequence of encoding antibody light variable region, described antibody chain variable region comprises the aminoacid sequence being selected from a group:

(i) SEQIDNO:4-6；

(ii) SEQIDNO:10-12；

(iii) SEQIDNO:16-18；

(iv) SEQIDNO:21,17,18.

The nucleic acid molecules of any one according to a third aspect of the present invention, described antibody chain variable region comprises the aminoacid sequence being selected from a group:

Several amino acid whose replacements are arrived containing one in SEQIDNO:48, SEQIDNO:50, SEQIDNO:52, SEQIDNO:55, SEQIDNO:56 or above-mentioned sequence framework region.

In embodiments of the invention, described nucleic acid molecules comprises selected from the sequence as shown in SEQIDNO:62-66.

In embodiments of the invention, also comprise can the nucleotide sequence of encoding antibody light constant region for described nucleic acid molecules；Described constant region of light chain is κ type or λ type.

In specific embodiments of the present invention, the nucleotide sequence of κ type constant region of light chain is such as shown in SEQIDNO:70.

Fourth aspect present invention relates to carrier, and it contains the nucleic acid molecules of any one of second or third aspect of the present invention.

The carrier of any one according to a fourth aspect of the present invention, the nucleic acid molecules of its nucleic acid molecules containing any one of second aspect present invention and any one of the third aspect.

Fifth aspect present invention relates to host cell, the carrier of its nucleic acid molecules containing any one of second or third aspect of the present invention or any one of fourth aspect present invention.

Sixth aspect present invention relates to conjugate, its anti-PD-L1 antibody containing any one of first aspect present invention or its antigen-binding portion thereof, and other bioactive substance, described anti-PD-L1 antibody or its antigen-binding portion thereof are directly or by junction fragment and other bioactive substance coupling.

nullIn embodiments of the invention，Other bioactive substance described is selected from can direct or indirect cell growth inhibiting or kill cell、Or by activating organism immune response thus suppressing or killing cell，Thus reaching the chemical substance for the treatment of tumor、Toxin、Polypeptide、Enzyme、Isotope、Cytokine or other there is bioactive one matter or compounding substances，Such as AuristatinMMAE、AuristatinMMAF、MaytansineDM1、MaytansineDM4、Calicheamicin (calicheamicin)、duocarmycinMGBA、Amycin (doxorubicin)、Ricin、The toxin such as diphtheria toxin, diphtherotoxin、I131、Interleukin class、Tumor necrosis factor、Chemotactic factor、Nano-particle etc..

Seventh aspect present invention relates to compositions (such as pharmaceutical composition), the conjugate of its anti-PD-L1 antibody containing any one of first aspect present invention or its antigen-binding portion thereof, second aspect or the nucleic acid molecules of any one of the third aspect, the carrier of any one of fourth aspect, the host cell of the 5th any one of aspect or any one of sixth aspect present invention, and optional pharmaceutically acceptable carrier or excipient, and other optional bioactive substance.

The compositions (such as pharmaceutical composition) of any one according to a seventh aspect of the present invention, other bioactive substance described includes but not limited to other antibody, fusion protein or medicine (such as antitumor drug, such as Radiotherapy chemotherapy medicine).

The invention still further relates to reagent or test kit, its anti-PD-L1 antibody containing any one of first aspect present invention or its antigen-binding portion thereof, described detectable or test kit are used for detecting whether PD-L1 albumen or derivatives thereof exists.

The invention still further relates to diagnostic reagent or test kit, its anti-PD-L1 antibody containing any one of first aspect present invention or its antigen-binding portion thereof, described diagnostic reagent or test kit diagnose the disease (such as tumor or viral infection, for instance the viral infection of PD-L1 high expressed or the tumor of PD-L1 high expressed) relevant to PD-L1 for (such as cell or tissue) or internal (such as human or animal's model) in vitro.

In embodiments of the invention, described anti-PD-L1 antibody or the also coupling of its antigen-binding portion thereof have and can be used for detection or the fluorescent dye that can be detected by other reagent, chemical substance, polypeptide, enzyme, isotope, label etc..

In embodiments of the invention, described tumor includes but not limited to pulmonary carcinoma, ovarian cancer, colon and rectum carcinoma, melanoma, renal carcinoma, bladder cancer, breast carcinoma, hepatocarcinoma, lymphoma, malignant hematologic disease, head and neck cancer, glioma, gastric cancer, nasopharyngeal carcinoma, laryngeal carcinoma, cervical cancer, carcinoma of uterine body, osteosarcoma, thyroid carcinoma, carcinoma of prostate.

In embodiments of the invention, described viral infection includes but not limited to acute, subacute or Chronic HBV, HCV, HIV.

The invention still further relates to the anti-PD-L1 antibody of any one of first aspect present invention or its antigen-binding portion thereof, second aspect or the nucleic acid molecules of any one of the third aspect, the carrier of any one of fourth aspect, the host cell of the 5th any one of aspect, the conjugate of the 6th any one of aspect or the 7th aspect any one compositions for preparing the purposes of the medicine of prevention or the treatment disease (such as tumor or viral infection, for instance the tumor of PD-L1 high expressed or the viral infection of PD-L1 high expressed) relevant to PD-L1.

In embodiments of the invention, described tumor refers to the tumor relevant to PD-L1, for instance refer to the tumor of high expressed PD-L1.

In specific embodiments of the present invention, wherein said tumor includes but not limited to pulmonary carcinoma, ovarian cancer, colon and rectum carcinoma, melanoma, renal carcinoma, bladder cancer, breast carcinoma, hepatocarcinoma, lymphoma, malignant hematologic disease, head and neck cancer, glioma, gastric cancer, nasopharyngeal carcinoma, laryngeal carcinoma, cervical cancer, carcinoma of uterine body, osteosarcoma, thyroid carcinoma, carcinoma of prostate.

The invention still further relates to the anti-PD-L1 antibody of any one of first aspect present invention or its antigen-binding portion thereof for preparing the purposes in the reagent or test kit diagnosing the disease (such as tumor or viral infection, for instance the tumor of PD-L1 high expressed or the viral infection of PD-L1 high expressed) relevant to PD-L1.

In embodiments of the invention, wherein said anti-PD-L1 antibody or the also coupling of its antigen-binding portion thereof have and can be used for detection or the fluorescent dye that can be detected by other reagent, chemical substance, polypeptide, enzyme, isotope, label etc..

The invention still further relates to the anti-PD-L1 antibody of any one of first aspect present invention or its antigen-binding portion thereof for preparing the purposes of the medicine preventing or treating the disease relevant to CD80.

In the present invention, relevant for described CD80 disease is such as the disease relevant to CD80 high expressed.

The invention still further relates to prevention or treat disease (the such as tumor or viral infection relevant to PD-L1, the tumor of such as PD-L1 high expressed or the viral infection of PD-L1 high expressed) method, described method includes the anti-PD-L1 antibody to experimenter in need prevention or any one of first aspect present invention of therapeutically effective amount or its antigen-binding portion thereof, the nucleic acid molecules of second aspect or any one of the third aspect, the carrier of any one of fourth aspect, the host cell of the 5th any one of aspect, the conjugate of the 6th any one of aspect or the 7th aspect any one compositions, and it is optional with radiation therapy (such as roentgen radiation x) method coupling.

The invention still further relates to prevention or the method treating the disease relevant to CD80, described method includes the anti-PD-L1 antibody to experimenter in need prevention or any one of first aspect present invention of therapeutically effective amount or its antigen-binding portion thereof.

In the present invention, the described disease relevant to CD80 is such as the disease relevant with CD80 high expressed.

Hereinafter the present invention is described further:

In the present invention, unless otherwise stated, Science and Technology noun used herein has the implication that those skilled in the art are generally understood that.Further, protein used herein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology relational language and laboratory operation step are in corresponding field widely used term and conventional steps.Meanwhile, in order to be more fully understood that the present invention, provide below definition and the explanation of relational language.

In the present invention, term " antibody " refers to the immunoglobulin molecules being generally made up of two pairs of identical polypeptide chains (every pair has " gently " (L) chain and " weight " (H) chain).Light chain of antibody can be categorized as κ and lambda light chain.Heavy chain can be categorized as μ, δ, γ, α or ε, and respectively the isotype of antibody is defined as IgM, IgD, IgG, IgA and IgE.In light chain and heavy chain, variable region and constant region are connected by about 12 or more amino acid whose " J " district, and heavy chain also comprises about 3 or more amino acid whose " D " district.Each heavy chain is by variable region of heavy chain (V_H) and CH (C_H) composition.CH is by 3 domain (C_H1、C_H2 and C_H3) composition.Each light chain is by variable region of light chain (V_L) and constant region of light chain (C_L) composition.Constant region of light chain is by a domain C_LComposition.The constant region of antibody can mediated immunity globulin and host tissue or the factor, including the combination of immune various cells (such as, effector lymphocyte) and first component (C1q) of classics complement system.V_HAnd V_LDistrict also can be subdivided into has denatured region (being called complementary determining region (CDR)), is interspersed with the more conservative region being called framework region (FR).Each V_HAnd V_LBy in the following order: 3 CDR and 4 FR that FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 arrange from amino terminal to carboxyl terminal form.Variable region (the V of each heavy chain/light chain pair_HAnd V_L) form antibody combining site respectively.Aminoacid follows KabatSequencesofProteinsofImmunologicalInterest (NationalInstitutesofHealth to the distribution of each region or domain, Bethesda, (1987and1991)), or Chothia&Lesk (1987) J.Mol.Biol.196:901-917 Md.；The definition of Chothia et al. (1989) Nature342:878-883.Term " antibody " not method by any specific generation antibody is limited.Such as, it includes, especially, and recombinant antibodies, monoclonal antibody and polyclonal antibody.Antibody can be the antibody of different isotype, for instance, IgG (such as, IgG1, IgG2, IgG3 or IgG4 hypotype), IgA1, IgA2, IgD, IgE or IgM antibody.

In the present invention, " antigen-binding portion thereof " of term antibody refers to one or more parts of full length antibody, described part keeps the ability of same antigen (such as, PD-L1) that binding antibody combines, with specific binding to antigen of complete antibody competition.Generally referring to, FundamentalImmunology, (Paul, W., ed., second edition, RavenPress, N.Y. (1989), it integrates with by reference in full herein Ch.7 with it, for all purposes.Recombinant DNA technology can be passed through or enzymatic or chemical disruption by complete antibody produce antigen-binding portion thereof.In some cases, antigen-binding portion thereof includes Fab, Fab', F (ab')_2、Fd, Fv, dAb and complementary determining region (CDR) fragment, single-chain antibody are (such as, scFv), chimeric antibody, double antibody (diabody) and such polypeptide, it comprises at least some of of the antibody that is enough to give polypeptid specificity antigen binding capacity.

In the present invention, term " Fd fragment " means by V_HAnd C_HThe antibody fragment of 1 domain composition；Term " Fv fragment " means by the V of the single armed of antibody_LAnd V_HThe antibody fragment of domain composition；Term " dAb fragment " means by V_HThe antibody fragment (Ward et al., Nature341:544-546 (1989)) of domain composition；Term " Fab fragment " means by V_L、V_H、C_LAnd C_HThe antibody fragment of 1 domain composition；Term " F (ab')₂Fragment " mean to comprise the antibody fragment of two the Fab fragments connected by the disulphide bridges on hinge region.

In some cases, the antigen-binding portion thereof of antibody is single-chain antibody (such as, scFv), wherein V_LAnd V_HDomain by can be produced as single polypeptide chain connector pairing formed monovalent molecule (referring to, such as, Bird et al., Science242:423-426 (1988) and Huston et al., Proc.Natl.Acad.Sci.USA85:5879-5883 (1988)).This type of scFv molecule can have general structure: NH₂-V_L-joint-V_H-COOH or NH₂-V_H-joint-V_L-COOH.Suitable prior art joint (connection peptides) is made up of the GGGGS aminoacid sequence repeated or its variant.Such as, can use there is aminoacid sequence (GGGGS)₄Joint, but be used as its variant (Holliger et al. (1993), Proc.Natl.Acad.Sci.USA90:6444-6448).Can be used for other joints of the present invention by Alfthan et al. (1995), ProteinEng.8:725-731, Choi et al. (2001), Eur.J.Immunol.31:94-106, Hu et al. (1996), CancerRes.56:3055-3061, Kipriyanov et al. (1999), J.Mol.Biol.293:41-56 and Roovers et al. (2001), CancerImmunol. describe.In embodiments of the invention, the sequence of described connection peptides is (GGGGS)₃。

In some cases, antibody is bi-specific antibody, it can respectively with two kinds of antigens or epitope combine, it includes the light chain of antibody of specific binding first antigen, heavy chain or its antigen-binding portion thereof and the light chain of antibody of specific binding second antigen, heavy chain or its antigen-binding portion thereof.In embodiments of the invention, can being antibody or its antigen-binding portion thereof of any one of the present invention in conjunction with the light chain of the antibody of the first antigen, heavy chain or its antigen-binding portion thereof in described bi-specific antibody, the light chain of antibody of described specific binding second antigen, heavy chain or its antigen-binding portion thereof can be other anti-PD-L1 antibody or its antigen-binding portion thereof or for the antibody of other antigen or its antigen-binding portion thereof.

In some cases, antibody is double antibody, i.e. bivalent antibody, wherein V_HAnd V_LDomain is expressed on single polypeptide chain, but use too short connector so that matching not between two domains of same chain, thus force domain and another chain complementary domain pairing and produce two antigen-binding sites (referring to, such as, HolligerP. et al., Proc.Natl.Acad.Sci.USA90:6444-6448 (1993), and PoljakR.J. et al., Structure2:1121-1123 (1994)).

Routine techniques well known by persons skilled in the art can be used (such as, recombinant DNA technology or enzymatic or chemical disruption method) obtain the antigen-binding portion thereof of antibody (such as from given antibody (such as monoclonal antibody 2E12), above-mentioned antibody fragment), and by with for complete antibody in the way of identical mode with regard to the antigen-binding portion thereof of specificity screening antibody.

In the present invention, described antigen-binding portion thereof includes single-chain antibody (scFv), chimeric antibody, double antibody, scFv-Fc bivalent molecule, dAb and complementary determining region (CDR) fragment, Fab fragment, Fd fragment, Fab' fragment, Fv and F (ab')₂Fragment.

In the present invention, described IgG1 CH includes various allotype, as G1m (f), G1m (z), G1m (z, a) or G1m (z, a, x).In embodiments of the invention, described IgG1 CH is G1m (f) type.

In the present invention, described κ constant region of light chain includes various allotype, such as Km1, Km1, and 2 or Km3.The present invention embodiment in, described κ constant region of light chain is Km3 type.

In the present invention, described lambda light chain constant region includes various allotype, such as λ I, λ II, λ III, λ VI.The present invention embodiment in, described lambda light chain constant region is λ II type.

The antibody nucleic acids molecule that the present invention relates to can also utilize traditional genetic engineering recombinant technique or chemical synthesis process to obtain.On the one hand, the sequence of the antibody nucleic acids molecule that the present invention relates to contains the variable region of heavy chain of anti-PD-L1 antibody or the partial nucleic acid sequence of antibody molecule.On the other hand, the sequence of the antibody nucleic acids molecule that the present invention relates to also includes the variable region of light chain of anti-PD-L1 antibody or the partial nucleic acid sequence of antibody molecule.On the other hand, the sequence of the antibody nucleic acids molecule that the present invention relates to also includes the CDR sequence of heavy chain or variable region of light chain.Complementary determining region (complementarydeterminantregion, CDR) being the position being combined with epitope, the CDR sequence in the present invention is determined by IMGT/V-QUEST (http://imgt.cines.fr/textes/vquest/).But the CDR sequence that different division methods obtains is slightly different.

An aspect of of the present present invention relates to encoding antibody B60-55, B II nucleic acid molecules of 61-62, B50-6, B60, B II 61 and B50 heavy chain and light-chain variable sequence.Antibody B60-55, B II the nucleic acid molecules of 61-62, B50-6, B60, B II 61 and B50 weight chain variabl area sequence correspond respectively to SEQIDN0:57, SEQIDNO:58, SEQIDNO:59, SEQIDNO:60, SEQIDNO:61 and SEQIDN0:59.Antibody B60-55, B II the nucleic acid molecules of 61-62, B50-6, B60, B II 61 and B50 light-chain variable sequence correspond respectively to SEQIDN0:62, SEQIDNO:63, SEQIDN0:64, SEQIDNO:62, SEQIDNO:65 and SEQIDNO:66.The invention still further relates to contain antibody B60-55, B II 61-62, B50-6, B60, B II 61 and B50 heavy chain and light-chain variable sequence nucleic acid molecules variant or analog.

On the other hand, the invention still further relates to the variant of the nucleic acid molecules of various separation, specifically, the sequence of nucleic acid variants and following nucleic acid sequence SEQ ID N0:57, SEQIDNO:58, SEQIDNO:59, SEQIDNO:60, SEQIDNO:61, SEQIDN0:59, SEQIDN0:62, SEQIDNO:63, SEQIDN0:64, SEQIDNO:62, SEQIDNO:65 and SEQIDNO:66 homogeny at least up to 70%, preferably at least reach 75%, more preferably at reaching 80%, more preferably at reaching 85%, more preferably at reaching 90%, most preferably at least up to 95%.

The present invention further further relates to encoding antibody B60-55, B II 61-62, B50-6, B60, B II 61 and corresponding separated nucleic acid molecules that B50 variable region of heavy chain is aminoacid sequence SEQIDNO:47,49,51,53,54,51.The invention still further relates to encoding antibody B60-55, B II 61-62, B50-6, B60, B II 61 and corresponding nucleic acid molecules that B50 chain variable region amino acid sequence is SEQIDNO:48,50,52,48,55,56.

The present invention relates to the recombinant expression carrier containing described nucleic acid molecules, be directed to convert the host cell of these molecules.And, the invention still further relates to and utilize the host cell containing described nucleic acid molecules cultivate under given conditions and separate the method obtaining inventing described antibody.

Antibody amino acids sequence

Monoclonal antibody B60-55, B II 61-62, B50-6, B60, B II 61 and B50 heavy chain and chain variable region amino acid sequence can be derived by from corresponding nucleotide sequence.Antibody B60-55, B II 61-62, B50-6, B60, B II 61 and B50 heavy chain variable amino acid sequence be SEQIDNO:47,49,51,53,54,51 respectively.Antibody B60-55, B II 61-62, B50-6, B60, B II 61 and B50 chain variable region amino acid sequence be SEQIDNO:48,50,52,48,55,56 respectively.

On the other hand, the aminoacid sequence of the variable region of heavy chain of antibody provided by the invention and the sequence similarity of SEQIDNO:47,49,51,53,54 or 51 are at least up to 70%, it will be preferred that at least 75%, it is preferably at least 80%, preferably 85%, be further preferably at least 90%, it is desirable to be at least 95%.

On the other hand, the aminoacid sequence of the variable region of light chain of antibody provided by the invention and the sequence similarity of SEQIDNO:48,50,52,48,55 or 56 are at least up to 70%, it will be preferred that at least 75%, it is preferably at least 80%, preferably 85%, be further preferably at least 90%, it is desirable to be at least 95%.

Antibody B60-55, B II 61-62, B50-6, B60, B II 61 and B50 heavy chain and the aminoacid sequence of CDR of variable region of light chain be defined below:

The CDR1 of antibody B60-55 heavy chain, CDR2 and CDR3 aminoacid sequence respectively SEQIDNO:1-3.The CDR1 of antibody B60-55 light chain, CDR2 and CDR3 aminoacid sequence respectively SEQIDNO:4-6.

The CDR1 of antibody B II 61-62 heavy chain, CDR2 and CDR3 aminoacid sequence respectively SEQIDNO:7-9.The CDR1 of antibody B II 61-62 light chain, CDR2 and CDR3 aminoacid sequence respectively SEQIDNO:10-12.

The CDR1 of antibody B50-6 heavy chain, CDR2 and CDR3 aminoacid sequence respectively SEQIDNO:13-15.The CDR1 of antibody B50-6 light chain, CDR2 and CDR3 aminoacid sequence respectively SEQIDNO:16-18.

On the other hand, the aminoacid sequence that the anti-heavy chain of PD-L1 antibody or the CDR of fragment contain is probably to be occurred one or more amino acid whose sudden change on SEQIDNO:1-3,7-9,13-15,19,20 or increases or lack.Preferably, the aminoacid of sudden change or interpolation or disappearance is less than 3 aminoacid.It is further preferred that the aminoacid of sudden change or interpolation or disappearance is less than 2 aminoacid.Most preferably, the aminoacid of sudden change or interpolation or disappearance is less than 1 aminoacid.

On the other hand, the aminoacid sequence that the anti-light chain of PD-L1 antibody or the CDR of fragment contain is probably at SEQIDNO:4-6,10-12,16-18, one or more amino acid whose sudden change occurs on 21, increase or lack.Preferably, the aminoacid of sudden change, interpolation or disappearance is less than 3 aminoacid.It is further preferred that the aminoacid of sudden change, interpolation or disappearance is less than 2 aminoacid.Most preferably, the aminoacid of sudden change, interpolation or disappearance is less than 1 aminoacid.

Antibody B60-55, B II 61-62, B50-6, B60, B II 61 and B50 heavy chain and the aminoacid sequence of FR of variable region of light chain be defined below:

The sequence respectively SEQIDNO:22-25 of antibody B60-55, B60 variable region of heavy chain FR1, FR2, FR3, FR4.The sequence of variable region of light chain FR1, FR2, FR3, FR4 respectively SEQIDNO:26-29.

The sequence respectively SEQIDNO:30-33 of antibody B II 61-62 variable region of heavy chain FR1, FR2, FR3, FR4.The sequence of variable region of light chain FR1, FR2, FR3, FR4 respectively SEQIDNO:34-37.

The sequence respectively SEQIDNO:38-41 of antibody B50-6, B50 variable region of heavy chain FR1, FR2, FR3, FR4.The sequence of variable region of light chain FR1, FR2, FR3, FR4 respectively SEQIDNO:42-45.

The sequence respectively SEQIDNO:30-33 of antibody B II 61 variable region of heavy chain FR1, FR2, FR3, FR4.The sequence of variable region of light chain FR1, FR2, FR3, FR4 respectively SEQIDNO:34,46,36,37.

On the other hand, the heavy chain of anti-PD-L1 antibody or the aminoacid sequence of variable region of light chain FR are probably and occur one or more amino acid whose sudden change on SEQIDNO:22-46, increase or lack.Preferably, the aminoacid of sudden change, interpolation or disappearance is less than 3 aminoacid.It is further preferred that the aminoacid of sudden change, interpolation or disappearance is less than 2 aminoacid.Most preferably, the aminoacid of sudden change, interpolation or disappearance is less than 1 aminoacid.

The aminoacid of above-mentioned antibody or CDR region or framework region undergo mutation, add or lack after variant still retain the ability of specific binding human PD-L 1.The present invention also comprises the variant of such antigen-binding portion thereof.

The monoclonal antibody variant of the present invention can be obtained by traditional gene engineering method.Those skilled in the art has full knowledge that the method utilizing nucleic acid mutation transformation DNA molecular.It addition, the nucleic acid molecules of encoding heavy chain and light chain variant can also be obtained by chemosynthesis.

In the present invention, for determining that the algorithm of sequence iden and sequence similarity percent is such as BLAST and BLAST2.0 algorithm, they are respectively described at (1990) J.Mol.Biol.215:403-410 such as (1977) Nucl.Acid.Res.25:3389-3402 and Altschul such as Altschul.Adopting such as described in document or default parameters, BLAST and BLAST2.0 is determined for the amino acid sequence identity percent of the present invention.Perform the BLAST software analyzed to be obtained by the public by National Biotechnology information centre.

In the present invention, the aminoacid sequence of the described sequence iden with aminoacid sequence with at least 70% includes the peptide sequence substantially same with described aminoacid sequence, such as when adopting methods described herein (BLAST for example with canonical parameter analyzes), contain compared with peptide sequence of the present invention at least 70% sequence iden, preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher those sequences of sequence iden.

In the present invention, term " carrier " refers to and can be inserted by the polynucleotide encoding certain albumen and make albumen obtain a kind of nucleic acid vehicle expressed.Carrier can pass through to convert, transduce or transfection host cell so that it is the hereditary material element carried is expressed at host cell inner expression.For example, carrier includes: plasmid；Phasmid；Coemid；The artificial chromosome (PAC) in artificial chromosome such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1 source；Phage such as bacteriophage lambda or M13 phage and animal virus etc..Animal virus kind as carrier has retrovirus (including slow virus), adenovirus, adeno-associated virus, herpesvirus (such as herpes simplex virus), poxvirus, baculovirus, human papillomavirus, papova viruses (such as SV40).A kind of carrier is likely to the element expressed containing various control, including promoter sequence, transcriptional initiation sequence, enhancer sequence, selection element and reporter gene.It addition, carrier also can contain replication origin.Carrier is it is also possible to include and assist it to enter the composition of cell, such as virion, liposome or protein coat, but not only only has these materials.

In the present invention, term " host cell " refers to the cell importing carrier, including following many cell types, such as prokaryotic cells such as escherichia coli or hay bacterium, such as the fungal cell such as yeast cells or aspergillosis, such as insect cells such as S2 drosophila cell or Sf9, or such as fibroblast, Chinese hamster ovary celI, COS cell, NSO cell, HeLa cell, bhk cell, the zooblast of HEK293 cell or people's cell.

The antibody molecule that the antibody fragment of the present invention can utilize hydrolysis complete obtains (referring to Morimotoetal., J.Biochem.Biophys.Methods24:107-117 (1992) andBrennanetal., Science229:81 (1985)).It addition, these antibody fragments directly can also be produced (reviewedinHudson, Curr.Opin.Immunol.11:548-557 (1999) by recombinant host cell；Littleetal.,Immunol.Today,21:364-370(2000)).Such as, Fab' fragment can directly obtain or chemistry Rhizoma Nelumbinis connection formation F (ab') 2 fragment (Carteretal., Bio/Technology, 10:163-167 (1992)) from E.coli cell.For another example, F (ab')₂Fragment can be used leucine zipper GCN4 to connect and obtain.It addition, Fv, Fab or F (ab')₂Fragment directly can also be directly separating from recombinant host cell culture fluid and obtain.Those of ordinary skill in the art has full knowledge that other technology preparing antibody fragment.

In the present invention, " specific binding " refers to two intermolecular nonrandom association reactions, as antibody and produce this antibody antigen between reaction.Herein, it is can't detect or very weak in conjunction with the antibody of the first antigen to the binding affinity of the second antigen.In some embodiments, certain antigen-specific antibodies refers to affinity (KD)≤10^-5M is (such as 10^-6M、10^-7M、10^-8M、10^-9M、10^-10M etc.) in conjunction with this antigen, wherein KD refers to the ratio (koff/kon) of dissociation yield and combination rate, and it can adopt the method that those skilled in the art are familiar with to be measured.

In embodiments of the invention, the anti-PD-L1 antibody of the present invention specifically with human PD-L 1 simultaneously or be combined with mice PD-L1, and can not be combined with PD-L2 and B7H3.

In embodiments of the invention, the anti-PD-L1 antibody of the present invention can with hPD-1 competition binding hPD-L1.

In the present invention, the disease relevant to PD-L1 such as includes the tumor relevant with PD-L1 or viral infection, particularly relevant to PD-L1 high expressed tumor or viral infection.

In the present invention, common usage is deferred in 20 kinds of conventional amino acid and its abbreviation.Referring to Immunology-ASynthesis (second edition, E.S.Golub and D.R.Gren, Eds., SinauerAssociates, Sunderland, Mass. (1991)), it is integrated with herein by reference.

The beneficial effect of the invention

The present invention utilizes yeast display, the anti-PD-L1 human antibody with good specificity, higher affinity and stability is obtained with further affinity maturation by screening, this antibody can specifically with human PD-L 1 simultaneously or be combined with Mus PD-L1, and be not combined with B7H3 and PD-L2, it can pass through and the T cell of activation combines and then strengthens the activation of T cell, and tumor growth has significant inhibitory action.

Accompanying drawing explanation

Fig. 1: the anti-hPD-L1scFv of the purification suppression that hPD-L1/hPD-1 ligand-receptor is combined.

X-axle represents the fluorescence intensity of EGFP, and Y-axle represents the fluorescence intensity of SA-PE.A is blank, and B is negative control, and C is B50scFv, D be B60scFv, E is B II 61scFv.

The yeast improved with hPD-L1 affinity is obtained after the screening of Fig. 2 affinity maturation

Wherein X-axle represents the fluorescence intensity (the myc positive represents the yeast expressing complete antibody segment) of myc, and Y-axle represents the fluorescence intensity of the SA-APC of conjugated antigen ability.

The comparison of the ability of the antibody obtained after Fig. 3 affinity maturation and hPD-1 competition binding hPD-L1

Wherein abscissa is antibody concentration (unit: ng/ml), and vertical coordinate is OD value.

Wherein A is the comparison of B II 61 and B II 61-62, and B is the comparison of B50 and B50-6, and C is the comparison of B60 and B60-55.

Fig. 4 ELISA method detection anti-hPD-L1 antibody and hPD-L1 binding ability

The ability of Fig. 5 competitive ELISA detection anti-hPD-L1 antibody and hPD-1 competition binding hPD-L1

Wherein Graph#5 is B II 61-62mAb, Graph#2 be B50-6mAb, Graph#3 is B60-55mAb.

Fig. 6 is detected by competitive ELISA methodAnti-hPD-L1 antibody and CD80 competition knot Close the ability of hPD-L1

Fig. 7 anti-hPD-L1 antibody specificity detects

Wherein X-axle is EGFP fluorescence intensity, and Y-axle is the fluorescence intensity of corresponding antibodies, and A is blank, and B is negative control, and C is B II 61-62mAb, D be B60-55mAb, E is B50-6mAb；

(1) for hPD-L1-EGFP albumen, (2) are hB7H3-EGFP, and (3) are hPD-L2-EGFP albumen.

The ability that Fig. 8 anti-hPD-L1 antibody is combined with mPD-L1

Wherein X-axle is EGFP fluorescence intensity, and Y-axle is the fluorescence intensity of corresponding antibodies, and A is blank, and B is negative control, and C is B60-55mAb, D be B II 61-62mAb, E is B50-6mAb；

(1) for hPD-L1-EGFP albumen, (2) are mPD-L1-EGFP albumen.

Fig. 9 anti-hPD-L1 antibody and the machin PD-L1 ability being combined

Figure 10 anti-hPD-L1 antibody is to CD4⁺The activation of T cell

Figure 11 anti-hPD-L1 antibody B50-6 inhibitory activity to tumor growth

Figure 12 anti-hPD-L1 antibody B60-55, B II 61-62 inhibitory activity to tumor growth

Wherein A is dosage when being 3mg/kg, B II 61-62mAb and the B60-55 inhibitory action to tumor growth；B is B II 61-62mAb of the different dosage inhibitory action to tumor growth.

The stability of Figure 13 B60-55 and antibody 2.41H90P compares

Wherein A is B60-55 and the time dependent IC50 value of antibody 2.41H90P；

B is the time dependent ratio of antibody dimer；

C is the competitive ELISA result of B60-55 acceleration for stabilization experiment.

Detailed description of the invention

Below in conjunction with embodiment, embodiment of the present invention are described in detail, it will be appreciated by those skilled in the art that the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Unreceipted actual conditions person in embodiment, conventionally the condition of condition or manufacturer's suggestion carries out.Agents useful for same or the unreceipted production firm person of instrument, be can pass through city available from conventional products.

Prepared by embodiment 1 recombined human PD-L1, the expression of PD-1 and relevant EGFP cell

According to the aminoacid sequence (Q9NZQ7) of human PD-L 1 on albumen database Uniprot, obtain the aminoacid sequence (namely in Q9NZQ7 the 1st residue to 238 residues) of human PD-L 1 ectodomain；According to the amino acid constant region sequence (P01857) of human normal immunoglobulin gamma1 (IgG1) on albumen database Uniprot, obtain the domain amino acid sequence (namely in P01857 the 104th residue to 330 residues) of human IgG1-Fc；According to the amino acid constant region sequence (P01868) of human normal immunoglobulin gamma1 (IgG1) on albumen database Uniprot, obtain the domain amino acid sequence (namely in P01868 the 98th residue to 324 residues) of human IgG1-Fc.The DNA sequences encoding utilizing DNAworks online tool (http://helixweb.nih.gov/dnaworks/) design corresponding obtains the gene of hPD-L1-Fc, hPD-L1-muFc fusion protein, obtains the gene of hPD-1-Fc after the same method.Enhanced green fluorescence protein EGFP aminoacid sequence (C5MKY7) is obtained according to information on albumen database Uniprot, the aminoacid sequence (Q9NZQ7) of human PD-L 1, the aminoacid sequence (Q9EP73) of Mus PD-L1, the aminoacid sequence (Q15116) of people PD1.The DNA sequences encoding utilizing DNAworks online tool (http://helixweb.nih.gov/dnaworks/) design corresponding obtains the gene of PD-L1-EGFP fusion protein, obtains the gene of hPD-1-EGFP and mPD-L1-EGFP after the same method.Its DNA fragmentation is obtained by the mode of synthetic.Synthetic gene order is subcloned into commercialization carrier pcDNA4/myc-HisA (Invitrogen through HindIII and the EcoRI double digestion of Fermentas company respectively, V863-20) in, sequence verification build plasmid accuracy, it is thus achieved that recombinant plasmid dna namely: pcDNA4-hPD-L1-Fc, pcDNA4-hPD-L1-muFc, pcDNA4-hPD1-Fc, pcDNA4-hPD-L1-EGFP, pcDNA4-hPD1-EGFP, pcDNA4-mPD-L1-EGFP.

Utilizing reverse transcription-polymerase chain reaction RT-PCR technology PD-L2, B7H3 gene of amplification people from the dendritic cell (DC cell) (this DC cell is ripe through TNF-α by the mononuclear cell separated PBMC) of laboratory cultures, amplimer is as follows: PDL2-FHindIII:

GCGCAAGCTTGCCACCATGATCTTCCTCCTGCTAATG (SEQIDNO:74), PDL2-REcoI:

GCCGAATTCGATAGCACTGTTCACTTCCCTC (SEQIDNO:75)；HB7H3-FHindIII:

GCGCAAGCTTGCCACCATGCTGCGTCGGCGGGGCAGC (SEQIDNO:76), hB7H3-RBamHI:

GCGCGAATTCGGCTATTTCTTGTCCATCATCTTC (SEQIDNO:77)；The PCR primer obtained is subcloned in the pcDNA4-hPD-L1-EGFP built through HindIII and the EcoRI double digestion of Fermentas company, sequence verification build plasmid accuracy, it is thus achieved that recombinant plasmid dna namely: pcDNA4-hPD-L2-EGFP, pcDNA4-hB7H3-EGFP.

By relevant EGFP Transfected Recombinant Plasmid to HEK293 (ATCC, CRL-1573^TM) in cell, after transfection 48h, confirmed the expression of hPD1, hPD-L1, mPD-L1, hPD-L2, hB7H3 by fluorescence-activation signal sorting (FACS).

Cell in pcDNA4-hPD-L1-Fc, pcDNA4-hPD-L1-muFc, pcDNA4-hPD1-Fc transient transfection to HEK293 is used for protein production.Recombinant expression plasmid Freestyle293 culture medium diluted and adds required PEI (Polyethylenimine) solution of conversion, will often organize plasmid/PEI mixture and be separately added in cell suspension, and be placed on 37 DEG C, 10%CO₂, 90rpm cultivates；Add 50 μ g/L IGF-1 (insulin-likegrowthfactor-1, IGF-1) simultaneously.Adding EX293 culture medium after four hours again, 2mMGlutamine and 50 μ g/LIGF-1,135rpm cultivate.3.8mM sodium valproate (VPA) is added after twenty four hours.After cultivating 5～6 days, collecting transient expression culture supernatant, by ProteinA affinity chromatography, preliminary purification obtains hPD-L1-Fc, hPD-L1-muFc and hPD-1-Fc protein sample, for following embodiment.The protein sample obtained utilizes SDS-PAGE to carry out preliminary detection, it is possible to be clearly visible that purpose band.

Embodiment 2: screen anti-hPD-L1 antibody, clonal expression and qualification from yeast display library

Adopt yeast display screening for the human antibody of PD-L1.By cloning from the spleen of 21 Healthy Peoples and lymph node IgM, VH and VL gene in IgGcDNA is to specific support, building scFV yeast display library (catenation sequence in the middle of VH and VL is GGGGSGGGGSGGGGS (SEQIDNO:67) connection peptides), storage capacity is 5 × 10⁸.Being recovered in the yeast storehouse of 10 times of storage capacity, induction yeast surface expresses antibody, utilizes the mode of magnetic bead sorting to be enriched with secondary with the biotinylated hPD-L1 antigen of 100nM, then does airflow classification with anti-myc antibody and biotinylated hPD-L1 and be enriched with twice again.The yeast coated plate obtained, picking monoclonal.Monoclonal yeast uses anti-myc antibody and biotinylated hPD-L1 or comparison antigen hPD-1 staining analysis after amplification and abduction delivering, and the yeast of antigen positive/comparison yeast-negative is positive yeast.

The yeast clone confirmed through FACS is carried out yeast colony PCR and order-checking, and PCR primer is: pNL6-F:GTACGAGCTAAAAGTACAGTG (SEQIDNO:78)；PNL6-R:TAGATACCCATACGACGTTC (SEQIDNO:79)；The primer of order-checking is pNL6-R.With BioEdit software, sequence is compared after obtaining sequencing result.

HindIII and EcoRI double digestion through Fermentas company after single-chain antibody scFv gene obtained above and human IgG1's-Fc gene fusion of being previously mentioned is cloned in commercialization carrier pEE6.4 (Lonza), carries out clone according to the standard operation of molecular cloning and plasmid is little carries.Plasmid after extraction is transient expression in HEK293 cell, and by proteinA column purification.

Taking hPD-L1-EGFP cell, be resuspended in 0.5%PBS-BSABuffer, add the anti-hPD-L1scFv antibody after above-mentioned purification, arrange related control simultaneously, negative control is the hIgG1 albumen of 2 μ g, and positive control adds hPD-1-Fc.Two resist the anti-hIg-PE for eBioscience.After dyeing, flow cytometer detects.Identifying in this approach can in conjunction with the antibody of cell surface PD-L1 antigen.

Take hPD-L1-EGFP cell, it is resuspended in 0.5%PBS-BSABuffer, add anti-hPD-L1scFv antibody after above-mentioned purification, negative control is set simultaneously, negative control is the hIgG1 albumen of 2 μ g, and all samples add 0.3 μ ghPD-1-Fc-biotin, and two resist the SA-PE for eBioscience, after dyeing, flow cytometer detects, and result is shown in Fig. 1.Identify the antibody that can block cell surface PD-L1 antigen and PD-1 combination in this approach.

Obtaining the three good antibody of strain characteristic after screening and qualification is B50, B60, B II 61 respectively.By result it can be seen that the antibody of the three anti-hPD-L1 of strain all can block the combination of itself and receptor hPD-1.

Containing connection peptides sequence GGGGSGGGGSGGGGS (SEQIDNO:67) between above-mentioned heavy chain of antibody and variable region of light chain.

Wherein the heavy chain variable amino acid sequence of B50 is:

QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSTKAAWYWIRQSPSRGLEWLGRTYFRSKWYNDYADSVKSRLTINPDTSKNQFSLQLKSVSPEDTAVYYCARGQYTAFDIWGQGTMVTVSS (SEQIDNO:51)；

Wherein horizontal line part respectively CDR1,2,3, its sequence numbers respectively SEQIDNO:13-15；Not drawing horizontal line part respectively FR1,2,3,4, its sequence numbers respectively SEQIDNO:38-41；

The DNA sequence of its correspondence is:

nullCAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTC ACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCACCAAGGCTGCTTGGTACT GGATCAGGCAGTCCCCTTCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTTCCGG TCCAAGTGGTATAATGACTATGCCGACTCTGTGAAAAGTCGATTAACCATCAACCC AGACACATCCAAGAACCAGTTCTCCCTGCAACTTAAGTCTGTGAGTCCCGAGGACA CGGCTGTGTATTACTGTGCAAGAGGGCAATACACTGCTTTTGATATCTGGGGCCAA GGGACAATGGTCACCGTCTCTTCA (SEQIDNO:59)；

Its chain variable region amino acid sequence is:

QSALIQPASVSGSPGQSITISCTGTSSDVGGYDLVSWYQQYPGQAPRLIIYEVIKRPSGISDRFSGSKSGNTASLTISGLQAEDEADYYCSSYAGRRLHGVFGGGTQLTVL (SEQIDNO:56)；

Wherein horizontal line part respectively CDR1,2,3, its sequence numbers respectively SEQIDNO:21,17,18；Not drawing horizontal line part respectively FR1,2,3,4, its sequence numbers respectively SEQIDNO:42-45；

The DNA sequence of its correspondence is:

nullCAGTCTGCTCTGATTCAGCCTGCCTCCGTGTCTGGGTCCCCTGGACAGTCGATCAC TATCTCCTGTACTGGCACCAGTAGTGATGTTGGAGGTTATGACCTTGTCTCCTGGT ACCAACAGTACCCGGGCCAAGCCCCCAGACTCATCATTTATGAGGTCATTAAGCGG CCCTCAGGGATTTCTGATCGCTTCTCTGGTTCCAAGTCTGGCAACACGGCCTCCCT GACAATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTATTGCAGCTCATATG CAGGTAGACGTCTTCATGGTGTGTTCGGAGGAGGCACCCAGCTGACCGTCCTC (SEQIDNO:66).

The heavy chain variable amino acid sequence of B60 is:

QVQLVQSGAEVKKPASSVKVSCTASGGSFSTYAISWVRQAPGQGLEWMGGIIPIFGTTKYAQRFQGRVTITADESTTTAYMELSSLISDDTALYYCTTSRGFSYGWFDYWGQGTLVTVSS (SEQIDNO:53)；

Wherein horizontal line part respectively CDR1,2,3, its sequence numbers respectively SEQIDNO:1,2,19；Not drawing horizontal line part respectively FR1,2,3,4, its sequence numbers respectively SEQIDNO:22-25；

The DNA sequence of its correspondence is:

nullCAGGTCCAGCTTGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGCGTCCTCGGTCAA AGTCTCCTGCACGGCTTCTGGCGGCTCCTTCAGCACCTATGCTATCAGTTGGGTGC GACAGGCTCCTGGACAAGGGCTTGAATGGATGGGCGGGATCATCCCCATCTTTGGT ACAACTAAGTACGCACAGAGGTTCCAGGGCAGGGTCACGATTACCGCGGACGAATC GACGACCACAGCCTACATGGAGCTGAGCAGCCTGATATCTGACGACACGGCCCTGT ATTATTGTACGACGTCTCGTGGATTCAGCTATGGCTGGTTTGACTACTGGGGCCAG GGTACCCTGGTCACCGTCTCCTCA (SEQIDNO:60)；

Its chain variable region amino acid sequence is:

EIVMTQSPATLSLSPGERATLSCRASQSVGIHLAWYQQKLGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPRTFGQGTKVEIK (SEQIDNO:48)；

Wherein horizontal line part respectively CDR1,2,3, its sequence numbers respectively SEQIDNO:4-6；Not drawing horizontal line part respectively FR1,2,3,4, its sequence numbers respectively SEQIDNO:26-29；

The DNA sequence of its correspondence is:

nullGAAATTGTAATGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGC CACCCTCTCCTGTAGGGCCAGTCAGAGTGTTGGCATACACTTAGCCTGGTACCAAC AGAAACTTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGTAGGGCCACT GGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCAT CAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTTCTT TACCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (SEQIDNO:62).

The heavy chain variable amino acid sequence of B II 61 is:

QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSASWNWIRQSPSRGLEWLGRTYYRSKWYDDYAVSVKSRISINPDTSKNQFSLQLNSVTPEDTAVYYCARSQGRYFVNYGMDVWGQGTTVTVSS (SEQIDNO:54)；

Wherein horizontal line part respectively CDR1,2,3, its sequence numbers respectively SEQIDNO:7,20,9；Not drawing horizontal line part respectively FR1,2,3,4, its sequence numbers respectively SEQIDNO:30-33；

The DNA sequence of its correspondence is:

nullCAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTC ACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTTCTTGGAACT GGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATATTACAGG TCCAAATGGTATGATGATTATGCAGTATCTGTGAAAAGTCGAATCAGCATCAACCC AGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACA CGGCTGTGTATTACTGTGCAAGAAGCCAGGGACGATATTTTGTCAACTACGGTATG GACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA (SEQIDNO:61)；

Its chain variable region amino acid sequence is:

DIRLTQSPSSLSASVGDRITITCRASQSISSYLNWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQ SYFTPRGITFGPGTKVDIK (SEQIDNO:55)；

Wherein horizontal line part respectively CDR1,2,3, its sequence numbers respectively SEQIDNO:10-12；Not drawing horizontal line part respectively FR1,2,3,4, its sequence numbers respectively SEQIDNO:34,46,36,37；

The DNA sequence of its correspondence is:

nullGACATCCGGTTGACCCAGTCTCCATCTTCCCTGTCTGCATCTGTAGGAGACAGAAT CACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGTTATTTAAATTGGTATCAAC AGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGTGCATCCAGTTTGCAAAGT GGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCAT CAGCAGTCTGCAACCTGAAGATGTTGCAACTTACTACTGTCAACAGAGTTACTTTA CCCCCCGCGGGATCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA (SEQIDNO:65).

The Yeast libraries that embodiment 3:anti-hPD-L1scFv affinity improves builds

Respectively with pEE6.4-B50-Fc, pEE6.4-B60-Fc, pEE6.4-B II 61-Fc plasmid is template, and pEE6.4-F:TCTGGTGGTGGTGGTTCTGCTAGC (SEQIDNO:80) and cMyc-BBXhoI:GCCAGATCTCGAGCTATTACAAGTCTTCTTCAGAAATAAGCTTT TGTTCTAGAATTCCG (SEQIDNO:81) carries out standard PCR reaction for primer.The PCR primer obtained is cloned in commercialization carrier pCT302 (addgene:#41845) through NheI and the BglII of Fermentas company, obtains pCT302-B50, pCT302-B60, pCT302-BII61 recombiant plasmid.With reference next to (2006) NatProtoc1 (2) such as document Ginger: the method for 755-68, the method for errorpronePCR is utilized to obtain the PCR primer of scFv random mutation.Primer used is T7proshort:TAATACGACTCACTATAGGG (SEQIDNO:82) and Splice4/L:GGCAGCCCCATAAACACACAGTAT (SEQIDNO:83).The PCR primer obtained after Fermentas company GeneJETDNApurificationKit purification again ethanol precipitation concentration to concentration more than 1 μ g/ μ l.Utilize Fermentas company NheI and BamHI double digestion commercialization carrier pCT302, simultaneously with Fermentas company FastAP dephosphorylation enzyme to carrier dephosphorylation after enzyme action, after purifying with Fermentas company GeneJETDNApurificationKit again, ethanol precipitation concentration to concentration more than 1 μ g/ μ l.(2006) NatProtoc1 (2) such as list of references Ginger: the method for 755-68, utilize yeast electricity to convert the method with In vivo recombination and obtain the yeast storehouse of affinity maturation.

Embodiment 4: produce the yeast anti-hPD-L1scFv screening that affinity improves

By the hPD-L1-Fc albumen of yeast storehouse 10nM and the 1nM after affinity maturation obtained above through two-wheeled airflow classification, the yeast product coated plate that sorting obtains, picking monoclonal is identified.Utilizing the method that low concentration antigen dyes, using the wild-type yeast that obtains before as comparison, the yeast monoclonal that affinity improves is determined in streaming dyeing.

The yeast clone confirmed through FACS is carried out yeast colony PCR and order-checking, and method is ibid.HindIII and EcoRI double digestion through Fermentas company after scFv gene after affinity maturation and human IgG1's-Fc gene fusion before is cloned in commercialization carrier pEE6.4, carries out clone according to the standard operation of molecular cloning and plasmid is little carries.Plasmid after extraction is transient expression in HEK293 cell, and by proteinA column purification.

The detection combining (binding) ability and (blocking) ability of blocking-up is carried out according to the method antagonist in embodiment 2.

The experimental result of binding ability is shown in Fig. 2, it can be seen that the affinity of three strain antibodies obtained after affinity maturation significantly improves.

The experimental result of blocking ability is shown in Fig. 3, can be seen that, it is 0.837 μ g/ml (B II 61 is 0.884 μ g/ml) that the three strain antibodies competition PD-1 obtained after affinity maturation are respectively as follows: B II 61-62 in conjunction with the IC50 of PD-L1, B50-6 is 4.56 μ g/ml (B50 is 5.63 μ g/ml), and B60-55 is 1.14 μ g/ml (B60 is 16.8 μ g/ml)

Obtain after affinity maturation B50-6, B60-55, B II 61-62 tri-strain affinity improve anti-hPD-L1scFv antibody sequence.Compared with B50, B50-6 there occurs the sudden change of aminoacid D to N in the CDR1 district of VL；Compared with B60, B60-55 there occurs the sudden change of aminoacid S to N in the CDR3 district of VH；Compared with BII61, BII61-62 there occurs the sudden change of aminoacid S to G in the CDR2 district of VH, there occurs the sudden change of aminoacid I to V in VLFR2 district.Containing connection peptides sequence GGGGSGGGGSGGGGS (SEQIDNO:67) between above-mentioned heavy chain of antibody and variable region of light chain.

Wherein the heavy chain variable amino acid sequence of B50-6 is:

The DNA sequence of its correspondence is:

Its chain variable region amino acid sequence is:

QSALIQPASVSGSPGQSITISCTGTSSNVGGYDLVSWYQQYPGQAPRLIIYEVIKRPSGISDRFSGSKSGNTASLTISGLQAEDEADYYCSSYAGRRLHGVFGGGTQLTVL (SEQIDNO:52)；

Wherein horizontal line part respectively CDR1,2,3, its sequence numbers respectively SEQIDNO:16-18；Not drawing horizontal line part respectively FR1,2,3,4, its sequence numbers respectively SEQIDNO:42-45；

The DNA sequence of its correspondence is:

nullCAGTCTGCTCTGATTCAGCCTGCCTCCGTGTCTGGGTCCCCTGGACAGTCGATCAC TATCTCCTGTACTGGCACCAGTAGTAATGTTGGAGGTTATGACCTTGTCTCCTGGT ACCAACAGTACCCGGGCCAAGCCCCCAGACTCATCATTTATGAGGTCATTAAGCGG CCCTCAGGGATTTCTGATCGCTTCTCTGGTTCCAAGTCTGGCAACACGGCCTCCCT GACAATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTATTGCAGCTCATATG CAGGTAGACGTCTTCATGGTGTGTTCGGAGGAGGCACCCAGCTGACCGTCCTC (SEQIDNO:64)；

The heavy chain variable amino acid sequence of B60-55 is:

QVQLVQSGAEVKKPASSVKVSCTASGGSFSTYAISWVRQAPGQGLEWMGGIIPIFGTTKYAQRFQGRVTITADESTTTAYMELSSLISDDTALYYCTTSRGFNYGWFDYWGQGTLVTVSS (SEQIDNO:47)；

Wherein horizontal line part respectively CDR1,2,3, its sequence numbers respectively SEQIDNO:1-3；Not drawing horizontal line part respectively FR1,2,3,4, its sequence numbers respectively SEQIDNO:22-25；

The DNA sequence of its correspondence is:

nullCAGGTCCAGCTTGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGCGTCCTCGGTCAA AGTCTCCTGCACGGCTTCTGGCGGCTCCTTCAGCACCTATGCTATCAGTTGGGTGC GACAGGCTCCTGGACAAGGGCTTGAATGGATGGGCGGGATCATCCCCATCTTTGGT ACAACTAAGTACGCACAGAGGTTCCAGGGCAGGGTCACGATTACCGCGGACGAATC GACGACCACAGCCTACATGGAGCTGAGCAGCCTGATATCTGACGACACGGCCCTGT ATTATTGTACGACGTCTCGTGGATTCAACTATGGCTGGTTTGACTACTGGGGCCAG GGTACCCTGGTCACCGTCTCCTCA (SEQIDNO:57)；

Its chain variable region amino acid sequence is:

The DNA sequence of its correspondence is:

nullGAAATTGTAATGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGC CACCCTCTCCTGTAGGGCCAGTCAGAGTGTTGGCATACACTTAGCCTGGTACCAAC AGAAACTTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGTAGGGCCACT GGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCAT CAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTTCTT TACCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (SEQIDNO:62)；

The heavy chain variable amino acid sequence of B II 61-62 is:

QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSASWNWIRQSPSRGLEWLGRTYYRSKWYDDYAVSVKGRISINPDTSKNQFSLQLNSVTPEDTAVYYCARSQGRYFVNYGMDVWGQGTTVTVSS (SEQIDNO:49)；

Wherein horizontal line part respectively CDR1,2,3, its sequence numbers respectively SEQIDNO:7-9；Not drawing horizontal line part respectively FR1,2,3,4, its sequence numbers respectively SEQIDNO:30-33；

The DNA sequence of its correspondence is:

nullCAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTC ACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTTCTTGGAACT GGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATATTACAGG TCCAAATGGTATGATGATTATGCAGTATCTGTGAAAGGTCGAATCAGCATCAACCC AGACACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCCGAGGACA CGGCTGTGTATTACTGTGCAAGAAGCCAGGGACGATATTTTGTCAACTACGGTATG GACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA (SEQIDNO:58)；

Its chain variable region amino acid sequence is:

DIRLTQSPSSLSASVGDRITITCRASQSISSYLNWYQQKPGKAPKLLVYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQ SYFTPRGITFGPGTKVDIK (SEQIDNO:50)；

Wherein horizontal line part respectively CDR1,2,3, its sequence numbers respectively SEQIDNO:10-12；Not drawing horizontal line part respectively FR1,2,3,4, its sequence numbers respectively SEQIDNO:34-37；

The DNA sequence of its correspondence is:

nullGACATCCGGTTGACCCAGTCTCCATCTTCCCTGTCTGCATCTGTAGGAGACAGAAT CACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGTTATTTAAATTGGTATCAAC AGAAACCAGGGAAAGCCCCTAAGCTCCTGGTCTATGGTGCATCCAGTTTGCAAAGT GGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCAT CAGCAGTCTGCAACCTGAAGATGTTGCAACTTACTACTGTCAACAGAGTTACTTTA CCCCCCGCGGGATCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA (SEQIDNO:63).

Embodiment 5:scFv type antibody is formatted as IgG type antibody

According to the amino acid constant region sequence (P01857) of human normal immunoglobulin gamma1 (IgG1) on albumen database Uniprot, obtain human IgG1's amino acid constant region sequence.The DNA sequences encoding utilizing DNAworks online tool (http://helixweb.nih.gov/dnaworks/) design corresponding obtains human IgG1's constant region gene, the B50-6 that screening is obtained, B60-55, the variable region of heavy chain VH sequence of BII61-62 and human IgG1's constant region gene sequences are stitched together, and add signal peptide sequence: ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCAC CGGT (SEQIDNO:84) at the 5 ' ends of VH simultaneously；The gene chemical synthesis that will have spliced, obtains pEE6.4-B50-6HC through Fermentas company HindIII and EcoRI double digestion sub-clone to carrier pEE6.4；pEE6.4-B60-55HC；pEE6.4-BⅡ61-62HC.According to the amino acid constant region sequence (P01834) of human normal immunoglobulin Kappa on albumen database Uniprot, obtain people's Kappa chain constant region amino acid sequence.The DNA sequences encoding utilizing DNAworks online tool (http://helixweb.nih.gov/dnaworks/) design corresponding obtains people's Kappa light chain constant region gene, the B60-55 that screening is obtained, the variable region of light chain VL sequence of B II 61-62, together with people's Kappa light chain constant region gene sequence assembly, adds signal peptide sequence: ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCAC CGGT (SEQIDNO:84) at the 5 ' ends of VL simultaneously；The DNA sequences encoding utilizing DNAworks online tool (http://helixweb.nih.gov/dnaworks/) design corresponding obtains people lambda (λ) light chain constant region gene, the variable region of light chain VL sequence of B50-6 screening obtained, together with people's lambda light chain constant region gene sequence assembly, adds signal peptide sequence: ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCAC CGGT (SEQIDNO:84) at the 5 ' ends of VL simultaneously；The gene chemical synthesis that will have spliced, obtains pEE12.4-B50-6LC through Fermentas company HindIII and EcoRI double digestion sub-clone to carrier pEE12.4 (Lonza)；pEE12.4-B60-55LC；pEE12.4-BⅡ61-62LC.

Utilize the big extraction reagent kit of the plasmid (PL14) that AidLab company provides that heavy chain obtained above and light chain plasmids are carried out plasmid to carry greatly.The light chain of recombination to construct and heavy chain plasmid cotransfection HEK293 cell are carried out antibody expression.Recombinant expression plasmid Freestyle293 culture medium diluted and adds required PEI (Polyethylenimine) solution of conversion, will often organize plasmid/PEI mixture and be separately added in cell suspension, and be placed on 37 DEG C, 10%CO₂, 90rpm cultivates；Add 50 μ g/LIGF-1 simultaneously.Adding EX293 culture medium, 2mMGlutamine and 50ug/LIGF-1 after four hours again, 135rpm cultivates.3.8mMVPA is added after twenty four hours.After cultivating 5～6 days, collecting transient expression culture supernatant, by ProteinA affinity chromatography, purification obtains anti-hPD-L1B50-6, B60-55, B II 61-62mAb antibody.

Wherein IgG1 chain amino acid constant region sequence is:

nullASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQIDNO:68)；

IgG1 chain constant region nucleotide sequence is:

nullGCCAGCACTAAGGGGCCCTCTGTGTTTCCACTCGCCCCTTCTAGCAAAAGCACTTCCGGAGGCACTGCAGCACTCGGGTGTCTGGTCAAAGATTATTTCCCTGAGCCAGTCACCGTGAGCTGGAACTCTGGCGCCCTCACCTCCGGGGTTCACACCTTTCCAGCCGTCCTGCAGTCCTCCGGCCTGTACTCCCTGAGCAGCGTCGTTACCGTGCCATCCTCTTCTCTGGGGACCCAGACATACATCTGCAATGTCAACCATAAGCCTAGCAACACCAAGGTGGACAAAAAGGTCGAGCCAAAGAGCTGCGATAAGACACACACCTGCCCTCCATGCCCCGCACCTGAACTCCTGGGCGGGCCTTCCGTTTTCCTGTTTCCTCCCAAGCCCAAGGATACACTGATGATTAGCCGCACCCCCGAAGTCACTTGCGTGGTGGTGGATGTGAGCCATGAAGATCCAGAAGTTAAGTTTAACTGGTATGTGGACGGGGTCGAGGTGCACAATGCTAAAACAAAGCCCAGGGAGGAGCAATATAACTCCACATACAGAGTGGTGTCCGTTCTGACAGTCCTGCACCAGGACTGGCTGAACGGGAAGGAATACAAGTGCAAGGTGTCTAATAAGGCACTGCCAGCCCCCATAGAGAAGACAATCTCTAAAGCTAAAGGCCAACCACGCGAGCCTCAGGTCTACACACTGCCACCATCCAGGGACGAACTGACCAAGAATCAGGTGAGCCTGACTTGTCTCGTCAAAGGATTCTACCCAAGCGACATCGCCGTGGAGTGGGAATCCAACGGCCAACCAGAGAACAACTACAAGACCACCCCACCAGTCCTGGACTCTGATGGGAGCTTTTTCCTGTATTCCAAGCTGACAGTGGACAAGTCTCGGTGGCAACAGGGCAACGTGTTCAGCTGCTCCGTGATGCATGAAGCCCTGCATAACCACTATACCCAGAAAAGCCTCAGCCTGTCCCCCGGGAAATAATGA ( SEQIDNO：69 )；

Kappa chain amino acid constant region sequence is:

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQIDNO:70)；

Kappa chain constant region nucleotide sequence is:

nullCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA ATCTGGTACCGCTAGCGTTGTGTGCCTGCTGAATAACTTTTATCCACGGGAGGCTA AGGTGCAGTGGAAAGTGGACAATGCCCTCCAGAGCGGAAATAGCCAAGAGTCCGTT ACCGAACAGGACTCTAAAGACTCTACATACTCCCTGTCCTCCACACTGACCCTCTC CAAGGCCGACTATGAGAAACACAAGGTTTACGCATGCGAGGTCACACACCAGGGAC TCTCCTCTCCCGTGACCAAGAGCTTCAACCGGGGAGAATGC (SEQIDNO:71)；

B50-6 light chain (lambda) amino acid constant region sequence is:

GQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPVKVGVETT KPSKQSNNKYAASSYLSLTPEQWKSHRSYSCRVTHEGSTVEKTVAPAECS (SEQIDNO:72)；

B50-6 light chain (lambda) constant region nucleotide sequence is:

nullGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCACCCTCCTCTGAGGAGCT TCAAGCCAACAAGGCCACACTGGTGTGTCTCGTAAGTGACTTCTACCCGGGAGCCG TGACAGTGGCCTGGAAGGCAGATGGCAGCCCCGTCAAGGTGGGAGTGGAGACCACC AAACCCTCCAAACAAAGCAACAACAAGTATGCGGCCAGCAGCTACCTGAGCCTGAC GCCCGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCGGGTCACGCATGAAGGGA GCACCGTGGAGAAGACAGTGGCCCCTGCAGAATGCTCT (SEQIDNO:73).

Embodiment 6:anti-hPD-L1mAb CHARACTERISTICS IDENTIFICATION

The anti-hPD-L1 antibody of purification and hPD-L1 binding ability detection (ELISA method):

To be coated buffer (50mMNa₂CO₃, NaHCO₃PH9.6) dilution hPD-L1-muFc to 2 μ g/ml, 100 μ L/well, 4 DEG C overnight.Getting rid of liquid in plate, after PBST (pH7.4,0.05%Tween-20, V/V) washes plate, 3%BSA-PBS closes 1h.By antibody B50-6mAb, B60-55mAb and B μ 61-62mAb respectively from 2000ng/ml, carrying out 2 times of gradient dilutions, totally 11 concentration, diluent (1%BSA-PBS) compares, and hatches 2h for 37 DEG C.Add goat anti-human igg-HRP (Goatanti-humanIgG-HRPconjugated), hatch 1h.Add solubility one pack system tmb substrate nitrite ion, room temperature lucifuge colour developing 5-10min.2NH₂SO₄50 μ L/well, color development stopping is reacted.Putting reading OD450nm-650nm value in MDSpectraMaxPlus384 microplate reader, application software SotfMaxProv5.4 carries out data process and mapping analysis, and result is shown in Fig. 4.

The conjugated antigen EC50 respectively 40 μ g/ml (B60-55mAb), 18.3 μ g/ml (B II 61-62mAb) of three strain antibodies and 28.1 μ g/ml (B50-6mAb) are determined by the method.

The anti-hPD-L1 antibody of purification and hPD-L1 binding kinetics detection (SPR):

Anti-PD-L1 antibody B50-6mAb, BII61-62mAb and B60-55mAb pass through surface plasmon resonance (surfaceplasmonresonance for the binding kinetics of the human PD-L 1 of restructuring, SRP) method, uses BIAcoreX100 apparatus measures.Restructuring hPD-L1-Fc direct coated on CM5 biologic sensor chip to obtain about 1000 response units (responseunits, RU).For kinetic measurement, by antibody HBS-EP+1 × three times of serial dilutions (1.37nm to 1000nm) of (GE, cat#BR-1006-69) buffer, at 25 DEG C of sample introduction 120s, Dissociation time is that 30min, 10mMglycine-HCl (pH2.0) regenerate 120s.Use simple Languir combination model (BIAcore evaluation software 3.2 editions (BIAcoreEvaluationSoftwareversion3.2)) calculations incorporated speed (kon) and dissociation rate (koff) one to one.Equilibrium dissociation constant (kD) calculates with ratio koff/kon.

The binding affinity of the anti-PD-L1 antibody measured is in Table 1.

Table 1anti-hPD-L1 antibody and the detection of hPD-L1 binding kinetics

Title	Kon(1/Ms)	Koff(1/s)	KD(M)
				B50-6mAb	1.672E+5	1.370E-2	8.193E-8
B60-55mAb	1.295E+6	2.222E-4	1.716E-10
				BⅡ61-62mAb	9.795E+4	4.264E-4	4.353E-9

The anti-hPD-L1 antibody of purification and the detection of hPD-1 competition binding hPD-L1 ability:

To be coated buffer (50mMNa₂CO₃, NaHCO₃PH9.6) dilution hPD-L1-hIgG to 5 μ g/ml, 4 DEG C overnight.After PBST (pH7.4,0.05%Tween-20, V/V) washes plate, 3%BSA-PBS closes 1h.It is 100 μ g/ml, 1%BSA-PBST-0.05%Tween-20 (hPD-1-hIgG-biotin containing 10 μ g/ml) 1:6 gradient dilution, totally 9 dilution factors by the concentration dilution of anti-hPD-L1mAb to be measured, 37 DEG C of 2h.Streptavidin (SA-HRPconjugated) the incubated at room 1.5h of horseradish peroxidase-labeled is added after washing plate.Add solubility one pack system tmb substrate nitrite ion room temperature lucifuge colour developing 5-10min, 2NH₂SO₄Color development stopping is reacted.Put reading OD in MDSpectraMaxPlus384 microplate reader_450nm-650nmValue, application software SotfMaxProv5.4 carries out data process and mapping analysis, analyzes antibody competition ability according to detection data and IC50 value strong and weak, and result is shown in Fig. 5.

The three strain antibody competition PD-1 IC50 respectively 0.255 μ g/ml1.7nM (B60-55), 0.24 μ g/ml1.6nM (B II 61-62) in conjunction with PD-L1 and 1.76 μ g/ml11.7nM (B50-6) are determined by the method.

The anti-hPD-L1 antibody of purification and the detection of CD80 competition binding hPD-L1 ability:

Take three strain antibody B60-55, B II 61-62 and B50-6 that screening obtains, by the method for competitive ELISA, evaluate its combination that whether can block PD-L1 and CD80.Concrete grammar is as follows: to be coated buffer (50mMNa₂CO₃, NaHCO₃PH9.6) dilution hPD-L1-hFc to 5 μ g/ml, 4 DEG C overnight.After PBST (pH7.4,0.05%Tween-20, V/V) washes plate, 3%BSA-PBS closes 1h.It is 100 μ g/ml, 1%BSA-PBST-0.05%Tween-20 (hCD80-hFc-biotin, R&D:140-B1-100 containing 100 μ g/ml) 1:6 gradient dilution, totally 9 dilution factors by the concentration dilution of anti-hPD-L1mAb to be measured, 37 DEG C of 2h.Streptavidin (SA-HRPconjugated) the incubated at room 1.5h of horseradish peroxidase-labeled is added after washing plate.Add solubility one pack system tmb substrate nitrite ion room temperature lucifuge colour developing 5-10min, 2NH₂SO₄Color development stopping is reacted.Putting reading OD450nm-650nm value in MDSpectraMaxPlus384 microplate reader, application software SotfMaxProv5.4 carries out data process and mapping analysis, analyzes antibody competition ability according to detection data and IC50 value strong and weak, and result is shown in Fig. 6.

The three strain antibody competition CD80 IC50 respectively 0.543 μ g/ml (B60-55), 0.709 μ g/ml (B II 61-62) in conjunction with PD-L1 and 0.553 μ g/ml (B50-6) is determined by the method.

The qualification of antibody whether specific recognition PD-L1: anti-hPD-L1 and the hPD-L1 of purification, The combination of hPD-L2, hB7H3

The HEK293 cell containing hPD-L1-EGFP, hB7H3-EGFP, hPD-L2-EGFP that Example 1 builds, is resuspended in 0.5%PBS-BSABuffer, adds anti-hPD-L1mAb albumen, and negative control is hIgGFc albumen, hatches 20min on ice.The anti-anti-hIg-PE of eBioscience bis-, 20min on ice is added after washing.Being resuspended in by cell in 500 μ l0.5%PBS-BSABuffer after washing, flow cytometer detects.Result is as shown in Figure 6.It can be seen from the results that our three strain antibodies all with hB7H3-EGFP, hPD-L2-EGFP Cell binding, can not show good specificity with hPD-L1-EGFP Cell binding.

The combination of the anti-hPD-L1 of purification and mice PD-L1 (mPD-L1):

The HEK293 cell containing hPD-L1-EGFP, mPD-L1-EGFP that Example 1 builds, it is resuspended in 0.5%PBS-BSABuffer, add anti-hPD-L1mAb to be detected, negative control is hIgGFc albumen, hatch 20min on ice, add the anti-anti-hIg-PE of eBioscience bis-after washing, hatch 20min on ice.Being resuspended in 0.5%PBS-BSABuffer by cell after washing, flow cytometer detects.Result is as shown in Figure 7.Be can be seen that B50-6mAb can combine with the PD-L1 (mPD-L1) of mice by result, and B60-55 and BII61-62 can not be combined with mPD-L1.

The combination of the anti-hPD-L1 of purification and machin PD-L1:

Human lymphocyte separating medium (Tianjin Hao ocean) is utilized to separate machin peripheral blood PBMC, re-suspended cell is in RPMI complete medium, adjustment cell concentration is 1,000,000/milliliter, then in 24 orifice plates, add 2,000,000 cell machin PBMC cells, being simultaneously introduced phytohaemagglutinin (phytohaemagglutinin, PHA), final concentration of 2 μ g/ml stimulate cell 48 hours, then collect cell, FACS buffer adds after washed cell antibody staining.Using Isotypectrl (anti-KLH) as negative control, the anti human PD-L 1 antibody (Biolegend:329705) of commercialization PE labelling is as positive control.Our antibody, as primary antibodie, adds anti-hIg-PE and anti-dyes as two after scrubbed.Each step dyeing 4 DEG C is hatched 30 minutes, and dyeing uses FACS buffer by centrifugation washed cell twice after terminating, and adds after two anti-or direct 2% paraformaldehydes are fixed and analyzes with Guava.Result is as shown in Figure 8.Result shows that machin T cell stimulates through PHA and expresses PD-L1, and our three strain antibodies can combine with the machin T cell of activation.

Embodiment 7: measure PD-L1 antibody to CD4 in dendritic cell-T cell mixing lymph reaction⁺The activation of T cell

Utilize human lymphocyte separating medium (Tianjin Hao ocean) density gradient centrifugation separating peripheral blood mononuclear cells PBMC from healthy donor's peripheral blood White Blood Cells Concentrate.Then it is resuspended in the RPMI1640 of serum-free, cultivates 1-2 hour in 10cm culture dish, remove not adherent cell, and cell is incubated in the RPMI containing 10%FBS.Cytokine is added, the fresh culture adding factor-containing in every 2-3 days with the final concentration of 250ng/mlGM-CSF (Shanghai is general glad: 102-03) and 100ng/mlIL-4 (Shanghai is general glad: 101-04).At the 6th day cultivated, make cell maturation with the TNF-alpha (Shanghai is general glad: 103-01) of 50ng/ml and make it hatch 24 hours.With HLA-DR antibody staining, the dendritic cell that results are ripe, determine that it is ripe.Be resuspended in RPMI complete medium, 200,000/ml. then in 96 holes U-shaped base plate (Costar:3799) every hole add 50 μ l, put in incubator cultivate.

Magnetic bead separation kit (MiltenyiBiotec:130-096-533) method to specifications is utilized to separate CD4 from another donor PBMC⁺T cell.Counting is resuspended in RPMI complete medium, and concentration is 2,000,000/ml, is then added to the 96 hole U-shaped base plates containing dendritic cell, and every hole adds 50 μ l.Every hole adds gradient dilution PD-L1 antibody 100 μ l in RPMI complete medium, and antibody final concentration is 100,10,1,0.1,0.01,0.001,0 μ g/ml respectively.Take supernatant after cultivating five days, utilize IFN-rELISA detection kit (ebioscience) to detect the level of IFN-r in supernatant.Result is shown in Fig. 9.Visible PD-L1 antibody can strengthen CD4 in mixed lymphocyte reaction⁺T cell secretion gamma interferon, say, that PD-L1 antibody enhances the activation of T cell.The EC50 value of B II 61-62 is the EC50 value of 0.078 μ g/ml (being equivalent to 0.5nM), B60-55 is that 0.189 μ g/ml (is equivalent to 1.2nM.

The inhibitory activity of embodiment 7:anti-hPD-L1 antibodies on tumor growth

It has become clear that a lot of tumors utilize expresses PD-1 part as the method weakening Anti-tumor t cell response.In the tumor of several people and tumor-infiltrating leukocyte, find to express characteristically the PD-L1 of elevated levels, and the PD-L1 of this rising expresses and is often associated with worse prognosis.Mouse tumor model shows that in tumor, PD-L1 expression has similar increase, and shows the effect in suppressing tumour immunity of the PD-1/PD-L1 approach.

We provide and experiments show that blocking-up PD-L1 is on the impact of the tumor growth of MC38 cell (Mus colorectal cancer cell) in syngeneic C57B6 mice herein.

When the 0th day with 1,000,000 MC38 cells (Chicago University professor Fu Yangxin gives) subcutaneous vaccination C57B6 mice, the 0th, 3,7,10 day with 10mg/kganti-PD-L1 (B50-6) or PBS intraperitoneal injection of mice.Within every three days, measure tumor major diameter and minor axis, calculate gross tumor volume, draw tumor growth curve figure (referring to Figure 10), it can be seen that anti-PD-L1 (B50-6) can significantly inhibit tumor growth.

For not can recognise that antibody B60-55 and the BII61-62 of mice PD-L1, adopt immunodeficiency NOD/SCID (non-obese patients with type Ⅰ DM/severe combined immunodeficiency) its activity in vivo of mice study.By NOD/SCID mouse subcutaneous transplanting being expressed the K-1735 of human PD-L 1_A375(ATCC, CRL-1619^TM) and the experiment of PERIPHERAL BLOOD MONONUCLEAR CELL PBMC of people realize this research purpose.A375 and PBMC mixes before the injection in the ratio of 5:1, cumulative volume 100 μ l subcutaneous injection (containing 5,000,000 A375,1,000,000 PBMC), and at the 0th, 7,14,21,28 day intraperitoneal injection of tumor inoculation, (Figure 11 A antibody dosage is 3mg/kg to antibody；Figure 11 B antibody dosage is shown in Figure 11), PBS is as negative control.4-6 mice of each experimental group.The formation that twice is observed tumor weekly, and with vernier caliper measurement tumor major diameter and minor axis, calculate gross tumor volume, draw tumor growth curve figure (referring to Figure 11), it can be seen that antibody B60-55 and BII-61-62 can significantly inhibit tumor growth.

Embodiment 8B60-55 and antibody 2.41H90P (MedimmuneLLC company) stability compares

nullThe antibody 2.41H90P of antagonism PD-L1 antibody B60-55 and MedImmuneLLC has carried out 45 DEG C of accelerated stability experiments，Specific experiment method is: (prepared according to the method for 2.14H9 in patent US20130034559 by the anti-PD-L1 antibody 2.41H90P of anti-PD-L1 antibody B60-55 and MedImmuneLLC company，And renamed as 2.41H90P) it is concentrated into 10mg/ml，Take 100 μ g antibody to put in 200 μ lPCR pipes，45 DEG C of water-baths，In the 0th day，10th day，20th day，30th day receive sample be at war with ELISA and SEC-HPLC analyze experiment，Competitive ELISA method is with described in previous embodiment 6，Try to achieve IC50.SEC-HPLC adopts Shimadzu LC20ATHPLC chromatograph of liquid to be analyzed experiment, and by sample concentration to 1mg/ml, flow velocity is 0.5ml/min loading, and total applied sample amount is 50ug, carries out isocratic elution 30min after loading, and result is shown in Figure 12.

Wherein A is B60-55 and the time dependent IC50 value comparison diagram of antibody 2.41H90P, it can be seen that the simple competitive power that different time is checked and accepted does not have significant change；B is the time dependent ratio of antibody dimer, it can be seen that increase over time, and B60-55 and 2.41H90P can present the situation that dimer ratio declines, but the speed that 2.41H90P declines is faster than B60-55, it was shown that the stability of B60-55 is better；C is the competitive ELISA curve of B60-55 acceleration for stabilization experiment, it can be seen that B60-55 can keep activity and stability preferably.

Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, it is possible to those details carry out various amendment and replacement, and these change all within protection scope of the present invention.The four corner of the present invention is provided by claims and any equivalent thereof.

Claims

1. anti-PD-L1 antibody or its antigen-binding portion thereof, it includes being selected from such as the CDR region of next group:

2. the anti-PD-L1 antibody of claim 1 or its antigen-binding portion thereof, it includes being selected from such as the variable region of heavy chain of next group:

3. the anti-PD-L1 antibody of claim 1 or its antigen-binding portion thereof, it includes being selected from such as the variable region of light chain of next group:

4. any one of claim 1-3-anti-PD-L1 antibody or its antigen-binding portion thereof, it is whole antibody, bi-specific antibody, scFv, Fab, Fab', F (ab')₂Or Fv.

5. the anti-PD-L1 antibody of claim 4 or its antigen-binding portion thereof, when it is scFv, can contain connection peptides between its heavy chain and variable region of light chain.

6. the anti-PD-L1 antibody of claim 5 or its antigen-binding portion thereof, the sequence of described connection peptides is such as shown in SEQIDNO:67.

7. the anti-PD-L1 antibody of any one of claim 1-4 or its antigen-binding portion thereof, its CH is selected from IgG, IgM, IgE, IgD and IgA.

8. the anti-PD-L1 antibody of claim 7 or its antigen-binding portion thereof, its CH is selected from IgG1, IgG2, IgG3 and IgG4.

9. the anti-PD-L1 antibody of any one of claim 6-8 or its antigen-binding portion thereof, its constant region of light chain is κ or λ.

10. nucleic acid molecules, its comprise can the nucleotide sequence of encoding antibody heavy variable region, described antibody heavy chain variable region comprises the aminoacid sequence being selected from a group:

(i) SEQIDNO:1-3；

(ii) SEQIDNO:7-9；

(iii) SEQIDNO:13-15；

(iv) SEQIDNO:1,2,19；

(v) SEQIDNO:7,20,9.

11. the nucleic acid molecules of claim 10, described antibody heavy chain variable region comprises the aminoacid sequence being selected from a group:

12. nucleic acid molecules, its comprise can the nucleotide sequence of encoding antibody light variable region, described antibody chain variable region comprises the aminoacid sequence being selected from a group:

(i) SEQIDNO:4-6；

(ii) SEQIDNO:10-12；

(iii) SEQIDNO:16-18；

(iv) SEQIDNO:21,17,18.

13. the nucleic acid molecules of claim 12, described antibody chain variable region comprises the aminoacid sequence being selected from a group:

14. carrier, it contains the nucleic acid molecules of any one of claim 10-13.

15. the carrier of claim 14, the nucleic acid molecules of its nucleic acid molecules containing any one of claim 10-11 and any one of claim 12-13.

16. host cell, the carrier of its nucleic acid molecules containing any one of claim 10-13 or claims 14 or 15.

17. conjugate, its anti-PD-L1 antibody containing any one of claim 1-9 or its antigen-binding portion thereof, and other bioactive substance, described anti-PD-L1 antibody or its antigen-binding portion thereof are directly or by junction fragment and other bioactive substance coupling.

null18. the conjugate of claim 17，Other bioactive substance described is selected from can direct or indirect cell growth inhibiting or kill cell、Or by activating organism immune response thus suppressing or killing cell，Thus reaching the chemical substance for the treatment of tumor、Toxin、Polypeptide、Enzyme、Isotope、Cytokine、Antibody or other there is bioactive one matter or compounding substances，Such as AuristatinMMAE、AuristatinMMAF、MaytansineDM1、MaytansineDM4、Calicheamicin (calicheamicin)、duocarmycinMGBA、Amycin (doxorubicin)、Ricin、The toxin such as diphtheria toxin, diphtherotoxin、I131、Interleukin class、Tumor necrosis factor、Chemotactic factor、Nano-particle etc..

19. compositions (such as pharmaceutical composition), the conjugate of its anti-PD-L1 antibody containing any one of claim 1-9 or its antigen-binding portion thereof, the nucleic acid molecules of claim 10-13, the carrier of claims 14 or 15, the host cell of claim 16 or claim 17 or 18, and optional pharmaceutically acceptable carrier or excipient, and other optional bioactive substance.

20. the compositions of claim 19 (such as pharmaceutical composition), other bioactive substance described includes but not limited to other antibody, fusion protein or medicine (such as antitumor drug, such as chemotherapeutics).

21. detectable or test kit, its anti-PD-L1 antibody containing any one of claim 1-9 or its antigen-binding portion thereof, described detectable or test kit are used for detecting whether PD-L1 albumen or derivatives thereof exists.

22. diagnostic reagent or test kit, its anti-PD-L1 antibody containing any one of claim 1-9 or its antigen-binding portion thereof, described diagnostic reagent or test kit diagnose the disease (such as tumor or viral infection, for instance the viral infection of PD-L1 high expressed or the tumor of PD-L1 high expressed) relevant to PD-L1 for (such as cell or tissue) or internal (such as human or animal's model) in vitro.

23. the diagnosis of claim 22 or diagnostic kit, described anti-PD-L1 antibody or the also coupling of its antigen-binding portion thereof have can be used for detection or the fluorescent dye that can be detected by other reagent, chemical substance, polypeptide, enzyme, isotope, label etc..

24. the anti-PD-L1 antibody of any one of claim 1-9 or its antigen-binding portion thereof, the nucleic acid molecules of claim 10-13, the carrier of claims 14 or 15, the host cell of claim 16, the conjugate of claim 17 or 18 or the compositions of claim 19 or 20 are for preparing the purposes of the medicine preventing or treating the disease (such as tumor or viral infection, for instance the tumor of PD-L1 high expressed or the viral infection of PD-L1 high expressed) relevant to PD-L1.

25. the purposes of claim 24, wherein said tumor includes but not limited to pulmonary carcinoma, ovarian cancer, colon and rectum carcinoma, melanoma, renal carcinoma, bladder cancer, breast carcinoma, hepatocarcinoma, lymphoma, malignant hematologic disease, head and neck cancer, glioma, gastric cancer, nasopharyngeal carcinoma, laryngeal carcinoma, cervical cancer, carcinoma of uterine body, osteosarcoma.

26. the purposes of claim 24, wherein said viral infection includes but not limited to acute, subacute or Chronic HBV, HCV, HIV.

27. the anti-PD-L1 antibody of any one of claim 1-9 or its antigen-binding portion thereof are for preparing the purposes in the reagent or test kit diagnosing the disease (such as tumor or viral infection, for instance the tumor of PD-L1 high expressed or the viral infection of PD-L1 high expressed) relevant to PD-L1.

28. the purposes of claim 27, wherein said tumor includes but not limited to pulmonary carcinoma, ovarian cancer, colon and rectum carcinoma, melanoma, renal carcinoma, bladder cancer, breast carcinoma, hepatocarcinoma, lymphoma, malignant hematologic disease, head and neck cancer, glioma, gastric cancer, nasopharyngeal carcinoma, laryngeal carcinoma, cervical cancer, carcinoma of uterine body, osteosarcoma.

29. the purposes of claim 27, wherein said viral infection includes but not limited to acute, subacute or Chronic HBV, HCV, HIV.

30. the purposes of any one of claim 27-29, wherein said anti-PD-L1 antibody or the also coupling of its antigen-binding portion thereof have can be used for detection or the fluorescent dye that can be detected by other reagent, chemical substance, polypeptide, enzyme, isotope, label etc..

31. the anti-PD-L1 antibody of any one of claim 1-9 or its antigen-binding portion thereof are for preparing the purposes of the medicine preventing or treating the disease relevant to CD80.