CN106478819B

CN106478819B - A kind of monoclonal antibody or antibody fragment for PD-L1

Info

Publication number: CN106478819B
Application number: CN201610950752.3A
Authority: CN
Inventors: 徐强; 徐圣涛
Original assignee: Changzhou Velox Pharmaceutical Science & Technology Co Ltd
Current assignee: Changzhou Velox Pharmaceutical Science & Technology Co Ltd
Priority date: 2016-10-27
Filing date: 2016-10-27
Publication date: 2018-12-07
Anticipated expiration: 2036-10-27
Also published as: CN106478819A

Abstract

The invention discloses a kind of monoclonal antibodies or antibody fragment for PD-L1, and the antibody or antibody fragment include: heavy chain and light chain.Heavy chain and light chain include variable region, and variable region includes complementary determining region.Complementary determining region CDR1, CDR2 and CDR3 of heavy chain are indicated with HCDR1, HCDR2 and HCDR3 respectively.Complementary determining region CDR1, CDR2 and CDR3 of light chain are indicated with LCDR1, LCDR2 and LCDR3 respectively.The amino acid sequence of HCDR1 includes SEQ ID NO:3, the amino acid sequence of HCDR2 includes SEQ ID NO:4, the amino acid sequence of HCDR3 includes SEQ ID NO:5, the amino acid sequence of LCDR1 includes SEQ ID NO:6, the amino acid sequence of LCDR2 includes that the amino acid sequence of SEQ ID NO:7, LCDR3 include SEQ ID NO:8.Antibody or antibody fragment of the invention can enhance immune system, improve lymphocytic emiocytosis interleukin 2, have as the purposes for preparing antitumor, anti-inflammatory and anti-infection drug.

Description

A kind of monoclonal antibody or antibody fragment for PD-L1

Technical field

The present invention relates to a kind of mankind's programmed death receptors ligand 1(PD-L1) antibody, and in particular to a kind of needle PD- The monoclonal antibody or antibody fragment of L1.

Background technique

Programmed death ligand 1(PD-L1) in chronic infectious disease, pregnancy, tissue transplantation, autoimmune disease and cancer During play immunosuppressive effect.PD-L1 passes through with programmed death receptor 1(PD-1) it combines to regulate and control siberian crabapple The response of system.PD-1 is mainly expressed on T cell, the surface of B cell and monocyte.PD-L1 can also by with it in addition Receptor B7-1 interaction lower the function of T cell.The activation energy of PD-L1/ PD-1 signal path lowers the work of T cell The secretion of property and cell factor, inhibits the anticancer activity of immune system.PD-L1 has very high table in many cancerous tissues It reaches, such as bladder cancer, breast cancer, colon cancer, lung cancer, melanoma, oophoroma, salivary-gland carcinoma, gastric cancer, thyroid cancer etc..PD- Overexpression of the L1 in cancer cell is possible to related with the invasion of acceleration cancer cell and very low prognosis.Anti-human PD-L1 Dan Ke Grand antibody can be effectively in conjunction with the PD-L1 of people, the signal path for blocking PD-L1 to mediate, thus the siberian crabapple of human activin System, achievees the purpose that treating cancer or other related diseases.

It is directed to the monoclonal antibody medicine of PD-L1 receptor PD-1 at present, such as the drug that Keytruda(Mo Shadong is researched and developed, first by FDA The PD-1 inhibitor of approval), the drugs of Opdivo(Bristol Myers Squibb research and development, PD-1 inhibitor) and sieve Atezolizumab( The drug of family name's research and development, PD-L1 inhibitor) it has listed, and significant treatment effect is shown in the treatment of kinds cancer Fruit.

Summary of the invention

The present invention provides a kind of monoclonal antibody or antibody fragment for PD-L1, the antibody or antigen in connection Section be capable of high specific with human PD-L 1 ining conjunction with, can be improved lymphocytic emiocytosis interleukin 2 (IL-2), enhancing be immunized System.

In order to achieve the above object, the present invention provides a kind of monoclonal antibodies or antibody fragment for PD-L1, this is anti- Body or antibody fragment include: heavy chain and light chain, and the antibody fragment is segment of the antigen in conjunction with the antibody.

Wherein, the heavy chain and light chain include variable region, and the variable region includes complementary determining region.

Wherein, complementary determining region CDR1, CDR2 and CDR3 of the heavy chain are indicated with HCDR1, HCDR2 and HCDR3 respectively； Complementary determining region CDR1, CDR2 and CDR3 of the light chain are indicated with LCDR1, LCDR2 and LCDR3 respectively.

Wherein, the amino acid sequence of the HCDR1 includes one or a few ammonia in SEQ ID NO:3 or the sequence Base acid is by the SEQ ID NO:3 of other amino acid simple substitutes.

Wherein, the amino acid sequence of the HCDR2 includes one or a few ammonia in SEQ ID NO:4 or the sequence Base acid is by the SEQ ID NO:4 of other amino acid simple substitutes.

Wherein, the amino acid sequence of the HCDR3 includes one or a few ammonia in SEQ ID NO:5 or the sequence Base acid is by the SEQ ID NO:5 of other amino acid simple substitutes.

Wherein, the amino acid sequence of the LCDR1 includes one or a few ammonia in SEQ ID NO:6 or the sequence Base acid is by the SEQ ID NO:6 of other amino acid simple substitutes.

Wherein, the amino acid sequence of the LCDR2 includes one or a few ammonia in SEQ ID NO:7 or the sequence Base acid is by the SEQ ID NO:7 of other amino acid simple substitutes.

Wherein, the amino acid sequence of the LCDR3 includes one or a few ammonia in SEQ ID NO:8 or the sequence Base acid is by the SEQ ID NO:8 of other amino acid simple substitutes.

The heavy chain variable amino acid sequence includes SEQ ID NO:1 or the amino acid in SEQ ID NO:1 The amino acid sequence of at least 80% homology of composition.

The chain variable region amino acid sequence includes SEQ ID NO:2 or the amino acid in SEQ ID NO:2 The amino acid sequence of at least 80% homology of composition.

The antibody or antibody fragment is with 2 × 10^-9M or lower K_DIn conjunction with people PD- L1, and with people PD-L2 In conjunction with not significant or do not combine.

The antibody or antibody fragment are as follows: chimeric antibody, humanized antibody, human antibody or selected from the following anti- Body segment: Fab, F(ab ')₂, Fv, dAb and scFv.

The human antibody is any one in IgG1, IgG2 or IgG4 full length antibody.

The heavy chain variable region of the antibody or antibody fragment is by nucleotide sequence coded in SEQ ID NO:9； The light chain variable region of the antibody or antibody fragment is by nucleotide sequence coded in SEQ ID NO:10.

A kind of expression vector containing the nucleotide sequence.

A kind of host cell containing the expression vector.

The full human monoclonal antibody or antibody fragment for PD-L1 is preparing immune conjugate or combinations thereof object In purposes, the immune conjugate is by therapeutic agent and the antibody or antigen segment covalent bond in connection；It is described Composition by the immune conjugate and pharmaceutically available support combines.

The therapeutic agent includes: cytotoxic agent, radioactive isotope or immunosuppressor.

The full human monoclonal antibody or antibody fragment for PD-L1 is preparing anti-tumor drug or combinations thereof object In purposes.

The tumour includes: osteocarcinoma, lung cancer, cutaneum carcinoma, head and neck cancer, breast cancer, uterine cancer, oophoroma, carcinoma of fallopian tube, Carcinoma of endometrium, cervical carcinoma, carcinoma of vagina, prostate cancer, carcinoma of testis, carcinoma of penis, bladder cancer, carcinoma of ureter, kidney, carcinoma of renal pelvis, Colon cancer, the carcinoma of the rectum, gastric cancer, cancer of the esophagus, cancer of pancreas, carcinoma of small intestine, thyroid cancer, parathyroid carcinoma, adrenal, lymthoma, Soft tissue sarcoma, malignant mela noma, chronic or acute leukemia, any one or two kinds in central nerve neuroma with On；The cutaneum carcinoma is squamous cell carcinoma；The lymthoma is Hodgkin lymphoma, non-Hodgkin lymphoma or T cell Lymthoma；The chronic or acute leukemia is acute myeloid lymph cancer, chronic myelocytic lymph cancer, acute lymphoblastic Cancer or chronic lymphocytic cancer；The central nerve neuroma is glioma or pituitary adenoma.

The full human monoclonal antibody or antibody fragment for PD-L1 is preparing anti-infection drug or its group The purposes in object is closed, the infectious disease includes: by any one in virus, bacterium, fungi or helminth or two or more to draw The infectious disease risen；

Wherein, the virus include: HIV, hepatitis virus, influenza virus, herpesviral, adenovirus, arboviruse, angstrom It can virus, rhinovirus, Coxsackie virus, coronavirus, Respiratory Syncytial Virus(RSV), mumps virus, rotavirus, fiber crops Exanthema virus, rubella virus, parvovirus, vaccinia virus, human T-cell virus, dengue fever virus, papillomavirus, molluscum Virus, poliovirus, rabies viruses, JC virus and arboviruse encephalitis viruses in any one or it is two or more.

Wherein, the bacterium includes: mycobacteria, staphylococcus, streptococcus, Neisseria, Klebsiella, deformation Bacterium, Serratieae, pseudomonad, Legionnella, corynebacterium diphtheriae, salmonella, bacillus, cholera bacteria, tetanolysin, meat poisoning In bacillus and yersinia pestis any one or it is two or more；The staphylococcus is staphylococcus aureus, the chain Coccus is streptococcus pneumonia；The Neisseria is meningococcus or gonococcus, and the pseudomonad is Pseudomonas aeruginosa, The bacillus is bacillus anthracis.

Wherein, the fungi includes: Candida, Cryptococcus neoformans, aspergillus, mucor (such as), Sporothrix schenckii Any one or two kinds in bacterium, Blastomyces dermatitidis, Paracoccidioides brasiliensis, posadasis spheriforme and histoplasma capsulatum with On；The Candida be candida albicans, candida krusei, Candida glabrata or candida tropicalis, it is described Aspergillus be aspergillus fumigatus or aspergillus niger, the mucor is that Mucor, colter be mould or head mold.

Wherein, the helminth include: Entamoeba histolytica, balantidium Coli, naegleria fowleri, Acanthamoeba, Leishmania, giardia lamblia, Cryptosporidium, Pneumocystis carinii, plasmodium, babesia microti, trypanosoma bocagei, gram Family name's trypanosome, Leishmania donovani, toxoplasma gondii, nippostrongylus brasiliensis；The giardia lamblia is giardia lamblia, the malaria Protozoon is Plasmodium vivax.

Described full human monoclonal antibody or antibody fragment for PD-L1 in the drug for preparing anti-inflammatory disease or Purposes in its composition, the diseases associated with inflammation refer to the diseases associated with inflammation caused by lowering lymphocyte immunity.

The diseases associated with inflammation includes: lichen planus tinea.

A kind of monoclonal antibody or antigen segment in connection for PD-L1 provided by the invention, has the advantage that

Antibody and antibody fragment of the invention is not combined with people PD-L2 or not in conjunction with the high-affinity of human PD-L 1 Significant to combine, the specificity for showing antibody and antibody fragment of the invention is high；

Antibody and antibody fragment of the invention can enhance the ability of IL-2 secretion in mixed lymphocyte reaction (MLP), and enhancing is exempted from Epidemic disease response；

Antibody and antibody fragment blocks of the invention PD-L1 stimulates the ability of antibody response in conjunction with PD-1 receptor, The holddown of regulatory T-cell is reversed, to be at state of activation.

Detailed description of the invention

Fig. 1 is the bar chart that PD-L1 antigen result is screened in experimental example 1.

Fig. 2 is antibody and human PD-L 1 combination degree figure of the invention in experimental example 4.

Fig. 3 is the affinity curve figure of antibody of the invention in experimental example 5 in conjunction with human PD-L 1.

Fig. 4 is the influence curve figure of antibody of the invention in experimental example 6 to lymphocytic emiocytosis IL-2.

Specific embodiment

Definition

" immune response " refers to immunity-associated cell (such as lymphocyte, antigen presenting cell, macrophage or granulocyte Deng) after receiving antigenic stimulus, a series of signal transduction process of generation and resulting immune-related biology The disease that macromolecular (such as generation antibody, cell factor or complement) leads to selective injury, destroys or remove from human body intrusion It is substance, thin by the cell or tissue of pathogenic infection, cancer cell or normal person in autoimmunity or pathological inflammatory Born of the same parents or tissue.

" signal transduction pathway " refers to signal from a part of a cell into the transmission of another part of a cell Biochemical relationship between the multi-signal transduction molecule of effect.

" cell surface receptor " includes the molecule and molecule that can be received signal and propagate this signal across cytoplasma membrane Compound.PD-L1 receptor of the present invention is a kind of cell surface receptor.

" antibody " refers at least two weight (H) chains and two light (L) chain comprising being mutually connected to each other by disulfide bond Glycoprotein or its antigen-binding portion thereof.Antibody includes complete antibody and its any antigen-binding fragment or single-chain antibody.

" heavy chain " is made of heavy chain variable region (VH) and heavy chain constant region.

" heavy chain constant region " is made of three domain Cs H1, CH2 and CH3.

" light chain " is made of light chain variable region (VL) and constant region of light chain (CL).

" constant region of light chain " is made of a structural domain.

" heavy chain variable region " and " light chain variable region " can be further subdivided into hypervariable region, referred to as " complementary determining region " (CDR), CDR is dispersed in the referred to as more conservative region of " framework region " (FR).Each VH and VL is by three CDR and four FR Composition, they are arranged in the following order from aminoterminal to c-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, heavy chain Containing with the variable region of light chain can be with the binding structural domain of antigen interactions.

" constant region " can include with the combination of mediated immunity globulin and host tissue or the factor, the host tissue or the factor The first composition (C1q) of the various cells (such as effector cell) of immune system and classical complement system.

" antigen-binding portion thereof " refers to one or more of the antibody of the ability of reservation and antigen (such as PD-L1) specific binding A segment.The antigen binding function of being proved antibody can be exercised by the segment of full length antibody.It is wrapped in " antigen-binding portion thereof " The binding fragment included includes: (1) Fab segment, i.e., the monovalent fragment being made of VL, VH, CL and CH1 structural domain；(2)F(ab')₂ Segment includes two Fab segments that hinge area (region between the functional areas heavy chain CHl and CH2) is connected by disulfide bond Divalent fragments thereof；(3) the Fd segment being made of VH and CH1 structural domain；(4) Fv being made of VL the and VH structural domain of antibody single armed Segment；(5) the dAb segment being made of VH structural domain；(6) complementary determining region (CDR) separated.In addition, although the two of Fv segment A structural domain VL and VH is encoded by individual gene, but they can use recombination method and link together, and can be made one Protein chain, wherein the area VL and VH pairing composition monovalent molecule is known as scFv (i.e. scFv), and this single-chain antibody is also included within In term " antigen-binding portion thereof ".These antibody fragments with well known to a person skilled in the art routine techniques to obtain, and with it is complete The identical method of whole antibody screens the practicability of these segments.

" isolated antibody " refer to be substantially free of the antibody for specifically binding other antigens (as described in the present invention with PD- The isolated antibody of L1 specific binding, other antibody being substantially free of in conjunction with the antigentic specificity in addition to PD-L1). But may have with the isolated antibody of PD-L1 specific binding and the PD-L1 molecule (other antigens) etc. from other species There is cross reactivity.Isolated antibody is substantially free of other cell materials and/or chemical substance.

" monoclonal antibody " or " monoclonal antibody combination " refers to the antibody molecule preparation by single molecular composition.Dan Ke Grand antibody compositions show specific binding and parent to defined epitope (antigen part identified by antigen receptor specificity) And property.

" human antibody " includes the antibody with following variable region, and in the variable region, framework region and CDR region are derived from ethnic group It is immunoglobulin sequences.Moreover, constant region also originates from human germline immunoglobulin's sequence if the antibody contains constant region Column.Human antibody of the invention may include not by the amino acid residue of human germline immunoglobulin's sequential coding (as by external Random or site-specific mutagenesis or the mutation introduced by internal somatic mutation).But " people is anti-for terms used herein Body " does not include wherein being transplanted on people's frame sequence from the CDR sequence of another mammalian germ line (such as mouse germline) Antibody.

" human monoclonal antibodies " refer to that the antibody for showing single binding specificity, framework region and CDR region are derived from ethnic group It is the variable region of immunoglobulin sequences.

" recombinant human antibody " includes by recombination method preparation, expresses, all human antibodies of generation or separation, such as: (1) Divide from by the animal (such as mouse) of human immunoglobulin gene's transgenosis or transfection chromosome or the hybridoma prepared by the animal From antibody；(2) antibody separated from the host cell (such as transfectoma) of translation table intelligent's antibody；(3) people is combined from recombination The antibody separated in antibody library；(4) by by human immunoglobulin gene's sequence montage be other DNA sequence dnas any other Method preparation, expression, generation or isolated antibody.There is these recombinant human antibodies framework region and CDR region to be derived from ethnic group system and exempt from The variable region of epidemic disease globin sequence.But in certain embodiments, these recombinant human antibodies can undergo mutagenesis in vitro, or Undergo internal somatic mutagenesis when using the transgenic animals of human immunoglobulin(HIg) (Ig) sequence, thus the VH of recombinant antibodies and The amino acid sequence in the area VL may not be natural in vivo despite being originated from ethnic group system VH and VL sequence and associated sequence It is present in the repertoire of human antibody set.

" isotype " refers to the Antibody types (such as IgM or IgG1) encoded by weight chain constant area gene.

" antibody of identification antigen " and " antigen-specific antibodies " can be with term " antibody with antigen, specific binding " It is used interchangeably.

" human antibody derivative " refers to any modified forms (such as coupling of antibody and other reagents or antibody of human antibody Object).

" humanized antibody " refers to the CDR sequence quilt for wherein deriving from another mammalian germ line (such as mouse germline) The antibody being transplanted on people's frame sequence can also carry out other framework region modifications in people's frame sequence.

" chimeric antibody " refers to that wherein variable region sequences derive from a species and constant-region sequences derive from another object Kind antibody (as wherein variable region sequences from mouse antibodies and constant-region sequences derive from the antibody of human antibody).

The antibody of " specifically binding with human PD-L 1 " refers to 1 × 10^-8Mole (M) lower K_DIn conjunction with human PD-L 1 Antibody.

" ka " refers to the combination of specific antibodies-antigen interactions, and " kd " refers to specific antibodies-antigen interactions solution From " K_D" referring to dissociation constant, it is to obtain (i.e. kd/ka) by the ratio of kd and ka, is indicated with molar concentration (M).The K of antibody_D The conventional method that value can be established with this field measures, and measures antibody K_DA kind of preferred method be surface plasmon resonance method, It is preferable to use bio-sensor systems.

" high-affinity " of IgG antibody refers to antibody for the K of target antigen_DIt is 10^-8M or lower, but it is anti-for other For body isotype, " high-affinity " combination may be different.

" regulating and controlling sequence " includes other expression control members of promoter, enhancer and the transcription of control antibody gene or translation Part.

" subject " includes anyone or non-human animal, and " non-human animal " includes (such as inhuman spirit of all vertebrates Long class animal, sheep, dog, cat, horse, ox, chicken, amphibian animal, reptile etc.).

" effective dose " or " effective quantity " refers to can generate function or active and can be received by subject to subject Amount.

" pharmaceutically acceptable carrier " is suitable for subject and without excessive adverse reaction (such as toxicity, stimulation and metamorphosis Reaction), i.e., with the carrier of reasonable Benefit Risk ratio.

Presently in connection with the following drawings and embodiment, the following further describes the technical solution of the present invention.

The present invention provides a kind of monoclonal antibodies or antibody fragment for PD-L1, and the antibody or antibody fragment include: Heavy chain and light chain, antibody fragment are segment of the antigen in conjunction with antibody.Heavy chain and light chain include variable region, the variable region packet Include complementary determining region.

Wherein, complementary determining region CDR1, CDR2 and CDR3 of heavy chain are indicated with HCDR1, HCDR2 and HCDR3 respectively, light chain Complementary determining region CDR1, CDR2 and CDR3 indicated respectively with LCDR1, LCDR2 and LCDR3.The amino acid sequence of HCDR1 includes The amino acid sequence of SEQ ID NO:3 or SEQ the ID NO:3, HCDR2 of conservative modification include SEQ ID NO:4 or conservative modification SEQ ID NO:4, HCDR3 amino acid sequence include SEQ ID NO:5 or conservative modification SEQ ID NO:5, LCDR1's Amino acid sequence includes that the amino acid sequence of SEQ the ID NO:6, LCDR2 of SEQ ID NO:6 or conservative modification include SEQ ID The amino acid sequence of NO:7 or SEQ the ID NO:7, LCDR3 of conservative modification include the SEQ of SEQ ID NO:8 or conservative modification ID NO:8.

The present invention also provides the preparation methods of monoclonal antibody or antibody fragment for PD-L1, in order to express antibody Or its antibody fragment, it can be by the DNA of standard molecular biological technique coded portion or full-length light chains and heavy chain, and by the DNA It is inserted into expression vector, so that the DNA and the regulating and controlling sequence of transcription and translation effectively connect.Selection and expressive host used The expression vector and expression regulation sequence that cell matches, antibody light chain gene and antibody heavy chain gene can be inserted into separated In carrier, by being inserted into the heavy chain constant region of encoded desired isotype and the expression vector of constant region of light chain, make VH section is obtained effectively to connect with the CH section in carrier, and VL section is effectively connect with the CL section in carrier, can use this The light chain and heavy chain variable region of the antibody of invention generate the full length antibody gene of any antibody isotype.

It will be appreciated by those skilled in the art that the design of expression vector, the selection including regulating and controlling sequence, the host to be converted The selection of cell and desired protein expression level, depend primarily on the selection of the host cell of conversion.It is dynamic for lactation The regulating and controlling sequence of object host cell expression is preferably able to instruct the virus member of protein high level expression in mammalian cells Part, such as from cytomegalovirus (CMV), vacuolating virus of monkey 40(SV40), the promoter and/or enhancing of adenovirus polyomavirus Son.

Recombinant expression carrier can also encode the signal peptide for being conducive to host cell secretory antibody chain, can be by antibody chain gene It is cloned into carrier, so that signal peptide is connect with the amino terminal of the antibody chain gene meets reading frame, signal peptide, which can be, exempts from Epidemic disease Immunoglobulin Signal Peptide or heterologous signal peptide.

In order to express light chain and heavy chain, it is thin that the expression vector of encoding heavy chain and light chain is transfected by host by standard technique In born of the same parents.The various forms of term " transfection " includes the various skills being usually used in Exogenous DNA transfered protokaryon or eukaryotic host cell Art, such as electroporation, calcium phosphate precipitation or liposome transfection.Although theoretically can be in protokaryon or eukaryotic host cell Antibody or antibody fragment of the invention are expressed, it is preferred that in eukaryocyte, the table most preferably in mammalian host cell Up to the antibody, because eukaryocyte (especially mammalian cell) is easier the tool assembled and secretion correctly folds than prokaryotic cell There is immunocompetent antibody.It is reported that the expression antibody gene of prokaryotic cell can not high productivity generation active antibodies.

Preferred mammalian host cell for expressing antibody of the invention include: Chinese hamster ovary cell (CHO), NSO myeloma cell, African green monkey kidney cell (COS) and myeloma cell (SP2).Particularly, for CHO myeloma cell's Another preferred expression system is disclosed pFUSE expression system, this serial expression vector is that Invivogen company releases one group Antibody expression vector can be used to express the antibody or antibody fragment of various overall lengths.When by the recombination table of encoding antibody genes When up in vector introduction mammalian host cell, by being enough host cell culture express that antibody in host cell, Or it is highly preferred that culture is enough that antibody-secreting is made to generate antibody into the culture medium of host cell growth.

The preparation method of antibody or antibody fragment provided by the invention is specific as follows:

Firstly, being obtained using recombinant DNA technology well known in the art and gene transfection method, or in host cell transfectoma Obtain antibody or antibody fragment of the invention；

Secondly, passing through the side of gene chemical synthesis after the DNA sequence dna for obtaining monoclonal antibody or antibody fragment of the invention Formula obtains the weight chain variabl area sequence and light-chain variable sequence of the monoclonal antibody；

Then, this two sections of sequences are inserted by two pFUSE-IgG1 antibody expressions by the molecule clone technology of standard respectively It in carrier, by the two obtained carriers while being transfected into 293F cell, suspend culture；

Finally, collecting supernatant, isolate and purify to obtain antibody or antibody fragment of the invention.

The present invention also provides the purposes of monoclonal antibody or antibody fragment for PD-L1:

(1) as the purposes for preparing immune conjugate or combinations thereof object

Antibody or antibody fragment of the invention can be with such as cytotoxic agent, drug (such as immunosuppressor) or radioactive poison The therapeutic agents such as element are coupled to obtain conjugate, these conjugates are referred to herein as " immune conjugate ", including one or more cells The immune conjugate of toxin is referred to as " immunotoxin ".

Cytotoxic agent includes any reagent of (such as killing cell) harmful to cell, comprising: taxol, cytochalasin B, Gramicidin D, Ethidum Eremide, ipecine, mitomycin, etioposide, teniposide, Changchun are new Alkali, vincaleukoblastinum, colchicine, adriamycin, daunorubicin, dihydroxy anthracin diketone, mitoxantrone, mithramycin, unwrapping wire Rhzomorph D, 1- dehydrogenation testosterone, glucocorticoid, procaine, totokaine, lidocaine, Propranolol, puromycin and its class Like object or homologue.

Therapeutic agent further include: antimetabolite (such as methotrexate (MTX), Ismipur, 6-thioguanine, cytarabine, ammonia Alkene miaow amine (decarbazine), 5 FU 5 fluorouracil and hydroxycarbamide), alkylating agent (such as mustargen, Chlorambucil, phenylalanine nitrogen Mustard, Carmustine, lomustine, cyclophosphamide, busulfan, dibromo mannitol, streptozotocin, mitomycin C and cis-platinum), Antibiotic (such as actinomycin D, bleomycin, mithramycin, Anthramycin, daunorubicin and adriamycin) and antimitotic Agent.

The therapeutic cells toxic agent being coupled with antibody of the present invention or antibody fragment further include: times carcinomycin, calicheamycin, beauty Smooth life, Ali's statin (Auristatin) and its derivative.

Connection body technique that this field uses be can use by cytotoxin or drug and antibody coupling of the invention, the company Junctor includes but is not limited to: hydrazone, thioether, ester, disulphide and the connector containing peptide, or in lysosomal compartment it is easily low Connector pH cutting or easily cut by protease.Wherein, which is the protease of the priority expression in tumor tissues, such as Cathepsin (cathepsin B, C, D).

Antibody or antibody fragment of the invention can also be coupled with radioactive isotope, generate cytotoxicity radioactivity medicine Object, also referred to as " radioimmunoassay conjugate ".The radioactive isotope includes but is not limited to: iodine 131, indium 111, Y90 and Lutetium 177.The method of radioimmunoassay conjugate is prepared in the art it has been established that this hair can be used using similar method Bright antibody or antibody fragment and radioactive isotope prepares radioimmunoassay conjugate.

Immune conjugate of the invention can be used for modifying specific biologically, and drug moiety should not be construed as limiting to In classical chemotherapeutant.For example, drug moiety can be the protein or polypeptide for having bioactivity in need, including (but being not limited to): with enzymatic activity toxin or its active fragment (such as abrin, ricin A, outside pseudomonad Toxin or diphtheria toxin), tumor necrosis factor, interferon-γ and biologically instrumentality.The biologically instrumentality packet It includes (but being not limited to): lymphokine, interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin-6 (IL- 6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF) and other growth because Son.

(2) as the purposes for preparing anti-tumor drug or combinations thereof object

PD-L1 is not expressed in normal cell, the high expression in a variety of mankind tumor tissue's cells.PD-1 and PD-L1 Interaction cause infiltrate tumour lymphocyte reduce, T cell receptor mediate proliferation reduce, lead to exempting from for tumour cell Epidemic disease is escaped.Immunosupress can be reversed by inhibiting the local interaction of PD-L1 and PD-1, when the interaction of PD-L2 and PD-1 When being also blocked, effect is adduction.Therefore, the blocking of PD-L1 can be enhanced in patient to tumour cell in PD-L1 antibody Immune response.

Currently, bone-marrow transplantation can be used to treat the diseases such as neoplastic hematologic disorder, graft versus host disease(GVH disease) is the one of this treatment Kind consequence, but graft can obtain therapeutic benefit to the response of tumour cell, by being implanted using blocking PD-L1 raising The validity of the donor of tumor specific T cells.

Anti- PD-L1 antibody can be used alone, to inhibit peaceful coexistence or the anti-PD-L1 antibody of cancerous tumour can be with With other immunogenic agents, standard cancer treatments or other antibody combined uses.

The tumour that antibody of the invention or the anti-tumor drug of antibody fragment preparation or combinations thereof object can treat or prevents Generally to there is the tumour of response to immunization therapy.Above-mentioned tumour includes but is not limited to: osteocarcinoma, cutaneum carcinoma, head and neck cancer, uterus Cancer, oophoroma, uterine cancer, carcinoma of fallopian tube, carcinoma of endometrium, cervix cancer, carcinoma of vagina, carcinoma of penis, carcinoma of testis, carcinoma of urethra, wing Guang cancer, kidney or carcinoma of ureter, carcinoma of renal pelvis, cancer of the esophagus, carcinoma of small intestine, the carcinoma of the rectum, cancer of the anal region, internal system cancer, thyroid cancer, first Gland cancer by shape, adrenal, kidney, gastric cancer, cancer of pancreas, melanoma, soft tissue sarcoma, lymthoma, chronic or acute white blood Disease, childhood solid tumor, central nervous system (CNS) tumour, any one in Kaposi sarcoma or two or more.

Wherein, melanoma is skin or intraocular malignant melanoma, and cutaneum carcinoma is epidermis shape cancer or squamous cell carcinoma, leaching Bar tumor is Hodgkin lymphoma, what non-Hodgkin lymphoma or lymphocytic lymphoma, and lymphocytic lymphoma is T cell Lymthoma, chronic or acute leukemia are acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblast Property leukaemia or chronic lymphocytic leukemia, central nervous system (CNS) tumour is that primary CNS lymphoma, backbone are swollen Tumor, brain stem glioma, pituitary adenoma.

Above-mentioned tumour further include: by the tumour (tumour of such as Induced by Asbestos) and metastatic carcinoma of ambient induced, especially Express the metastatic carcinoma of PD-L1.

Preferably, tumour includes: any one in melanoma, kidney, prostate cancer, breast cancer, colon cancer and lung cancer Kind is two or more.

PD-L1 antibody of the invention can also can be by table with bispecific antibody use in conjunction, the bispecific antibody Up to the effector cell of Fc α receptor or Fc γ receptor targeted to tumour cell, and it can use bispecific antibody targeting two The different antigen of kind.For example, using anti-Fc receptor/antitumor antigens (such as Her-2/neu) bispecific antibody that macrophage is thin Born of the same parents' target tumor position, this targeting can more effectively activate tumour-specific response.It can reinforce this using PD-L1 blocking The T cell of a little responses, or can use the bispecific antibody of tumour antigen and dendritic cells specific cell surface markers In conjunction with antigen to be directly delivered to Dendritic Cells (DC).

Tumour escapes the immunosurveillance of host by number of mechanisms, but can pass through the inhibitive ability of immunity of inactivation tumour expression Protein overcomes.Every kind of individual antibody can be combined with anti-PD-L1, to resist the effect of inhibitive ability of immunity protein, and And be conducive to the tumour cell immune response of host.

Antibody or antibody fragment of the invention can also with for activating other antibody of host cell immune response to be combined. Wherein, including the molecule on the surface DC, these molecular activations DC function and antigen presentation；Anti-CD 40 antibodies, can be effectively Substitute T cell auxiliary activity；To T cell costimulatory molecules (activating antibodies of such as TNFRSF4), T cell activation can be improved It is horizontal；Block negative costimulatory molecules (such as Cytotoxic T lymphocyte-associated antigen -4(CTLA-4), B and T lymphocyte decaying Protein antibodies (BTLA)) active antibody.

(3) as the purposes for preparing anti-infection drug or combinations thereof object

Antibody or antibody fragment of the invention can treat and prevent the infectious disease as caused by particular toxin or pathogen.Class The application being similar in above-mentioned tumour, the antibody or antibody fragment can be used alone, and can also be used as adjuvant or and vaccine group It closes and uses, lymphocyte can be stimulated to the immune response of pathogen, toxin and autoantigen.

The antibody or antibody fragment, which are specifically for use in pathogen or conventional vaccine currently without effective vaccine, not exclusively to be had The pathogen of effect.Pathogen includes virus, Chlamydia, rickettsia, Leptospira, bacterium, fungi and helminth.

Wherein, virus includes but is not limited to: HIV, hepatitis virus (first, second, third), influenza virus, herpesviral are (such as VZV, HSV-1, HAV-6, HSV-II, CMV and EB), adenovirus, arboviruse, echovirus, rhinovirus, Coxsackie virus, hat Shape virus, Respiratory Syncytial Virus(RSV), mumps virus, rotavirus, measles virus, rubella virus, parvovirus, acne It is seedling diseases poison, human T-cell virus (HTLV), dengue fever virus, papillomavirus, contagiosum, poliovirus, mad Dog disease poison, JC virus and arboviruse encephalitis viruses in any one or it is two or more.

Wherein, bacterium includes but is not limited to: mycobacteria, staphylococcus (such as staphylococcus aureus), streptococcus are (such as Streptococcus pneumonia), Neisseria (such as meningococcus and gonococcus), Klebsiella, mycetozoan, Serratieae, false unit cell Bacterium (such as Pseudomonas aeruginosa), Legionnella, corynebacterium diphtheriae, salmonella, bacillus (such as bacillus anthracis), cholera bacteria, tetanus In bacterium, clostridium botulinum and yersinia pestis any one or it is two or more.

Wherein, fungi includes but is not limited to: Candida (such as candida albicans, candida krusei, smooth false silk Yeast and candida tropicalis), Cryptococcus neoformans, aspergillus (such as aspergillus fumigatus and aspergillus niger), (such as Mucor, colter is mould for mucor And head mold), Sporothrix schenckii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, in posadasis spheriforme and histoplasma capsulatum Any one is two or more.

Wherein, helminth includes but is not limited to: Entamoeba histolytica, balantidium Coli, Fu Shi Nai Gelia Meter Ba, Acanthamoeba, Leishmania, giardia lamblia (such as giardia lamblia), Cryptosporidium, Pneumocystis carinii, plasmodium (every other day Plasmodium), babesia microti, trypanosoma bocagei, schizotrypanum cruzi, Leishmania donovani, toxoplasma gondii, nippostrongylus brasiliensis.

The antibody or antibody fragment are applied especially to confrontation infection as caused by HIV, and HIV is that escape is exempted from course of infection The antigen changed is presented in epidemic disease monitoring.Antibody or antibody fragment of the invention can block the signal pathway of PD-L1, keep these new Negative signal when epitope is identified as allogene not by PD-L1 is influenced, to enhance the response of T cell.

In addition, antibody or antibody fragment of the invention can combine with the immunotherapy of other forms, as cell factor is controlled (such as interferon, GM-CSF, G-CSF or IL-2) and bispecific antibody treatment are treated, bispecific antibody treatment can enhance tumour The presentation of antigen.

(4) purposes as the drug for preparing anti-inflammatory disease or combinations thereof object

Antibody or antibody fragment of the invention can treat or prevent chronic inflammatory disease, as lichen planus or T cell are situated between The chronic inflammatory skin mucosal disease led.Lichen planus shows immunologic hypofunction, and there are body fluid immunologic derangements, therefore can lead to Cross the immune response treatment of anti-PD-L1 antibody enhancing T cell.

(5) other purposes

Body can generate immune response to tumour cell or peptide vaccine, can generate autoreactivity, anti-PD- in the process Autoimmune response can be enhanced in L1 antibody, therefore can use antibody or antibody fragment of the invention and combine a variety of oneself proteins Matter designs vaccination regimen, with the immune response for these oneself protein matter that effectively create antagonism, for treating and these itself eggs The relevant disease of white matter.It deposits for example, Alzheimer disease is related to beta-amyloid protein (A β), is answered for the antibody of A β in brain Answer the A β that can remove deposition；It is closed for treating the immunoglobulin E (IgE) of allergy and asthma, or for rheumatoid Save scorching tumor necrosis factor-alpha (TNF-α).

Antibody or antibody fragment of the invention can also enhance response of the autoantibody to various hormones, and it is anti-such as to enhance itself Body can be used for practising contraception to the response of reproductive hormone, other needed for enhancing antibody grows other hormones or specific tumors are solvable The response of sex factor.

In preparing above-mentioned " drug or combinations thereof object ", " drug " be antibody or antibody fragment comprising effective dose, or The immune conjugate prepared by antibody or antibody fragment, " composition " are antibody or antibody fragment, pharmaceutically acceptable carrier Combined with therapeutic agent, therapeutic agent includes: vaccine, polyspecific or bispecific antibody, pharmaceutically acceptable carrier include (but It is not limited to): water, salt water, glucose, buffer, ethyl alcohol, glycerol and combinations thereof.Medicine of the invention is applied in vivo or in vitro The appropriate approach of object or combinations thereof object by those skilled in the art it is well known in the art that can be selected, such as injection (vein or skin Under) administration, the optimal dose used is depending on the age of subject and the concentration and/or preparation of weight and antibody compositions.

Antibody or antibody fragment of the invention can in vitro or the cell of in vitro culture, or be applied in subject's body With thus enhancing or up-regulation immune response.Preferably, subject is the human patients for needing to enhance immune response, is especially suitable for The patient of disease is treated by the immune response that enhancing T cell mediates.In order to realize the antigen-specific of self immune system antibody Property enhancing, anti-PD-L1 antibody can be administered together with target antigen.

Antibody or antibody fragment of the invention can also with therapeutic agent medication, antibody can before therapeutic agent, it It afterwards or is administered simultaneously, or can be answered jointly with other known treatment methods (radiotherapy carried out in such as antineoplaston) With.Antitumor cytotoxic agent is only effective when having the level of toxicity or inferior toxicity to patient.Wherein, cis-platinum is with 100mg/ agent Dosage by intravenous injection administration, every 4 weeks 1 time, adriamycin with the dosage of 60-75mg/mL by intravenous injection is administered, every 21 It is 1 time.Antibody or antibody fragment and chemotherapeutics co-administered of the invention, since the mechanism of action of two kinds of different pharmaceuticals is different, The mode functioned is different, thus such administration mode is able to solve drug resistance or tumor-cell antigen sexually revises (this general Make they to antibody without reactivity) cause the problem of.

Some materials source is illustrated in this:

PD-L1 albumen: Human PD-L1/CD274(is purchased from Acrobiosysterms, article No. PD1-H82F3).

The phage antibody library of full people's single-chain antibody: construction method is shown in that experimental example 1(is that Changzhou expense Loews medicine company science and technology is limited Company's building).

Streptavidin MagneSphere: (being purchased from Invitrogen company).

Enzyme linked immunological microwell plate: the low flat bottom microtiter plate of 96 half bore (is purchased from Corning company).

Anti-M13 HRP antibody: (being purchased from Sai Mofei company).

M13 helper phage: (being purchased from Invitrogen company).

SOC culture medium: (purchased from the raw work in Shanghai).

PCGMT phagemid vector: (being purchased from Addgene company).

XL1-blue bacterium: (being purchased from agilent company, article No. 200228).

LB solid medium tablets: by 5g yeast extract, 10g peptone, 10g sodium chloride, 10g agar powder is dissolved in 1L's In distilled water, then the high pressure sterilization under 121 DEG C of high temperature is poured on plate.

Developer solution ABTS solution: (being purchased from Sai Mofei company, article No. 002024).

Gelred nucleic acid dye: (being purchased from Sai Mofei company).

PFUSE-IgG1 expression vector: (being purchased from Invivogen company).

Plasmid extraction kit: (being purchased from Qiagen company).

Restriction enzyme: (being purchased from Takara company).

Recombinase: (being purchased from Novoprotein company).

293Fectin transfection reagent: (being purchased from Invitrogen company, article No. 12347500).

293Freestyle suspension cell: (being purchased from Sai Mofei company).

Immunoglobulin IgG1 constant region fc section: (being purchased from Nanjing Jin Sirui company).

BCA method protein quantification kit: (being purchased from Pierce, article No. 23252).

CBS antigen fixed solution: by 1.59g Na₂CO₃With 2.93g NaHCO₃It is dissolved in 1L water, adjustment pH value is 9.6.

Anti-Human Fc HRP secondary antibody: (being purchased from Sai Mofei company).

The saturating plate of 96 bottom holes: (being purchased from Corning company).

Biacore instrument: (being purchased from GE company, T200).

Protein A chip: (being purchased from GE company).

CD4+ positive selection kit: (being purchased from DynalBiotech).

GM-CSF:(is purchased from R&D Biosystems).

Monocyte negative selection kit: (being purchased from MitenyiBiotech).

Human IL-2 HTRF kit kit: (being purchased from Cisbio company).

AlphsLISA kit: (being purchased from PerkinElmer company, article No. AL356C).

Envision^®Multiple labeling micropore board detector platform: (being purchased from PerkinElmer company).

Proxiplate^®Microwell plate: (being purchased from PerkinElmer company, article No. 6008280).

The screening of the anti-PD-L1 antibody of experimental example 1

1. the phage antibody library of full people's single-chain antibody: the heavy chain variable region and light chain variable of design primer amplification antibody Heavy chain variable region and light chain variable region are connected by way of Overlap extension PCR with Linker, obtain overall length by area Connection converted product electrotransformation is entered XL1-blue impression with SfiI digestion PCR product and pCGMT phagemid vector by PCR product Then the SOC culture medium of 3mL is added in state cell into competent cell, after 30 DEG C of culture 1h, the final concentration of 50 μ g/mL of addition Ampicillin, the tetracycline 20mL, 37 DEG C of shaken cultivation 2h of 10 μ g/mL.Then it is 10 that concentration, which is added,¹³/ mL's VCSM13 helper phage 50uL is incubated at room temperature 1h, intermediate primary every 10min jog.37 DEG C of shaken cultivation 2h, add end Concentration is that the card of 70 μ g/mL receives mycin, and 30 DEG C are incubated overnight.Supernatant is collected by centrifugation, the PEG-8000/ chlorine that concentration is 10% is added Change sodium solution (PEG, that is, polyethylene glycol), be placed in 1h, 8000rpm, 4 DEG C of ice bath centrifugations, abandon supernatant, is 1% with 2mL concentration BSA(bovine serum albumin(BSA)) PBS solution (phosphate buffer) sufficiently dissolution precipitating, supernatant is collected by centrifugation, as full people is single The phage antibody library of chain antibody.

2. antibody screening

The 200 μ L(of phage antibody library for expressing full people's single-chain antibody is taken to contain bacteriophage 1 × 10¹²A/mL) and 5 μ g PD- L1 antigen mixes, and the Streptavidin MagneSphere of 50 μ L is added after 30 min of incubation at room temperature, the bacteriophage with antigen binding is by strepto- Avidin magnetic bead capture, unbonded bacteriophage is removed after the rinsing of the PBS solution of 0.5% Tween-20, with the sweet ammonia of hydrochloric acid Sour (pH 2.2) solution will be stand-by with the bacteriophage elution of magnetic bead stable bond.

Be inoculated with XL1-Blue bacterium 200mL, after OD(optical density) reach 0.6 after, the bacteriophage of above-mentioned elution is added, with XL1-Blue bacterium is coated on amicillin resistance plate in 37 DEG C of standing 30min, bacterium solution, and it is anti-that ammonia benzyl is collected in elution in second day Thallus on mild-natured plate is infecting 1 × 10¹²After the VCSM13 helper phage of pfu/mL, next round screening is carried out after amplification, altogether Carry out 3 wheel screenings.

The XL1-Blue bacterium solution for infecting bacteriophage is sufficiently diluted, it is that 15cm resistance is that this bacterium solution, which is then coated on diameter, The LB solid medium tablets of ampicillin have 100-500 clone, picking monoclonal antibody, by biting on each plate Thallus enzyme linked immunoassay verifies each wheel phage library after elutriation.

3. bacteriophage enzyme linked immunoassay

The 96 hole Bacteria Culture plates that the XL1-Blue monoclonal bacterium for transfecting bacteriophage is inoculated into 2mL are (public purchased from Corning Department) in, the SB culture medium that 500 μ L contain tetracyclin resistance is added, the lower 37 DEG C of shaking tables 4-6 h of 200 rpm revolving speeds detects OD₆₀₀ The helper phage of 1 μ L is added close to after 0.6 in (light absorption value at 600nm wavelength) value, and shaking table is stayed overnight at 30 DEG C, and second day 3000g centrifuging and taking supernatant is stand-by.

Enzyme linked immunological microwell plate is taken, 4 DEG C of envelope antigen overnight, third day, then the rear enclosed of PBST elution is added upper one Walk the bacteriophage supernatant for preparing, be incubated at room temperature 2h, PBST adds Anti-M13 HRP antibody incubation 30min, after use PBST It washes 3 times, 50 μ L developer solution ABTS is added.

4. experimental result:

As shown in Figure 1, PD-L1 antigen is screened in human antibody phage display library, and after 3 wheel screenings, PD- The signal strength of L1 antigen enzyme linked immunoassay is up to more than 100 times of control antigen, and above-mentioned control antigen is immunoglobulin IgG1 constant region fc section (human IgG1-Fc) shows to express in conjunction with PD-L1 antigentic specificity after 3 wheel screenings The bacteriophage of antibody is constantly enriched with.196 monoclonals of picking carry out enzyme-linked from the phage library obtained after the 3rd wheel screening Immune response verifying, the signal strength for finally determining enzyme linked immunoassay are twice of antigen of control or more of monoclonals as positive Positive monoclonal is sequenced in monoclonal, obtains nucleotide coding sequence, the sequence of acquisition is compared, and repeats Clone is more, illustrates that the affinity of the antibody sequence is stronger, the nucleotide coding sequence being effectively enriched with is determined with this.

Experimental example 2 constructs the pFUSE-IgG1 expression vector of anti-PD-L1 full length antibody

It is template by the nucleotide coding sequence for the bacteriophage screened in experimental example 1, designs PCR primer, expand respectively Increase this constant region for going out heavy chain variable region and human IgG1, by Overlap extension PCR method by the IgG1 of weight chain variabl area sequence and people Area is stitched together, and segment is then accessed pFUSE expression vector by the method for recombination, and (pFUSE expression vector passes through restricted Restriction endonuclease BamH1 and BglII act on restriction enzyme site) in, thus to obtain the pFUSE-IgG1 of full length antibody heavy chain of the invention Expression vector.Equally, PCR primer is designed, expands the light chain variable region and people's Lambda chain of monoclonal antibody of the present invention respectively This two sections are stitched together by Overlap extension PCR method, the segment of splicing are recombined by the constant region of (light chain of IgG1) PFUSE expression vector, thus to obtain the pFUSE-IgG1 expression vector of full length antibody light chain of the invention.

Experimental result:

The pFUSE-IgG1 expression vector of the full length antibody heavy chain of above-mentioned building and full length antibody light chain of the invention PFUSE-IgG1 expression vector, wherein the amino acid sequence of heavy chain variable region is SEQ ID NO:1, the amino acid of light chain variable region Sequence is SEQ ID NO:2.

The preparation (expression and purifying) of the 3 full human monoclonal antibody of anti-PD-L1 of experimental example

According to volume mass ratio it is 30 μ L by the eukaryon antibody expression vector of 293Fectin transfection reagent and above-mentioned acquisition: The ratio of 30 μ g mixes, and the 293Freestyle suspension cell of 30 μ L is added, and 37 DEG C of shaking tables are stayed overnight under 125rpm revolving speed, centrifugation After collect supernatant, resisted on KTApurifier100 protein purification instrument using HiTrap Protein A HP columns Body protein purifying detects antibody concentration referring finally to the specification of BCA method protein quantification kit.

4 enzyme linked immunoassay of experimental example (ELISA) detects the combination of anti-PD-L1 full human monoclonal antibody and human PD-L 1

The 50 μ L diluted human PD-L 1 of CBS antigen fixed solution, Mei Gekong is added in each Kong Zhongjun of the saturating plate of 96 bottom holes Middle 0.05 μ g of addition antigen, 4 DEG C are incubated overnight.PBST is rinsed is added the PBST confining liquid for being 5% containing milk concn three times, 37 1h is closed under the conditions of DEG C.PBST is rinsed three times, the full human monoclonal antibody of anti-PD-L1 that experimental example 3 obtains is added, in 37 DEG C of items It is incubated for 1h under part, HRP(horseradish peroxidase is added after rinsing is dry) the Anti-Human Fc secondary antibody of label, room temperature concussion It is incubated for 30min, is rinsed 3 times with PBS, development substrate A BST solution is added, finally uses microplate reader reading numerical values.

Experimental result:

As shown in Fig. 2, abscissa is the absorbance value that antibody identifies PD-L1 antigen, ordinate is the identification pair of same antibody According to the absorbance value of antigen I gG1-Fc, the OD value of the enzyme linked immunoassay of control group (human IgG1-Fc) 0.2 hereinafter, and The OD value of positive group (the i.e. anti-full human monoclonal antibody of PD-L1) shows PD-L1 antibody identification PD-L1 and fails to see 0.4 or more Other human IgG1-Fc.

Experimental example 5 detects the binding specificity and binding kinetics feature of the full human monoclonal antibody of anti-PD-L1

Using the dynamic (dynamical) method detection antibody of multi-cycle and antigen affinity and dynamic characteristic, fixed use of antibody is caught Method progress is obtained, first the full human monoclonal antibody of anti-PD-L1 is coupled on Protein A chip, then by PD-L1 sample gradient Chip surface is flowed through after dilution, diluted initial concentration is 40nM, and extension rate is 2 times, dilutes 6 concentration points altogether, PD-L1 is just The full human monoclonal antibody of anti-PD-L1 that can be coupled captures, and antigen rear signal in conjunction with antibody is detected and records, Finally the antibody of Protein A chip surface and antigen samples are all eluted with regenerative agent (glycine solution of pH1.5), And carry out new round detection.

Experimental result:

As shown in figure 3, each line represents different PD-L1 antibody concentrations, PD-L1 antibody concentration is higher, in conjunction with more Fastly, signal is stronger, illustrates that PD-L1 antibody is good with PD-L1 affinity, changes in PBS solution in 120 seconds postpositions, by antigen and resists Body dissociation proves the anti-full human monoclonal antibody of PD-L1 and PD-L1 protein-interacting by Biacore detection, of the invention The K of the anti-full human monoclonal antibody of PD-L1_DIt is 5 × 10^-10。

Experimental example 6 detects the full human monoclonal antibody of anti-PD-L1 and generates in mixed lymphocyte reaction (MLP) to cell factor Influence

The influence that blocking PD-L1/PD-1 approach pairing effect lymphocyte is proved using mixed lymphocyte reaction (MLP), is existed Or there is no the full human monoclonal antibody of anti-PD-L1 in the case where detect the secretion of IL-2 in the test.

Using CD4+ positive selection kit from PBMC(peripheral blood mononuclear cells) in Purification of Human CD4+T cell, dendron is thin The source of born of the same parents is to pass through 1000U/mL IL-4(interleukin 4) and 500U/mL GM-CSF is cultivated 7 days and the monokaryon of purifying Cell (monocyte is prepared with monocyte negative selection kit).Every kind of culture contains 10 in 200 μ l total volumes⁵It is a pure The people CD4+T cell of change and 10⁴A dendritic cells.Anti- PD-L1 monoclonal antibody adds to every kind of culture with different antibody concentrations In object.Antibody is not added or uses the human antibody Fc Isotype control antibodies without antigen binding ability as negative control.Carefully Born of the same parents cultivate 5 days at 37 DEG C, and 100 μ L culture mediums are taken out from each culture and carry out cytokine assay.IL-2 content Human IL-2 HTRF kit kit measurement, the anti-full human monoclonal antibody of PD-L1 promote IL-2 to secrete in a manner of concentration dependant.

Experimental result:

As shown in figure 4, with the increase of the full human monoclonal antibody concentration of anti-PD-L1, mixed lymphocyte reaction (MLP) detection To IL-2 secretion also increase, illustrate that the antibody can identify PD-L1 ligand and block it with PD1 ining conjunction with, so promotion T cell Activation secretion IL-2.

Experimental example 7 detects the combination that the full human monoclonal antibody of anti-PD-L1 blocks PD-L1 ligand and PD-1

The anti-full human monoclonal antibody of PD-L1 to be detected is subjected to concentration gradient dilution with 3 times of extension rates, is originated dense Degree point is 5 μ g/ μ L, amounts to 10 concentration points of dilution.Detecting reaction system is respectively 5 μ of measuring samples antibody protein or inhibitor L, biotinylation PD-1 5 μ L of 5 μ L, histidine tag (His-tag) PD-L1, 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 according to saying Bright book is by each reagent diluent preparing, respectively 4X histidine tag PD-L1 (20nM), 4X biotinylation PD-1 (20nM), the anti-6X histidine AlphaLISA of 4X^®Acceptor bead (Anti-6xHis AlphaLISA^®Acceptor beads) The donor bead (Streptavidin Donor beads) (80 μ g/mL) of (40 μ g/mL) and marked by streptavidin mixes Liquid.Above-mentioned four kinds of liquid is admixed together according to 20 μ L reaction systems, and room temperature, which is protected from light, is incubated for 90min, reads on Envision Take the numerical value at 615nm.

Experimental result:

The full human monoclonal antibody of the anti-PD-L1 of the present invention can effectively inhibit PD-1 albumen and its ligand PD-L1 in vitro Combination, and it is this inhibit to be in dose dependent, with the raising of the full human monoclonal antibody concentration of anti-PD-L1, PD-1 with The combination of its ligand D-L1 is gradually suppressed, the inhibition constant IC detected₅₀For 77.57 nmol/L.

In conclusion the present invention is for providing a kind of monoclonal antibody or antibody fragment for PD-L1, the antibody or anti- Body segment be capable of high specific in conjunction with human PD-L 1, blocked PD-L1 in conjunction with PD-1 receptor, stimulated the energy of antibody response Power has reversed the holddown of regulatory T-cell, enhances immune response.

It is discussed in detail although the contents of the present invention have passed through above preferred embodiment, but it should be appreciated that above-mentioned Description is not considered as limitation of the present invention.After those skilled in the art have read above content, for of the invention A variety of modifications and substitutions all will be apparent.Therefore, protection scope of the present invention should be limited to the appended claims.

<110>Loews medicine company Science and Technology Ltd. is taken in Changzhou

<120>a kind of monoclonal antibody or antibody fragment for PD-L1

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 119

<212> PRT

<213>artificial sequence

<400> 1

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Ala Tyr Tyr Tyr Pro Tyr Gly Met Asp Val Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 2

<211> 111

<212> PRT

<213>artificial sequence

<400> 2

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

1 5 10 15

Thr Val Thr Ile Pro Cys Thr Gly Thr Gly Ser Asn Val Gly Ala Gly

20 25 30

Phe Asp Leu His Trp Tyr Gln Gln Phe Pro Gly Ser Ala Pro Lys Leu

35 40 45

Leu Leu Ser Gly Asn His Asn Arg Pro Ser Gly Val Pro Glu Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Gly Ser Leu Val Ile Ser Gly Leu

65 70 75 80

Gln Thr Glu Asp Glu Ala Ile Tyr Tyr Cys Gln Ser Tyr Asp Thr Arg

85 90 95

Leu Ser Gly Pro Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu

100 105 110

<210> 3

<211> 8

<212> PRT

<213>artificial sequence

<400> 3

Gly Gly Thr Phe Ser Ser Tyr Ala

1 5

<210> 4

<211> 8

<212> PRT

<213>artificial sequence

<400> 4

Ile Ile Pro Ile Leu Gly Ile Ala

1 5

<210> 5

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<212> PRT

<213>artificial sequence

<400> 5

Ala Arg Ala Tyr Tyr Tyr Pro Tyr Gly Met Asp Val

1 5 10

<210> 6

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<212> PRT

<213>artificial sequence

<400> 6

Gly Ser Asn Val Gly Ala Gly Phe Asp

1 5

<210> 7

<211> 3

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<213>artificial sequence

<400> 7

Ser Gly Asn His

1

<210> 8

<211> 11

<212> PRT

<213>artificial sequence

<400> 8

Gln Ser Tyr Asp Thr Arg Leu Ser Gly Pro Val

1 5 10

<210> 9

<211> 357

<212> DNA

<213>artificial sequence

<400> 9

caggtacagc tggttcaatc gggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60

tcctgcaagg cttctggagg caccttcagc agctatgcta tcagctgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggaagg atcatcccta tccttggtat agcaaactac 180

gcacagaagt tccagggcag agtcacgatt accgcggaca aatccacgag cacagcctac 240

atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagggcgtat 300

tactacccct atggtatgga cgtctggggc caaggaaccc tggtcaccgt ctcctca 357

<210> 10

<211> 333

<212> DNA

<213>artificial sequence

<400> 10

caggcagtgt tgacgcagcc gccctcagtg tctggggtcc cagggcagac ggtcaccatc 60

ccctgcactg ggaccggttc caacgtcggg gctggttttg acctccattg gtaccagcag 120

tttccaggga gtgcccccaa actcctcctc tctggaaacc acaatcgccc ctcaggggtc 180

cctgaacgat tctctggctc caagtctggc acttcaggtt ccctggtcat ctctggcctc 240

cagactgaag atgaggctat ttattactgc cagtcctatg atactcgcct cagtggtccg 300

gtcttcggcg gagggaccaa ggtcaccgtc cta 333

Claims

1. a kind of monoclonal antibody or antibody fragment for PD-L1, which is characterized in that the antibody or antibody fragment include: weight Chain and light chain, the antibody fragment are segment of the antigen in conjunction with the antibody；

The heavy chain and light chain includes variable region, and the variable region includes complementary determining region；

Complementary determining region CDR1, CDR2 and CDR3 of the heavy chain are indicated with HCDR1, HCDR2 and HCDR3 respectively；

Complementary determining region CDR1, CDR2 and CDR3 of the light chain are indicated with LCDR1, LCDR2 and LCDR3 respectively；

The amino acid sequence of the HCDR1 is SEQ ID NO:3；

The amino acid sequence of the HCDR2 is SEQ ID NO:4；

The amino acid sequence of the HCDR3 is SEQ ID NO:5；

The amino acid sequence of the LCDR1 is SEQ ID NO:6；

The amino acid sequence of the LCDR2 is SEQ ID NO:7；

The amino acid sequence of the LCDR3 is SEQ ID NO:8.

2. the monoclonal antibody or antibody fragment according to claim 1 for PD-L1, which is characterized in that the weight Chain variable region amino acid sequence is SEQ ID NO:1.

3. the monoclonal antibody or antibody fragment according to claim 2 for PD-L1, which is characterized in that described is light Chain variable region amino acid sequence is SEQ ID NO:2.

4. the monoclonal antibody or antibody fragment according to claim 3 for PD-L1, which is characterized in that described is anti- Body or antibody fragment are with 2 × 10^-9M or lower K_DIn conjunction with human PD-L 1, and it is not significant in conjunction with people PD-L2 or do not combine.

5. the monoclonal antibody or antibody fragment according to claim 4 for PD-L1, which is characterized in that described is anti- Body or antibody fragment are as follows: chimeric antibody, humanized antibody, human antibody or antibody fragment selected from the following: Fab, F (ab’)₂, Fv, dAb and scFv.

6. the monoclonal antibody or antibody fragment according to claim 5 for PD-L1, which is characterized in that described is complete Human antibody is any one in IgG1, IgG2 or IgG4 full length antibody.

7. being directed to the monoclonal antibody or antibody fragment of PD-L1 described in any one of -6 according to claim 1, feature exists In the heavy chain variable region of the antibody or antibody fragment is by the nucleotide sequence coded of SEQ ID NO:9；The antibody or The light chain variable region of antibody fragment is nucleotide sequence coded by SEQ ID NO:10's.

8. a kind of expression vector containing nucleotide sequence as claimed in claim 7.

9. a kind of host cell containing expression vector as claimed in claim 8.

10. a kind of full human monoclonal antibody or antibody piece for being directed to PD-L1 described in any one of -6 according to claim 1 Section is preparing the purposes in immune conjugate or combinations thereof object, which is characterized in that the immune conjugate is by therapeutic agent and institute The antibody or antigen stated segment covalent bond in connection；The composition can be used by the immune conjugate and pharmaceutically Carrier combination.

11. according to claim 10 preparing the purposes in immune conjugate or combinations thereof object, which is characterized in that described Therapeutic agent include: cytotoxic agent, radioactive isotope or immunosuppressor.

12. a kind of full human monoclonal antibody or antibody piece for being directed to PD-L1 described in any one of -6 according to claim 1 Section is preparing the purposes in anti-tumor drug or combinations thereof object；

The tumour includes: osteocarcinoma, lung cancer, cutaneum carcinoma, head and neck cancer, breast cancer, uterine cancer, oophoroma, carcinoma of fallopian tube, uterus Endometrial carcinomas, cervical carcinoma, carcinoma of vagina, prostate cancer, carcinoma of testis, carcinoma of penis, bladder cancer, carcinoma of ureter, kidney, carcinoma of renal pelvis, colon Cancer, the carcinoma of the rectum, gastric cancer, cancer of the esophagus, cancer of pancreas, carcinoma of small intestine, thyroid cancer, parathyroid carcinoma, adrenal, soft tissue sarcoma, Malignant mela noma, in central nerve neuroma any one or it is two or more；

The cutaneum carcinoma is squamous cell carcinoma；

The central nerve neuroma is glioma or pituitary adenoma.