CN110156895A

CN110156895A - A kind of anti-PD-L1 antibody or its functional fragment and application thereof

Info

Publication number: CN110156895A
Application number: CN201910460477.0A
Authority: CN
Inventors: 张明; 原野
Original assignee: Shanghai Jiabei Biomedical Technology Co Ltd
Current assignee: Ningbo Jiabei Medical Technology Co ltd; Shanghai Jiabei Biomedical Technology Co ltd; Shanghai Jiabei Pharmaceutical Technology Co ltd
Priority date: 2019-05-30
Filing date: 2019-05-30
Publication date: 2019-08-23
Anticipated expiration: 2039-05-30
Also published as: CN110156895B

Abstract

The application belongs to biomedicine field, discloses following several respects: a kind of anti-PD-L1 antibody or its functional fragment；Herein described anti-PD-L1 antibody or the nucleic acid molecules of its functional fragment are encoded, for expressing the expression vector and host cell of the anti-PD-L1 antibody or its functional fragment；The preparation method of the anti-PD-L1 antibody or its functional fragment；Immunoconjugates comprising the anti-PD-L1 antibody or its functional fragment and its pharmaceutical composition；And the anti-PD-L1 antibody or its functional fragment are preparing the purposes for treating the drug of T cell dysfunctional disease.

Description

A kind of anti-PD-L1 antibody or its functional fragment and application thereof

Technical field

The application belongs to biomedicine field, is related to the following aspect of anti-PD-L1 antibody: a kind of anti-PD-L1 antibody or its Functional fragment；Herein described antibody or the nucleic acid molecules of its functional fragment are encoded, for expressing the antibody or its function The expression vector and host cell of energy property segment；The preparation method of herein described anti-PD-L1 antibody or its functional fragment；Packet Immunoconjugates and its pharmaceutical composition containing herein described anti-PD-L1 antibody or its functional fragment；And the application The anti-PD-L1 antibody or its functional fragment are preparing the purposes for treating the drug of T cell dysfunctional disease.

Background technique

Programmed death ligand 1 (Programmed death-ligand 1, PD-L1), also referred to as differentiation cluster 274 (cluster of differentiation 274, CD274) or B7 homologous protein 1 (B7 homolog 1, B7-H1) belongs to In tumor necrosis factor superfamily, the I type transmembrane glycoprotein being made of 290 amino acid residues, comprising an area GeIgVYang, One area GeIgCYang, a cross-film hydrophobic region and 30 amino acid tail portion intracellular, intact molecular weight 40kDa.Almost institute In organized, PD-L1 mRNA has expression, but PD-L1 albumen continuous expression in small part tissue, for example, liver, lung Dirty, tonsillotome and immune special pardon tissue eye, placenta etc..The T cell, B cell, monokaryon that PD-L1 is also expressed in activation are thin Born of the same parents, Dendritic Cells, macrophage etc..

The receptor of PD-L1 is programmed death receptor 1(Programmed death 1, PD-1).PD-1 is mainly expressed in The immunocytes surfaces such as CD4+T cell, CD8+T cell, NKT cell, B cell and the monocyte of activation.PD-L1 and PD-1 is tied Conjunction can star PD-1 cytoplasmic domain immunity receptor Tyrosine Inhibitory Motifs (Immuno receptor tyrosine-based Inhibitory motif, ITIM), the phosphorylation of tyrosine residue promotes tyrosine phosphatidase in conjunction with SHP2, activation SHP2 makes downstream Syk and PI3K that dephosphorylation occur to transmit termination signal, and limited antigen is thin in delivery cell or dendron shape The interaction of born of the same parents and T cell.This combination can also further suppress the metabolism of T cell, inhibit anti-apoptotic proteins Bcl-X2 Secretion, reduce effector cell's factor Human Inter Leukin-2 (Interleukin-2, IL-2) and r-interferon The secretion of (Interferon-r, IFN-r), inducing T cell is exhausted and apoptosis, to reduce immune the answering of immune t-cell participation It answers, exercises negative immune regulatory function.

PD-L1 is expressed in tumor tissues height, for example, gastric cancer, lung cancer, liver cancer, intrahepatic cholangiocarcinoma, colon cancer, cancer of pancreas, ovum Nest cancer, breast cancer, cervix cancer, Head and neck squamous cell carcinoma, nasopharyngeal carcinoma, the cancer of the esophagus, bladder cancer, clear-cell carcinoma, cutaneum carcinoma, oral cavity Squamous cell carcinoma etc..The expression of tumor locus PD-L1 can protect tumour cell to escape injury through a variety of ways, PD-L1's Negative immune regulatory function plays an important role in tumour immunity.Therefore, using PD-1/PD-L1 as the immunological regulation of target spot There is important meaning to antitumor, and a variety of chronic and acute virus is also escaped human immunity using PD-L1 signal and detected.

In recent years, more and more preclinical and clinical study results show to target immunologic test point becoming and most have The method of desired treating cancer patient.PD-1 is one of immunologic test point albumen, passes through what is expressed in the T cell of activation The interaction of the PD-1 and PD-L1 expressed on tumour cell adjusts immune response negativity and weakens anti-tumor immunity Expression of the PD-L1 in tumour, it is related to the decline of the survival rate of the cancer of the esophagus, cancer of pancreas and other a plurality of types of cancers, it should Access has been used as new promising immunotherapy of tumors target spot.There is drugmaker to develop a variety of for PD-1/PD-L1 The drug of access, and clinical test statistics indicate that this kind of drug clinical activity lasting in the patient of kinds of tumors type and Good safety.

However, existing treatment method is not all fully up to expectations, it is still desirable to the human antibody of new anti-PD-L1.

Summary of the invention

In view of above-mentioned insufficient and anti-PD-L1 antibody the clinical application demand of the prior art, inventor herein one Aspect is intended to provide the antibody and its functional fragment of a kind of anti human PD-L 1；On the other hand, it is desirable to provide the antibody or its function It can purposes of the property segment in terms for the treatment of T cell dysfunctional disease；Another further aspect, it is desirable to provide the preparation side of the antibody Method and detection method.

In order to achieve the above object, in a first aspect, this application provides a kind of anti-PD-L1 human antibody, the anti-PD- L1 antibody is monoclonal antibody, the anti-PD-L1 antibody include at least one of heavy chain hypervariable region CDR1, CDR2 and CDR3 or One of a variety of and/or light chain hypervariable region CDR1, CDR2 and CDR3 or a variety of；Wherein:

The amino acid sequence of the light chain hypervariable region CDR1 is for sequence shown in SEQ ID No.3 or through replacement, missing or addition One or more amino acids formed amino acid sequences with sequence shown in SEQ ID No.3 with same function are described light The amino acid sequence of chain hypervariable region CDR2 is one or more for sequence shown in SEQ ID No.4 or through replacement, missing or addition The amino acids formed amino acid sequence with sequence shown in SEQ ID No.4 with same function and the light chain hypervariable region The amino acid sequence of CDR3 is for sequence shown in SEQ ID No.5 or through replacement, missing or adds one or more amino acid shapes At with sequence shown in SEQ ID No.5 have same function amino acid sequence；And

The amino acid sequence of the heavy chain hypervariable region CDR1 is for sequence shown in SEQ IDNo.6 or through replacement, missing or addition one A or multiple amino acids formed amino acid sequences with sequence shown in SEQ IDNo.6 with same function, the heavy chain are high Become the amino acid sequence of area CDR2 into sequence shown in SEQ ID No.7 or through replacement, missing or the one or more amino of addition The amino acid sequence and the heavy chain hypervariable region CDR3 with sequence shown in SEQ ID No.7 with same function that acid is formed Amino acid sequence be sequence shown in SEQ ID No.8 or through replacement, missing or addition it is one or more it is amino acids formed with Sequence shown in SEQ ID No.8 has the amino acid sequence of same function.

Wherein: described through SEQ ID in replacement, missing or the one or more amino acids formed and sequence tables of addition Any shown sequence is with the ammonia referred in the amino acid sequence of same function with herein described anti-PD-L1 antibody in No.3-8 Base acid sequence is compared, and has at most 10, and preferably at most 8, more preferably at most 5, most preferably at most 3 amino acid are by property Similar or similar amino acid substitution and the polypeptide formed.The sequence of this variant and its derived sequences have at least 95%, 96%, 97%, 98% or 99% consistency.

Further, the amino acid sequence of the light chain variable region of the anti-PD-L1 antibody includes at least SEQID No.1 institute The sequence shown or through replacement, missing or addition it is one or more it is amino acids formed have with sequence shown in SEQID No.1 it is same The amino acid sequence of function, the amino acid sequence of heavy chain variable region include at least sequence shown in SEQ ID No.2 or through replacement, Missing or the one or more amino acids formed amino acid sequences with sequence shown in SEQ ID No.2 with same function of addition Column.

Further, the light chain framework region (Framework region, FR) of the anti-PD-L1 antibody includes FR1, FR2 With one of FR3 or a variety of, wherein the amino acid sequence of the light chain FR1 is sequence or sequence shown in SEQ ID No.9 Column have same function through sequence shown in replacement, missing or the one or more amino acids formed and SEQ ID No.9 of addition Amino acid sequence, the amino acid sequence of the light chain FR2 is for sequence shown in SEQ ID No.10 or through replacement, missing or addition One or more amino acids formed amino acid sequences with sequence shown in SEQ ID No.10 with same function and described light The amino acid sequence of chain FR3 is for sequence shown in SEQ ID No.11 or through replacement, missing or adds one or more amino acid What is formed has the amino acid sequence of same function with sequence shown in SEQ ID No.11；

Further, the heavy chain framework regions include one of FR1, FR2 and FR3 or a variety of, wherein the heavy chain FR1's Amino acid sequence be sequence shown in SEQ ID No.12 or through replacement, missing or addition it is one or more it is amino acids formed with Sequence shown in SEQ ID No.12 has the amino acid sequence of same function, and the amino acid sequence of the heavy chain FR2 is SEQ ID Sequence shown in No.13 or through replacement, missing or addition is one or more amino acids formed has and SEQ ID No.13 institute The amino acid sequence of the amino acid sequence and the heavy chain FR3 that show the same function of sequence is SEQ ID No.14 or is replaced, lacked Lose or add one or more amino acids formed amino acid sequences with sequence shown in SEQ ID No.14 with same function.

Preferably, the nucleotides sequence of the coding light chain variable region in the anti-PD-L1 antibody is classified as SEQ ID No.15 institute The sequence shown or the sequence through replacement, missing or add that one or more nucleotide are formed with sequence shown in SEQ ID No.15 The nucleotides sequence of nucleotide sequence with same function, the encoding heavy chain variable region in the anti-PD-L1 antibody is classified as SEQ Sequence shown in ID No.16 or the sequence through replacement, missing or add that one or more nucleotide are formed with SEQ ID Sequence shown in No.16 has the nucleotide sequence of same function.

Second aspect, present invention also provides a kind of expression vector, the expression vector includes at least following amino acid sequences One of column: sequence shown in SEQ ID No.15 or the sequence through replacement, missing or add one or more nucleotide shapes At with sequence shown in SEQ ID No.15 have same function nucleotide sequence；And sequence shown in SEQ ID No.16 Or the sequence through replacement, missing or adds having on an equal basis with sequence shown in SEQ ID No.16 for one or more nucleotide formation The nucleotide sequence of function.Meanwhile present invention also provides a kind of host cell, the host cell is carried comprising above-mentioned expression Body.Preferably, the host cell selects mammalian cell.

The third aspect, present invention also provides the preparation methods of the anti-PD-L1 antibody or its functional fragment.

Fourth aspect, present invention also provides the immunoconjugates comprising the anti-PD-L1 antibody or its functional fragment And its pharmaceutical composition.

5th aspect, herein described anti-PD-L1 antibody or its functional fragment lose in preparation for treating T cell function The purposes of the drug of tonality disease.

In a kind of embodiment of the application first aspect, the anti-PD-L1 antibody of humanization is monoclonal antibody, institute State light chain hypervariable region CDR1, CDR2 and CDR3 of antibody amino acid sequence be respectively SEQ ID No.3, SEQ ID No.4, Sequence shown in SEQ ID No.5；The amino acid sequence of heavy chain hypervariable region CDR1, CDR2 and CDR3 of the antibody is respectively SEQ ID No.6, SEQ ID No.7, sequence shown in SEQ ID No.8.

Further, the amino acid sequence of the light chain variable region of the antibody is sequence shown in SEQ ID No.1, or should Sequence has same function through sequence shown in replacement, missing or the one or more amino acids formed and SEQ ID No.1 of addition Amino acid sequence；The amino acid sequence of the heavy chain variable region of the antibody is sequence or sequence shown in SEQ ID No.2 Through replacement, missing or the one or more amino acids formed ammonia with sequence shown in SEQ ID No.2 with same function of addition Base acid sequence.

The amino acid sequence of light chain framework region FR1, FR2 and FR3 of the antibody are respectively SEQ ID No.9, SEQ Sequence shown in IDNo.10, SEQ ID No.11；The amino acid sequence of heavy chain framework regions FR1, FR2 and FR3 of the antibody point It Wei not SEQ D No.12, SEQID No.13, sequence shown in SEQ ID No.14.

In the embodiment of the application second aspect, this application provides two kinds of DNA fragmentations.These described DNA fragmentations Encode the anti-PD-L1 antibody as described in first aspect above.The DNA fragmentation includes light chain variable region coded sequence and heavy chain can Become area's coded sequence；The nucleotide sequence of the light chain variable region and the heavy chain variable region is encoded respectively such as SEQ ID Shown in No.15, SEQ IDNo.16.

In the embodiment of the application third aspect, present invention also provides two kinds of expression vectors, the expression vectors Comprising at least one copy such as the DNA fragmentation in second aspect above.Meanwhile this application provides a kind of host is thin Born of the same parents, the host cell include the expression vector as described in the third aspect above.

In the embodiment of the application fourth aspect, present invention also provides include the anti-PD-L1 antibody or its function The immunoconjugates and its pharmaceutical composition of energy property segment.Preferably, described pharmaceutical composition further includes that toxin, radioactivity are same Position element, drug or cytotoxic agent.In other embodiments, the application is further related to comprising the anti-PD-L1 antibody or its function The pharmaceutical composition of property segment and pharmaceutical acceptable carrier.The described pharmaceutical composition at least anti-PD-L1 containing effective dose Antibody or its functional fragment and its pharmaceutically acceptable carrier.

In the embodiment of the 5th aspect of the application, this application provides the anti-PD-L1 antibody or its functional pieces Section is preparing the purposes for treating the drug of T cell dysfunctional disease.Specifically, pass through elimination, inhibition or reduction PD- L1 activity includes to described in the object application effective dose for having this to need to prevent or treat disease or alleviate the method for illness Anti- PD-L1 antibody or its functional fragment, nucleic acid, expression vector, host cell, immunoconjugates or pharmaceutical composition.Into one Step ground, herein described purposes include the anti-PD-L1 antibody or its functional fragment and correspondingly nucleic acid, expression vector, Host cell, immunoconjugates or pharmaceutical composition are preparing the purposes in the drug for treating disease or illness.

In human body, the affinity KD of the antibody and PD-L1 are less than or equal to herein described anti-PD-L1 antibody sources 1.0 x 10^-8M.Specifically, the anti-PD-L1 antibody is with about 9x10^-9M or smaller, about 8x10^-9M or smaller, about 7x10^-9M or Smaller, about 6x10^-9M or smaller, about 5x10^-9M or smaller, about 4x10^-9M or smaller, about 3x10^-9M or smaller, about 2x10^-9M or It is smaller, about 10^-9Any KD value in M or smaller binding affinity KD is specifically in conjunction with human PD-L 1.Wherein, at one In embodiment, the affinity KD of the anti-PD-L1 antibody and PD-L1 are 7.1nM, can be good at inhibiting PD-L1 and PD-1 Combination, by elimination, inhibition or reduce PD-L1 activity and prevent or treat disease or illness, promote T cell proliferation, secretion IL-2 and IFN-γ, can enhance the function of T cell, to raise cell-mediated immune response for treating T cell function Dysfunctional disease.

[Detailed description of the invention]

Fig. 1 a shows the association reaction that FACs detects anti-the PD-L1 antibody and HEK293 PD-L1.

Fig. 1 b shows FACs and detects different antibodies concentration in the association reaction of the PD-L1 antibody and HEK293 PD-L1 Average fluorescent strength.

Fig. 2 shows the AlphaLISA detection anti-PD-L1 antibody to inhibit PD-L1 in conjunction with PD-1.

Fig. 3 shows the result that anti-PD-L1 antibody described in mixed lymphocyte reaction (MLP) promotes T cell secretion IL-2.

Fig. 4 shows the result that anti-PD-L1 antibody described in mixed lymphocyte reaction (MLP) promotes T cell secretion of gamma-IFN.

Fig. 5 shows the result that anti-PD-L1 antibody described in mixed lymphocyte reaction (MLP) promotes T cell proliferation.

[specific embodiment]

It, hereafter will be further by specific embodiments and the drawings to better understand present invention conception and technical scheme It explains the application, wherein being only preferred embodiment about the technical solution in embodiment, is not necessarily to be construed as limitation originally The range of application.To those skilled in the art, it under the premise of not departing from the technical principle of the application, can also make Several improvement and deformations, these improvement and deformations also should be regarded as falling among the protection scope of the application.

Unless otherwise defined, all scientific and technical terminologies used herein should be regarded as being understood with those of ordinary skill in the art Identical meanings.Definition and term about this field, professional specifically refer to Current Protocols in Molecular Biology (Ausubel).The abbreviation of amino acid residue is 20 common L- ammonia of reference used in this field 3 letter of standard of base acid and/or 1 alphanumeric codes.

" Quan Renyuan " or " humanization " antibody that refer in the application or when its functional fragment or antigen-binding fragment are Refer to that the antibody or antigen-binding fragment have certain amino acid sequence or is made of the amino acid sequence, the amino acid sequence Corresponding to by people or the production of people's immunocyte or from for example utilizing the inhuman source such as the transgenic nonhuman animal of human antibody library The sequence of the amino acid sequence of derivative antibody or other coding human antibodies.In some embodiments, human antibody Not comprising the amino acid residue (especially antigen binding residues) for deriving from non-human antibody.

The light chain hypervariable region or heavy chain hypervariable region referred in the application, wherein " hypervariable region " is referred to as complementary determining region again (complementarity- determining region, CDR) 。

" sequence " referred in the application can refer to comprising the equivalent amino acid sequence of certain biological functions or " conservative Substitution ", " other sequences " may include the aniso- amino acid of function or " non-conservation substitution ", be genetically engineered with Improve the characteristic of CDR or the antibody containing CDR.Under the premise of substantially not influencing antibody activity, those skilled in the art can be with Sequence in the application is operated, that is, replaces, add and/or missing one or more (such as 1,2,3,4,5,6,7,8,9 Or 10 or multiple) amino acid, to obtain the variant of the antibody or its functional fragment sequence.They should be deemed to be included in In the range of the application protection.For example, the amino acid with similarity is replaced in variable region.It is referred in the application The sequence of variant can have the consistency of at least 95%, 96%, 97%, 98% or 99% with its derived sequences.Sequence is consistent Property can be used sequence analysis software measurement.Such as the computer program BLAST using default parameter, especially BLASTP or TBLASTN.A variety of amino acid sequences as described herein are detailed in sequence table.

The antibody referred in the application is human monoclonal antibody.The anti-PD-L1 antibody includes that single-chain antibody, double-strand are anti- Body, chimeric antibody or derivatives thereof.The anti-PD-L1 antibody or its antigen-binding fragment can be good at combining human PD-L 1. The antibody can be (for example, IgG1 or IgG4 antibody) of overall length or can only include antigen-binding portion thereof (for example, Fab, F (ab ') 2 or scFv segment), or can be modified to influence function.Antibody described herein further includes the glycosyl with modification The anti-PD-L1 antibody of change mode.In some applications, opening can be modified to remove undesirable glycosylation site, for example, Fucose moiety is not present, on oligonucleotide chain to enhance the cytotoxicity (antibody- of antibody dependent cellular mediation Dependent cell-mediated cytotoxicity, ADCC) function antibody.In other applications, half can be carried out Glycosylation modified cytotoxicity (the complement dependent to change complement-dependent cytotoxicity Complement Dependent of cream Cytotoxicity, CDC).

Term as used herein " functional fragment " refers in particular to antibody fragment, as Fv, scFv (sc refers to single-stranded), Fab, F (ab ') 2, Fab ', scFv-Fc segment, double antibody (diabody) or by chemical modification or by incorporation liposome In can increase any segment of half-life period, wherein these chemical modifications, for example, poly- (alkylidene) glycol of addition, such as poly- second two Alcohol (" Pegylation, i.e., PEGylated "), the segment through chemical modification is referred to as Fv-PEG, scFv-PEG, Fab-PEG, F (ab') The pegylated fragments of 2-PEG or Fab'-PEG), the segment has EGF-R ELISA (Epidermal Growth Factor Receptor, EGFR) combine activity.Preferably, the functional fragment will be by the weight of its derived antibodies Or the partial sequence of light variable chains constitutes or comprising them, which is enough to retain combination identical with its derived antibodies Specific and sufficient affinity.For PD-L1, preferably at least equal to or greater than the 1/100 of its derived antibodies affinity, more The 1/10 of its derived antibodies affinity is at least equal to or greater than in preferred embodiment.This functional fragment will include minimum 5 ammonia Base acid, preferably the 10,15,25,50 and 100 of the antibody sequence in its source continuous amino acid.

Terms used herein " pharmaceutical composition " include at least one drug and optional pharmaceutical acceptable carrier or auxiliary material, To realize certain specific purpose.In some embodiments, described pharmaceutical composition further include in these ingredients in the time and/or Spatially separated combination, if its can collective effect to realize the purpose of the application.For example, institute in described pharmaceutical composition The ingredient (for example, according to antibody described herein, nucleic acid molecules, nucleic acid molecules combination and/or conjugate) contained can be with whole Body is applied to object or separate administration in object.The ingredient contained in the described pharmaceutical composition is dividually applied to object When, the ingredient can simultaneously or sequentially be applied to object.Preferably, described pharmaceutical acceptable carrier be water, it is buffered aqueous solution, isotonic Salting liquid such as PBS (phosphate buffer), glucose, mannitol, D-glucose, lactose, starch, magnesium stearate, cellulose, Magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethyl alcohol or polyalkylene glycol such as polypropylene glycol, triglycerides etc..Specifically, it selects The type of pharmaceutical acceptable carrier be whether to be formulated for taking orally according to the composition of the application, is nose, intradermal, subcutaneous, intramuscular or quiet Arteries and veins application and it is different.Composition according to the application may include wetting agent, emulsifier or buffer as additive.The application's Pharmaceutical composition can be applied by any suitable approach, such as orally available, nose, intradermal, subcutaneous, intramuscular or intravenous application.

" by elimination, inhibition or reduce PD-L1 activity to prevent or treat disease or alleviate illness " that the application refers to Symptom/feature disease or illness are expressed as caused by indicating by PD-L1 expression or with PD-L1.In some embodiments, The disease or illness are selected from cancer or inflammatory disease.

" nucleotide sequence " that the application refers to is detailed in sequence table.It, can by using polynucleotides described herein Effectively synthesize anti-PD-L1 antibody or antigen-binding fragment.Can polynucleotides described above effectively synthesize it is herein described Anti- PD-L1 antibody or antigen-binding fragment.The preparation method of the antibody can use technology well known to those skilled in the art It carries out, does not do herein specifically limited.

In view of the degeneracy of codon, for example, can be right under conditions of not changing amino acid sequence in its code area The gene order of encoding such antibodies is transformed, and obtains the gene for encoding antibody with the same function.Those skilled in the art Member can be according to the codon-bias of expression antibody host, artificial synthesized modifying gene, to improve the expression efficiency of antibody.

Further, the application recombinates the light chain variable region of the anti-PD-L1 antibody and heavy chain variable region, can obtain The smaller single-chain antibody of molecular weight (ScFv) is obtained, which can equally combine PD-L1.The single-chain antibody penetration power is strong, is easy to It plays a role into local organization.Can be by the gene of above-mentioned encoding antibody, ScFv gene cloning into expression vector, and then convert Or transfection host cell, obtain the antibody and single-chain antibody.In addition, the light chain variable region of antibody noted earlier can be encoded Gene and heavy chain variable region gene are cloned into complete anti-expression vector, and are imported in host cell, are obtained and are expressed the complete of anti-PD-L1 Panimmunity globulin.

In a first aspect, anti-PD-L1 human antibody is monoclonal antibody in the application, which is included at least One in one of heavy chain hypervariable region CDR1, CDR2 and CDR3 or a variety of and/or light chain hypervariable region CDR1, CDR2 and CDR3 Kind is a variety of；Wherein:

The amino acid sequence of light chain hypervariable region CDR1 is for sequence shown in SEQ ID No.3 or through replacement, missing or addition one Or multiple amino acids formed amino acid sequences with sequence shown in SEQ ID No.3 with same function, light chain hypervariable region The amino acid sequence of CDR2 is for sequence shown in SEQ ID No.4 or through replacement, missing or adds one or more amino acid shapes At with sequence shown in SEQ ID No.4 have same function amino acid sequence and light chain hypervariable region CDR3 amino acid Sequence is for sequence shown in SEQ ID No.5 or through replacement, missing or the one or more amino acids formed and SEQ ID of addition Sequence shown in No.5 has the amino acid sequence of same function；And

The amino acid sequence of heavy chain hypervariable region CDR1 be SEQ IDNo.6 shown in sequence or through replacement, missing or addition one or Multiple amino acids formed amino acid sequences with sequence shown in SEQ IDNo.6 with same function, heavy chain hypervariable region CDR2 Amino acid sequence be sequence shown in SEQ ID No.7 or one or more amino acids formed through replacement, missing or addition There is the amino acid sequence of same function with sequence shown in SEQ ID No.7 and the amino acid sequence of heavy chain hypervariable region CDR3 is Sequence shown in SEQ ID No.8 is one or more amino acids formed with SEQ ID No.8 institutes through replacement, missing or addition The sequence shown has the amino acid sequence of same function.

Wherein: through in SEQ ID No.3-8 in replacement, missing or the one or more amino acids formed and sequence tables of addition Sequence shown in any is with the amino acid sequence phase referred in the amino acid sequence of same function with the anti-PD-L1 antibody of the application Than having at most 10, preferably at most 8, more preferably at most 5, most preferably at most 3 amino acid are similar or close by property Amino acid substitution and the polypeptide that is formed.The sequence of this variant and its derived sequences have at least 95%, 96%, 97%, 98% or 99% consistency.

Further, the amino acid sequence of the light chain variable region of the anti-PD-L1 antibody includes at least shown in SEQID No.1 Sequence or through replacement, missing or addition is one or more amino acids formed has same function with sequence shown in SEQID No.1 The amino acid sequence of energy, the amino acid sequence of heavy chain variable region include at least sequence shown in SEQ ID No.2 or are replaced, lacked Lose or add one or more amino acids formed amino acid sequences with sequence shown in SEQ ID No.2 with same function.

Further, the light chain framework region (Framework region, FR) of the anti-PD-L1 antibody include FR1, FR2 and One of FR3 or a variety of, wherein the amino acid sequence of light chain FR1 is sequence shown in SEQ ID No.9 or sequence warp Replacement, missing or the one or more amino acids formed amino with sequence shown in SEQ ID No.9 with same function of addition Acid sequence, the amino acid sequence of light chain FR2 be sequence shown in SEQ ID No.10 or through replacement, missing or addition one or Multiple amino acids formed amino acid sequences and light chain FR3 with sequence shown in SEQ ID No.10 with same function Amino acid sequence be sequence shown in SEQ ID No.11 or through replacement, missing or addition it is one or more it is amino acids formed with Sequence shown in SEQ ID No.11 has the amino acid sequence of same function；

Further, which includes one of FR1, FR2 and FR3 or a variety of, wherein the amino of heavy chain FR1 Acid sequence is for sequence shown in SEQ ID No.12 or through replacement, missing or the addition amino acids formed and SEQ of one or more Sequence shown in ID No.12 has the amino acid sequence of same function, and the amino acid sequence of heavy chain FR2 is SEQ ID No.13 Shown in sequence or through replacement, missing or addition is one or more amino acids formed has and sequence shown in SEQ ID No.13 The amino acid sequence of same function and the amino acid sequence of heavy chain FR3 are SEQ ID No.14 or through replacement, missing or addition One or more amino acids formed amino acid sequences with sequence shown in SEQ ID No.14 with same function.

Preferably, the nucleotides sequence of the coding light chain variable region in the anti-PD-L1 antibody is classified as shown in SEQ ID No.15 Sequence or the sequence is through replacement, missing or that adds that one or more nucleotide are formed have with sequence shown in SEQ ID No.15 There is the nucleotide sequence of same function, the nucleotides sequence of the encoding heavy chain variable region in the anti-PD-L1 antibody is classified as SEQ ID Sequence shown in No.16 or the sequence through replacement, missing or add that one or more nucleotide are formed with SEQ ID No.16 Shown sequence has the nucleotide sequence of same function.

Second aspect, present invention also provides a kind of expression vector, which includes at least following amino acid sequences One of: sequence shown in SEQ ID No.15 or the sequence are formed through replacement, missing or the one or more nucleotide of addition With sequence shown in SEQ ID No.15 have same function nucleotide sequence；And sequence shown in SEQ ID No.16 or The sequence has same function with sequence shown in SEQ ID No.16 through what replacement, missing or the one or more nucleotide of addition were formed The nucleotide sequence of energy.Meanwhile present invention also provides a kind of host cell, which includes above-mentioned expression vector.It is excellent Selection of land, the host cell are mammalian cell.

The third aspect, the application have also correspondingly provided the anti-PD-L1 antibody or the preparation method of its functional fragment.

Fourth aspect, present invention also provides the immunoconjugates comprising the anti-PD-L1 antibody or its functional fragment with And its pharmaceutical composition.

5th aspect, the application anti-PD-L1 antibody or its functional fragment are in preparation for treating T cell functional disturbance The purposes of the drug of property disease.

In a kind of embodiment of the application first aspect, the anti-PD-L1 antibody of the humanization is monoclonal antibody, this is anti- The amino acid sequence of light chain hypervariable region CDR1, CDR2 and CDR3 of body are respectively SEQ ID No.3, SEQ ID No.4, SEQ ID Sequence shown in No.5；The amino acid sequence of heavy chain hypervariable region CDR1, CDR2 and CDR3 of the antibody are respectively SEQ ID No.6, SEQ ID No.7, sequence shown in SEQ ID No.8.

Further, the amino acid sequence of the light chain variable region of the antibody is sequence or sequence shown in SEQ ID No.1 Column have same function through sequence shown in replacement, missing or the one or more amino acids formed and SEQ ID No.1 of addition Amino acid sequence；The amino acid sequence of the heavy chain variable region of the antibody is sequence shown in SEQ ID No.2 or the sequence through replacing Change, lack or add one or more amino acids formed amino acid with sequence shown in SEQ ID No.2 with same function Sequence.

The amino acid sequence of light chain framework region FR1, FR2 and FR3 of the antibody are respectively SEQ ID No.9, SEQ Sequence shown in IDNo.10, SEQ ID No.11；The amino acid sequence of heavy chain framework regions FR1, FR2 and FR3 of the antibody are distinguished For sequence shown in SEQ D No.12, SEQID No.13, SEQ ID No.14.

In the embodiment of the application second aspect, this application provides two kinds of DNA fragmentations.These DNA fragmentations are compiled The anti-PD-L1 antibody that code such as first aspect above is somebody's turn to do.The DNA fragmentation includes that light chain variable region coded sequence and heavy chain variable region are compiled Code sequence；The nucleotide sequence of the light chain variable region and the heavy chain variable region is encoded respectively such as SEQ ID No.15, SEQ Shown in IDNo.16.

In the embodiment of the application third aspect, present invention also provides two kinds of expression vectors, the expression vector packets Containing at least one copy such as the DNA fragmentation in second aspect above.Meanwhile this application provides a kind of host cells, it should Host cell includes the expression vector as described in the third aspect above.

In the embodiment of the application fourth aspect, present invention also provides include the anti-PD-L1 antibody or its function The immunoconjugates and its pharmaceutical composition of property segment.Preferably, which further includes toxin, the same position of radioactivity Element, drug or cytotoxic agent.In other embodiments, the application is further related to comprising the anti-PD-L1 antibody or its functional piece The pharmaceutical composition of section and pharmaceutical acceptable carrier.The anti-PD-L1 antibody at least containing effective dose of the pharmaceutical composition or its Functional fragment and its pharmaceutically acceptable carrier.

In the embodiment of the 5th aspect of the application, this application provides the anti-PD-L1 antibody or its functional fragments Preparing the purposes for treating the drug of T cell dysfunctional disease.Specifically, pass through elimination, inhibition or reduction PD-L1 Activity includes the anti-PD- to the object application effective dose for having this to need to prevent or treat disease or alleviate the method for illness L1 antibody or its functional fragment, nucleic acid, expression vector, host cell, immunoconjugates or pharmaceutical composition.Further, Herein described purposes includes the anti-PD-L1 antibody or its functional fragment and correspondingly nucleic acid, expression vector, Su Zhuxi Born of the same parents, immunoconjugates or pharmaceutical composition are preparing the purposes in the drug for treating disease or illness.

Extracellular structural area -6His albumen the preparation of 1. human PD-L 1 of embodiment

Embodiment 1.1. chemical synthesis human PD-L 1 extracellular region -6His gene

Chemical synthesis human PD-L 1 extracellular region -6His gene (NCBI Reference Sequence:NM_014143.4), clone Into eukaryon expression plasmid pcDNA3.1, PEI transfected HEK 293 after 6 days, collects medium supernatant, and pass through nickel column Affinitive layer purification human PD-L 1 extracellular region protein.

Human PD-L 1 extracellular region -6His amino acid sequence: MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIE CKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMI SYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNV TSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERHHHHHH

Embodiment 1.2. biotinylated antigen Bio-PD-L1-6His preparation:

Biotinylation is carried out to human PD-L 1 extracellular region -6His molecule using NHS-PEG4-Biotin, after obtaining biotinylation Antigen Bio-PD-L1-6His.

Extracellular structural area -6His albumen the preparation of 2. people PD-1 of embodiment

Embodiment 2.1. chemical synthesis people's PD-1 extracellular region -6His gene

Chemical synthesis people PD-1 extracellular region -6His gene (NCBI Reference Sequence:NM_005018.3), clone Into eukaryon expression plasmid pcDNA3.1, PEI transfected HEK 293 after 6 days, collects medium supernatant, passes through nickel column parent With chromatographic purifying people's PD-1 extracellular region protein.

People's PD-1 extracellular region -6His amino acid sequence: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPA LLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRND SGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVHHHHHH

Embodiment 2.2. biotinylated protein Bio-PD-1-6His preparation:

Biotinylation is carried out to PD-L1 extracellular region -6His molecule using NHS-PEG4-Biotin, it is anti-after obtaining biotinylation Former Bio-PD-L1-6His.

The foundation of 3. people's ScFv phage library of embodiment

20 parts of healthy human peripheral bloods are collected, density gradient centrifugation separation obtains 5 × 10⁹Lymphocyte, Triziol extract total serum IgE, According to bibliography (Andris-Widhopf, J., Steinberger, P., Fuller, R., Rader, C., & Barbas, C. F., 3rd. (2011). Generation of human scFv antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences. Cold Spring Harb Protoc, 2011 (9) doi:10.1101/pdb.prot065573) method, construct people ScFV bacteriophage Library, bacteriophage primary Kuku hold up to 2 × 10⁹。

4. paramagnetic particle method of embodiment screens anti human PD-L 1 ScFv

The 200 μ L(of phage antibody library for choosing the full people's single-chain antibody of expression contains bacteriophage 1 × 10¹²A/mL) it is biological with 5 μ g The Bio-PD-L1-6His antigen of elementization mixes, and is incubated at room temperature 30 min, the Streptavidin MagneSphere of 50 μ L is then added, and anti- The bacteriophage that original combines is captured by Streptavidin MagneSphere, PBS solution (phosphorus of the unbonded bacteriophage through 0.5% Tween-20 Acid buffer) rinsing after be removed, will be waited for the bacteriophage elution of magnetic bead stable bond with glycine hydrochloride (pH 2.2) solution With.

It is inoculated with XL1-Blue bacterium 200mL, the light absorption value at OD600nm(600nm wavelength) close to after 0.6, in addition The bacteriophage for stating elution, with XL1-Blue bacterium in 37 DEG C of standing 30min, bacterium solution is coated on amicillin resistance plate, the The thallus on amicillin resistance plate is collected in elution in two days, is infecting 1 × 10¹²The VCSM13 helper phage of pfu/ml Afterwards, next round screening is carried out after amplification, carries out 3 wheel screenings altogether.

The XL1-Blue bacterium solution for infecting bacteriophage is sufficiently diluted, it is that 15cm ammonia benzyl is green that this bacterium solution, which is then coated on diameter, The LB solid medium tablets of chloramphenicol resistance have 100-500 clone on each plate, and picking monoclonal antibody passes through phagocytosis Body enzyme linked immunoassay verifies each wheel phage library after screening.

Bacteriophage enzyme linked immunoassay

The XL1-Blue monoclonal bacterium for transfecting bacteriophage is inoculated into the 96 hole Bacteria Culture plates of 2mL, 500 μ L are added and contain four The SB culture medium of ring element resistance, the lower 37 DEG C of shaking tables 4-6 h of 200 rpm revolving speeds detect OD600nm value close to after 0.6,1 μ L are added Helper phage, at 30 DEG C shaking table stay overnight, 3000g centrifuging and taking supernatant is stand-by within second day.

Enzyme linked immunological microwell plate is taken, 4 DEG C of envelope antigen overnight, and PBST washs rear enclosed, and previous step is then added and prepares Bacteriophage supernatant, be incubated at room temperature 2h, PBST adds Anti-M13 HRP antibody incubation 30min, after washed 3 times with PBST, add Enter 50 μ L developer solution ABTS [side 2,2'- nitrogen base-bis- (- 6 sulfonic acid of 3- ethyl benzo thiophene pyrrolin)].Detect OD405nm value.

After 3 wheel screenings, expresses and be enriched with the bacteriophage of antigentic specificity binding antibody.From the 3rd wheel after screening 700 monoclonals of picking carry out enzyme linked immunoassay verifying in library, finally, the readings for determining enzyme linked immunoassay is control group two Times or more clone as positive colony.The analysis of 100 positive colony sequencings, analyze by comparing, and picking repeat number is most Positive colony.Resulting clone's phasmid is sequenced, the nucleotide sequence of its heavy chain variable region and light chain variable region is analyzed The nucleotide sequence of information, encoding heavy chain variable region and light chain variable region is respectively such as SEQ ID No.15 and SEQ ID in sequence table Shown in No.16, orresponding amino acid sequence is as shown in SEQ ID No.1 and SEQ ID No.2.

Antibody variable region DNA sequence dna

The antibody's light chain variable region nucleic acid sequence is as shown in SEQ ID No.15 in sequence table.This states antibody heavy chain variable region nucleic acid Sequence is as shown in SEQ ID No.16 in sequence table.

Antibody variable region amino acid sequence

The amino acid sequence of antibody's light chain variable region is as shown in SEQ ID No.1.The amino acid sequence of antibody heavy chain variable region is such as Shown in SEQ ID No.2.

The expression of 5. complete antibody of embodiment

Embodiment 5.1. constructs the expression vector of anti-PD-L1 full length antibody

According to weight chain variabl area sequence synthetic primer, both ends add restriction enzyme BamH1 and BglII restriction enzyme site, above The heavy chain variable region nucleotide coding sequence for stating the antibody screened in experimental example is template, then standard PCR amplification leads to Segment is accessed pFUSEss-CHIg-hG1 expression vector by the method for crossing digestion connection, thus to obtain the full length antibody weight of the application The pFUSE-IgG1 expression vector of chain.

According to light-chain variable sequence synthetic primer, both ends take EcoRI and AvrII restriction enzyme site, to sieve in experimental example The light chain variable region nucleotide coding sequence for selecting obtained antibody is template, and standard PCR amplification is inserted by digestion connection method Light chain pFUSE2ss-CLIg-hl2 carrier, thus to obtain the pFUSE-CLIg expression vector of the full length antibody light chain of the application.

The preparation of the full human monoclonal antibody of the anti-PD-L1 of embodiment 5.2.

By 293 Fectin transfection reagents and 2 eukaryon antibody expression vectors of above-mentioned acquisition according to volume: mass ratio is 30 μ L: The ratio of 30 μ g:30 μ g mixes, and 30 milliliters of 293 Freestyle suspension cells, 37 DEG C of shaking tables under 125rpm revolving speed is added Overnight, supernatant is collected after centrifugation, using HiTrap Protein A HP columns in KTApurifier100 protein purification Antibody protein purifying is carried out on instrument, finally, the specification referring to BCA method protein quantification kit detects antibody concentration.

The combination of 6. SRP method of embodiment detection antibody and PD-L1

Antibody and antigen affinity and dynamic characteristic are detected by the dynamic (dynamical) method of multi-cycle.Antibody is fixed to use prize law It carries out: first the full human monoclonal antibody of anti-PD-L1 being coupled on Protein A chip, then dilutes PD-L1 sample gradient After flow through chip surface, diluted initial concentration is 40nM, and extension rate is 2 times, dilutes 6 concentration points altogether, and PD-L1 will be by The full human monoclonal antibody of anti-PD-L1 being coupled is captured, and antigen rear signal in conjunction with antibody is detected and records, most Afterwards, the antibody of Protein A chip surface and antigen samples are all washed using regenerative agent (glycine solution of pH1.5) It is de-, and carry out new round detection.

The interaction between the full human monoclonal antibody of anti-PD-L1 and PD-L1 albumen is detected by Biacore, through surveying Fixed, the KD of the full human monoclonal antibody of anti-PD-L1 of the application is 7.1 nM.

The combination of 7. cell-based assay of embodiment anti-PD-L1 antibody and PD-L1

Chemical synthesis human PD-L 1 full-length gene (NCBI Reference Sequence:NM_014143.4), is cloned into purine In mycin (Puromycin) screening system in eukaryon expression plasmid, PEI transfected HEK 293.After transfection 24 hours, pass through Puromycin (2 μ g/ml) is screened, until forming 293F PD-L1 surely turns cell bank.Simultaneously by limiting dilution assay, press 0.8, every hole cell spreads 96 orifice plates, after 15 days, picks out HEK293 PD-L1 monoclonal, and passed on, and forms expression PD- The HEK293 PD-L1 stable cell strain of L1.

HEK293 PD-L1 stable cell strain is digested, it is about 2.5 by cell concentration that centrifugation, which is resuspended in FACS buffer, ×10⁴, volume be 50 μ L be added in 1.5ml EP pipe, be added 50 μ L various concentrations antibody diluent mix, incubation at room temperature 30min；Antibody initial concentration 0.15ug/ml, 5 times of concentration gradients dilute, totally 8 concentration.

Fluidic cell fluorescence sorting technology (Fluorescence activated Cell Sorting, FACS) detection is anti- The association reaction of body and PD-L1 expression cell.Cell is washed with FACS buffer, and 100 μ L Goat Anti- are added afterwards twice Human IgG-FITC antibody is protected from light and is incubated for 30min；FACS is carried out after being washed twice with FACS buffer detects 8 concentration values Antibody, testing result is as illustrated in figs. 1A and ib.Fig. 1 a and Fig. 1 b show that the anti-PD-L1 antibody of the application can be with HEK293 PD-L1 specific binding on cell.

8. AlphaLISA of embodiment detects the combination that the full human monoclonal antibody of anti-PD-L1 inhibits PD-L1 and PD-1

The anti-full human monoclonal antibody of PD-L1 to be detected is subjected to concentration gradient dilution, initial concentration value with 3 times of extension rates For 5 μ g/ μ L, amount to 10 concentration values of dilution.Detecting reaction system is respectively 5 μ L of measuring samples antibody protein or inhibitor, raw 55 μ L of μ L, PD-L1-6His of object element PD-1, Streptavidin donor bead and acceptor bead (SA-Donor beads and Acceptor beads) 5 μ L of mixture, add up to 20 μ L.1X dilution is prepared first, then, to specifications by each reagent With diluent preparing, respectively 4X histidine tag PD-L1 (20nM), the anti-6X histidine of 4X biotinylation PD-1 (20nM), 4X AlphaLISA acceptor bead (Anti-6xHis AlphaLISA Acceptor beads) (40 μ g/mL) and strepto- are affine Donor bead (Streptavidin Donor beads) (80 μ g/mL) mixed liquor of element label.By above-mentioned four kinds of liquid according to 20 μ L reaction system is admixed together, and room temperature, which is protected from light, is incubated for 90min, the numerical value at 615nm is read on Envision, as a result such as Shown in Fig. 2.Fig. 2 the result shows that, the anti-PD-L1 antibody of the application is able to suppress the combination of PD-1/PD-L1, and IC50 is 37.18ng/ml。

The anti-PD-L1 antibody of 9. people of embodiment cell proliferation and cell factor in mixed lymphocyte reaction (MLP) generate It influences

Using mixed lymphocyte reaction (MLP) (mixed lymphocyte reaction, MLR) detection PD-L1 antibody to CD4+ T The influence that the proliferation and IFN-γ and IL-2 of lymphocyte generate.

Separation, culture and the induction of embodiment 9.1. cell

From the new blood of healthy volunteer, by being carried out outside the isolated people of density gradient centrifugation using Ficoll-Paque All blood mononuclear cells (Human peripheral blood mononuclear cell, PBMCs).PBMC is incubated at containing 10% In the RPMI-1640 complete culture solution of fetal calf serum and 1% Pen .- Strep, while 100U recombinant human il-2 is added.

Monocyte is separated from PBMC using person monocytic cell's enrichment kit, and is incubated at containing 10% fetal calf serum In the RPMI-1640 complete culture solution of 1% Pen .- Strep, adjustment cell density to 2 × 10⁶/ml.Simultaneously in culture solution Middle addition rhGM-CSF and IL-4, final concentration are respectively 800U/ml and 50ug/ml.Cell is inoculated in every hole 2.5ml 6 orifice plates, culture induce it to become Dendritic Cells (dendritic cells, DC) after 5-7 days.Discarded half within period every 2-3 days Culture solution, and supplement the fresh medium containing cell factor.In mixing lymph reaction preceding 18-24 hour in culture solution add Enter 1ug/ml LPS, culture obtains mature dendritic cell (mature DC, mDC).

User's CD4+ T cell enrichment kit separates CD4+T lymphocyte from PBMCs.

The reaction of embodiment 9.2. mixing lymph

The CD4+T lymphocyte of purifying and the mDC cell of xenogenic origin co-culture.Wherein CD4+T lymphocyte is with 1 × 10⁵Or 2 ×10⁵The density in/hole is added 50 μ L(and is respectively used to detection IFN-γ and IL-2), the antibody of 50 μ L various concentrations is then added. Then, the 100 μ L of mDC of different proportion is added, 96 orifice plates are placed in 37 DEG C, in 5% CO2 incubator.Antibody in cultivating system Initial concentration is 10 μ g/mL, carries out concentration gradient dilution with 3 times of extension rates.96 parallel orifice plates are set, the 96 of 3 days are cultivated ELISA of the supernatant of orifice plate for IL-2 is detected, and ELISA of the supernatant in 96 orifice plates of culture 5 days for IFN-γ is detected.Altogether CD4+T lymphocyte cell proliferation is detected with the 3H-TDR of the cell after culture 5 days.

The detection of embodiment 9.3. cell factor

With the people's IFN-γ and IL-2 level in ELISA detection supernatant.Standard items recombinanthumanifn-γ and IL-2 are respectively used to mark The measurement of quasi- concentration curve.The specific antibody of employment IFN-γ or the preparatory coated elisa plate of the antibody of IL-2, it is secondary daily to contain 2% The PBS solution of BSA is closed 1 hour.100 μ L standard items and sample to be tested are added in each reacting hole, is incubated for 2 hours at room temperature. After washing be added biotin labeling IFN-γ antibody or IL-2 antibody incubation 1 hour.Marked by streptavidin is added after washing HRP(Horseradish Peroxidase) be incubated at room temperature 30 minutes.Tmb substrate colour developing is added after washing again, to The color of reaction product from light to dark after, with 2M HCl terminate react.Microplate reader (Molecular is used in 15 minutes Devices SpectraMax M5e Plate Reader) absorbance value is read at 450nm wavelength.According to standard curve meter The cytokine content in supernatant to be measured is calculated, testing result is as shown in Figure 3 and Figure 4, the results showed that, the anti-PD-L1 of the application is anti- Body can promote T cell secretion IL-2 and IFN-γ.

The detection of embodiment 9.4. cell Proliferation

With 0.9%NaCl solution with 20 times of volume dilution 3H-thymidine, 96 orifice plates are added, make its final concentration of 0.5 uCi/ well.96 orifice plates are continued to be placed in 5%CO2, are cultivated 16-18 hours in 37 DEG C of incubator.After culture, it is centrifuged and removes Then supernatant in GF/B plate washed once with distilled water.96 orifice plates are placed on cell harvestor (PerkinElmer), very It is washed 5 times after empty pump filter with the distilled water of pre-cooling, is placed in 45 DEG C of ovens and dries.Every hole is added 50 μ L scintillation solutions and is dodged with liquid Bright calculating instrument (Top Count NXT HTS Reader) is counted, and testing result is as shown in Figure 5, the results showed that, the application's is anti- PD-L1 antibody can promote T cell to be proliferated.

The above results show that the full human monoclonal antibody of anti-PD-L1 of the application promotes IL-2 in a manner of concentration dependant Secretion, IFN-γ secretion and T cell proliferation.Based in embodiment as a result, those skilled in the art can definitely realize the antibody In fourth aspect described above and the 5th aspect, especially in preparing the drug for treating T cell dysfunctional disease Purposes.

Unless otherwise indicated, practice of the invention use chemistry in those of ordinary skill in the art's limit of power, Molecular biology, microbiology, recombinant DNA technology and chemical method routine techniques.Although this has been disclosed in detail herein The particular implementation of application, but this is only through exemplary mode and implements, and for illustration purposes only.Above-mentioned implementation Mode is not intended to limit scope of the appended claims, does not also mean that the application must rely on above-mentioned detailed construction feature It can implement.

Those skilled in the art should know, to any equivalent replacement of the application or improvement, such as to selected by the application With any equivalence replacement of method, sequence, reagent etc. or improvement and the increase of slave part, the selection of concrete mode etc., It falls within the protection scope and the open scope of the application.Each particular technique described in above-mentioned specific embodiment is special Sign, in the case of no contradiction, can be combined in any appropriate way, in order to avoid unnecessary repetition, this Shen Please to various combinations of possible ways, no further explanation will be given.

SEQUENCE LISTING

<110>Shanghai Jia Bei biological medicine technology limited liability company

<120>a kind of anti-PD-L1 antibody or its functional fragment and application thereof

<130> 2018591-1

<160> 18

<170> PatentIn version 3.5

<210> 1

<211> 110

<212> PRT

<213> Artificial Sequence

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<223>antibody variable region chain variable region amino acid sequence

<400> 1

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

1 5 10 15

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

20 25 30

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

35 40 45

Met Ile Tyr Glu Val Thr Lys Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 2

<211> 119

<212> PRT

<213> Artificial Sequence

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<223>antibody variable heavy variable region amino acid sequence

<400> 2

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ser Ser Ser Tyr Arg His Phe Ala Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

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<211> 9

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<223>light chain variable region CDR1 amino acid sequence

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Ser Ser Asp Val Gly Ser Tyr Asn Leu

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<223>light chain variable region CDR2 amino acid sequence

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Glu Val Thr

1

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<223>light chain variable region CDR3 amino acid sequence

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Ser Ser Tyr Ala Gly Ser Asn Asn Leu Val

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<223>heavy chain variable region CDR1 amino acid sequence

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Gly Gly Thr Phe Ser Ser Tyr Ala

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<223>heavy chain variable region CDR2 amino acid sequence

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Ile Ile Pro Ile Phe Gly Thr Ala

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<223>heavy chain variable region CDR3 amino acid sequence

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Ala Ser Ser Ser Tyr Arg His Phe Ala Phe Asp Ile

1 5 10

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<220>

<223>light chain variable region FR1 amino acid sequence

<400> 9

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

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Ser Ile Thr Ile Ser Cys Thr Gly Thr

20 25

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<212> PRT

<213> Artificial Sequence

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<223>light chain variable region FR2 amino acid sequence

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Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile

1 5 10 15

Tyr

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<223>light chain variable region FR3 amino acid sequence

<400> 11

Lys Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly

1 5 10 15

Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala

20 25 30

Asp Tyr Tyr Cys

35

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<223>heavy chain variable region FR1 amino acid sequence

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser

20 25

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<223>heavy chain variable region FR2 amino acid sequence

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

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Arg

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<223>heavy chain variable region FR3 amino acid sequence

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Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu

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

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 15

<211> 330

<212> DNA

<213> Artificial Sequence

<220>

<223>antibody variable region DNA sequence dna light chain variable region

<400> 15

cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60

tcctgcactg gaaccagcag tgatgttggg agttataacc ttgtctcctg gtaccaacag 120

cacccaggca aagctcccaa actcatgatt tatgaggtca ctaagcggcc ctcaggggtt 180

tctaatcgct tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc 240

caggctgagg acgaggctga ttattactgc agttcatatg caggcagcaa caatttggtc 300

tttggcggag ggaccaaggt caccgtccta 330

<210> 16

<211> 357

<212> DNA

<213> Artificial Sequence

<220>

<223>heavy chain variable region nucleic acid sequence

<400> 16

caggtgcagc tggtgcaatc tggaggtgag ctgaagcggc ctgggtcttc ggtgaaggtc 60

tcctgcaagg cttctggagg caccttcagc agctatgcta tcagctgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggaagg atcatcccta tctttggtac agcaaactac 180

gcacagaagt tccagggcag agtcacgatt accgcggacg aatccacgag cacagcctac 240

atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagtagtagt 300

taccggcatt ttgcttttga tatctggggc caagggacca cggtcaccgt ctcctca 357

<210> 17

<211> 244

<212> PRT

<213> Artificial Sequence

<220>

<223>human PD-L 1 extracellular region -6His amino acid sequence

<400> 17

Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu

1 5 10 15

Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr

20 25 30

Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu

35 40 45

Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile

50 55 60

Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser

65 70 75 80

Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn

85 90 95

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

100 105 110

Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val

115 120 125

Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val

130 135 140

Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr

145 150 155 160

Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser

165 170 175

Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn

180 185 190

Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr

195 200 205

Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu

210 215 220

Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg His His

225 230 235 240

His His His His

<210> 18

<211> 176

<212> PRT

<213> Artificial Sequence

<220>

<223>people PD-1 extracellular region -6His amino acid sequence

<400> 18

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

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

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

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

165 170 175

Claims

1. a kind of anti-PD-L1 human antibody, the anti-PD-L1 antibody is monoclonal antibody, which is characterized in that the anti-PD- L1 antibody include at least one of heavy chain hypervariable region CDR1, CDR2 and CDR3 or a variety of and/or light chain hypervariable region CDR1, One of CDR2 and CDR3 or a variety of；Wherein:

2. anti-PD-L1 antibody according to claim 1, which is characterized in that the anti-PD-L1 antibody includes light chain hypervariable region CDR1, CDR2 and CDR3 and heavy chain hypervariable region CDR1, CDR2 and CDR3, in which:

The amino acid sequence of light chain hypervariable region CDR1, CDR2 and CDR3 of the antibody are respectively SEQ ID No.3, SEQ ID Sequence shown in No.4, SEQ ID No.5；

The amino acid sequence of heavy chain hypervariable region CDR1, CDR2 and CDR3 of the antibody are respectively SEQ ID No.6, SEQ ID Sequence shown in No.7, SEQ ID No.8.

3. anti-PD-L1 antibody according to claim 1, which is characterized in that the anti-PD-L1 antibody further includes light chain variable Area and heavy chain variable region, in which: the amino acid sequence of the light chain variable region of the anti-PD-L1 antibody includes at least SEQ ID No. Sequence shown in 1 or through replacement, missing or addition is one or more amino acids formed has with sequence shown in SEQID No.1 The amino acid sequence of the amino acid sequence of same function, the heavy chain variable region of the anti-PD-L1 antibody includes at least SEQ ID Sequence shown in No.2 is one or more amino acids formed with sequence shown in SEQ ID No.2 through replacement, missing or addition Amino acid sequence with same function.

4. anti-PD-L1 antibody according to claim 1, which is characterized in that the anti-PD-L1 antibody further includes light chain framework Area (Framework region, FR) and heavy chain framework regions, wherein；

The light chain framework region of the anti-PD-L1 antibody includes one of light chain FR1, FR2 and FR3 or a variety of, in which: described The amino acid sequence of light chain FR1 is that sequence shown in SEQ ID No.9 or the sequence are one or more through replacement, missing or addition The amino acids formed amino acid sequence with sequence shown in SEQ ID No.9 with same function；The amino acid of the light chain FR2 Sequence is for sequence shown in SEQ ID No.10 or through replacement, missing or the one or more amino acids formed and SEQ ID of addition Sequence shown in No.10 has the amino acid sequence of same function；The amino acid sequence of the light chain FR3 is SEQ ID No.11 institute The sequence shown or through replacement, missing or addition it is one or more it is amino acids formed have with sequence shown in SEQ ID No.11 it is same Etc. functions amino acid sequence；And

The heavy chain framework regions of the anti-PD-L1 antibody include one of heavy chain FR1, FR2 and FR3 or a variety of, in which: described heavy The amino acid sequence of chain FR1 is for sequence shown in SEQ ID No.12 or through replacement, missing or adds one or more amino acid What is formed has the amino acid sequence of same function with sequence shown in SEQ ID No.12；The amino acid sequence of the heavy chain FR2 For sequence shown in SEQ ID No.13 or through replacement, missing or addition is one or more amino acids formed has and SEQ ID The amino acid sequence of the same function of sequence shown in No.13；The amino acid sequence of the heavy chain FR3 is SEQ ID No.14 or through replacing Change, lack or add one or more amino acids formed amino acid with sequence shown in SEQ ID No.14 with same function Sequence.

5. anti-PD-L1 antibody according to claim 3, which is characterized in that encoding light chain in the anti-PD-L1 antibody can The nucleotides sequence for becoming area is classified as sequence shown in SEQ ID No.15 or the sequence through replacement, missing or adds one or more cores The nucleotide sequence with sequence shown in SEQ ID No.15 with same function that thuja acid is formed, is compiled in the anti-PD-L1 antibody The nucleotides sequence of code heavy chain variable region is classified as sequence shown in SEQ ID No.16 or the sequence through replacement, missing or addition one Or the nucleotide sequence with sequence shown in SEQ ID No.16 with same function that multiple nucleotide are formed.

6. anti-PD-L1 antibody according to any one of claims 1-5, which is characterized in that the parent of the antibody and PD-L1 It is less than or equal to 1.0 x10 with power KD^-8M。

7. a kind of expression vector, which is characterized in that the carrier includes at least one of following amino acid sequences: SEQ ID Sequence shown in No.15 or the sequence through replacement, missing or add that one or more nucleotide are formed with SEQ ID No.15 Shown sequence has the nucleotide sequence of same function；And sequence shown in SEQ ID No.16 or the sequence are replaced, are lacked Lose or add the nucleotide sequence with sequence shown in SEQ ID No.16 with same function that one or more nucleotide are formed.

8. a kind of host cell comprising carrier as claimed in claim 7, the host cell are mammalian cells.

9. a kind of immunoconjugates comprising anti-PD-L1 antibody of any of claims 1-6 or its functional fragment And its pharmaceutical composition, which is characterized in that the described pharmaceutical composition at least anti-PD-L1 antibody containing effective dose or Its functional fragment and its pharmaceutically acceptable carrier.

10. a kind of anti-PD-L1 antibody of any of claims 1-6 or its functional fragment are in preparation for treating T The purposes of the drug of cellular malfunction disease.