CN106243225A - Novel anti-PD-L1 antibody - Google Patents

Abstract

This application provides the monoclonal antibody for albumen programmed death ligand 1 (PD L1), it can block the combination of PD L1 and Programmed death 1 (PD 1), therefore blocks the inhibitory action of the PD L1 T cell to expressing PD 1.The antibody of the present invention provides the very effective reagent by regulation human immunity function treatment kinds cancer.

Description

Novel anti-PD-L1 antibody

Invention field

The present invention relates to new anti-PD-L1 antibody.

Background technology

Increasing clinical front and clinical effectiveness evidence shows, targeting immunologic test point is becoming most promising controlling The method treating cancer patient.Apoptosis molecule 1 is one of immunologic test point albumen, and it is lived in restricted T cells Playing a major role in property, described T cell provides main immune resistance mechanisms, by this restriction effect tumor cell Immune surveillance can be escaped.The phase of the PD-1 expressed in the T cell of activation and the PD-L1 expressed on tumor cell Immunne response is played negative regulator and weakens antineoplastic immune power by interaction.PD-L1 expression in tumor and the esophageal carcinoma, Cancer of pancreas declines relevant with the survival rate of other type of cancer, and highlighting this path can be as new promising tumor Immunotherapeutic targets.Drugmaker has been developed for the multiple medicine for PD-1 path, such as Bristol-Myers Squibb Co. (BMS), Merck & Co., Inc., Roche Holding Ag and GlaxoSmithKline PLC (GSK) company.The data of clinical trial show The early stage evidence of clinical activity lasting in the patient of various tumor types and good safety.Nivolumab is BMS The PD-1 medicine of exploitation, it is just being put to the center stage in field of future generation.At present in 6 later stage research, In in 5 cancer groups of research 3, treatment has promoted reducing of tumor, including in 72 example patients with lung cancer 18%, in 98 example melanoma patients close to 1/3rd and 33 in example patients with renal cell carcinoma 27%.Ground by Merck & Co., Inc. The lambrolizumab of system is Humanized monoclonal IgG4 antibody, and it acts on PD-1, and it is obtaining for skin carcinoma Impressive IB data after caught the new breakthrough index of FDA.The result of interim IB research shows The objective antitumor having 51% in 85 example cancer patients reacts, and occurs reacting completely in the patient of 9%.Sieve The experimental MPDL3280A of family name company demonstrates in the patient with advanced cancer of its tumor carrying all size in 140 examples Reduce the tumor of 29 examples (21%) patient.

But, existing Therapeutic Method is not all fully up to expectations, therefore need nonetheless remain for the antibody of new anti-PD-L1.

Invention summary

This application provides new anti-PD-L1 monoclonal antibody, encode its polynucleotide and the method using it.

In one aspect, this application provides monoclonal antibody or its Fab of a kind of separation, it can be with not More than 10^-9M (such as ,≤9x10^-10M、≤8x10^-10M、≤7x10^-10M、≤6x10^-10M、≤5x10^-10 M、≤4x10^-10M、≤3x10^-10M、≤2x10^-10M、or≤10^-10M) Kd value specifically with people PD-L1 combines, and described Kd value is measured by plasma resonance combined techniques.

In some embodiments, described antibody or its Fab with less than 10nM (as less than 1nM, 0.9nM、0.8nM、0.7nM、0.6nM、0.5nM、0.4nM、0.3nM、0.2nM、0.1nM、0.09nM、0.08nM、 0.07nM, 0.06nM, 0.05nM, 0.04nM, 0.03nM, 0.02nM or 0.01nM) EC₅₀With monkey PD-L1 In conjunction with.In some embodiments, described antibody or its Fab are not combined with mice PD-L1, but Be combined with monkey PD-L1 with the binding affinity that human PD-L 1 is similar.In some embodiments, described antibody or its Fab with less than 100nM (as less than 50nM, 40nM, 30nM, 20nM, 10nM, 9nM、8nM、7nM、6nM、5nM、4nM、3nM、2nM、1nM、0.9nM、0.8nM、0.7nM、 0.6nM、0.5nM、0.4nM、0.3nM、0.2nM、0.1nM、0.09nM、0.08nM、0.07nM、0.06nM、 0.05nM, 0.04nM, 0.03nM, 0.02nM or 0.01nM) IC₅₀Suppression people or monkey PD-L1 are subject to it efficiently The combination of body (such as PD-1).In some embodiments, described EC₅₀Or IC₅₀By the cell sorting of fluorescence-activation (FACS) analysis is measured.

In some embodiments, described antibody or its Fab have the effector function substantially reduced.? In some embodiment, described antibody or its Fab do not mediate ADCC or CDC or both of which does not mediates.

In some embodiments, antibody described herein or its Fab include heavy CDR sequences, institute State sequence to be selected from: SEQ ID NO:1,2,3.

In some embodiments, antibody described herein or its Fab include CDR sequence, institute State sequence to be selected from: SEQ ID NO:4,5,6.

In some embodiments, antibody described herein or its Fab include at least 1,2,3 Individual, 4,5 or 6 CDR selected from SEQ ID NO:1,2,3,4,5 and 6.

In some embodiments, antibody described herein or its Fab include such as SEQ ID NO:1, SEQ ID The variable region of heavy chain of NO:2 and/or SEQ ID NO:3.

In some embodiments, antibody described herein or its Fab include such as SEQ ID NO:4, SEQ ID NO:5 and/or the variable region of light chain of SEQ ID NO:6.

In some embodiments, antibody described herein or its Fab include such as SEQ ID NO:1, SEQ ID NO:2 and/or the variable region of heavy chain of SEQ ID NO:3；With such as SEQ ID NO:4, SEQ ID NO:5 and/or The variable region of light chain of SEQ ID NO:6.

In some embodiments, antibody described herein or its Fab include variable region of heavy chain, Qi Zhongsuo State variable region of heavy chain selected from SEQ ID NO:7, SEQ ID NO:11 and SEQ ID NO:18.

In some embodiments, antibody described herein or its Fab include variable region of light chain, Qi Zhongsuo State variable region of light chain selected from SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:16 and SEQ ID NO:22。

In some embodiments, antibody described herein or its Fab include:

A) variable region of heavy chain, it includes SEQ ID NO:7；And variable region of light chain, it includes SEQ ID NO:9；

B) variable region of heavy chain, it includes SEQ ID NO:11；And variable region of light chain, it includes SEQ ID NO:13；

C) variable region of heavy chain, it includes SEQ ID NO:11；And variable region of light chain, it includes SEQ ID NO:16；

D) variable region of heavy chain, it includes SEQ ID NO:18；And variable region of light chain, it includes SEQ ID NO:13；

And

E) variable region of heavy chain, it includes SEQ ID NO:18；And variable region of light chain, it includes SEQ ID NO:22.

In some embodiments, antibody described herein or its Fab include such as 2.74.15, 2.74.15.hAb4,2.74.15.hAb5,2.74.15.hAb6,2.74.15.hAb7 and 2.74.15.hAb8.

In some embodiments, antibody described herein or its Fab and antibody 2.74.15, 2.74.15.hAb4,2.74.15.hAb5,2.74.15.hAb6,2.74.15.hAb7 or 2.74.15.hAb8 The epi-position that competing phase is same.

In some embodiments, antibody described herein or its Fab can block human PD-L 1 and its Receptor combines, and therefore provides at least one in following activity:

A) at CD4⁺T cell is induced the generation of IL-2；

B) at CD4⁺T cell is induced the generation of IFN γ；

C) induction CD4⁺The propagation of T cell；And

D) reverse T reg and suppress function.

In some embodiments, herein described antibody is monoclonal antibody, humanized antibody, chimeric antibody, weight Group antibody, bispecific antibody, traget antibody, bivalent antibody or anti-idiotype antibody.

In some embodiments, Fab provided herein is camelised single domain antibody (camelized Single chain domain antibody), bifunctional antibody (diabody), scFv, scFv dimer, BsFv, dsFv, (dsFv) 2, dsFv-dsFv', Fv fragment, Fab, Fab', F (ab') 2, ds bifunctional antibody (ds diabody), receive Meter Kang Ti, domain antibodies or bivalent domain antibodies.

In some embodiments, antibody described herein or its Fab farther include immunoglobulin perseverance Determine district.

In some embodiments, antibody described herein or its Fab farther include conjugate.

In some embodiments, described conjugate can be detectable label, pharmacokinetics modification part or purification portion Point.

On the other hand, this application provides the polynucleotide of separation, it encodes antibody as described in the present application or its antigen Binding fragment.In some embodiments, the application provides polynucleotide encoding antibody as described in the present application or its resist The aminoacid sequence of former binding fragment.In some embodiments, this application provides and include the carrier of these polynucleotide. In some embodiments, this application provides and express one or more antibody described herein or Fab Method, it is thin by cultivating host under conditions of expressing the antibody by polynucleotide encoding or Fab in the carrier Born of the same parents realize.In some embodiments, the application provide polynucleotide in the carrier with promoter such as SV40 promoter It is operably connected.In some embodiments, the host cell of the carrier provided including the application is Chinese hamster ovary Cell, or 293 cells.

On the other hand, this application provides and include the test kit of antibody described herein or its Fab.

On the other hand, this application provides in biological sample detect PD-L1 (such as people or monkey) existence situation or The method of level, contacts with antibody described herein or its Fab including by described biological sample, and in institute State existence situation or the level determining people or monkey PD-L1 in sample.

On the other hand, this application provides discriminating and suffer from disease or the individuality of situation that PD-L1 antagonist is responded by possibility Method, comprising: with antibody described herein or its Fab at the to be measured biological sample from described individuality Product determine existence situation or the level of PD-L1 (such as people or monkey), wherein PD-L1 in described biological sample Exist or level raises the probability representing response.In some embodiments, described method farther includes described Body uses antibody described herein or its Fab of effective dose, and described individuality is identified suffers from possibility pair The disease of PD-L1 antagonist response or situation.

The application further provides and monitors therapeutic response or progression of disease in the experimenter with PD-L1 antagonist for treating Method, it includes with antibody described herein or its Fab in the biological sample to be measured of described individuality Determine existence situation or the level of PD-L1 (such as people or monkey).

On the other hand, this application provides pharmaceutical composition, including antibody described herein or its Fab And one or more pharmaceutically acceptable carriers.In some embodiments, described pharmaceutical carriers can be the dilutest Release agent, antioxidant, adjuvant, excipient or nontoxic auxiliary substance.

On the other hand, the method that this application provides the situation of the experimenter that treatment meeting benefits from the immune response raised, Including described experimenter is used effective dose antibody described herein or its Fab.At some embodiment In, described experimenter has the PD-L1 of rise and expresses.

On the other hand, it is provided that antibody described herein or its Fab are used for treating meeting from rise in preparation Immune response in benefit situation medicine in purposes.In some embodiments, described situation is cancer or chronic Virus infects.

Accompanying drawing is sketched

Fig. 1 shows facs analysis the Muridae Anti-Human's PD-L1 antibody measured and the Chinese hamster ovary celI expressing PD-L1 Combination.EC₅₀Represent with nM.

Fig. 2 shows humanized PD-L1 antibody that facs analysis measures and the Chinese hamster ovary celI expressing PD-L1 In conjunction with.

Fig. 3 shows the dendron of the facs analysis humanized PD-L1 antibody measured and the activation expressing PD-L1 The combination of cell (DC) cell.EC₅₀Represent with nM.

Fig. 4 shows the humanized PD-L1 antibody blocking that facs analysis measures PD-1 and has transfected PD-L1 The combination of Chinese hamster ovary celI.EC₅₀Represent with nM.

Fig. 5 shows that the humanized PD-L1 antibody that facs analysis measures is specific binding with PD-L1, and not with PD-L2 combines.

Fig. 6 shows that PD-L1 antibody is combined with machin PD-L1.EC₅₀Represent with nM.

Fig. 7 shows the Holonomic Dynamics of PD-L1 antibody and human PD-L 1 binding affinity.

Fig. 8 shows the humanization anti-PD-L1 antibody shadow to the production of IL-2 in the lymphocyte reaction (MLR) of mixing Ring.

Fig. 9 shows the humanization anti-PD-L1 antibody impact on the production of IFN γ in MLR.

Figure 10 shows that humanization anti-PD-L1 antibody promotes T cell propagation in MLR.

Figure 11 shows that humanization PD-L1 antibody adds the generation of IFN γ in specific T-cells responds.Described spy Specific T cell response is to be total to by the DC of cytomegalovirus (CMV) specific T-cells with load C MV-pp65 peptide Cultivate generation in 5 days.

Figure 12 shows that humanization PD-L1 antibody promotes T cell propagation in specific T-cells responds.

Figure 13 shows that anti-PD-L1 antibody has reversed Treg suppression function.

Figure 14 shows that anti-PD-L1 antibody lacks ADCC in mDC.

Figure 15 shows that anti-PD-L1 antibody lacks CDC in the T cell of activation.

Detailed Description Of The Invention

The following description of the application is only for the numerous embodiments of explanation the application.Therefore, concrete modification side discussed herein Formula should not be construed as the restriction to application range.Those skilled in the art is in the case of without departing from the application scope Draw multiple equivalent way easily, change and modifications, it should be understood that such equivalent embodiments is included in model of the present invention In enclosing.The all documents quoted in this application, all pass through the side quoted including public publication, patents and patent applications Formula is incorporated by.

Definition

" antibody " word in the present invention includes arbitrarily can be in conjunction with the immunoglobulin of certain specific antigen, monoclonal antibody, many Clonal antibody, multi-specificity antibody or bispecific (bivalent) antibody.One natural complete antibody comprises two heavy chains With two light chains.Every heavy chain is made up of a variable region and first, second, third constant region；Every light chain is variable by one District and constant region composition.The heavy chain of mammal can be divided into α, δ, ε, γ and μ, and the light chain of mammal can be divided into λ Or κ.Antibody is Y-shaped, the cervical region of Y-shaped structure by two articles of heavy chains second and the 3rd constant region form, its pass through two Sulfide linkage combines.Every arm of Y-shaped structure includes variable region and first constant region of a wherein heavy chain, and it is light with one The variable region of chain and constant region combine.The variable region of light chain and heavy chain determines the combination of antigen.The variable region of every chain all contains Have three hypervariable regions, claim complementary determining region (CDR) (CDR of light chain (L) comprises LCDR1, LCDR2, LCDR3, The CDR of heavy chain (H) comprises HCDR1, HCDR2, HCDR3).Antibody disclosed in the present invention and antigen binding fragment The CDR border of section can be named by Kabat, Chothia or Al-Lazikani nomenclature or identify.(Al-Lazikani, B.,Chothia,C.,Lesk,A.M.,J.Mol.Biol.,273(4),927(1997)；Chothia, C. etc., J Mol Biol. Dec 5；186(3):651-63(1985)；Chothia,C.and Lesk,A.M.,J.Mol.Biol.,196,901(1987)； Chothia, C. etc., Nature.Dec 21-28；342(6252):877-83(1989)；Kabat E.A. etc., National Institutes of Health,Bethesda,Md.(1991)).Wherein, three CDR are by being referred to as framework region (FR) Side continuous part is spaced apart, and framework region is than CDR more high conservative and forms a support support hypermutation ring.Heavy chain Combine with antigen unrelated with the constant region of light chain, but there is multiple effector function.Antibody is according to the aminoacid of CH Sequence is segmented into a few class.According to whether containing α, δ, ε, γ and μ heavy chain, antibody can be respectively divided into five main Classification or isomer: IgA, IgD, IgE, IgG and IgM.Several main antibody classifications also can be divided into subclass, as IgG1 (γ 1 heavy chain), IgG2 (γ 2 heavy chain), IgG3 (γ 3 heavy chain), IgG4 (γ 4 heavy chain), IgA1 (α 1 Heavy chain) or IgA2 (α 2 heavy chain) etc..

" Fab " word in the application, refers to by the antibody moiety containing one or more CDR or any Other conjugated antigens but not there is a kind of antibody fragment that the antibody fragment of complete antibody structure is formed.Fab Example include, but not limited to such as bifunctional antibody (diabody), Fab, Fab', F (ab')₂, Fv fragment, two The stable Fv fragment (dsFv) of sulfide linkage, (dsFv)₂, bispecific dsFv (dsFv-dsFv'), disulfide bond stable Bifunctional antibody (ds diabody), single-chain antibody molecules (scFv), scFv dimer (bivalent difunctional anti- Body), Bivalent single-chain antibody (BsFv), multi-specificity antibody, camelised single domain antibody (camelized single domain Antibody), nano antibody, domain antibodies and bivalent domain antibodies.Fab can be combined identical with maternal antibody Antigen.In some embodiments, Fab can one or more containing from certain particular person antibody CDR, transposing is to the framework region from one or more different people antibody.

" Fab " fragment of antibody refer to the variable region by a light chain (including variable region and constant region) and a heavy chain and The part antibody molecule got up through disulfide-bonded in constant region.

" Fab' " fragment refers to contain the Fab fragment in part hinge district.

“F(ab')₂" refer to the dimer of Fab.

" Fc " of antibody refers to the part antibody being made up of second, third constant region of heavy chain through disulfide-bonded.Anti- The Fc section of body is responsible for multiple different effector functions such as ADCC and CDC, but is not involved in the combination of antigen.

" Fv " section of antibody refers to the minimum antibody fragment containing complete antigen binding site.Fv fragment is by a light chain Variable region and a heavy chain variable region composition.

" single-chain Fv antibody " or " scFv " refers to be joined directly together by variable region of light chain and variable region of heavy chain or by a peptide chain The engineered antibody (Huston JS etc., Proc Natl Acad Sci USA, 85:5879 (1988)) being formed by connecting.

" single-chain antibody Fv-Fc " or " scFv-Fc " refer to the engineered antibody being made up of the scFv being connected to certain antibody Fc section.

" camelised single domain antibody (Camelized single domain antibody) ", " heavy chain antibody " or " HCAb (Heavy-chain-only antibodies, HCAb) " all referring to containing two V_HTerritory and do not contain the antibody of light chain (Riechmann L. and Muyldermans S., J Immunol Methods.Dec 10；231(1-2):25-38(1999)； Muyldermans S.,J Biotechnol.Jun；74(4):277-302(2001)；WO94/04678；WO94/25591；U.S. Patent No.6,005,079).Heavy chain antibody initially derives from camelidae (camel, one-humped camel and yamma) and obtains.Although Disappearance light chain, camelised antibodies (camelized antibodies) has the antigen of confirmation to combine repertoire (Hamers-Casterman C. etc., Nature.Jun 3；363(6428):446-8(1993)；Nguyen VK. etc., “Heavy-chain antibodies in Camelidae；a case of evolutionary innovation,”Immunogenetics. Apr；54(1):39-47(2002)；Nguyen VK. etc., Immunology.May；109(1):93-101(2003)).Heavy chain The variable region (VHH territory) of antibody is the antigen-binding units (Koch-Nolte that minimum known acquired immunity produces F. etc., FASEB J.Nov；21(13):3490-8.Epub 2007Jun 15(2007)).

" nano antibody " refers to a kind of antibody fragment, and it is by a VHH territory from heavy chain antibody and two constant regions CH2 and CH3 forms.

" bifunctional antibody (diabody) " includes the little antibody fragment with two antigen binding sites, and wherein this fragment contains There is the V being connected on same polypeptide chain_HTerritory and V_LTerritory (V_H-V_LOr V_H-V_L) (refer to, Holliger P. etc., Proc Natl Acad Sci U S A.Jul 15；90(14):6444-8(1993)；EP404097；WO93/11161).Two Between territory, linker is the shortest, makes on same chain two territories not match mutually, thus forces two territories and another chain Complementary domain pairing, form two antibody combining sites.The two antibody combining site can combine identical or different by targeting Antigen (or epitope).

" domain antibodies " refers to contain only a variable region of heavy chain or the antibody fragment of a variable region of light chain.In some situation Under, two or more V_HTerritory is by a peptide linker covalent bond and forms bivalent domain antibodies.The two of bivalent domain antibodies Individual V_HTerritory can targeting in identical or different antigen.

In some embodiments, " (dsFv)₂" containing three peptide chains: two V_HBy a peptide linker between group It is connected, and by disulfide bond and two V_LGroup combines.

In some embodiments, " bispecific ds bifunctional antibody " contains V_L1-V_H2(by a peptide linker phase Connect) and V_H1-V_L2(being also to be connected by a peptide linker), both are at V_H1And V_L1Between pass through disulfide-bonded.

" bispecific dsFv " or " dsFv-dsFv " contains three polypeptide chain: V_H1-V_H2Group, wherein both heavy chains lead to Cross peptide linker (such as: long elastic linker) to be connected, and by disulfide bond respectively with V_L1And V_L2Group combines, Every pair of heavy chain light chain matched by disulfide bond has different antigenic specificities.

In some embodiments, " scFv dimer " is bivalent bifunctional antibody or Bivalent single-chain antibody (BsFv), contains There are two V of dimerization_H-V_L(being connected by peptide linker) group, the V of one of them group_HWith another group V_LCooperation formed two basic change site, the two binding site can targeting combine same antigen (or epitope) or Not synantigen (or epitope).In other embodiments, " scFv dimer " is Bispecific diabodies, Containing interconnective V_L1-V_H2(being connected by peptide linker) and V_H1-V_L2(being connected by peptide linker), its Middle V_H1And V_L1Cooperation, V_H2And V_L2Cooperate, and the pairing of each cooperation has different antigenic specificities.

Term " humanization " used herein, when for antibody or Fab, refers to derive from non- The CDR of people animal, derive from Ren FR district, and derive from antibody or the antigen of the constant region (as applicable) of people Binding fragment.Owing to humanized antibody or Fab have the immunogenicity of reduction, it is at some embodiment In can be used as the therapeutic agent of people.In some embodiments, described non-human animal be mammal such as mice, rat, Rabbit, goat, sheep, Cavia porcellus or hamster.In some embodiments, described humanized antibody or Fab remove Beyond CDR sequence inhuman source, substantially all it is made up of people's source sequence.In some embodiments, described Derive from Ren FR district can include with its from the identical aminoacid sequence of human antibody, or it can include Amino acid change, such as, less than 10,9,8,7,6,5,4,3,2 or 1 amino acid changes.At some In embodiment, this amino acid change can be only present in heavy chain FR district, exists only in light chain FR district or exist simultaneously In two chains.In some preferred implementations, described humanized antibody includes people source FR1-3 and people source JH and J κ.

Term used herein " is fitted together to " part referring to have heavy chain and/or the light chain deriving from a kind of species, With antibody or the Fab that described heavy chain and/or light chain remainder derive from different plant species.Exemplary at one Example in, chimeric antibody can include deriving from the constant region of people and derive from the variable region of non-human animal such as mice.

" PD-L1 " used herein refers to that (PD-L1 see for example Freeman et to programmed cell death ligand 1 al.(2000)J.Exp.Med.192:1027).The aminoacid sequence of representational people source PD-L1 is NCBI registration number: NP_054862.1, and the nucleotide sequence of representational people source PD-L1 is NCBI registration number: NM_014143.3.PD-L1 Being expressed in Placenta Hominis, spleen, lymph node, thymus, heart, fetus liver is, and also discovery is present in many tumors or cancer is thin On born of the same parents.Receptor PD-1 or B7-1 that PD-L1 expresses on T cell, B cell and the medullary cell in activation is combined. The combination of PD-L1 and its receptor priming signal transduction can suppress the activation to cytokine production and the T that TCR mediates Cell proliferation.Therefore, PD-L1 is in particular event, such as right in pregnancy, autoimmune disease, tissue transplantation Suppression immune system plays Main Function, and it is considered to allow tumor or cancerous cell evades immunologic test point and escape is exempted from Epidemic disease response.

" anti-PD-L1 antibody " used herein refers to be enough to provide the affine of diagnosis and/or therapeutic use Property with the specific binding antibody of PD-L1 (such as people or monkey PD-L1).

" specific binding " or " specific combination " in the application refers to, refers to that two intermolecular nonrandom combinations are anti- Should, such as the reaction between antibody and antigen.In some embodiments, the antibody of the application or its Fab and people PD-L1 is specific binding, and its binding affinity (K_D)≤10^-6M is (such as :≤5x10^-7M ,≤2x10^-7M ,≤10^-7 M ,≤5x10^-8M ,≤2x10^-8M ,≤10^-8M ,≤5x10^-9M ,≤2x10^-9M ,≤10^-9M ,≤10^-10M).This KD in application refers to the ratio (koff/kon) dissociating speed with combining speed, can be by surface plasma resonance Method measures, such as, use such as the instrument of Biacore.

The ability of " block and combine " or " competitive same epi-position " in the application refers to antibody or its Fab The interaction of two intermolecular combinations (such as human PD-L 1 and anti-PD-L1 antibody) is suppressed to any detectable The ability of degree.In some embodiments, the antibody or the Fab that block two intermolecular combinations can be by two The interaction suppression at least 50% of intermolecular combination.In some embodiments, such inhibitory action can be more than 60%, more than 70%, more than 80%, or more than 90%.

" epi-position " used herein refers to part aminoacid or the atomic radical in antigen molecule with antibodies. If two kinds of antibody exhibits go out the competitive binding to antigen, the then identical epi-position on possible conjugated antigen.Such as, if The antibody of the application offer or its Fab blocking-up Example antibody, such as 2.74.15.hAb4,2.74.15.hAb5, 2.74.15.hAb6, the combination of 2.74.15.hAb7 and 2.74.15.hAb8 and human PD-L 1, then described antibody or its resist Former binding fragment may be considered that the epi-position identical with the antibodies of those examples.

Herein described " 2.74.15 " refers to murine monoclonal antibodies, and it includes respectively by SEQ ID NO:1,2 and 3 heavy chain CDR of 3 compositions, and respectively by 4,5 and 63 the light chain CDR formed.

" 2.74.15.hAb4 " used herein refer to have the variable region of heavy chain as shown in SEQ ID NO:7, as Variable region of light chain shown in SEQ ID NO:9 and the humanized antibody of humanized IgG 4 isotype constant region.

" 2.74.15.hAb5 " used herein refer to have the variable region of heavy chain as shown in SEQ ID NO:11, as Variable region of light chain shown in SEQ ID NO:13 and the humanized antibody of humanized IgG 4 isotype constant region.

" 2.74.15.hAb6 " used herein refer to have the variable region of heavy chain as shown in SEQ ID NO:11, as Variable region of light chain shown in SEQ ID NO:16 and the humanized antibody of humanized IgG 4 isotype constant region.

" 2.74.15.hAb7 " used herein refer to have the variable region of heavy chain as shown in SEQ ID NO:18, as Variable region of light chain shown in SEQ ID NO:13 and the humanized antibody of humanized IgG 4 isotype constant region.

" 2.74.15.hAb8 " used herein refer to have the variable region of heavy chain as shown in SEQ ID NO:18, as Variable region of light chain shown in SEQ ID NO:22 and the humanized antibody of humanized IgG 4 isotype constant region.

In this application when " conservative replacement " is for aminoacid sequence, refer to had with another by an amino acid residue The amino acid residue having the side chain of similar physicochemical properties substitutes.For example, it is possible between hydrophobic side chain amino acid residue (such as Met, Ala, Val, Leu and Ile), (such as Cys, Ser, Thr, Asn and Gln) between neutral hydrophilic side chains residue, (such as His, Lys and Arg) or side, direction between (such as Asp, Glu), basic side chain aminoacid between acid side-chain residue Between chain residue, (such as Trp, Tyr and Phe) carries out conservative replacement.Conservative replacement known in the art generally will not cause albumen The notable change of conformational structure, therefore, it is possible to the biological activity of retaining protein.

When " Percent sequence identity " is for aminoacid sequence (or nucleotide sequence), refer to carrying out sequence alignment, And after introducing interval makes same amino acid (or nucleic acid) number reach at most if desired, in candidate sequence, with reference Aminoacid (or nucleic acid) residue that sequence is identical accounts for the percentage ratio of aminoacid (or nucleic acid) residue of described candidate sequence. The conservative replacement of described amino acid residue is it is believed that maybe can be not considered as identical residue.Can be by disclosed in this area Instrument, such as BLASTN, BLASTp (American National Biotechnology Information center website (NCBI), it is possible to see, Altschul S.F. etc., J.Mol.Biol., 215:403 410 (1990)；Stephen F. etc., Nucleic Acids Res., 25:3389 3402 (1997)), (European Bioinformatics institute website, can be found in ClustalW2, Higgins D.G. Deng, Methods in Enzymology, 266:383-402 (1996)；Larkin M.A. etc., Bioinformatics (Oxford, England), 23 (21): 2947-8 (2007)) and ALIGN or Megalign (DNASTAR) software, sequence is entered Row comparison is to determine the Percent sequence identity of aminoacid (or nucleic acid) sequence.Those skilled in the art can use institute State the default parameters of instrument or suitably adjust parameter, such as by selecting suitable algorithm according to the needs of comparison.

" T cell " used herein includes CD4⁺T cell, CD8⁺T cell, T assist 1 type T cell, T assists 2 type T cell, T to assist 17 type T cell and suppressor T lymphocytes.

" effector function " used herein refers to that the Fc district of antibody is subject to its effector such as C1 complex and Fc The biological activity that body combines.Exemplary effector function includes antibody and the C1q interaction induction on C1 complex The antibody-dependant of the Fc receptor zygotic induction on CDC (CDC), the Fc district of antibody and effector lymphocyte The cytotoxicity (ADCC) of sexual cell mediation and phagocytosis.

" cancer " or " cancer state " in the application refer to any by tumor or Malignant cellular growth, breed or shift institute Mediation, and cause solid tumor and the most leukemic medical condition of non-physical tumor." tumor " in the present invention refer to tumor and / or the solid substance of malignant cell.

" treatment " or " therapy " of certain state is included prevention or alleviates certain state, reduce certain state rise or The speed of development, reduces the risk developing certain state, prevents or postpone the symptom development relevant to certain state, subtract Less or terminate the symptom relevant to certain state, produce the reverse wholly or in part of certain state, cure certain state, Or above combination.For cancer, " treatment " or " therapy " can refer to suppression or slow down tumor or malignant cell Growth, breeding, or transfer, or some above combination.For tumor, " treatment " or " therapy " includes clearly Except all or part of tumor, suppress or slow down growth and metastasis of tumours, preventing or delay the development of tumor, or above Some combination.

The material of " by separating " is through manually being changed by naturalness.If nature occurs certain " by separating " Material or composition, then it has changed by or departs from its initial condition, or both has generation.Such as, a certain work In body animal body, naturally occurring polynucleotide or polypeptide are not separated, if but these polynucleotide or polypeptide therewith The material coexisted under native state is sufficiently separated and exists with sufficiently pure state, then may be considered " by separating ". In some embodiments, the purity of antibody and Fab be at least 90%, 93%, 95%, 96%, 97%, 98%, 99%, it is by electrophoresis method (such as SDS-PAGE, isoelectrofocusing, capillary electrophoresis), or chromatography (as Ion exchange chromatography or reversed-phase HPLC) determine.

In the present invention, " carrier " refers to, can will be inserted and make this albumen with encoding the polynucleotide manipulation of certain albumen Obtain a kind of vehicle expressed.Carrier can be used for converting, transduceing or transfection host cell so that it is the hereditary thing carried Matter element is expressed in host cell.For example, carrier includes: plasmid, phasmid, coemid, artificial The artificial chromosome that chromosome such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1 are derivative (PAC), phage such as bacteriophage lambda or M13 phage, and animal virus etc..Animal virus as carrier Kind have retrovirus (including slow virus), adenovirus, adeno-associated virus, herpesvirus (such as herpes simplex virus), Poxvirus, baculovirus, human papillomavirus, papova viruses (such as SV40).Carrier can contain multiple control The element that tabulation reaches, including promoter sequence, transcriptional initiation sequence, enhancer sequence, selection element and reporter gene. It addition, carrier also can contain replication origin.Carrier may also include assist its enter cell composition, including but do not limit In, virion, liposome or protein coat.

In the present invention, " host cell " refers to import exogenous polynucleotide and/or the cell of carrier.

" relevant to PD-L1 or related disease " in the present invention refers to, any due to PD-L1 (such as: human PD-L 1) Express or activity is raised and lowered and causes, aggravates or other states being correlated with.

" therapeutically effective amount " or " effective dose " in the present invention refers to, certain medicine is effectively treated and human PD-L 1 Relevant disease or the dosage of state or concentration.Such as, for the antibody disclosed in the present invention or the use of its Fab For Tu, therapeutically effective amount is under this dosage or concentration, and this antibody or antigen conjugates can clear all or part is swollen Tumor, suppress or slow down tumor growth, the growth of cell of suppression mediation cancer state or breeding, suppression Nasopharyngeal neoplasms, Alleviate any symptom relevant to tumor or cancer state or labelling, prevent or delay tumor or the development of cancer state, or more than Some combination.

" medicinal acceptable " refers to the supporting agent of indication, solvent, diluent, adjuvant and/or salt, is generally speaking changing On and/or mutually compatible with other dispensings in preparation physically, and physiologically mutually compatible with receiver.

Anti-PD-L1 antibody

In one aspect, the invention provides anti-PD-L1 antibody and its Fab.PD-1, also referred to as B7-1, Being the known critical immune checkpoint receptor expressed by activating T cell, it regulates immunosuppressive action.PD-1 part 1 (PD-L1) is to express at kinds of tumor cells, stromal cell or the transmembrane protein of the 40kDa on both, itself and PD-1 In conjunction with.Interaction between known PD-1 and PD-L1 can improve t cell response and thus mediate active anticancer.

In some embodiments, this application provides exemplary murine monoclonal antibodies 2.74.15, its CDR sequence As shown in table 1.

Table 1

In some embodiments, this application provides exemplary humanized antibody 2.74.15, including: 2.74.15.hAb4, 2.74.15.hAb5,2.74.15.hAb6,2.74.15.hAb7 and 2.74.15.hAb8, its heavy chain or variable region of light chain amino Acid sequence and nucleic acid sequence encoding are as follows.

2.74.15.hAb4-VH (SEQ ID NO:7 be aminoacid SEQ ID NO:8 be nucleic acid):

V section: IGHV4-59*01

D section: IGHD3-16*01

J section: IGHJ1*01

2.74.15.hAb4-VL (SEQ ID NO:9 be aminoacid SEQ ID NO:10 be nucleic acid):

V section: IGKV1-27*01

J section: IGKJ2*01

2.74.15hAb5-VH (SEQ ID NO:11 be aminoacid SEQ ID NO:12 be nucleic acid):

V section: IGHV4-59*06

D section: IGHD1-26*01

J section: IGHJ1*01

2.74.15hAb5-VL (SEQ ID NO:13 be aminoacid SEQ ID NO:14 be nucleic acid):

V section: IGKV1-9*01

J section: IGKJ2*01

2.74.15hAb6-VH (SEQ ID NO:11 be aminoacid SEQ ID NO:15 be nucleic acid):

V section: IGHV4-59*06

D section: IGHD1-26*01

J section: IGHJ1*01

2.74.15hAb6-VL (SEQ ID NO:16 be aminoacid SEQ ID NO:17 be nucleic acid):

V section: IGKV1-27*01

J section: IGKJ2*01

2.74.15hAb7-VH (SEQ ID NO:18 be aminoacid SEQ ID NO:19 be nucleic acid):

V section: IGHV4-59*01

D section: IGHD3-16*01

J section: IGHJ1*01

2.74.15hAb7-VL (SEQ ID NO:13 be aminoacid SEQ ID NO:20 be nucleic acid):

V section: IGKV1-9*01

J section: IGKJ2*01

2.74.15hAb8-VH (SEQ ID NO:18 be aminoacid SEQ ID NO:21 be nucleic acid):

V section: IGHV4-59*01

D section: IGHD3-16*01

J section: IGHJ1*01

2.74.15hAb8-VL (SEQ ID NO:22 be aminoacid SEQ ID NO:23 be nucleic acid):

V section: IGKV1-27*01

J section: IGKJ2*01

In some embodiments, described anti-PD-L1 antibody and its Fab include heavy CDR sequences, institute State sequence to be selected from: SEQ ID NOs:1,2 and 3.In some embodiments, described anti-PD-L1 antibody and its antigen Binding fragment includes that CDR sequence, described sequence are selected from: SEQ ID NOs:4,5 and 6.

In some embodiments, described anti-PD-L1 antibody and its Fab include variable region of heavy chain, wherein Described variable region of heavy chain includes SEQ ID NO:1, SEQ ID NO:2 and/or SEQ ID NO:3.

In some embodiments, described anti-PD-L1 antibody and its Fab include variable region of light chain, wherein Described variable region of light chain includes SEQ ID NO:4, SEQ ID NO:5 and/or SEQ ID NO:6.

In some embodiments, described anti-PD-L1 antibody and its Fab include that it includes such as SEQ ID NO:1, SEQ ID NO:2 and/or the variable region of heavy chain of SEQ ID NO:3；With such as SEQ ID NO:4, SEQ ID NO: The variable region of light chain of 5 and/or SEQ ID NO:6.

It will be understood by those skilled in the art that can carry out modifying by the CDR sequence provided in table 1 comprising one or more Amino acid whose replacement, the binding affinity with human PD-L 1 that the biologic activity being thus improved such as improves.Example As, it is possible to use display technique of bacteriophage produces and expresses antibody variants storehouse (such as Fab or FcFv variant), with Rear screening and human PD-L 1 have the antibody of affinity.In another example, can be described with computer software simulation The combination of antibody and human PD-L 1 also differentiates to be formed on antibody the amino acid residue of combination interface.These residues can be avoided Replacement to prevent binding affinity from reducing, or can carry out substituting to form higher combination with these residues of targeting.At certain In a little embodiments, it is conservative replacement that at least one (or whole) in CDR sequence replace.

In some embodiments, described antibody and Fab include one or more CDR sequence, these sequences Row have the sequence listed with table 1 at least 80% (for example, at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity, and remain and it simultaneously Parental antibody is similar or even above its binding affinity with human PD-L 1, and described parental antibody has essentially identical Sequence, but its corresponding CDR sequence and sequence listed by table 1 have 100% sequence identity.

In some embodiments, described anti-PD-L1 antibody and its Fab are humanized.Described people source Change antibody and there is compared with the parental antibody before its humanization in human body the immunogenicity of reduction, but remain parental antibody Binding affinity.In some embodiment, described anti-PD-L1 antibody and its Fab include inhuman source CDR Sequence, Ren Yuan framework region and optionally Ren Yuan constant region.In some embodiment, in described Ren Yuan framework region, use CDR One or more amino acid residues of the non-human source antibodies (such as mice framework region) that sequence is derived from replace, such as to improve Or reservation binding affinity.

In some embodiments, described humanization anti-PD-L1 antibody and its Fab include variable region of heavy chain, Wherein said variable region of heavy chain is selected from SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:18, and has therewith At least 80% (for example, at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homologous sequence of sequence identity；And/or variable region of light chain, wherein said variable region of light chain is selected from SEQ ID NO: 9, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:22, and have at least 80% therewith (for example, at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence is consistent The homologous sequence of property.These humanized antibodies remain the binding affinity with human PD-L 1, preferably anti-with exemplary Body: the level phase of 2.74.15.hAb4,2.74.15.hAb5,2.74.15.hAb6,2.74.15.hAb7 and 2.74.15.hAb8 Seemingly.

In some embodiments, described humanization anti-PD-L1 antibody and its Fab include a) weight chain variable District, it includes SEQ ID NO:7；And variable region of light chain, it includes SEQ ID NO:9；B) variable region of heavy chain, its Including SEQ ID NO:11；And variable region of light chain, it includes SEQ ID NO:13；C) variable region of heavy chain, it includes SEQ ID NO:11；And variable region of light chain, it includes SEQ ID NO:16；D) variable region of heavy chain, it includes SEQ ID NO:18；And variable region of light chain, it includes SEQ ID NO:13；And e) variable region of heavy chain, it includes SEQ ID NO: 18；And variable region of light chain, it includes SEQ ID NO:22.

The application also contemplate PD-L1 antibody anti-with the application and its Fab competing phase with the antibody of epi-position and Its Fab.In some embodiments, described antibody is with less than 10^-6M, less than 10^-7M, less than 10^-7.5 M, less than 10^-8M, less than 10^-8.5M or less than 10^-9M or less than 10^-10The IC of M₅₀Value (i.e. half suppression Concentration) block 2.74.15,2.74.15.hAb4,2.74.15.hAb5,2.74.15.hAb6,2.74.15.hAb7 or

2.74.15.hAb8 with people or the combination of monkey PD-L1.IC₅₀Value is tested such as ELISA by competitiveness and is measured, radiation Property Ligand Competition binding assay, and facs analysis determines.

In some embodiments, herein described anti-PD-L1 antibody and its Fab can be with≤10^-6 M(e.g.,≤5x10^-7M,≤2x10^-7M,≤10^-7M,≤5x10^-8M,≤2x10^-8M,≤10^-8M,≤5x10^-9M, ≤2x10^-9M,≤10^-9M,10^-10M) binding affinity (Kd) is specific binding with human PD-L 1, and it passes through Ion resonance combined techniques is measured.Binding affinity can use K_DValue represents, it is by dividing when antigen and antigen combine The dissociation rate when combination of son reaches balance is calculated with the ratio (koff/kon) of association rate.Described antigen Binding affinity (such as K_D) can be determined aptly by proper method known in the art, described method bag Include and use instrument (to participate in such as Murphy, M.et al, Current such as the plasma resonance combined techniques such as Biacore protocols in protein science,Chapter 19,unit 19.14,2006)。

In some embodiments, herein described antibody and its Fab and human PD-L 1 with 0.02nM-100nM (such as 0.02nM-50nM, 0.02nM-30nM, 0.02nM-20nM or 0.02nM-10nM, 0.02nM-1nM or 0.02nM-0.1nM) EC₅₀(i.e. half combines concentration) combines.Described antibody and human PD-L 1 Combination can pass through methods known in the art such as sandwich assay such as ELISA, western blot, FACS or its He measures by binding tests.In exemplary example, by test antibodies (i.e. resists) and immobilized human PD-L 1 Or express the Cell binding of human PD-L 1, and to wash uncombined antibody subsequently off, introduce the two of labelling and resist, it can be with One anti-binding is therefore, it is possible to detect that the one of combination resists.Can be on microplate reader plate when using immobilized PD-L1 Carry out described detection, maybe facs analysis can be used to carry out described detection when using the cell expressing human PD-L 1.

In some embodiments, herein described antibody and its Fab are with 0.2nM-100nM (example Such as 0.2nM-50nM, 0.2nM-30nM, 0.2nM-20nM or 0.2nM-10nM) IC₅₀Suppression human PD-L 1 With the combination of its receptor, it is recorded by competitiveness.

In some embodiments, herein described antibody and its Fab suppression human PD-L 1 are subject to it The combination of body, and thus provide include such as induced activation T cell produce cytokine (such as CD4⁺T is thin Born of the same parents and CD8⁺T cell), the propagation of the T cell of induced activation is (such as CD4⁺T cell and CD8⁺T cell) Biologic activity with the inhibition function reversing regulatory T reg.Exemplary cytokine include IL-2 and IFNγ.Term " IL-2 " refers to interleukin II, and it is cytokine signaling transduction molecule, in immune system The activity of the leukocyte (such as leukocyte) of middle regulation.Term " interferon gamma (IFN γ) " is by sky So kill (NK) cell, NK T cell, CD4⁺And CD8⁺The cytokine that T cell produces, it is huge Phagocytal important activator and the induction of MHC inductor (MHC) developed by molecule Agent.The generation of cytokine can be determined by methods known in the art, such as ELISA.These methods also may be used To be used for detecting T cell propagation, including [³H] thymidine incorporation mensuration.

Described anti-PD-L1 antibody and its Fab are that human PD-L 1 is specific.At some embodiment In, described antibody and its Fab are not combined (such as people PD-L2) with PD-L2.Such as, with PD-L2 Binding affinity than human PD-L 1 the 15% of binding affinity, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% is also low.

In some embodiments, described antibody and its Fab are with not higher than 10nM, such as, the highest In 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM, 0.1nM, 0.09nM, 0.08nM, 0.07nM, 0.06nM, 0.05nM, 0.04nM, 0.03nM, 0.02nM or 0.01nM's EC₅₀(being measured by ELISA) is combined with monkey PD-L1.

In some embodiments, described antibody and its Fab are not combined with Mus PD-L1, but and monkey PD-L1 is combined with the binding affinity similar with human PD-L 1.Such as, exemplary antibodies 2.74.15, 2.74.15.hAb4,2.74.15.hAb5,2.74.15.hAb6,2.74.15.hAb7 or 2.74.15.hAb8 and Mus PD-L1 Combination measure such as ELISA or facs analysis cannot detect with conventional combination, and ELISA or FACS detects this A little antibody to monkey PD-L1 with the affinity similar to human PD-L 1 or EC₅₀Value combines.

In some embodiments, that described anti-PD-L1 antibody and its Fab have a reduction or consumption The effector function exhausted.In some embodiments, described anti-PD-L1 antibody and its Fab have The constant region of IgG4 isotype, effector function that is that it has reduction or that exhaust.Such as ADCC and CDC etc. Effector function can result in the cytotoxicity to the cell expressing PD-L1.Many cells include health or normal Cell can express PD-L1.In order to avoid the unwanted toxicity healthy or the most possible to these, the present invention Effect merit that is that some embodiment of described antibody and its Fab has reduction or that even exhaust Energy.Known have many tests for estimating ADCC or CDC activity, such as Fc receptor binding assays, complement C1q Binding experiment and cell cracking process, those skilled in the art can easily select.It is not intended to be restrainted by theory Tie up, it is believed that the antibody of effector function such as ADCC and CDC that be that have reduction or that exhaust will not cause PD-L1 The cytotoxicity of express cell (such as those healthy or normal cells) or it is reduced to minimum degree, because of This avoids unwanted side effect.And meanwhile, the tumor cell expressing PD-L1 can resist with anti-PD-L1 Body combines, and therefore cannot escape from immunologic test point, and thus it can be identified and eliminated by immune system.

Anti-PD-L1 antibody described herein and its Fab can be monoclonal antibody, Anti-TNF-α Body, humanized antibody, chimeric antibody, recombinant antibodies, bi-specific antibody, traget antibody, bivalent antibody or Anti-idiotype antibody.Recombinant antibodies is to use recombination method in vitro and antibody prepared by non-animal.Bispecific Antibody or bivalent antibody are the artificial antibodies of the fragment with two kinds of different monoclonal antibodies, and they can be in conjunction with two kinds Different antigen.The antibody of " bivalence " and its Fab include two antigen binding sites.Two resist Former binding site in conjunction with same antigen, or can be each coupled to different antigen, in this case, Antibody or Fab are " bispecific ".

In some embodiments, anti-PD-L1 antibody described herein and its Fab are camelised Single domain antibody (camelized single chain domain antibody), bifunctional antibody (diabody), scFv, scFv Dimer, BsFv, dsFv, (dsFv) 2, dsFv-dsFv', Fv fragment, Fab, Fab', F (ab') 2, ds are difunctional anti- Body (ds diabody), nano antibody, domain antibodies or bivalent domain antibodies.

In some embodiments, anti-PD-L1 antibody described herein and its Fab wrap further Include constant region for immunoglobulin.In some embodiments, constant region for immunoglobulin includes heavy chain and/or chain constant District.Described CH includes CH1, CH1-CH2 or CH1-CH3 district.In some embodiments, Constant region for immunoglobulin may further include one or more modification character needed for obtaining.For example, it is possible to by institute State constant region modify with reduce or exhaust one or more effector functions with strengthen FcRn receptor combine or introduce one Individual or multiple cysteine.

In some embodiments, described anti-PD-L1 antibody and Fab thereof comprise further and put together Thing.It is contemplated that antibody or its Fab in the present invention can be connected with multiple conjugates (see such as "Conjugate Vaccines"、Contributions to Microbiology and Immunology、J.M.Cruse and R.E.Lewis、Jr.(eds.)、Carger Press、New York、(1989)).These conjugates can lead to Cross covalent bond, affine combination, embedding, equal combine (coordinate binding), complexation, combine, mixed Other modes such as conjunction or addition are connected with described antibody or antigen conjugates.In some embodiments, the present invention Disclosed antibody and Fab can make it contain the spy beyond epi-position bound fraction by the method for engineering Anchor point, these sites can be used to combine one or more conjugates.Such as, such site can comprise one Or multiple reactive amino acid residues, such as cysteine residues and histidine residues, for assisting and conjugate Covalently bound.In some embodiments, antibody can be connected in conjugate indirectly, or by another conjugate It is connected.Such as, described antibody or its Fab biotin-binding, the most indirectly combine second and sew Compound, it is connected with Avidin.Described conjugate can be detectable labelling, pharmacokinetics modification part, Purification part or cytotoxic moieties.The example of detectable labelling can include fluorescent labeling (such as fluorescein, Rhodamine, dansyl, phycoerythrin or texas Red), enzyme-substrate label (such as horseradish peroxidase, Alkali phosphatase, luciferase, glucoamylase, lysozyme, carbohydrate oxidase or beta-D-galactosidase), Radiosiotope is (such as,¹²³I、¹²⁴I、¹²⁵I、¹³¹I、³⁵S、³H、¹¹¹In、¹¹²In、¹⁴C、⁶⁴Cu、⁶⁷Cu、⁸⁶Y、⁸⁸Y、⁹⁰Y、¹⁷⁷Lu、²¹¹At、¹⁸⁶Re、¹⁸⁸Re、¹⁵³Sm、²¹²Bi、and ³²P、 Other lanthanide series, luminescent marking), chromophoric moiety, digoxin, biotin/avidin, DNA molecular or Gold is to detect.In some embodiments, described conjugate can be pharmacokinetics modification part such as PEG, its half-life helping to extend antibody.Other suitable polymer include that such as carboxymethyl cellulose, Portugal are poly- Sugar, polyvinyl alcohol, polyvinylpyrrolidone, ethylene glycol/propylene glycol copolymers etc..In some embodiments, Described conjugate can be purification part such as magnetic bead." cytotoxic moieties " can be harmful to cell or May damage or kill any reagent of cell.The example of cytotoxic moieties include, but not limited to paclitaxel, Cytochalasin B, Gramicidin D, ethidium bromide, ipecine, mitomycin, etoposide, teniposide, Vincristine, vinblastine, colchicine, amycin, daunorubicin, dihydroxy anthracin diketone, meter Tuo Anthraquinone, mithramycin, actinomycin D, 1-boldenone, glucocorticoid, procaine, tetracaine, profit (such as, methotrexate, 6-sulfydryl are fast for many caines, Propranolol, puromycin and the like, antimetabolite Purine, 6-thioguanine, cytosine arabinoside, 5-fluorouracil Dacca bar), alkylating agent (such as chlormethine, phosphinothioylidynetrisaziridine benzene Butanoic acid chlormethine, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, disappear in vain Peace, mitobronitol, streptozotocin, ametycin and cis-dichlorodiamine platinum (II) (DDP) cisplatin), Anthracycline antibiotics (such as daunorubicin (daunomycin in the past) and amycin), antibiotic are (the most more Mildew element (being formerly referred to as D actinomycin D), bleomycin, mithramycin and anthramycin (AMC)) and Antimitotic agent (such as vincristine and vinblastine).

Polynucleotide and recombination method

This application provides the polynucleotide of the separation encoding anti-PD-L1 antibody and its Fab.At certain In a little embodiments, the polynucleotide of described separation include the sequence of the CDR in one or more coding such as table 1, Such as those sequences provided in the polynucleotide sequence of encoding variable regions.

In some embodiments, the polynucleotide encoding variable region of heavy chain of described separation include the sequence selected from lower group Row: SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO: 21, and have at least 80% therewith (for example, at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homologous sequence of sequence identity.In some embodiments, institute State the polynucleotide encoding variable region of light chain of separation and include the sequence selected from lower group: SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, and have therewith at least 80% (for example, at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homologous sequence of sequence identity.In some embodiments, described conforming percentage ratio is derived from The degeneracy of genetic code, and the protein sequence encoded keeps constant.

Use recombinant technique well known in the art, described anti-PD-L1 antibody can be encoded by including and its antigen is tied The carrier of the polynucleotide closing fragment (such as including the sequence shown in table 1) introduces host cell and is used for cloning (expansion Increase DNA) or gene expression.In another embodiment, described antibody can be by homology weight well known in the art The method of group prepares.Encode the DNA of described monoclonal antibody can be separated by conventional method and order-checking (as Can use oligonucleotide probe, this probe can be tied with the heavy chain of encoding said antibody and the gene of light chain by specificity Close).Variety carrier is available.Carrier component generally includes, but is not limited to, following one or more: Such as: SV40, signal sequence, replication origin, one or more marker gene, enhancement sequences, promoter are ( CMV, EF-1 α) and transcription terminator.

In some embodiments, described carrier system includes mammal, antibacterial, Yeast system etc., and will bag Include plasmid such as but not limited to pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI、pCMV、pEGFP、pEGFT、pSV2、pFUSE、pVITRO,pVIVO、pMAL、pMONO、 pSELECT、pUNO、pDUO、Psg5L、pBABE、pWPXL、pBI、p15TV-L、pPro18、 PTD, pRS420, pLexA, pACT2 etc. can obtain or commercially available carrier other from laboratory.Suitable load Body can include plasmid or viral vector (such as, replication defect type retrovirus, adenovirus and gland related diseases Poison).

The carrier including the polynucleotide of encoding said antibody and its Fab can be introduced host cell For clone or gene expression.Be applicable to the host cell of the DNA cloning or expressing described carrier in the present invention For prokaryotic cell, yeast or above-mentioned higher eukaryotes.The prokaryotic cell being applicable to purposes of the present invention includes the thinnest Bacterium such as, gram negative bacteria or gram positive bacteria, such as, enterobacteriaceae, e.g., escherichia coli, enterobacteria Belong to, Erwinia, klebsiella, Proteus, Salmonella, e.g., mouse typhus sramana (family name) Bacillus, Serratia, e.g., serratia marcescens, and Shigella, and Bacillus is such as, hay bud Spore bacillus and Bacillus licheniformis, pseudomonas such as, bacillus pyocyaneus and streptomycete.

In addition to prokaryotic cell, eukaryotic microorganisms such as filamentous fungi or yeast also can be made host cell clone or express coding The carrier of anti-PD-L1 antibody.Saccharomyces cerevisiae, or bakery yeast is the most frequently used eucaryon host microorganism such as low.But, Other genus and species many and strain is the most the more commonly used and is suitable in the present invention, such as schizosaccharomyces pombe；Kluyveromyces place Lead such as, Kluyveromyces lactis, Kluyveromyces fragilis (ATCC 12,424), Bulgaria kluyveromyces (ATCC 16,045), Wei Shi kluyveromyces (ATCC 24,178), Crewe hero yeast (ATCC 56,500), fruit bat kluyveromyces (ATCC 36,906), Kluyveromyces thermotolerans and yeast Kluyveromyces marxianus；Yarrowia lipolytica (EP 402,226)；Bar This moral Pichia sp. (EP 183,070)；Candida mycoderma；Trichoderma reesei (EP 244,234)；Neurospora；Prosperous yeast is permitted in west, As: prosperous yeast is permitted in west；And filamentous fungi, such as: neurospora, penicillium, curved neck be mould and aspergillosis, such as: hook nest is bent Mould and aspergillus niger.

The host cell being applicable to express glycosylated antibodies or its Fab provided in the present invention is given birth to by many cells Thing is derivative to be obtained.Example without vertebrate cell includes plant and insect cell.Have been found that multiple baculovirus strain Permissive insect host cells (the permissive insect host of (baculoviral strains) and variant and correspondence Cells), such as following host is come from: fall army worm (caterpillar), Aedes Aegypti (mosquito), Aedes albopictus (mosquito Son), Drosophila melanogaster (fruit bat) and silkworm.The multiple Strain for transfection is public Ke get, such as Autographa night The Bm-5 mutation of moth nuclear polyhedrosis virus and bombyx mori nuclear polyhydrosis virus, these viruses all can use in the present invention, Especially for transfection Spodopterafrugiperda cells.Cotton Gossypii, Semen Maydis, Rhizoma Solani tuber osi, Semen sojae atricolor, petunia, Fructus Lycopersici esculenti and Nicotiana tabacum L. Culture plant cell also is used as host.

But, most interested is vertebrate cell, and the cultivation (tissue culture) of vertebrate cell has become as routine operation. Available mammalian host cell example has, monkey-kidney cells CV1 system (COS-7, the ATCC CRL that SV40 converts 1651)；Human embryonic kidney cell is (293 or 293 cell subclone of suspension culture, Graham et al., J.Gen Virol. 36:59(1977))；Baby hamster kidney cell (BHK, ATCC CCL 10)；Chinese hamster ovary cell/-DHFR (CHO, Urlaub et al.,Proc.Natl.Acad.Sci.USA 77:4216(1980))；Properties of Sertoli Cells Isolated from Mice Testis (TM4, Mather,Biol.Reprod.23:243-251(1980))；Monkey-kidney cells (CV1ATCC CCL 70)；Cercopithecus aethiops Nephrocyte (VERO-76, ATCC CRL-1587)；Human cervical carcinoma cell (HELA, ATCC CCL 2)；Dog Nephrocyte (MDCK, ATCC CCL 34)；Buffalo rats liver (BRL 3A, ATCC CRL 1442)； Human pneumonocyte (W138, ATCC CCL 75)；Human liver cell (Hep G2, HB 8065)；Mammary gland of mouse tumor (MMT 060562, ATCC CCL51)；TRI cell (Mather etc., Annals N.Y.Acad.Sci.383:44-68 (1982))； MRC 5 cell；FS4 cell；And Bel7402 (Hep G2).Some preferred embodiment in, institute Stating host cell is 293F cell.

With the above-mentioned expression producing anti-PD-L1 antibody or cloning vehicle transformed host cell, and by it conventional Nutrient medium is cultivated, is suitable for evoked promoter after described Nutrient medium is modified, selects to convert cell or amplification The gene of coding aim sequence.

The host cell being used for producing described antibody or its Fab in the present invention can be cultivated in multiple culture medium. Commercially available culture medium such as Ham's F10 (Sigma), minimum basic training liquid (MEM, (Sigma)), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle's Medium (DMEM), Sigma) to can be used for cultivating described host thin Born of the same parents.It addition, it is any at Ham et al., Meth.Enz.58:44 (1979), Barnes et al., Anal.Biochem.102:255 (1980), U.S. Patent number 4,767,704；4,657,866；4,927,762；4,560,655；Or 5,122,469；WO 90/03430；WO 87/00195；Or the culture medium of explanation can be used as described place in U.S. Patent application Re.30,985 The culture medium of chief cell.These culture medium all can add necessary hormone and/or other somatomedin (such as insulin, turn Ferritin or epidermal growth factor), salt (such as sodium chloride, calcium chloride, magnesium chloride and phosphate), buffer (as HEPES), nucleotide (such as adenylic acid and thymus pyrimidine), antibiotic (such as gentamycin), trace element (definition For final concentration generally at micro-molar range inorganic compound), and glucose or the energy source that is equal to therewith.Described culture medium Also can contain the additive of any other necessity of debita spissitudo well known in the art.The condition of described culture medium, as temperature, The conditions of similarities such as pH value, for selecting the previously used condition of host cell for expressing, for those of ordinary skill institute Know.

When using recombinant technique, described antibody can be in intracellular, the generation of wall film space, or direct secretion to culture medium. If described antibody intracellular generate, first remove host cell or cracking segment granule remains, such as, can by from The heart or ultrasonic method.Carter et al., Bio/Technology 10:163-167 (1992) describe and will be secreted into large intestine bar The method that the antibody in bacterium wall film space separates.In brief, at sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) melt cell under conditions of existing and stick with paste (cell paste) more than about 30 minutes.It is centrifuged off cell debris.As Described antibody-secreting is in culture medium, then generally first by commercially available protein concentration filter, such as Amicon or Millipore Pellicon ultrafiltration unit, concentrates the supernatant of this expression system.In any aforesaid step all Protease inhibitor such as PMSF can be added to suppress protein degradation, and antibiotic is to prevent the growth of accidental contamination thing.

The antibody prepared from described cell can use purification process to be purified, such as hydroxyapatite chromatography, gel electrophoresis, Dialysis, DEAE-cellulose ion exchange chromatography post, ammonium sulfate precipitation, saltout and affinity chromatography, wherein affinity chromatography For preferred purification technique.The kind of described antibody and described antibody exist the Fc domain of any immunoglobulin Determine protein A as affinity ligand if appropriate for.Protein A can be used for purification based on people γ 1, γ 2 or γ 4 heavy chain Antibody (Lindmark et al., J.Immunol.Meth.62:1-13 (1983)).Protein G is applicable to all Mus sources isomery Body and people γ 3 (Guss et al., EMBO J.5:15671575 (1986)).Agarose is the most frequently used affinity ligand attachment Substrate, but also can be selected for other substrate.Substrate such as controlled pore glass that mechanical force is stable or poly-(styrene) benzene and use Agarose is compared and can be realized faster flow velocity and shorter process time.As this antibody contains CH3 domain, then can use Bakerbond ABX.TM resin is purified (J.T.Baker, Phillipsburg, N.J.).Obtain also dependent on needs Antibody determine the technology of other protein purifications, such as the fractional distillation in ion exchange column, ethanol precipitation, reversed-phase HPLC, silicon Glue chromatograph, heparin sepharose chromatograph based on anion or cation exchange resin (such as poly-aspartate post), layer Analysis focusing, SDS-PAGE and ammonium sulfate precipitation.

After any any preliminary purification step, the method for available low pH hydrophobic interaction chromatograph processes containing interested (such as, antibody and the mixture of impurity, with the elution buffer of pH about 2.5-4.5, preferably carried out under low salt concn From about 0 to 0.25M salinity).

Test kit

This application provides the test kit including described anti-PD-L1 antibody and its Fab.Some embodiment party In formula, described test kit is for detecting existence situation or the level of the PD-L1 in biological sample.Described biological sample Cell or tissue can be included.

In some embodiments, described test kit includes anti-PD-L1 antibody and its antigen puted together with detectable label Binding fragment.In some embodiments, described test kit includes that unlabelled anti-PD-L1 antibody and its antigen combine Fragment, and farther include to resist with the two of unlabelled anti-PD-L1 antibody and its Fab incorporation of markings. Described test kit may further include operation instruction and the packaging separated by each assembly in test kit.

In some embodiments, described anti-PD-L1 antibody and its Fab are connected to substrate or instrument Sandwich assay such as ELISA or immune chromatograph measure.The substrate or the instrument that are suitable for can be such as microwell plate and reagent paper.

Pharmaceutical composition and Therapeutic Method

The application further provides the pharmaceutical composition and including described anti-PD-L1 antibody and its Fab Individual or multiple pharmaceutically acceptable carriers.

The medicinal acceptable supporting agent being used in pharmaceutical composition disclosed in the present application can include, such as, and medicinal acceptable liquid Body, gel or solid carriers, aqueous media, non-aqueous phase medium, antimicrobial material, isotonic material, buffer, anti- Oxygen agent, anesthetis, suspending agent/dispersant, chelating agen, diluent, adjuvant, adjuvant or non-toxic auxiliary substances, other Component well known in the art or above multiple combination.

Be suitable for component can include, such as, antioxidant, filler, binding agent, disintegrating agent, buffer, preservative, Lubricant, stir taste agent, thickening agent, coloring agent, emulsifying agent or stabilizer such as sugar and cyclodextrin.The antioxidant being suitable for can Including, such as, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, Cysteine, mercapto glycerol, TGA, sulfydryl sorbitol, butyl methyl methoxybenzene, Yoshinox BHT and/ Or propylgallate.As disclosed in the present invention, a kind of containing antibody disclosed by the invention or its Fab Compositions includes one or more antioxidant such as methionine, can will reduce described antibody or the oxygen of its Fab Change.Minimizing to Oxidation can prevent or reduce the reduction of binding affinity, thus improve Antibody stability and extend guarantor The matter phase.Therefore, in some embodiments, in the compositions that the present invention provides containing one or more described antibody or Its Fab and one or more antioxidant such as methionine.Invention further provides multiple method, By the antibody provided in the present invention or its Fab are mixed with one or more antioxidant, such as first sulfur ammonia Acid, can prevent described antibody or the oxidation of its Fab, extend its shelf-life and/or improve its activity.

Further, medicinal acceptable supporting agent can include, such as, and aqueous media such as sodium chloride injection, woods grignard Liquid injection, isotonic glucose injection, Sterile Water Injection or glucose and lactated Ringer's injection, non-aqueous media Such as: the fixed oil of plant origin, cottonseed oil, Semen Maydis oil, Oleum sesami or Oleum Arachidis hypogaeae semen, bacteriostatic or Antibiotic substance under fungus inhibition concentration, isotonic agent be such as: sodium chloride or glucose, buffer such as: phosphate or citric acid Phthalate buffer, antioxidant such as: sodium bisulfate, local anesthetic such as: procaine hydrochloride, suspending agent and dispersant Such as sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose or polyvinylpyrrolidone, emulsifying agent such as: polysorbate 80 (tween 80s), chelating reagent such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol bis (2-amino-ethyl ether) Tetraacethyl), ethanol, Polyethylene Glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid.Resisting as supporting agent Microbial inoculum can add in the pharmaceutical composition in multidose container, and it includes phenols or cresol, mercurial, benzyl alcohol, chlorine Spread for butanol, methyl and propyl para-hydroxybenzoate, thiophene hydrargyrum, Neotran ammonium and chlorine Benzethonium.The adjuvant being suitable for can Including, such as, water, salt, glucose, glycerol or ethanol.The non-toxic auxiliary substances being suitable for can include, such as, and emulsifying Agent, pH, stabilizer, solubilizing agent, or sodium acetate, sorbitan monolaurate, triethanolamine The material of oleate or cyclodextrin etc.

Described pharmaceutical composition can be liquid solution, suspension, Emulsion, pill, capsule, tablet, sustained release Preparation or powder.Oral formulations can include the mannitol of standard vector such as pharmaceutical grade, lactose, starch, magnesium stearate, Polyvinylpyrrolidone, saccharin sodium, cellulose, magnesium carbonate etc..

In some embodiments, described pharmaceutical composition is formulated to injectable compositions.Injectable drug regimen Thing can be prepared with the form of any routine, such as, liquid flux, suspending agent, emulsifying agent or be applicable to produce liquid flux, Suspending agent or the solid form of emulsifying agent.Ejection preparation is existing before can including used aseptic and/or pyrogen-free solution, use The aseptic dry soluble substance being combined with solvent, such as lyophilized powder, including subcutaneous, inject sterile suspensions i.e., make With the front existing aseptic dry insoluble product being combined with medium, and aseptic and/or pyrogen-free Emulsion.Solvent can be aqueous phase Or nonaqueous phase.

In some embodiments, the ejection preparation of unit dose is packaged in an ampoule, an arm or one with pin In syringe.This area is known, and the preparation of all drug administration by injection should be aseptic apyrogeneity.

In some embodiments, suitable molten by antibody disclosed in the present application or its Fab being dissolved in certain Agent can be prepared aseptic freeze-dried powder.Described solvent can improve powder or the restructuring solution prepared by powder containing a kind of Stability, or improve powder or other pharmacology components of restructuring solution.The adjuvant being suitable for includes, but are not limited to, water, Portugal Grape sugar, minashi sugar alcohol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose or other materials being suitable for. Solvent can contain buffer, such as citrate buffer, sodium phosphate or kaliumphosphate buffer or other this persons skilled in the art public affairs The buffer known, in one embodiment, the pH of buffer is neutral.Enter under standard conditions known in the art Row carries out filtration sterilization subsequently to described dissolving, and then lyophilizing prepares preferable preparation.In one embodiment, will The solvent subpackage of gained is to lyophilizing in tubule.Every tubule can accommodate the described anti-PD-L1 of single dose or multidose Antibody or its Fab or a combination thereof thing.It is required or many that charge weight in every tubule can be slightly higher than each dosage Secondary dosage required (such as 10% excess), thus ensure that sampling accurately and is administered accurately.Lyophilized powder can be in suitable condition Lower storage, as at about 4 DEG C to room temperature scope.

The preparation for drug administration by injection is obtained by molten for lyophilizing grain weight with water for injection.In one embodiment, can be by lyophilizing It is molten that powder adds to weight in aseptic apirogen water or other liquid carrier being suitable for.Accurate amount is determined by the therapy selected, can root Determine according to empirical value.

Additionally provide Therapeutic Method, use including by antibody described herein or its Fab of therapeutically effective amount Give the experimenter needing it, thus treat or prevent the situation relevant to PD-L1 or disease.On the other hand, also carry Supply the method for situation of the experimenter that treatment can benefit from the immune response raised, including to its experimenter of described needs The antibody described herein of administering therapeutic effective dose or its Fab.

The treatment effective dose of antibody provided herein or its Fab depends on well known in the art multiple Factor, such as body weight, age, passing medical history, existing treatment, the health status of object and the potentiality of cross infection, mistake Quick and side effect quick, super, and route of administration and the degree of tumor development.One skilled in the art (such as doctor or beast Doctor) can be according to these or other condition or require reduce in proportion or raise dosage.

In some embodiments, the present invention provides antibody or its Fab can be in treatment effective doses about 0.01 Between mg/kg to about 100mg/kg be administered (such as, about 0.01mg/kg, about 0.5mg/kg, about 1mg/kg, about 2 Mg/kg, about 5mg/kg, about 10mg/kg, about 15mg/kg, about 20mg/kg, about 25mg/kg, about 30mg/kg, About 35mg/kg, about 40mg/kg, about 45mg/kg, about 50mg/kg, about 55mg/kg, about 60mg/kg, about 65mg/kg, about 70mg/kg, about 75mg/kg, about 80mg/kg, about 85mg/kg, about 90mg/kg, about 95mg/kg Or about 100mg/kg).In some embodiments, described antibody or its Fab are with about 50mg/kg or more Few dosed administration, in some embodiments, dosage be 10mg/kg or less, 5mg/kg or less, 1 Mg/kg or less, 0.5mg/kg or less or 0.1mg/kg or less.Certain given dose can at multiple doses at intervals, The most once a day, twice daily or more, monthly twice or more, once in a week, once every two weeks, every three Mondays Secondary, monthly or per bimester or more moon once.In some embodiments, dosage can be with treatment process change. Such as, in some embodiments, initial dosages is high than subsequent dose dosage.In some embodiments, give Pharmaceutical quantities is adjusted according to the reaction being administered object in treatment process.

Dosage regimen can reach peak optimization reaction (such as therapeutic response) by adjustment.Such as, can carry out single dose administration or A period of time divides the dosed administration of multiple separation.

Antibody disclosed in the present invention and Fab can be administered by administering mode well known in the art, such as, inject Be administered (e.g., subcutaneous injection, lumbar injection, intravenous injection, including intravenous drip, intramuscular injection or intradermal injection) or Non-injection administration (e.g., oral administration, nasal-cavity administration, sublingual administration, rectally or topical administration).

The situation relevant to PD-L1 and disease can be Ia disease or disease.Such situation and disease bag Include tumor and cancer, such as nonsmall-cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal carcinoma, ovarian cancer, breast Cancer, pancreatic cancer, gastric cancer, bladder cancer, the esophageal carcinoma, mesothelioma, melanoma, incidence cancer, thyroid carcinoma, sarcoma, Carcinoma of prostate, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphoma, myeloma, cutaneous T cell lymphoma (mycoses fungoids), merkel's cells cancer and other malignant hematologic disease, as Classical Hodgkin Lymphoma (CHL), Primary Mediastinal large B cell lymphoid tumor, T cell/histiocytic rich B cell lymphoma, positive and negative PTLD of EBV Outer NK/T is thin for diffusivity large B cell lymphoid tumor (DLBCL) relevant with EBV, plasmablast lymphoma, knot Born of the same parents' lymphoma, nasopharyngeal carcinoma lymphoma primary effusion relevant with HHV8, Hodgkin lymphoma；Autoimmune disease, Such as systemic lupus erythematosus (sle) (SLE), Autoimmune Diabetes, chronic viral infection such as hepatitis B, hepatitis C, Herpesvirus, Epstein-Barr virus, HIV (human immunodeficiency virus), cytomegalovirus, herpes simplex virus I-type, herpes simplex Virus 2 types, human papillomavirus, the virus infection of adenovirus, the herpesvirus epidemic diseases that card ripple cc sarcoma is relevant, Thin circovirus virus (Torquetenovirus), JC virus or BK virus etc..

Using method

The application further provides the described anti-PD-L1 antibody of use or the method for its Fab.

In some embodiments, this application provides in biological sample, detect the existence situation of PD-L1 or level Method, contacts with anti-PD-L1 antibody described herein or its Fab including by described biological sample, and Existence situation or the level of PD-L1 is determined in described sample.Sample containing PD-L1 can derive from experimenter's Cell or tissue or body fluid.In some embodiments, the sample source containing PD-L1 is in tumor or cancerous cell or group Knit such as circulating tumor cell, tumor mass or the doubtful tissue containing tumor cell.In some embodiments, contain The sample of PD-L1 is the immunocyte of infiltration tumor, such as immunocyte around the immunocyte of intra-tumor, tumor or Other stromal cells such as fibroblast.The cell of described infiltration tumor can be T cell (such as CD4⁺T is thin Born of the same parents or CD8⁺T cell), B cell or other myeloid lineage cells, such as, macrophage, natural killer cell, Dendritic cell, mononuclear cell etc..In sample the existence situation of PD-L1 or level can by such as detection antibody with PD-L1 combines and determines, the method for use can be such as immunohistochemistry (IHC), ELISA, fluidic cell Instrument or other suitable method any as known in the art.

In target biological tissue, existence situation and the level of PD-L1 may indicate that the individuality that described biological sample is originated is No PD-L1 antagonist may be responded.Therefore, invention further provides discriminating and suffer from possibility to PD-L1 antagonism The disease of agent response or the individual method of situation, comprising: with described anti-PD-L1 antibody described herein or its Fab is determining existence situation or the level of PD-L1, wherein in the biological sample to be measured of described individuality In described biological sample to be measured, existence or the level of PD-L1 raises the probability representing response.At some embodiments In, described testing sample derives from cancerous cell or tissue, or enters the immunocyte of tumor.Term used in this application " on Adjust " refer to, compared with PD-L1 protein level in the reference sample using same antibody to detect, use described herein Total increase of the PD-L1 protein level that antibody or its Fab detect in testing sample no less than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or more.Described can be from health or without the control sample obtained the experimenter of disease with reference to sample, or The health that obtains or the sample without disease from the individuality in testing sample source.Such as, described permissible with reference to sample It is near testing sample (such as tumor) or adjacent without disease sample.In some embodiments, described side Method farther includes the identified disease likely responded PD-L1 antagonist or the individual administering therapeutic of situation The anti-PD-L1 antibody of effective dose or its Fab.

Antibody disclosed by the invention and Fab can individually dosed or with one or more other treatment means or thing Matter administering drug combinations.Such as, antibody disclosed by the invention and Fab can be with chemotherapy, radiotherapy, treatment of cancer operations The therapy of the complication that (such as tumorectomy), one or more Antiemetics or other chemotherapy cause or any other It is combined for the therapeutic substance of cancer or the therapeutic substance of any disease mediated by PD-L1.Such at some In embodiment, when antibody disclosed by the invention and Fab are combined with one or more therapeutic substances, can be with institute One or more therapeutic substances stated are administered simultaneously, and in some such embodiment, described antibody and antigen combine Fragment can be administered simultaneously as a part for same pharmaceutical composition.But, anti-with what other treatment material " was combined " Body and antigen conjugates need not be administered simultaneously or be administered in same compositions with this therapeutic substance.The present invention " is combined " Implication be additionally included in the antibody being administered before or after another therapeutic substance and antigen conjugates be also considered as with should Therapeutic substance " is combined ", even if described antibody or its Fab are given by different modes of administration with the second material Medicine.In the conceived case, with antibody disclosed by the invention or its Fab associated with other treatment material can join According to the method medication of the product description of this other treatment material, or with reference to surgical desk reference book 2003 (Physicians'Desk Reference, 57th Ed；Medical Economics Company；ISBN:1563634457； 57th edition (in November, 2002)), or with reference to other methods well known in the art.

In some embodiments, described therapeutic substance can induce or strengthen the immunoreation for cancer.Such as, swollen Tumor vaccine may be used for induction to some tumor or the immunne response of cancer.Cytokine therapy may be used for improving tumor Antigen is to immune submission.The example of cytokine therapy includes but not limited to interferon such as interferon-ALPHA, β and γ, Colony stimulating factor such as macrophage CSF, granular leukocyte macrophage CSF and granulocyte-CSF, interleukin such as IL-1, IL-1 α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 and IL-12, tumor Necrosin such as TNF-α and TNF-β.Can also use inactivation immunosuppressant target reagent, as TGF-β inhibitor, IL-10 inhibitor and FasL inhibitor.Another group reagent includes activating the immune response for tumor or cancerous cell Those reagent, such as, improve t cell activation (such as T cell costimulatory molecules agonist such as CTLA-4, ICOS and OX-40) those, and improve those of Dendritic Cell Function and antigen presentation.

The application further provides and monitors therapeutic response or progression of disease in the experimenter with PD-L1 antagonist for treating Method, including with to be measured from described individuality of herein described anti-PD-L1 antibody or its Fab Biological sample determines existence situation or the level of PD-L1.In some embodiments, described method farther includes By the PD-L1 level in biological sample to be measured and the PD-L1 level in the comparable sample obtained from same individuality before this Comparing, the increase of the PD-L1 wherein reducing or being slowed or stopped in test biological sample shows positive treatment Reaction or controlled progression of disease.Described can be same type of sample with testing sample than sample, but it is to control Obtain from same individual before treating or in treatment initial stage.

Following example are intended to be better described the present invention, and the scope that should not be construed as limiting the invention.All following Particular composition, material and method, it is within whole or in part.These specific compositionss, Material and method are not limited to the present invention, and simply for specific embodiment being described within the scope of the invention.This Skilled practitioner can without creative and develop without departing from the scope of the invention compositions of equivalent, material and Method.Should be understood that the multiple change making the method for the present invention can still be included in the scope of the present invention.Invention Such variation is being included within the scope of the invention by people's will.

Embodiment 1: the generation of antibody hybridoma

1.1 immunity :～the female Balb/c mice of 8 week old is injected at the human PD-L 1 in 10 μ g TiterMax via foot pad ECD protein sensitization, the PD-L1ECD albumen being used in Fosfalugel (Yamanouchi) adjuvant for the most every 3 days excite through foot pad until It is suitable for merging.Anti-PD-L1 antibody serum titer is detected every two weeks by ELISA or FACS.

1.2 cells merge: in fusion first 3 days, and animal via lumbar injection receives human PD-L 1 ECD in the PBS of 10g Last the exciting of albumen.Merging the same day, results lymph-node cell also prepares single cell suspension.The pouring that will obtain Bar cell mixes with the ratio of 1:1 with myeloma cell (P3).By cell mixture washing and in ECF solution with 2.0x 10⁶The density of cells/mL is resuspended.BTX 2000 electro' asion instrument is used to carry out cell fusion.

1.3 screen with second time doma supernatant for the first time: after cultivating 7-14 days in 37 DEG C, by a part of hybridoma Supernatant analyzes detection by Mirrorball.Briefly, doma supernatant 1XPBS is diluted 5 times.Express CHO-K1 cell (the 1.25x 10 of PD-L1⁵Cell/mL) with the antibody (400ng/mL) of secondary fluorescence labelling and DraQ5 (1:5000) mixes.The cell mixture of 20 μ L and the dilute of 20 μ L is added in each hole of 384 orifice plates The doma supernatant sample released at room temperature lucifuge are hatched at least 2 hours until preparingHigh It is analyzed on sensitivity microwell plate cell instrument.By using the CHO-K1 cell expressing PD-L1 through FACS Checking positive cell.With doma supernatant sample to cell dyeing, anti-with the goat being connected with FITC subsequently Mouse IgG Fc carries out two anti-bindings.With corresponding parental cells system as negative control.Use BD The cell combined is analyzed by the FACSCanto II and FlowJo version software of Biosciences.

1.4 sub-clones: use the hybridoma cell line being verified as PD-L1 combination positive to carry out sub-clone.Briefly Ground, for every strain of hybridoma system, by cell counting and be diluted to 5, every hole or 1 in Cloning Medium Individual cell.Bed board is carried out with 200 μ L/ holes in 96 orifice plates.Plate is placed in 37 DEG C, 5%CO₂Hatch until after Continuous analysis.

1.5 homotype tests: with goat anti mouse IgG1, IgG2a, IgG2b, IgG3, IgA and the IgM in 50 μ L/ holes Antibody is coated elisa plate respectively with 1 μ g/mL.After closure, the doma supernatant sample of 50 μ L is added Enter every hole, incubated at room temperature 2 hours.Use goat anti mouse kappa light chain-HRP as detection antibody.Make Hatch 10min colour developing with tmb substrate, terminate reaction with 2M HCl.In ELISA microplate reader at 450nm Read plate.

1.6 Binding experiment based on cell: for the combination activity of inspection guide's antibody with target, human PD-L 1 will be expressed CHO-K1 or mDC guide's antibodies, two be combined with the goat anti-human IgG Fc connected with FITC subsequently Anti-binding.Use the parental cells system of correspondence as negative control.Use the FACSCanto of BD Biosciences The cell combined is analyzed by II and FlowJo version software.

Total length human PD-L 1 has been transfected by the antibodies for human PD-L 1 from Muridae hybridoma Chinese hamster ovary celI, the two resistive connection merging FACS being combined with the goat anti-rat IgG Fc connected with FITC subsequently Analyze.As it is shown in figure 1, antibody 27.3；74.15 and 17.42 with the EC of about 1nM₅₀Specifically with CHO The PD-L1 expressed on cell combines.

Embodiment 2: humanization and purification

Humanization: select two kinds of mices from hybridoma according to the high-affinity being combined with PD-L1 and specificity Anti-Human's PD-L1 antibody (being respectively designated as 2.27.3 and 2.74.15) carries out humanization, is used for improving T cell and increases The cytokine IFN γ grown and increase in MLR and the secretion of IL-2.Described humanization uses and is referred to as CDR transplanting Technology carry out.FR and CDR region is defined according to Kabat system and IMGT system.By sequence homology result and knot The structure likelihood ratio, to combining, uses immediate people source FR 1-3 gene to replace mice FR 1-3 gene, uses closest People source JH and J kappa gene replace mice FR4 gene.After validation template sequence and optimizing codon, can by heavy chain Become district and variable region of light chain expands and clones into expression vector, and then express humanized antibody.By the 2.74.15's of acquisition Humanized antibody named 2.74.15.hAb4,2.74.15.hAb5,2.74.15.hAb6,2.74.15.hAb7 and 2.74.15.hAb8.Also it is prepared for the humanized antibody of 2.27.3.

Antibody purification: use the DNA vector transfection 293F cell containing humanized antibody, for antibody expression and life Produce.Antibody in 293F cell culture supernatant uses protein A affinity chromatography column purification.

Embodiment 3: the sign of humanized antibody

The humanized antibody selecting 2.74.15 further characterizes.According to the step of 1.6 chapters and sections in embodiment 1, By humanized antibody 2.74.15.hAb4,2.74.15.hAb5,2.74.15.hAb6,2.74.15.hAb7 and 2.74.15.hAb8 With its combination with the Chinese hamster ovary celI having transfected total length human PD-L 1 of facs analysis.

Fig. 2 shows and uses the humanized antibody for human PD-L 1 to carry out the Chinese hamster ovary celI having transfected PD-L1 Dyeing, and facs analysis display humanization PD-L1 antibody is with the EC of about 1nM₅₀Specifically with PD-L1 In conjunction with.Fig. 3 shows that the unicellular cultures by cultivating 7 days in GM-CSF and IL4 creates people's dendron Cell, its by humanization PD-L1 antibody (i.e. 2.74.15.hAb4,2.74.15.hAb5,2.74.15.hAb6, 2.74.15.hAb7 and 2.74.15.hAb8) dyeing.Facs analysis display humanized antibody specifically with on DC The natural PD-L1 expressed combines.

The competitive test that 3.1FACS measures: in order to check humanized antibody whether can block PD-L1 with The combination of PD-1, by express the CHO-K1 cell of human PD-L 1 at 4 DEG C with the humanized antibody of variable concentrations Hatch 1 hour.By unconjugated antibody elution, then the people PD-1 of mice Fc-labelling is added in cell. The goat anti mouse IgG detection people PD-1 using FITC to connect and the combination of the cell expressing PD-L1, subsequently The FACSCanto II and FlowJo version software using BD Biosciences is analyzed.

By express Chinese hamster ovary celI and the variable concentrations of human PD-L 1 humanized antibody (2.74.15.hAb4, 2.74.15.hAb5,2.74.15.hAb6,2.74.15.hAb7 and 2.74.15.hAb8) or control antibodies hatch, subsequently will The people PD-1 of mice Fc-labelling adds in cell.The goat anti mouse IgG using FITC to connect detects people PD-1 With the combination of the cell expressing PD-L1, pass through facs analysis subsequently.As shown in Figure 4, all people to be measured The combination of the PD-L1 expressed on the PD-L1 antibody blocking in source PD-1 and the Chinese hamster ovary celI of conversion.

The affinity test that 3.2 surface plasma body resonant vibrations (SPR) measure: use ProteOn by SPR method XPR36 (Bio-Rad) antagonist (2.74,2.74.15.cAb (chimeric antibody of 2.74.15), 2.74.15.hAb4, 2.74.15.hAb5,2.74.15.hAb6,2.74.15.hAb7 and 2.74.15.hAb8) with the affinity of PD-L1 and combination Kinetics characterizes.Protein A/Protein (Sigma) is fixed on GLM sensing chip by amine coupling (Bio-Rad).Make the antibody flows through sensor chip of purification and captured by protein A.By chip half-twist electricity consumption Swimming buffer solution (1XPBS/0.01%Tween20, Bio-Rad) is until baseline stability.Make the people of 5 concentration PD-L1 and electrophoretic buffer flow through described antibody flow unit with flow velocity 100 μ L/min, first flow mutually for combining 240s, phase 600s of dissociating subsequently.With the H of pH 1.7 after each run₃PO₄Regenerate described chip.Use ProteOn software will combine and the Langmiur combination model of dissociation curve matching to 1:1.

As it is shown in fig. 7, by using the humanization PD-L1 antibody of surface plasma body resonant vibration detection to recombined human The affinity of PD-L1 is from 1.55E-10 to 1.18E-11mol/L.

The affinity test that 3.3FACS measures: by using the CHO-K1 cell expressing human PD-L 1, warp Facs analysis, carries out the antibodies affinity with cell surface PD-L1 and measures.Test antibodies elution buffer Liquid is with 1:2 serial dilution (1XPBS/1%BSA) and hatches at 4 DEG C 1 hour.Add two anti-goat anti-human IgG Fc FITC (3.0 moles of FITC every mole IgG, (Jackson Immunoresearch Lab)) also keeps away at 4 DEG C Light hatches 1 hour.Washed once cell resuspended in 1XPBS/1%BSA subsequently, use flow cytometry (BD) Analyze.Based on quantitative beads QuantumTM MESF Kit (Bangs Laboratories, Inc.), glimmering Light intensity will be converted into relevant to molecule/cell.Graphpad Prism5 is used to calculate K_D。

3.4 external functional examinations: (include cytokine production to estimate the response of humanized antibody regulatory T-cell And cell proliferation) ability, use antibody 2.74.15.hAb4,2.74.15.hAb5,2.74.15.hAb6,2.74.15.hAb7 Three below experiment has been carried out with 2.74.15.hAb8 and control antibodies.

3.4.1 allogeneic MLR: use person monocytic cell's enrichment kit, divides from healthy donors according to explanation From mononuclear cell.Cell is cultivated 5-7 days to induce into dendritic cell (DC).Before using 18 to 24 hours, 1 μ g/mL LPS is added in cell culture and induce DC ripe.

In order to detect the effect to the production of the IL-2 in MLR of the described humanization anti-PD-L1 antibody, make employment CD4⁺T cell enrichment kit, separates people CD4 according to description⁺T cell, is being with or without humanization subsequently In the case of anti-PD-L1 antibody or control antibodies, ripe or jejune allochthonous DC is used to sting Swash.Measure the IL-2 level in culture supernatant with ELISA at the 3rd day.As shown in Figure 8, all to be measured Anti-PD-L1 antibody (2.74.15.hAb4,2.74.15.hAb5,2.74.15.hAb6,2.74.15.hAb7 and 2.74.15.hAb8) secretion of IL-2 is added in dose-dependent mode.

In order to detect the effect to the production of the IFN γ in MLR of the described humanization anti-PD-L1 antibody, make employment CD4⁺T cell enrichment kit separates people CD4 according to description⁺T cell, subsequently be with or without humanization resist In the case of-PD-L1 antibody or control antibodies, ripe or jejune allochthonous DC is used to stimulate. Measure the IFN γ level in culture supernatant with ELISA at the 5th day.As it is shown in figure 9, all to be measured resisting -PD-L1 antibody (2.74.15.hAb4,2.74.15.hAb5,2.74.15.hAb6,2.74.15.hAb7 and 2.74.15.hAb8) The secretion of IFN γ is added in dose-dependent mode.

In order to detect the effect that the T cell in MLR is bred by described humanization anti-PD-L1 antibody, make employment CD4⁺T cell enrichment kit separates people CD4 according to description⁺T cell, subsequently be with or without humanization resist In the case of-PD-L1 antibody or control antibodies, ripe or jejune allochthonous DC is used to stimulate. By [³H] thymidine incorporation mensuration T cell propagation.As shown in Figure 10, all anti-PD-L1 antibody to be measured (2.74.15.hAb4,2.74.15.hAb5,2.74.15.hAb6,2.74.15.hAb7 and 2.74.15.hAb8) all can increase Add the T cell propagation of concentration dependant.

3.4.2 self antigen specific immune response: from identical CMV⁺Donor separates CD4⁺T cell and list Nucleus.CD4 is cultivated in the presence of CMV pp65 peptide and low dosage IL2 (20U/ml)⁺T cell.Depend on simultaneously DC is produced by cultivating mononuclear cell according to preceding method.After 5 days, add DC by pp65 peptide pulsed, subsequently Under the conditions of humanized antibody or control antibodies presence or absence, DC is added to CD4⁺T cell.The 3rd It measures the IL-2 in culture supernatant and IFN γ level with ELISA.CMVpp65 specific C D4⁺T cell Propagation by [³H] thymidine incorporation mensuration.

As shown in figure 11, by humanization PD-L1 antibody (2.74.15.hAb4,2.74.15.hAb5,2.74.15.hAb6, 2.74.15.hAb7 and 2.74.15.hAb8) improve the generation of IFN γ in specific T-cells responds.Figure 12 shows Show that humanization PD-L1 antibody adds the CMV of the autologous DC concentration dependant using the load of CMV pp65 peptide⁺- CD4⁺T cell is bred.

3.4.3Treg inhibition test: regulatory T cells (Treg) is the subgroup of T cell, is critical immune regulation Son plays an important role in maintaining self tolerance.CD4⁺CD25⁺Treg is relevant to tumor, and this is due to many The property sent out cancer patient finding, Treg quantity increases, and relevant to poor prognosis.In order to directly estimate human PD-L 1 Humanized antibody effect in suppression Treg suppression function, exists at humanized antibody or control antibodies or does not deposits Under conditions, the function of Treg is compared.In short, CD4⁺CD25⁺Tregs and CD4⁺CD25^-T cell is led to Cross MACS to separate.Under the conditions of the humanized antibody or control antibodies presence or absence of variable concentrations, compare The function of Treg.In short, CD4⁺CD25⁺Tregs and CD4⁺CD25^-T cell is separated by MACS, CD4 is co-cultured by allogeneic maturation DC⁺CD25⁺Tregs and CD4⁺CD25^-T cell (Treg:Teff ratio For 1:1).Will be without antibody or Isotype antibody as negative control.With preceding method measure cytokine generation and T cell is bred.

Figure 13 shows through the CD4 that MACS separates⁺CD25⁺Tregs and CD4⁺CD25^-T cell.Not Under the conditions of the humanized antibody of concentration or control antibodies presence or absence, co-culture with allogeneic DC CD4⁺CD25⁺Tregs and CD4⁺CD25^-T cell (Treg:Teff ratio is 1:1).Result shows, PD-L1 Antibody abolished the suppression function of Treg, and recovered the propagation of reaction-ive T cell and the secretion of IFN γ.

3.5ADCC/CDC tests: owing to human PD-L 1 is expressed in various kinds of cell type, healthy thin with tumor In order to will be to healthy PD-L1 in born of the same parents⁺The unwanted toxicity of cell is reduced to minimum, demonstrates the anti-of selection -PD-L1 humanized antibody does not has ADCC and CDC function.

3.5.1ADCC: by humanized antibody preincubate in 96 orifice plates of target cell (mDC) and variable concentrations 30min, adds PBMC (effector) with ratio 50:1 of effector/target subsequently.By described plate 37 DEG C, 5%CO₂Couveuse is hatched 6 hours.The cracking of target cell is measured by citotoxicity detection kit (Roche). Molecular Devices SpectraMax M5e microwell plate detector is used to measure optical density (OD).Trastuzumab (sieve Family name) and MCF-7 BT474 (HER2 positive) as positive control.

Figure 14 shows, the PBMC that the IL-2-of use activates as NK cell (NK) cell source and will Express the mDC of PD-L1 of high-caliber cell surface as target cell, humanization PD-L1 antibody (2.74.15.hAb4,2.74.15.hAb5,2.74.15.hAb6,2.74.15.hAb7 and 2.74.15.hAb8) does not mediates ADCC。

3.5.2CDC: by target cell (mDC), the complement (Quidel-A112) of dilution and variable concentrations Humanized antibody mixes in 96 orifice plates.By described plate at 37 DEG C, 5%CO₂Couveuse is hatched 4 hours. CellTiter glo (Promega-G7573) is used to measure target cell lysis.Rituximab (Roche) and people B Cell lymphoma cell system Raji (CD20 is positive) is as positive control.

Figure 15 shows target cell (mDC), the complement (Quidel-A112) of dilution and the people of variable concentrations Source PD-L1 antibody mixing hatches 4 hours.CellTiter glo (Promega-G7573) is used to measure target cell Cracking.Data display humanization PD-L1 antibody does not mediate CDC.

3.6 classification determined by FACS (Binning) tests: this experiment is primarily to find out these antibody and be No combination epi-position identical, coincidence or diverse.In order to check whether are humanized antibody and control antibodies In conjunction with identical epi-position, the CHO-K1 cell of human PD-L 1 and the humanized antibody of variable concentrations will be expressed at 4 DEG C Hatch 1 hour.Eluting is not associated with antibody, adds biotin labeled control antibodies in cell.Use PE even The streptomycin connect detects the combination of biotin labeled control antibodies and PD-L1 express cell, uses BD subsequently The FACSCanto II and FlowJo version software of Biosciences is analyzed.

3.7 combine mensuration across species: the cross reaction of machin and Muridae PD-L1 is measured by antibody by ELISA. The PD-L1 of people, machin and mice is coated on ELISA flat board respectively.After closing, by humanized antibody Add in plate and at room temperature hatch at least 2 hours.With goat anti-human IgG Fc-HRP detection antibody with coated The combination of antigen.Use tmb substrate to hatch 10min colour developing, terminate reaction with 2M HCl.Use Molecular Plate is read at 450nm in Device M5e microplate reader.

As shown in Figure 6, ELISA test result indicate that humanization PD-L1 to be measured and machin PD-L1 is with agent The form that amount relies on combines.But, test antibodies (2.74.15.hAb4,2.74.15.hAb5,2.74.15.hAb6, 2.74.15.hAb7 and 2.74.15.hAb8) be not combined (data do not show) with Muridae PD-L1.Additionally, also survey Try the humanized antibody of 2.27.3, but result has shown that these humanized antibodies lost with monkey PD-L1's Binding affinity (data do not show).

3.8FACS combines across family and measures: active for combining across family of detection humanized antibody, uses humanization Antibodies expresses the cell line of PD-L2, carries out two with the goat anti-human IgG Fc being connected with FITC subsequently and resists In conjunction with.PD-L1 express cell is as positive control.Corresponding blast cell system is as negative control.Use BD The cell combined is analyzed by the FACSCanto II and FlowJo version software of Biosciences.

Use humanized PD-L1 antibody that the Chinese hamster ovary celI having transfected PD-L1 or PD-L2 is dyeed, And use facs analysis.As it is shown in figure 5, humanized PD-L1 antibody specificity combines PD-L1, but not with The PD-L2 of PD-1 ligand family combines.

Although the disclosure has specifically illustrated and carries out with reference to detailed description of the invention (some of them are preferred embodiment) Describe, but it should be understood by those skilled in the art that as shown in the application can be without departing from the spirit and scope of the present invention In, can carry out on various forms and change in details.

Claims (31)

1. the monoclonal antibody separated or its Fab, they can be with less than 10^-9The Kd value of M is specifically Being combined with human PD-L 1, described Kd value is measured by plasma resonance combined techniques.

Antibody the most according to claim 1 or its Fab, it is with less than 10nM, or less than 1nM's EC₅₀Be combined with monkey PD-L1, and/or be not combined with mice PD-L1.

3., according to the antibody described in aforementioned any one claim or its Fab, it is not combined with PD-L2.

4. according to the antibody described in aforementioned any one claim or its Fab, its do not mediate ADCC or CDC or Both of which does not mediates.

5., according to the antibody described in aforementioned any one claim or its Fab, it includes heavy CDR sequences, institute State sequence to be selected from: SEQ ID NOs:1,2 and 3.

6., according to the antibody described in aforementioned any one claim or its Fab, it includes CDR sequence, institute State sequence to be selected from: SEQ ID NOs:4,5 and 6.

7. according to the antibody described in aforementioned any one claim or its Fab, it include as SEQ ID NO:1, SEQ ID NO:2 and/or the variable region of heavy chain of SEQ ID NO:3.

8. according to the antibody described in aforementioned any one claim or its Fab, it include as SEQ ID NO:4, SEQ ID NO:5 and/or the variable region of light chain of SEQ ID NO:6,

9. according to the antibody described in aforementioned any one claim or its Fab, it include as SEQ ID NO:1, SEQ ID NO:2 and/or the variable region of heavy chain of SEQ ID NO:3；With as SEQ ID NO:4, SEQ ID NO:5 and/ Or the variable region of light chain of SEQ ID NO:6.

10., according to the antibody described in aforementioned any one claim or its Fab, it includes variable region of heavy chain, wherein Described variable region of heavy chain is selected from SEQ ID NO:7, SEQ ID NO:11 and SEQ ID NO:18.

11. according to the antibody described in aforementioned any one claim or its Fab, and it includes variable region of light chain, wherein Described variable region of light chain is selected from SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:16 and SEQ ID NO: 22。

12. according to the antibody described in aforementioned any one claim or its Fab, comprising:

A) variable region of heavy chain, it includes SEQ ID NO:7；And variable region of light chain, it includes SEQ ID NO:9；

B) variable region of heavy chain, it includes SEQ ID NO:11；And variable region of light chain, it includes SEQ ID NO:13；

C) variable region of heavy chain, it includes SEQ ID NO:11；And variable region of light chain, it includes SEQ ID NO:16；

D) variable region of heavy chain, it includes SEQ ID NO:18；And variable region of light chain, it includes SEQ ID NO:13；With And

E) variable region of heavy chain, it includes SEQ ID NO:18；And variable region of light chain, it includes SEQ ID NO:22.

13. 1 kinds of antibody or its Fab, it is tied with according to the antibody described in aforementioned any one claim or its antigen Close the epi-position that fragment competing phase is same.

14. according to the antibody described in aforementioned any one claim or its Fab, its can block human PD-L 1 and its Receptor combines, and therefore provides at least one in following activity:

A) at CD4⁺T cell is induced the generation of IL-2；

B) at CD4⁺T cell is induced the generation of IFN γ；

C) induction CD4⁺The propagation of T cell；And

D) reverse T reg and suppress function.

15. according to the antibody described in aforementioned any one claim or its Fab, and it is camelised single domain antibody (camelized single chain domain antibody), bifunctional antibody (diabody), scFv, scFv dimer, BsFv, dsFv, (dsFv) 2, dsFv-dsFv', Fv fragment, Fab, Fab', F (ab') 2, ds bifunctional antibody (ds diabody), Nano antibody, domain antibodies or bivalent domain antibodies.

16. according to the antibody described in aforementioned any one claim or its Fab, and it farther includes immunoglobulin Constant region.

17. according to the antibody described in aforementioned any one claim or its Fab, and it farther includes conjugate.

18. 1 kinds of polynucleotide separated, its coding combines according to the antibody described in any one in claim 1-16 or its antigen Fragment.

19. 1 kinds of carriers, it includes the polynucleotide of separation according to claim 18.

20. 1 kinds of host cells, it includes carrier according to claim 19.

Expressing according to the antibody described in any one in claim 1-16 or the method for its Fab for 21. 1 kinds, it includes Host according to claim 20 is cultivated under conditions of the polynucleotide expressing separation according to claim 18 Cell.

22. 1 kinds of test kits, it includes according to the antibody described in any one in claim 1-17 or its Fab.

23. 1 kinds are detected people or the existence situation of monkey PD-L1 or the method for level, including by described biological sample in biological sample Contact with according to the antibody described in any one in claim 1-17 or its Fab, and determine in described sample The existence situation of people or monkey PD-L1 or level.

24. 1 kinds of discriminatings suffer from disease or the individual method of situation that PD-L1 antagonist is responded by possibility, comprising: use basis In claim 1-17, antibody described in any one or its Fab are at the biological sample to be measured from described individuality The middle existence situation determining PD-L1 or level, wherein in described biological sample, existence or the level of PD-L1 raises and represents sound The probability answered.

25. methods according to claim 24, it farther includes wanting according to right described individual administering therapeutic effective dose Ask in 1-16 the antibody described in any one or its Fab.

Monitoring therapeutic response or the method for progression of disease in the experimenter with PD-L1 antagonist for treating for 26. 1 kinds, it includes with anti- -PD-L1 antibody or its Fab are in the existence feelings determining PD-L1 in the biological sample to be measured of described individuality Condition or level.

27. 1 kinds of pharmaceutical compositions, including according to the antibody described in any one in claim 1-17 or its Fab with And one or more pharmaceutically acceptable carriers.

The method of 28. 1 kinds of situations treating the experimenter that can benefit from the immune response raised, uses including to described experimenter Therapeutically effective amount according to the antibody described in any one in claim 1-17 or its Fab.

29. methods according to claim 28, wherein said experimenter has the PD-L1 of rise and expresses.

30. are used for treating meeting from rise in preparation according to the antibody described in any one in claim 1-17 or its Fab Immune response in benefit situation medicine in purposes.

31. purposes according to claim 30, wherein said situation is cancer.