CN106432501A

CN106432501A - Novel PD-L1 resisting antibody

Info

Publication number: CN106432501A
Application number: CN201610638134.5A
Authority: CN
Inventors: 郑勇; 李竞; 陈智胜
Original assignee: Wuxi Biologics Shanghai Co Ltd
Current assignee: CStone Pharmaceuticals Shanghai Co Ltd; CStone Pharmaceuticals Suzhou Co Ltd; CStone Pharmaceuticals
Priority date: 2015-08-06
Filing date: 2016-08-05
Publication date: 2017-02-22
Anticipated expiration: 2036-08-05
Also published as: CN106432501B

Abstract

The invention provides a monoclonal antibody for a protein programmed death ligand 1 (PD-L1). The monoclonal antibody can be used for blocking linkage between the PD-L1 and a programmed death molecule 1 (PD-1), and thus the inhibiting effect of the PD-L1 on T cells expressing the PD-1 is blocked. According to the antibody provided by the invention, very effective reagents for treating a variety of cancers through regulating human immunologic functions are provided.

Description

New anti-PD-L1 antibody

Invention field

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

Background technology

More and more preclinical and clinical effectiveness evidence shows, targeting immunologic test point is becoming most promising The method for the treatment of cancer patient.Apoptosis molecule 1 is one of immunologic test point albumen, and it is in restricted T cells activity In play a major role, described T cell provides main immune resistance mechanisms, can be hided by this restriction effect tumor cell Cross immune surveillance.In the T cell of activation, the PD-1 of expression and the interaction of the PD-L1 of expression on tumor cell are to immunity Response plays negative regulator and weakens antineoplastic immune power.Expression in tumor for the PD-L1 and the esophageal carcinoma, cancer of pancreas and other types of The survival rate of cancer declines correlation, and highlighting this path can be used as new promising immunotherapy of tumors target spot.Pharmacy is public Department has been developed for multiple medicines for PD-1 path, such as Bristol-Myers Squibb Co. (BMS), Merck ＆ Co., Inc., Roche Holding Ag With GlaxoSmithKline PLC (GSK) company.The data display of clinical trial is lasting in the patient of various tumor types clinical to live Property and the early stage evidence of good safety.Nivolumab is the PD-1 medicine of BMS exploitation, and it is just being put to neck of future generation The center stage in domain.At present in 6 later stage research, in 3 in 5 cancer groups of research, treatment has promoted tumor Reduce, including 18% in 72 patients with lung cancer, in 98 melanoma patients close to 1/3rd and 33 renal carcinomas In patient 27%.It is full people's resource monoclonal IgG4 antibody by the lambrolizumab that Merck ＆ Co., Inc. develops, it acts on PD- 1, it has caught the new breakthrough index of FDA after the impressive IB data obtaining for skin carcinoma.Interim IB grinds The result studied carefully is shown in the objective antitumor reaction having 51% in 85 cancer patients, and occurs completely in 9% patient Reaction.The experimental MPDL3280A of Roche Holding Ag demonstrates it and suffers from the terminal cancer of 140 tumors carrying all size The tumor of 29 (21%) patients is reduced in person.

However, existing Therapeutic Method is not all fully up to expectations, therefore there is still a need for the antibody of new anti-PD-L1.

Invention summary

This application provides new anti-PD-L1 monoclonal antibody (particularly human antibody), encoding its polynucleotide With using its method.

In one aspect, this application provides a kind of detached monoclonal antibody or its Fab, it can be with Less than 10^-9M (for example ,≤9x10^-10M、≤8x10^-10M、≤7x10^-10M、≤6x10^-10M、≤5x10^-10M、≤4x10^-10M、 ≤3x10^-10M、≤2x10^-10M、or≤10^-10M Kd value) is specifically combined with human PD-L 1, and described Kd value passes through plasma Resonance combined techniqueses measure.

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) EC₅₀Be combined with monkey PD-L1.In some embodiments In, described antibody or its Fab are not combined with mice PD-L1, but with the similar binding affinity of human PD-L 1 with Monkey PD-L1 combines.In some embodiments, described antibody or its Fab are (to be such as less than less than 100nM 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) IC₅₀The efficiently knot of suppression people or monkey PD-L1 and its receptor (as PD-1) Close.In some embodiments, described EC₅₀Or IC₅₀It is measured by cell sorting (FACS) analysis of fluorescence-activation.

In some embodiments, described antibody or its Fab have the effector function substantially reducing. In some embodiments, described antibody or its Fab does not mediate ADCC or CDC or both of which does not mediate.

In some embodiments, antibody described herein or its Fab include heavy CDR sequences, described Sequence is selected from：SEQ ID NO:1st, 3,5,13,15,17,25,27,29,37,39 and 41.

In some embodiments, antibody described herein or its Fab include CDR sequence, described Sequence is selected from：SEQ ID NO:7th, 9,11,19,21,23,31,33 and 35.

In some embodiments, antibody described herein or its Fab are included selected from SEQ ID NO:1、 3rd, 5,7,9 and 11；Or it is selected from SEQ ID NO:13rd, 15,17,19,21 and 23；Or it is selected from SEQ ID NO:25、27、29、31、33 With 35；Or it is selected from SEQ ID NO:37th, 39,41,19,21 and 23 at least one, 2,3,4,5 or 6 CDR.

In some embodiments, the heavy chain that antibody described herein or its Fab include being selected from the group can Become area：

A) weight chain variable district, it includes SEQ ID NO:1、SEQ ID NO:3 and/or SEQ ID NO:5；

B) weight chain variable district, it includes SEQ ID NO:13、SEQ ID NO:15 and/or SEQ ID NO:17；

C) weight chain variable district, it includes SEQ ID NO:25、SEQ ID NO:27 and/or SEQ ID NO:29；And

D) weight chain variable district, it includes SEQ ID NO:37、SEQ ID NO:39 and/or SEQ ID NO:41.

In some embodiments, antibody described herein or its Fab include light chain variable district, described Light chain variable district is selected from：

A) light chain variable district, it includes SEQ ID NO:7、SEQ ID NO:9 and/or SEQ ID NO:11；

B) light chain variable district, it includes SEQ ID NO:19、SEQ ID NO:21 and/or SEQ ID NO:23；And

C) light chain variable district, it includes SEQ ID NO:31、SEQ ID NO:33 and/or SEQ ID NO:35.

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

A) weight chain variable district, it includes SEQ ID NO:1、SEQ ID NO:3 and/or SEQ ID NO:5；And light chain variable Area, it includes SEQ ID NO:7、SEQ ID NO:9 and/or SEQ ID NO:11；

B) weight chain variable district, it includes SEQ ID NO:13、SEQ ID NO:15 and/or SEQ ID NO:17；And light chain Variable region, it includes SEQ ID NO:19、SEQ ID NO:21 and/or SEQ ID NO:23；

C) weight chain variable district, it includes SEQ ID NO:25、SEQ ID NO:27 and/or SEQ ID NO:29；And light chain Variable region, it includes SEQ ID NO:31、SEQ ID NO:33 and/or SEQ ID NO:35；Or

D) weight chain variable district, it includes SEQ ID NO:37、SEQ ID NO:39 and/or SEQ ID NO:41；And light chain Variable region, it includes SEQ ID NO:19、SEQ ID NO:21 and/or SEQ ID NO:23.

In some embodiments, antibody described herein or its Fab include weight chain variable district, described Weight chain variable district is selected from SEQ ID NO:43、SEQ ID NO:47、SEQ ID NO:51 and SEQ ID NO:55.

In some embodiments, antibody described herein or its Fab include light chain variable district, described Light chain variable district is selected from SEQ ID NO:45、SEQ ID NO:49 and SEQ ID NO:53.

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

A) weight chain variable district, it includes SEQ ID NO:43；And light chain variable district, it includes SEQ ID NO:45；

B) weight chain variable district, it includes SEQ ID NO:47；And light chain variable district, it includes SEQ ID NO:49；

C) weight chain variable district, it includes SEQ ID NO:51；And light chain variable district, it includes SEQ ID NO:53；Or

D) weight chain variable district, it includes SEQ ID NO:55；And light chain variable district, it includes SEQ ID NO:49.

In some embodiments, antibody described herein or its Fab include such as 1.4.1, 1.14.4,1.20.15 and 1.46.11.

In some embodiments, antibody described herein or its Fab and antibody 1.4.1,1.14.4, 1.20.15 with 1.46.11 competes identical epi-position.In some embodiments, antibody described herein or its antigen binding The epi-position that fragment combines includes at least one of following PD-L1 amino acid residue：E58, E60, D61, K62, N63 and R113.

In some embodiments, antibody described herein or its Fab can block human PD-L 1 and its Receptor binding, and at least one of following activity is therefore provided：

A) in CD4⁺The generation of IL-2 is induced in T cell；

B) in CD4⁺The generation of IFN γ is induced in T cell；

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

D) reverse T reg suppression function.

In some embodiments, herein described antibody is monoclonal antibody, human antibody, humanized antibody, embedding Close antibody, recombinant antibodies, 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), nano antibody, Domain antibodies or bivalent domain antibodies.

In some embodiments, antibody described herein or its Fab further include immunoglobulin Constant region.

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

In some embodiments, described conjugate can be detectable label, pharmacokineticss modification part or purification Part.

On the other hand, this application provides detached polynucleotide, its coding antibody as described in the present application or it is anti- Former binding fragment.In some embodiments, the application provides polynucleotide encoding antibody as described in the present application or it is anti- The aminoacid sequence of former binding fragment.In some embodiments, this application provides including the carrier of these polynucleotide.? In some embodiments, this application provides expressing the side of one or more antibody described herein or Fab Method, it passes through to express in the carrier real by culture host cell under conditions of the antibody of polynucleotide encoding or Fab Existing.In some embodiments, the polynucleotide that the application provides are in the carrier with promoter such as SV40 promoter operationally Connect.In some embodiments, the host cell of the carrier providing including the application is Chinese hamster ovary cell, or 293F Cell.

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

On the other hand, the PD-L1 antibody of the application, for example 1.4.1,1.14.4,1.20.15 and 1.46.11 are in animal In have good toleration and higher anti-tumor in vivo activity.In some embodiments, with only apply having of solvent The control animals of similar baseline gross tumor volume are compared, and apply the animal with tumor of PD-L1 antibody described herein Gross tumor volume reduce at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, extremely Few 90% or at least 95%.

On the other hand, this application provides detecting presence or the level of PD-L1 (such as people or monkey) in biological sample Method, contact with antibody described herein or its Fab including by described biological sample, and determine described The presence of the people in sample or monkey PD-L1 or level.

On the other hand, this application provides differentiate with may to PD-L1 antagonist response disease or situation The method of body, it includes：With antibody described herein or its Fab from described individual biology sample to be measured Presence situation or the level of PD-L1 (such as people or monkey), the presence of PD-L1 or water wherein in described biological sample is determined in product Flat rise represents the probability responding.In some embodiments, methods described further includes to described individual administration effectively The antibody described herein of amount or its Fab, described individuality identified with PD-L1 antagonist may being responded Disease or situation.

The application further provides monitoring treatment reaction or progression of disease in the experimenter with PD-L1 antagonist for treating Method, it is included with antibody described herein or its Fab in described individual biological sample to be measured Determine presence 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 antigen binding fragment Section and one or more pharmaceutically acceptable carrier.In some embodiments, described pharmaceutical carriers can be for example dilute Release agent, antioxidant, adjuvant, excipient or nontoxic auxiliary substance.

On the other hand, this application provides treatment can be from the side of the situation of the experimenter of the immune response benefit raising Method, including described experimenter is applied with effective dose antibody described herein or its Fab.In some embodiment party In formula, described experimenter has the PD-L1 expression of rise.

On the other hand, there is provided antibody described herein or its Fab can be from for treatment in preparation Purposes in the medicine of situation benefiting in the immune response adjusted.In some embodiments, described situation is cancer or chronic Virus infection.

Brief description

Fig. 1 shows the combination of the facs analysis full people source PD-L1 antibody measuring and the Chinese hamster ovary celI expressing PD-1.

Fig. 2 shows that the full people source PD-L1 antibody blocking that facs analysis measure PD-1 is thin with the CHO having transfected PD-L1 The combination of born of the same parents.

Fig. 3 shows that the full people source PD-L1 antibody that facs analysis measure is specifically bound with PD-L1, and does not tie with PD-L2 Close.

Fig. 4 shows the combination of full people source PD-L1 antibody and people and machin PD-L1.

Fig. 5 shows completely moving of the PD-L1 antibody measuring by plasmon resonance and human PD-L 1 binding affinity Mechanics is from 2.26E-10 to 4.78E-10mol/L.

Fig. 6 shows that full people source PD-L1 antibody increased the generation of IFN γ in specific T-cells response.

Fig. 7 shows that Quan Renyuan anti-PD-L1 antibody promotes specific T-cell proliferative.

Fig. 8 shows that in mixed lymphocyte reaction (MLR) full people source PD-L1 antibody increased the generation of IFN γ.

Fig. 9 shows the impact of the production to IL-2 for the full people source anti-PD-L1 antibody in MLR.

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

Figure 11 shows that anti-PD-L1 antibody has reversed Treg suppression function.

Figure 12 shows that anti-PD-L1 antibody lacks ADCC in the T cell of activation.

Figure 13 shows that anti-PD-L1 antibody lacks CDC in the T cell of activation.

Figure 14 A and 14B shows the cross reactivity of anti-PD-L1 antibody and people/mice PD-1.1.14.4 by 2 μ g/ml Antibody is coated on 96 orifice plates overnight and is incubated with hPD-L1-His albumen (Figure 14 A) and mPD-L1-His albumen (Figure 14 B), with Afterwards anti-for HRP- His antibody is added and be used for detecting.

Figure 15 shows the focus residue in hPD-L1 structure, and it is the binding site of antibody 1.14.4.Data is derived from table 3.In figure color is used for distinguishing the difference between epi-position.

Figure 16 shows hPD-L1 antibody 1.14.4 well tolerable property in vivo.1.14.4 antibody using three dosage (respectively 3mg/kg, 10mg/kg and 30mg/kg) is to the humanization B-hPD-1 mice being inoculated with MC38-B7H1 colon cancer cell After carrying out multiple intraperitoneal injection, the body weight of each group mice does not occur substantially to change during testing.

Figure 17 shows that hPD-L1 antibody 1.14.4 significantly inhibits effect to growth of tumour cell in vivo.Give in antibody After medicine 19 days, the 1.14.4 antibody (respectively 3mg/kg, 10mg/kg and 30mg/kg) of three dosage all shows tumour growth Suppression ratio (TGI)>40% significant antitumor action.

Detailed Description Of The Invention

The following description of the application is only the numerous embodiments that the application is described.Therefore, concrete modification discussed herein Mode 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 Easily draw multiple equivalent way, change and modifications it should be understood that such equivalent embodiments are included in the scope of the invention Interior.The all documents quoted in this application, including public publication, patents and patent applicationss all by way of reference in full It is incorporated to.

Definition

" antibody " one word in the present invention include arbitrarily can in conjunction with the immunoglobulin of certain specific antigen, monoclonal antibody, Polyclonal antibody, multi-specificity antibody or bispecific (bivalent) antibody.One natural complete antibody comprise two heavy chains and Two light chains.Every heavy chain is made up of a variable region and first, second, third constant region；Every light chain is by a variable region and one Constant region forms.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 in " Y " Type, the cervical region of Y-shaped structure by two articles of heavy chains second and the 3rd constant region form, it passes through disulfide-bonded.Y-shaped structure Every arm include the wherein variable region of a heavy chain and the first constant region, its variable region with a light chain and constant region knot Close.The variable region of light chain and heavy chain determines the combination of antigen.The variable region of every chain, all containing three hypervariable regions, claims complementary decision Area (CDR) (CDR of light chain (L) comprises LCDR1, LCDR2, LCDR3, and the CDR of heavy chain (H) comprises HCDR1, HCDR2, HCDR3). Kabat can be passed through in the CDR border of the antibody disclosed in the present invention and Fab, Chothia or Al-Lazikani names Method name or identification.(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.Dec21-28；342(6252):877-83 (1989)；Kabat E.A. etc., National Institutes of Health, Bethesda, Md. (1991)).Wherein, three Individual CDR is spaced apart by the side continuous part being referred to as framework region (FR), and framework region is more highly conserved than CDR and forms one Individual support supports hypermutation ring.The constant region of heavy chain and light chain is unrelated with antigen binding, but has multiple effector functions.Antibody foundation The aminoacid sequence of CH is segmented into several classes.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, such 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 " one word in the application, refers to by the antibody moiety containing one or more CDR or any Other conjugated antigens but there is no a kind of antibody fragment that the antibody fragment of complete antibody structure is formed.Fab Example includes, but not limited to as bifunctional antibody (diabody), Fab, Fab', F (ab')₂, Fv fragment, disulfide bond stable Fv fragment (dsFv), (dsFv)₂, the stable bifunctional antibody (ds of bispecific dsFv (dsFv-dsFv'), disulfide bond Diabody), single-chain antibody molecules (scFv), scFv dimer (bifunctional antibody of bivalent), Bivalent single-chain antibody (BsFv), Multi-specificity antibody, camelised single domain antibody (camelized single domain antibody), nano antibody, domain antibodies With bivalent domain antibodies.Fab can be combined identical antigen with maternal antibody.In some embodiments, antigen knot Closing fragment can be containing the one or more CDR from certain specific human antibody, and transposing is to from one or more difference human antibodies Framework region.

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

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

“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. The Fc section of antibody 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 light chain variable district and weight chain variable district or passes through a peptide Engineered antibody that chain is formed by connecting (Huston JS etc., Proc Natl Acad Sci USA, 85:5879(1988)).

" single-chain antibody Fv-Fc " or " scFv-Fc " refers 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) " is all referring to containing two V_HDomain 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 from camelidae (camel, one-humped camel and yamma) derive obtain.Although Disappearance light chain, camelised antibodies (camelized antibodies) have the antigen binding repertoire (Hamers- of confirmation 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)).The antigen-binding units that the known acquired immunity that the variable region (VHH domain) of heavy chain antibody is minimum produces (Koch-Nolte 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 domain being derived from heavy chain antibody and two constant region CH2 With CH3 composition.

" bifunctional antibody (diabody) " includes the little antibody fragment with two antigen binding sites, wherein this fragment Containing the V being connected on same polypeptide chain_HDomain and V_LDomain (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 domains it Between linker very short, so that on same chain two domains can not be mutually matched, thus forcing the complementation in two domains and another chain Domain is matched, and forms two antibody combining sites.This two antibody combining sites can targeting to combine identical or different antigen (or anti- Former epi-position).

" domain antibodies " refer to contain only the antibody fragment of a weight chain variable district or a light chain variable district.In some situations Under, two or more V_HDomain by a peptide linker covalent bond and forms bivalent domain antibodies.Two V of bivalent domain antibodies_HDomain Can targeting in identical or different antigen.

In some embodiments, " (dsFv)₂" contain three peptide chains：Two V_HPass through a peptide linker between group It is connected, and pass through 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 in V_H1And V_L1Between pass through disulfide-bonded.

" bispecific dsFv " or " dsFv-dsFv " contains three polypeptide chains：V_H1-V_H2Group, wherein both heavy chains lead to Cross peptide linker (such as：Long elastic linker) be connected, and pass through disulfide bond respectively with V_L1And V_L2Group combines, and each pair is led to The heavy chain light chain crossing disulfide bond pairing 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 group_HV with another group_LCooperation Form two basic change site, this two basic change site can targeting combine same antigen (or epitope) or not synantigen (or anti- Former epi-position).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), wherein V_H1And V_L1Cooperation, V_H2And V_L2Cooperation, and The pairing of each cooperation has different antigenic specificities.

Term " Quan Renyuan " used herein, when for antibody or Fab, refers to described antibody or anti- Former binding fragment has certain aminoacid sequence or is made up of described aminoacid sequence, and described aminoacid sequence corresponds to by people or people Ammonia that immunocyte produces or from the antibody derived from non-people source such as transgenic nonhuman animal for example utilizing human antibody library Base acid sequence, or the sequence of other coding human antibodies.In some embodiments, human antibody does not comprise from non- The amino acid residue (particularly antigen binding residues) of human antibody.

Term " humanization " used herein, when for antibody or Fab, refers to from non- The CDR of people animal, derive from Ren FR area, and from the antibody of constant region (as applicable) of people or antigen binding fragment Section.Because humanized antibody or Fab have the immunogenicity of reduction, it can be used as in some embodiments The therapeutic agent of people.In some embodiments, described non-human animal be mammal for example mice, rat, rabbit, goat, sheep, Cavia porcelluss or hamster.In some embodiments, described humanized antibody or Fab except CDR sequence be inhuman source In addition, substantially all it is made up of people's source sequence.In some embodiments, described can include and it from Ren FR area The human antibody identical aminoacid sequence being derived from, or it can include some amino acid changes, for example, less than 10,9,8, 7th, 6,5,4,3,2 or 1 amino acid changes.In some embodiments, this amino acid change can be only present in heavy chain FR area, Exist only in light chain FR area or be concurrently present in two chains.In some preferred implementations, described humanized antibody includes People source FR1-3 and people source JH and J κ.

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

" 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 is expressed in tire Disk, spleen, lymph node, thymus, heart, fetus liver, and also find to be present on many tumors or cancerous cell.PD-L1 with On the T cell of activation, B cell and medullary cell, receptor PD-1 or B7-1 of expression combines.PD-L1 can draw with the combination of its receptor Transduction of signaling is bred come the activation to cytokine production and T cell to suppress TCR mediation.Therefore, PD-L1 is in particular event In, such as in pregnancy, autoimmune disease, tissue transplantation, Main Function is played to suppression immune system, and it is recognized For allowing tumor or cancerous cell to evade immunologic test point escape from immune response.

" anti-PD-L1 antibody " used herein be refer to enough to provide diagnosis and/or therapeutic use affine Property the antibody that specifically binds with PD-L1 (such as people or monkey PD-L1).

" specific binding " or " specific combination " in the application refers to, refers to two intermolecular nonrandom combinations anti- Should, the such as reaction between antibody and antigen.In some embodiments, the antibody of the application or its Fab and people and/ Or monkey PD-L1 specific binding, and its binding affinity (K_D)≤10^-6M is (such as：≤5x10^-7M ,≤2x10^-7M ,≤10^-7M, ≤5x10^-8M ,≤2x10^-8M ,≤10^-8M ,≤5x10^-9M ,≤2x10^-9M ,≤10^-9M ,≤10^-10M, about 10^-10M、10^-10M is extremely 10^-9M、10^-10M to 10^-8.5M or 10^-10M to 10^-8M).KD in the application refers to dissociate speed and the ratio with reference to speed (koff/kon), can be measured by the method for surface plasma resonance, such as using the such as instrument of Biacore.

The ability of " block and combine " or " competitive same epi-position " in the application refers to antibody or its antigen binding fragment The interaction of two intermolecular combinations (such as human PD-L 1 and anti-PD-L1 antibody) is suppressed to any detectable degree by section Ability.In some embodiments, block the antibody of two intermolecular combinations or Fab can be intermolecular by two In conjunction with interaction suppression at least 50%.In some embodiments, such inhibitory action can be more than 60%, is more than 70%, more than 80%, or it is 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 same epitope on possible conjugated antigen.For example, if this Shen The antibody that please provide or its Fab block Example antibody, such as 1.4.1,1.14.4,1.20.15 and 1.46.11 with The combination of human PD-L 1, then described antibody or its Fab may be considered that identical with the antibodies of those examples Epi-position.

Specific amino acid residue will pass through such as Alanine scanning mutagenesis (alanine scanning in epi-position Mutagenesis) it is mutated, identify reduction or stop protein bound mutation." Alanine scanning mutagenesis " are permissible Some residues for identification impact epi-position and other compounds in connection or the protein of protein interaction or area The method in domain.Residue in protein or one group of target residues are passed through neutral or negative charge aminoacid replacement (most preferably third Propylhomoserin or Poly(Ala) Alanine homopolymer, or conservative aminoacid replacement).The amino acid residue mutation of any combination reducing described protein Or encode the degree of its codon mutation and exceed threshold value or maximize compared with other mutation and reduce what described protein combined Mutation is likely in described protein bound epi-position.In some embodiments of the application, important to PD-L1 antibody Epi-position includes following at least one amino acid residue E58, E60, D61, K62, N63 and R113.

" 1.4.1 " used herein refers to have as SEQ ID NO：Weight chain variable district shown in 43, such as SEQ ID NO：Light chain variable district shown in 45 and the full human monoclonal antibody of humanized IgG 4 isotype constant region.

" 1.14.4 " used herein refers to have as SEQ ID NO:Weight chain variable district shown in 47, such as SEQ ID NO:Light chain variable district shown in 49 and the full monoclonal human antibody of humanized IgG 4 isotype constant region.

" 1.20.15 " used herein refers to have as SEQ ID NO:Weight chain variable district shown in 51, such as SEQ ID NO:Light chain variable district shown in 53 and the full monoclonal human antibody of humanized IgG 4 isotype constant region.

" 1.46.11 " used herein refers to have as SEQ ID NO:Weight chain variable district shown in 55, such as SEQ ID NO:Light chain variable district shown in 49 and the full monoclonal human antibody of humanized IgG 4 isotype constant region.

In this application when " conservative replacement " is for aminoacid sequence, referring to, an amino acid residue is had with another 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), between neutral hydrophilic side chains residue between (such as Cys, Ser, Thr, Asn and Gln), acid side-chain residue (such as Trp, Tyr between (such as His, Lys and Arg) or direction side-chain residue between (such as Asp, Glu), basic side chain aminoacid And Phe) carry out conservative replacement.Conservative replacement known in the art generally will not cause the significant changes of protein conformation structure, therefore It is capable of the biological activity of retaining protein.

When " Percent sequence identity " is used 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 necessary, in candidate sequence, with reference sequence Identical aminoacid (or nucleic acid) residue accounts for the percentage ratio of aminoacid (or nucleic acid) residue of described candidate sequence.Described aminoacid The conservative replacement of residue can consider or can be not considered as identical residue.Instrument disclosed in this area can be passed through, for example BLASTN, BLASTp (American National Biotechnology Information center website (NCBI), also can be found in, 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. etc., 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 compared to determine aminoacid The Percent sequence identity of (or nucleic acid) sequence.Those skilled in the art can use described instrument default parameterss or according to The suitable adjusting parameter of needs comparing, such as by selecting suitable algorithm.

" T cell " used herein includes CD4⁺T cell, CD8⁺T cell, T assist 1 type T cell, T to assist 2 types T thin Born of the same parents, T assist 17 type T cell and suppressor T lymphocyte.

" effector function " used herein refers to Fc area and its effector such as the C1 complex and Fc receptor of antibody In conjunction with biological activity.The C1q that exemplary effector function is included on antibody and C1 complex interact induction complement according to The antibody dependent cellular mediation of the Fc receptor binding induction on bad sexual cell toxicity (CDC), the Fc area and effector lymphocyte of antibody Cytotoxicity (ADCC) and phagocytosis.

" cancer " or " cancer situation " in the application refers to any be situated between by tumor or Malignant cellular growth, propagation or transfer Lead, and cause solid tumor and for example leukemic medical condition of non-physical knurl." tumor " in the present invention refers to tumor and/or pernicious The solid substance of cell.

" treatment " or " therapy " of certain situation is included by prevention or mitigate certain situation, reduces certain situation and rise or send out The speed opened up, reduces the risk developing certain situation, prevents or postpones the symptom development related to certain situation, reduces or whole Only the symptom related to certain situation, produces the reverse wholly or in part of certain situation, cures certain situation, or more group Close.For cancer, " treatment " or " therapy " can refer to suppress or slow down tumor or Malignant cellular growth, breeding, or transfer, Or more some combinations.For tumor, " treatment " or " therapy " includes clearing all or partial tumor, suppresses or subtracts Slow growth and metastasis of tumours, prevents or delays the development of tumor, or more some combinations.

The material of " by separating " is through manually being changed by naturalness.If certain " by separating " occurs in nature Material or composition, then it has changed by or departs from its initial condition, or the two all has generation.For example, a certain living animal Naturally occurring polynucleotide or polypeptide are not detached in vivo, but if these polynucleotide or polypeptide are therewith in natural shape The material coexisting under state is sufficiently separated and is existed with sufficiently pure state, then may be considered " by separating ".In some embodiment party In formula, the purity of antibody and Fab is at least 90%, 93%, 95%, 96%, 97%, 98%, 99%, it is by electricity Swimming method (as SDS-PAGE, isoelectrofocusing, capillary electrophoresis), or chromatography (as ion exchange chromatography or reversed-phase HPLC) is really Fixed.

In the present invention, " carrier " refers to, can be inserted and make this albumen with encoding the polynucleotide manipulation of certain albumen Obtain a kind of vehicle of expression.Carrier can be used for converting, transduce or transfection host cell is so as to the hereditary material unit that carries Part is expressed in host cell.For example, carrier includes：Plasmid, phasmid, coemid, artificial chromosome such as ferment Female artificial chromosome (YAC), bacterial artificial chromosome (BAC) or artificial chromosome (PAC), phage such as λ phagocytosis derived from P1 Body or M13 phage, and animal viruss etc..Animal viruss species as carrier have retrovirus (include slow viruss), Adenoviruss, adeno-associated viruses, herpesviruss (as herpes simplex virus), poxvirus, baculoviruss, human papillomavirus, nipple are many Tumor vacuolating virus (as SV40).Carrier can containing various control expression element, including promoter sequence, transcriptional initiation sequence, Enhancer sequence, selection element and reporter gene.In addition, carrier also can contain replication origin.Carrier may also include assistance It enters the composition of cell, including but not limited to, virion, liposome or protein coat.

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

" related to PD-L1 or related disease " in the present invention refers to, any due to PD-L1 (such as：Human PD-L 1) table Reach or activity is raised and lowered and leads to, aggravates or situation that other are related.

" therapeutically effective amount " or " effective dose " in the present invention refers to, certain medicine effectively treatment is related to human PD-L 1 The dosage of disease or situation or concentration.For example, for the purposes of the antibody disclosed in the present invention or its Fab, Therapeutically effective amount is under this dosage or concentration, this antibody antigen conjugates can clear all or Partial tumors, suppression or Slow down tumour growth, the growth of cell of suppression mediation cancer situation or breeding, suppression Nasopharyngeal neoplasms, mitigate any and tumor Or the related symptom of cancer situation or labelling, prevent or delay tumor or the development of cancer situation, or more some combinations.

" medicinal acceptable " refers to supporting agent, solvent, diluent, adjuvant and/or the salt of indication, generally speaking in chemistry And/or physically mutually compatible with other dispensings in preparation, 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 CD279, It is the known critical immune checkpoint receptor expressed by activating T cell, it adjusts immunosuppressive action.PD-1 ligand 1 (PD- L1 it is) transmembrane protein expressed in kinds of tumor cells, stromal cell or the 40kDa on both, it is combined with PD-1.Suppression Interaction between PD-1 and PD-L1 can improve t cell response and thus mediate active anticancer.

In some embodiments, this application provides exemplary full human monoclonal antibody 1.4.1,1.14.4, 1.20.15 and 1.46.11, as shown in table 1, and heavy chain or light-chain variable sequence are also listed below its CDR sequence.

Table 1

1.4.1-VH(30511):(SEQ ID NO:43 is aminoacid, SEQ ID NO:44 is nucleic acid) heavy chain CDR1-3: SEQ ID NO:1st, 3,5 is aminoacid sequence and SEQ ID NO:2nd, 4,6 is nucleotide sequence.

V section：IGHV4-39*01

D section：IGHD1-26*01

J section：IGHJ4*02

1.4.1-VL(30027):(SEQ ID NO:45 is aminoacid and SEQ ID NO:46 is nucleic acid) light chain CDR1-3： SEQ ID NOs:7th, 9,11 is aminoacid sequence and SEQ ID NO:8th, 10,12 is nucleotide sequence：

V section：IGLV3-1*01

J section：IGLJ2*01

1.14.4-VH(29812):(SEQ ID NO:47 is aminoacid, SEQ ID NO:48 is nucleic acid) heavy chain CDR1-3： SEQ ID NOs:13rd, 15,17 is aminoacid sequence and SEQ ID NO:14th, 16,18 is nucleotide sequence：

V section：IGHV3-23*01

D section：IGHD5-5*01

J section：IGHJ4*02

1.14.4-VL with 1.46.11-VL (29841):(SEQ ID NO:49 is aminoacid, SEQ ID NO:50 is core Acid) light chain CDR1-3：SEQ ID NOs:19th, 21,23 is aminoacid sequence and SEQ ID NO:20th, 22,24 is nucleotide sequence：

V section：IGLV3-21*02

J section：IGLJ2*01

1.20.15-VH(30712):(SEQ ID NO:51 is aminoacid, SEQ ID NO:52 is nucleic acid) light chain CDR1- 3：SEQ ID NOs:25th, 27,29 is aminoacid sequence and SEQ ID NO:26th, 28,30 is nucleotide sequence：

V section：IGHV4-39*01

D section：Do not determine

J section：IGHJ4*02

1.20.15-VL(29907):(SEQ ID NO:53 is aminoacid, SEQ ID NO:54 is nucleic acid) light chain CDR1- 3：SEQ ID NOs:31st, 33,35 is aminoacid sequence and SEQ ID NO:32nd, 34,36 is nucleotide sequence：

V section：IGLV3-1*01

J section：IGLJ2*01

1.46.11-VH(30626):(SEQ ID NO:55 is aminoacid, SEQ ID NO:56 is nucleic acid) light chain CDR1- 3：SEQ ID NOs:37th, 39,41 is aminoacid sequence and SEQ ID NO:38th, 40,42 is nucleotide sequence：

V section：IGHV3-23*01

D section：IGHD5-5*01

J section：IGHJ4*02

1.46.11-VL(29841):(SEQ ID NO:49 is aminoacid, SEQ ID NO:50 is nucleic acid) light chain CDR1- 3：SEQ ID NOs:19th, 21,23 is aminoacid sequence and SEQ ID NO:20th, 22,24 is nucleotide sequence：

V section：IGLV3-21*02

J section：IGLJ2*01

In some embodiments, described anti-PD-1 antibody and its Fab include the heavy chain CDR being selected from the group Sequence：SEQ ID NO:1st, 3,5,13,15,17,25,27,29,37,39 and 41.In some embodiments, described anti-PD- L1 antibody and its Fab include the CDR sequence being selected from the group：SEQ ID NO:7、9、11、19、21、23、31、 33 and 35.

In some embodiments, the heavy chain that described anti-PD-L1 antibody and its Fab include being selected from the group can Become area：Weight chain variable district, it includes SEQ ID NO:1、SEQ ID NO:3 and/or SEQ ID NO:5；Weight chain variable district, its Including SEQ ID NO:13、SEQ ID NO:15 and/or SEQ ID NO:17；Weight chain variable district, it includes SEQ ID NO: 25、SEQ ID NO:27 and/or SEQ ID NO:29；And weight chain variable district, it includes SEQ ID NO:37、SEQ ID NO: 39 and/or SEQ ID NO:41.

In some embodiments, the light chain that described anti-PD-L1 antibody and its Fab include being selected from the group can Become area：Light chain variable district, it includes SEQ ID NO:7、SEQ ID NO:9 and/or SEQ ID NO:11；Light chain variable district, its Including SEQ ID NO:19、SEQ ID NO:21 and/or SEQ ID NO:23；And light chain variable district, it includes SEQ ID NO:31、SEQ ID NO:33 and/or SEQ ID NO:35.

In some embodiments, described anti-PD-L1 antibody and its Fab include：A) weight chain variable district, its Including SEQ ID NO:1、SEQ ID NO:3 and/or SEQ ID NO:5；And light chain variable district, it includes SEQ ID NO:7、 SEQ ID NO:9 and/or SEQ ID NO:11；B) weight chain variable district, it includes SEQ ID NO:13、SEQ ID NO:15 and/ Or SEQ ID NO:17；And light chain variable district, it includes SEQ ID NO:19、SEQ ID NO:21 and/or SEQ ID NO:23； C) weight chain variable district, it includes SEQ ID NO:25、SEQ ID NO:27 and/or SEQ ID NO:29；And light chain variable district, its Including SEQ ID NO:31、SEQ ID NO:33 and/or SEQ ID NO:35；And d) weight chain variable district, it includes SEQ ID NO:37、SEQ ID NO:39 and/or SEQ ID NO:41；And light chain variable district, it includes SEQ ID NO:19、SEQ ID NO:21 and/or SEQ ID NO:23.

It will be understood by those skilled in the art that the CDR sequence providing in table 1 can be modified one or more to comprise The replacement of aminoacid, the binding affinity with human PD-L 1 of the biologic activity being thus improved such as raising.For example, may be used To be produced using display technique of bacteriophage and to express antibody variants storehouse (such as Fab or FcFv variant), subsequently screening and human PD-L 1 There is the antibody of affinity.In another example, the combination of described antibody and human PD-L 1 can be simulated with computer software and differentiate The amino acid residue of combination interface is formed on antibody.The replacement of these residues can be avoided preventing binding affinity from reducing, or Can be substituted with these residues of targeting to form higher combination.In some embodiments, at least one of CDR sequence (or whole) replace is conservative replacement.

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

In some embodiments, described anti-PD-1 antibody and its Fab are full people sources.Described full people source Antibody does not have binding affinity of the immunogenicity or reduction such as often observed in humanized antibody etc. to ask in human body Topic.

In some embodiments, the anti-PD-L1 antibody in described full people source and its Fab include weight chain variable Area, wherein said weight chain variable district is selected from SEQ ID NO:43、SEQ ID NO:47、SEQ ID NO:51、SEQ ID NO:55, Have at least 80% therewith (for example, at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%th, 99%) homologous sequence of sequence iden；And/or light chain variable district, wherein said light chain variable district is selected from SEQ ID NO:45、SEQ ID NO:49、SEQ ID NO:53, and have at least 80% therewith (for example, at least 85%, 88%, 90%, 91%th, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homologous sequence of sequence iden.These full people sources Antibody remains the binding affinity with human PD-L 1, preferably with exemplary antibodies：1.4.1,1.14.4,1.20.15 and 1.46.11 level is similar.

In some embodiments, the anti-PD-L1 antibody in described full people source and its Fab include a) weight chain variable Area, it includes SEQ ID NO:43；And light chain variable district, it includes SEQ ID NO:45；B) weight chain variable district, it includes SEQ ID NO:47；And light chain variable district, it includes SEQ ID NO:49；C) weight chain variable district, it includes SEQ ID NO:51；With light Chain variable region, it includes SEQ ID NO:53；Or d) weight chain variable district, it includes SEQ ID NO:55；And light chain variable district, its Including SEQ ID NO:49.

The application further comprises PD-L1 antibody anti-with the application and its Fab competes the antibody of same epitope With its Fab.In some embodiments, described antibody is with less than 10^-6M, be less than 10^-7M, be less than 10^-7.5M, it is less than 10^-8M, be less than 10^-8.5M or be less than 10^-9M or be less than 10^-10The IC of M₅₀Value (i.e. half-inhibition concentration) block 1.4.1,1.14.4, 1.20.15 the combination with 1.46.11 and people or monkey PD-L1.IC₅₀Value is tested such as ELISA by competitiveness and is measured, and radioactivity is joined Body competition binding determination method, and facs analysis determination.

In some embodiments, herein described anti-PD-L1 antibody and its Fab can be with≤10^-6M (e.g.,≤5x10^-7M、≤2x10^-7M、≤10^-7M、≤5x10^-8M、≤2x10^-8M、≤10^-8M、≤5x10^-9M、≤2x10^-9M、 ≤10^-9M、10^-10M, about 10^-10M、10^-10M to 10^-8.5M or 10^-10M to 10^-8M binding affinity (Kd)) is special with human PD-L 1 Property combine, it is measured by plasma resonance combined techniqueses.Binding affinity can use K_DValue represents, it is by when antigen and antigen The combination of binding molecule is reached dissociation rate during balance and is calculated with the ratio (koff/kon) of association rate.Described antigen Binding affinity (such as K_D) suitably can be determined by proper method known in the art, methods described includes using instrument As such as the plasma resonance combined techniqueses of Biacore (participate in such as Murphy, M.et al, Current protocols in protein science,Chapter 19,unit19.14,2006).

In some embodiments, herein described antibody and its Fab and human PD-L 1 are with 0.1nM-100nM The EC of (such as 0.1nM-50nM, 0.1nM-30nM, 0.1nM-20nM or 0.1nM-10nM or 0.1nM-1nM)₅₀(i.e. half combines Concentration) combine.Described antibody can pass through methods known in the art such as sandwich assay such as ELISA with the combination of human PD-L 1, Western blot, FACS or other binding tests measure.In exemplary example, by test antibodies (i.e. resists) and fixation The human PD-L 1 changed or the cell of expression human PD-L 1 combine, and subsequently wash uncombined antibody off, and introduce labelling two resist, and it can be with One anti-binding therefore, it is possible to detect combination one resist.Can carry out described when using immobilized PD-L1 on microplate reader plate Detection, or described detection can be carried out using facs analysis when the cell using expression human PD-L 1.In some embodiments, Herein described antibody and its Fab are with the EC50 of 1nM to 10nM or 1nM to 5nM (being measured using facs analysis) (i.e. 50% valid density) is combined with human PD-L 1.

In some embodiments, herein described antibody and its Fab be with 0.2nM-100nM (for example 0.2nM-50nM, 0.2nM-30nM, 0.2nM-20nM, 0.2nM-10nM or 1nM-10nM) IC₅₀Suppression human PD-L 1 is subject to it The combination of body, it passes through competitiveness and records.

In some embodiments, herein described antibody and its Fab suppress human PD-L 1 and its receptor In conjunction with, and the T cell thus providing including such as induced activation produces cytokine (as CD4⁺T cell and CD8⁺T cell), The propagation of the T cell of induced activation is (as CD4⁺T cell and CD8⁺T cell) and reverse regulatory T reg suppression sexual function life Thing activity.Exemplary cytokine includes IL-2 and IFN γ.Term " IL-2 " refers to interleukin II, its be cell because Subsignal transduction molecule, the activity of leukocyte (such as leukocyte) adjusting in immune system.Term " interferon gamma (IFN γ) " is by NK cell (NK) cell, NK T cell, CD4⁺And CD8⁺The cytokine that T cell produces, it is huge biting carefully The important activator of born of the same parents and the inducing agent of MHC inductor (MHC) developed by molecule.Cytokine Generation can be determined by methods known in the art, such as ELISA.These methods may also be used for detecting T cell propagation, including [³H] thymidine incorporation mensure.

Described anti-PD-L1 antibody and its Fab are that human PD-L 1 is specific.In some embodiments, institute State antibody and its Fab is not combined (as people PD-L2) with PD-L2.For example, compare people with the binding affinity of PD-L2 The 15% of the binding affinity of PD-L1,10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% are also low.

In some embodiments, described antibody and its Fab to be to be not higher than 100nM, for example, not higher than 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 The EC of 0.01nM₅₀(being measured by ELISA) is combined with monkey PD-L1.In some embodiments, described antibody and its antigen binding Fragment is combined with monkey PD-L1 with the EC50 of about 1nM-10nM.

In some embodiments, described antibody and its Fab are not combined with Mus PD-L1, but with monkey PD-L1 Be combined with the binding affinity similar with human PD-L 1.For example, exemplary antibodies 1.4.1,1.14.4,1.20.15 and 1.46.11 Combination with Mus PD-L1 cannot be detected with conventional combination mensuration such as ELISA or facs analysis, and ELISA or FACS detects this A little antibody are to monkey PD-L1 with the affinity similar to human PD-L 1 or EC₅₀Value combines.

In some embodiments, described anti-PD-L1 antibody and its Fab have reduction or elimination Effector function.In some embodiments, described anti-PD-L1 antibody and its Fab have IgG4 isotype Constant region, it has reduction or elimination effector function.The effector functions such as such as ADCC and CDC can result in expression PD- The cytotoxicity of the cell of L1.Many cells include normal cell and can express PD-L1.In order to avoid normally thin to these Born of the same parents produce potentially undesirable toxicity, and some embodiments of antibody of the present invention and its Fab have fall Effector function that is low or even eliminating.Known have many tests for estimating ADCC or CDC activity, such as Fc receptor binding examination Test, C1Q. Binding experiment and cell cracking method, those skilled in the art can easily select.It is not intended to be restrainted by theoretical Tie up, it is believed that the antibody with reduction or elimination effector function such as ADCC and CDC will not cause the cell to expression PD-L1 The cytotoxicity of (such as those normal cells) or it is reduced to minimum degree, therefore avoids undesirable side effect. And meanwhile, the tumor cell of expression PD-L1 therefore can cannot escape from immunologic test point with anti-PD-L1 antibodies, by This its can be identified and eliminated by immune system.

In some embodiments, anti-PD-L1 antibody described herein and its Fab have the pair of reduction Effect.Anti- PD-L1 antibody and its Fab can have full humanized IgG sequence, therefore its immunogen to example as mentioned Property be less than humanized antibody.Again for example, described anti-PD-L1 antibody and its Fab can have IgG4 form To eliminate ADCC and CDC.

In some embodiments, the advantage of anti-PD-L1 antibody described herein and its Fab is it Can with there is the combination of immunogenic material, such as tumor cell, the tumor antigen of purification and transfected with encoding immune stimulating factor Cell, tumor vaccine.Additionally, described anti-PD-L1 antibody and its Fab can include in combination treatment, bag Include standard chemotherapeutic regimens and X-ray therapy, the small molecule therapy based on target spot, other emerging immunologic test points adjust agent therapy.? In some embodiments, described antibody and its Fab can serve as antibody-drug conjugates, bispecific or multivalence The base molecule of antibody.

Anti- PD-L1 antibody described herein and its Fab can be monoclonal antibody, polyclonal antibody, Human antibody, humanized antibody, chimeric antibody, recombinant antibodies, bi-specific antibody, traget antibody, bivalent antibody or anti-only Special type antibody.Recombinant antibodies are the antibody of the non-animal preparation using recombination method in vitro.Bi-specific antibody or bivalent resist Body is the artificial antibody of the fragment with two kinds of different monoclonal antibodies, and it can be in conjunction with two kinds of different antigens." bivalence " Antibody and its Fab include two antigen binding sites.Two antigen binding sites can in conjunction with same antigen, or Person can be each coupled to different antigens, and in this case, antibody or Fab are " bispecific ".

In some embodiments, anti-PD-1 antibody described herein and its Fab are human antibodies. In some embodiments, prepare described human antibody using recombination method.For example, it is possible to prepare transgenosis animal is for example little Mus so as to the transgenic of carrier source immunoglobulin gene or transfection chromosome, and therefore with suitable antigen such as people source PD- Human antibody can be produced after 1 immunity.Human antibody can separate from such transgenic animal, or alternatively, can To be prepared by hybridoma technology, the splenocyte of described transgenic animal and immortal cell line are merged described complete to generate secretion The hybridoma of human antibody.Exemplary transgenic animal include but is not limited to, Omni rat, its endogenous rat immunity The expression of globulin gene is deactivated and is genetically engineered to comprise functional recombination human source immunoglobulin gene simultaneously Seat；Omni mice, the expression of its endogenous mouse immunoglobulin genes is deactivated and is genetically engineered to comprise to have simultaneously There are J- locus disappearance and the recombination human source immunoglobulin locus of C-kappa mutation.OmniFilc, it is that transgenic is big Mus, the expression of its endogenous rat immunoglobulin gene is deactivated, and is genetically engineered single to comprise to have simultaneously VkJk light chain that is total, recombinating and the recombination human source immunoglobulin locus of feature heavy chain.Specifying information please be further Referring to：Osborn M.et al,Journal of Immunology,2013,190:1481-90；Ma B.et al,Journal of Immunological Methods 400–401(2013)78-86；Geurts A.et al,Science,2009,325: 433；United States Patent (USP) 8,907,157；European patent 2152880B1；European patent 2336329B1, it is all by quoting entirety simultaneously Enter the application.Also other suitable transgenic animal can be used, for example, HuMab mice is (referring specifically to Lonberg, N.et al.Nature 368(6474):856 859 (1994)), Xeno- mice (Mendez et al.Nat Genet., 1997,15: 146 156), TransChromo mice (Ishida et al.Cloning Stem Cells, 2002,4:91 102) and VelocImmune mice (Murphy et al.Proc Natl Acad Sci USA, 2014,111:5153–5158), Kymouse transgenic mice (Lee et al.Nat Biotechnol, 2014,32:, and transgene rabbit 356 363) (Flisikowska et al.PLoS One,2011,6:e21045).

In some embodiments, anti-PD-L1 antibody described herein and its Fab are camelised single domains Antibody (camelized single chain domain antibody), bifunctional antibody (diabody), scFv, scFv bis- Aggressiveness, 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.

In some embodiments, anti-PD-L1 antibody described herein and its Fab further include to exempt from Epidemic disease immunoglobulin constant area.In some embodiments, constant region for immunoglobulin includes heavy chain and/or constant region of light chain.Described CH includes CH1, CH1-CH2 or CH1-CH3 area.In some embodiments, constant region for immunoglobulin can enter One step includes one or more modifications to obtain required property.For example, it is possible to described constant region is modified with reducing or disappear Except one or more effector function is to strengthen FcRn receptor binding or to introduce one or more cysteine residues.

In some embodiments, described anti-PD-L1 antibody and its Fab comprise conjugate further.Can To envision, the antibody in the present invention or its Fab 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 by covalent bond, Other modes and the institutes such as affine combination, embedded, equal combination (coordinate binding), complexation, combination, mixing or addition State antibody or antigen conjugates connect.In some embodiments, antibody disclosed by the invention and Fab can lead to The method crossing engineering makes its specific site beyond containing epi-position bound fraction, and these sites can be used to sew with reference to one or more Compound.For example, such site can comprise one or more reactive amino acid residues, such as cysteine residues and histidine Residue, for assisting the covalent attachment with conjugate.In some embodiments, antibody can be connected in conjugate indirectly, or passes through Another conjugate is connected.For example, described antibody or its Fab biotin-binding, then combines second indirectly Conjugate, it is connected with Avidin.Described conjugate can be detectable labelling, pharmacokineticss modification part, purification portion Divide or cytotoxic moieties.The example of detectable labelling can include fluorescent labeling (such as fluorescein, rhodamine, dansyl, algae Lactoferrin or texas Red), enzyme-substrate label (such as horseradish peroxidase, alkali phosphatase, luciferase, glucose Amylase, lysozyme, carbohydrate oxidase or beta-D-galactosidase), radiosiotope (for example,¹²³I、¹²⁴I、¹²⁵I、¹³¹I、³⁵S 、³H、¹¹¹In、¹¹²In、¹⁴C、⁶⁴Cu、⁶⁷Cu、⁸⁶Y、⁸⁸Y、⁹⁰Y、¹⁷⁷Lu、²¹¹At、¹⁸⁶Re、¹⁸⁸Re、¹⁵³Sm、²¹²Bi、and³²P, other Lanthanide series, luminescent marking), chromophoric moiety, digoxin, biotin/avidin, DNA molecular or gold to be to be detected.At certain In a little embodiments, described conjugate can be that pharmacokineticss modify part such as PEG, and it helps extend the half-life of antibody. Other suitable polymer include such as carboxymethyl cellulose, glucosan, polyvinyl alcohol, Polyvinylpyrrolidone, ethylene glycol/the third Diol copolymer etc..In some embodiments, described conjugate can be purification part such as magnetic bead." cytotoxic moieties " It can be any reagent that cell is harmful to or may damage or kill with cell.The example of cytotoxic moieties includes, but not Be limited to, paclitaxel, cytochalasin B, Gramicidin D, ethidium bromide, ipecine, mitomycin, etoposide, teniposide, Vincristine, vinblastine, Colchicine, amycin, daunorubicin, dihydroxy anthracin diketone, mitoxantrone, light god are mould Element, actinomycin D, 1- boldenone, glucocorticoid, procaine, tetracaine, lignocaine, Propranolol, puromycin And the like, (for example, methotrexate, Ismipur, 6- thioguanine, cytosine arabinoside, 5-fluorouracil reach antimetabolite Kappa), alkylating agent (such as chlormethine, phosphinothioylidynetrisaziridine chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), Cyclophosphamide, busulfan, mitobronitol, streptozotocin, ametycin and cis- dichlorodiamine platinum (II) (DDP) cisplatin), anthracene Ring class antibiotic (such as daunorubicin (daunomycin in the past) and amycin), (for example dactinomycin (is formerly referred to as antibiotic D actinomycin D), bleomycin, mithramycin and anthramycin (AMC)) and antimitotic agent (such as vincristine and length Spring alkali).

Polynucleotide and recombination method

This application provides encoding the detached polynucleotide of anti-PD-L1 antibody and its Fab.In some realities Apply in mode, described detached polynucleotide include the nucleotide sequence in one or more such as tables 1, and its coding is as in table 1 CDR sequence.

In some embodiments, described detached polynucleotide encoding weight chain variable district include the sequence being selected from the group Row：SEQ ID NO:44、SEQ ID NO:48、SEQ ID NO:52、SEQ ID NO:56, and have at least 80% therewith The sequence of (for example, at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) is same The homologous sequence of one property.In some embodiments, described detached polynucleotide encoding light chain variable district include being selected from down The sequence of group：SEQ ID NO:46、SEQ ID NO:50、SEQ ID NO:54, and have at least 80% therewith (for example, at least 85%th, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence iden same Source sequence.In some embodiments, the percentage ratio of described homogeneity is derived from the degeneracy of genetic code, and the albumen encoding Sequence keeps constant.

Using recombinant technique well known in the art, can will include encoding described anti-PD-L1 antibody and its antigen binding fragment The carrier of the polynucleotide of section (for example including the sequence shown in table 1) introduces host cell and is used for cloning (DNA amplification) or gene Expression.In another embodiment, described antibody can be obtained by the method for homologous recombination well known in the art.Encode described list The DNA of clonal antibody can be separated by conventional method and sequencing (such as can use oligonucleotide probe, this probe can be special Property is combined with the heavy chain of encoding said antibody and the gene of light chain).Variety carrier is available.Carrier component generally includes, but It is not limited to, below one or more：Signal sequence, replication origin, one or more marker gene, enhancement sequences, startup Son is (for example：SV40, CMV, EF-1 α) and transcription terminator.

In some embodiments, described carrier system includes mammal, antibacterial, Yeast system etc., and will include matter Grain be 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 Carrier can include plasmid or viral vector (for example, replication defect type retrovirus, adenoviruss and adeno-associated viruses).

The carrier including the polynucleotide of encoding said antibody and its Fab can be introduced host cell to use In clone or gene expression.In the present invention be applied to clone or express described carrier DNA host cell be prokaryotic cell, Yeast or above-mentioned higher eukaryotes.The prokaryotic cell being applied to purposes of the present invention includes eubacteria such as, gram negative bacteria or Gram positive bacteria, for example, enterobacteriaceae, e.g., escherichia coli, Enterobacter, Erwinia, klebsiella, become Shape Bacillus, Salmonella, e.g., and mouse typhuss sramana (family name) bacillus, Serratia, e.g., serratia marcescens, and will he Bordetella, and Bacillus are such as, bacillus subtilises and Bacillus licheniformis, pseudomonass such as, bacillus pyocyaneus and streptomycete.

In addition to prokaryotic cell, eukaryotic microorganisms such as filamentous fungis or yeast also can make host cell clone or expression is compiled The carrier of the anti-PD-L1 antibody of code.Saccharomyces cerevisiae, or bakery yeast is the most frequently used low eucaryon host microorganism.But, many Other genus and species and strain are all the more commonly used and be suitable in the present invention, such as schizosaccharomyces pombe；Kluyveromyceses host is such as, newborn Sour kluyveromyces, Kluyveromyces fragilis (ATCC 12,424), Bulgarian kluyveromyces (ATCC 16,045), Wei Shi Kluyveromyces (ATCC 24,178), Crewe hero yeast (ATCC 56,500), fruit bat kluyveromyces (ATCC36,906), resistance to Hot kluyveromyces and yeast Kluyveromyces marxianus；Yarrowia lipolytica (EP 402,226)；Pichia pastoris phaff (EP 183,070)；Candida mycoderma；Trichoderma reesei (EP 244,234)；Neurospora；Prosperous yeast is permitted in west, such as：Prosperous yeast is permitted in west；With Filamentous fungis, such as：Neurospora, penicillium, curved neck be mould and aspergillosiss, such as：Hook nest aspergillosis and aspergillus niger.

There is provided in the present invention is applied to the host cell expressing glycosylated antibodies or its Fab by many cells Biologically-derived obtain.The example of no vertebrate cell includes plant and insect cell.Have been found that multiple baculoviruss strains (baculoviral strains) and its variant and corresponding permissive insect host cells (permissive insect Host cells), come from such as following host：Fall army worm (caterpillar), Aedes Aegypti (mosquito), Aedes albopictus (mosquito Son), Drosophila melanogaster (fruit bat) and silkworm.Multiple Strain for transfection are public Ke get, such as Autographa californica nuclear The Bm-5 mutation of polyhedrosis viruses and bombyx mori nuclear polyhydrosis virus, these viruses all can use in the present invention, particularly use In transfection Spodopterafrugiperda cells.The culture plant cell of Cotton Gossypii, Semen Maydiss, Rhizoma Solani tuber osi, Semen sojae atricolor, petunia, Fructus Lycopersici esculenti and Nicotiana tabacum L. Can be used as host.

But, most interested is vertebrate cell, and the culture (tissue culture) of vertebrate cell has become as routine operation. Available mammalian host cell example has, the monkey-kidney cells CV1 system (COS-7, ATCC CRL 1651) of SV40 conversion；People Embryonic kidney cell system (293 or suspension culture 293 cell subclone, 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)；African green monkey kidney cell (VERO- 76, ATCC CRL-1587)；Human cervical carcinoma cell (HELA, ATCC CCL 2)；Madin-Darby canine kidney(cell line) (MDCK, ATCC CCL 34)；Cloth Method sieve rat hepatocytes (BRL 3A, ATCC CRL 1442)；Human pneumonocyte (W138, ATCC CCL75)；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 In preferred embodiment, described host cell is 293F cell.

With the above-mentioned expression or the cloning vehicle transformed host cell that produce anti-PD-L1 antibody, and by it conventional Cultivate in Nutrient medium, be suitable for evoked promoter after described Nutrient medium is modified, select transformed cell or amplification to compile The gene of code aim sequence.

It is used in the present invention producing described antibody or the host cell of its Fab can be trained in multiple culture medium Support.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) can be used for cultivating described host cell.In addition, Any in 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 host cell in U.S. Patent application Re.30,985 Culture medium.These culture medium all can add necessary hormone and/or other somatomedin (as insulin, transferrinss or table Skin growth factor), salt (as sodium chloride, calcium chloride, magnesium chloride and phosphate), buffer (as HEPES), nucleotide is (as gland Thuja acid and thymus pyrimidine), antibiotic (as gentamycin), trace element (be defined as final concentration generally inorganic in micro-molar range Compound), and glucose or the energy source being equal to therewith.Described culture medium also can contain appointing of debita spissitudo well known in the art What his necessary additive.The condition of described culture medium, the such as conditions of similarity such as temperature, pH value, for selecting the place for expression The previously used condition of chief cell, is known to those of ordinary skill.

When using recombinant technique, described antibody can generate in intracellular, wall film space, or direct secretion is in culture medium. If described antibody intracellular generate, first remove host cell or cracking segment granule remains, for example, can by centrifugation or Ultrasonic method.Carter et al.,Bio/Technology 10:163-167 (1992) describes to be secreted into large intestine bar The detached method of antibody in bacterium wall film space.In brief, deposit in Sodium Acetate Trihydrate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) Melt cell paste (cell paste) under the conditions more than about 30 minutes.It is centrifuged off cell debriss.As described antibody-secreting To 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.Protease all can be added in any aforesaid step Inhibitor such as PMSF is suppressing protein degradation, and antibiotic is to prevent the growth of accidental contamination thing.

The antibody being obtained from described cell can carry out purification, such as hydroxyapatite chromatography, gel electricity using purification process Swimming, dialysis, DEAE- cellulose ion exchange chromatography post, ammonium sulfate precipitation, saltout and affinity chromatography, wherein affinity chromatography is Preferably purification technique.The Fc domain that there is any immunoglobulin in the species of described antibody and described antibody determines Protein A as affinity ligand if appropriate for.Protein A can be used for the antibody Ji Yu people γ 1, γ 2 or γ 4 heavy chain for the purification (Lindmark et al.,J.Immunol.Meth.62:1-13(1983)).Protein G is applied to all Mus source isomers and people γ3(Guss et al.,EMBO J.5:1567 1575(1986)).Agarose is the most frequently used affinity ligand attaching substratum, but Also can be selected for other substrate.The stable substrate of mechanical force such as controlled pore glass or poly- (styrene) benzene can compared with agarose Realize faster flow velocity and shorter process time.As this antibody contains CH3 domain, then can use Bakerbond ABX.TM tree Fat carries out purification (J.T.Baker, Phillipsburg, N.J.).Determine that other albumen are pure also dependent on the antibody needing to obtain The technology changed, as the fractional distillation in ion exchange column, ethanol precipitation, reversed-phase HPLC, silica gel chromatography, is based on anion or cation friendship Change heparin sepharose chromatograph (as poly-aspartate post), chromatofocusing, SDS-PAGE and the ammonium sulfate precipitation of resin.

After any any preliminary purification step, the method for low pH hydrophobic interaction chromatograph is can use to process containing interested Antibody and the mixture of impurity, with the elution buffer of pH about 2.5-4.5, are preferably carried out (for example, from about under low salt concn 0 arrives 0.25M salinity).

Test kit

This application provides including the test kit of described anti-PD-L1 antibody and its Fab.In some embodiment party In formula, described test kit is used for the presence situation of PD-L1 in biological sample for the detection or level.Described biological sample can wrap Include cell or tissue.

In some embodiments, described test kit includes anti-PD-L1 antibody and its antigen being conjugated with detectable label Binding fragment.In some embodiments, described test kit includes unlabelled anti-PD-L1 antibody and its Fab, And further include that can be combined labelling with unlabelled anti-PD-L1 antibody and its Fab two are resisted.Described reagent Box may further include operation instruction and the packaging separating 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.Applicable substrate or instrument can be such as microwell plate and reagent paper.

Pharmaceutical composition and Therapeutic Method

The application further provide including described anti-PD-L1 antibody and its Fab pharmaceutical composition and One or more pharmaceutically acceptable carriers.

Medicinal acceptable supporting agent in pharmaceutical composition disclosed in the present application may include, for example, medicinal acceptable Liquid, gel or solid carriers, aqueous media, non-aqueous phase medium, antimicrobial material, isotonic material, buffer, antioxidant, Anesthetis, suspending agent/dispersant, chelating agen, diluent, adjuvant, adjuvant or non-toxic auxiliary substances, other well known in the art group Point or more multiple combination.

Applicable component may include, for example, antioxidant, filler, binding agent, disintegrating agent, buffer, preservative, lubrication Agent, stir taste agent, thickening agent, coloring agent, emulsifying agent or stabilizer for example sugar and cyclodextrin.Applicable antioxidant may include, for example, Methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercapto glycerol, sulfydryl Acetic acid, sulfydryl Sorbitol, butyl methyl methoxybenzene, Yoshinox BHT and/or propylgallate.As public in present invention institute Open, include one or more antioxidant such as in a kind of compositionss containing antibody disclosed by the invention or its Fab Methionine, can will reduce the oxidation of described antibody or its Fab.Minimizing to Oxidation can prevent or reduce The reduction of binding affinity, thus improving Antibody stability and extending the shelf life.Therefore, in some embodiments, the present invention Containing the antibody described in one or more or its Fab and one or more antioxidant example in the compositionss providing As methionine.Invention further provides multiple methods, by by the antibody providing in the present invention or its antigen binding fragment Section is mixed with one or more antioxidant, such as methionine, can prevent described antibody or the oxidation of its Fab, extend Its shelf-life and/or improve its activity.

Further, medicinal acceptable supporting agent may include, for example, 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 are for example： The fixed oil of plant origin, cottonseed oil, Semen Maydis oil, Oleum sesami or Oleum Arachidis hypogaeae semen, bacteriostatic or funguses inhibition concentration Under antibiotic substance, isotonic agent such as：Sodium chloride or glucose, buffer are such as：Phosphate or citric acid phthalate buffer, antioxidation Agent is such as：Sodium bisulfate, local anesthetic is such as：Procaine hydrochloride, suspending agent and dispersant are such as：Sodium carboxymethyl cellulose, hydroxypropyl Ylmethyl cellulose or Polyvinylpyrrolidone, emulsifying agent is such as：Polysorbate80 (tween 80), chelating reagent such as EDTA (second Ethylenediamine tetraacetic acid (EDTA)) or EGTA (double (the 2- amino-ethyl ether) tetraacethyl of ethylene glycol), ethanol, Polyethylene Glycol, propylene glycol, hydroxide Sodium, hydrochloric acid, citric acid or lactic acid.Antibacterial as supporting agent can add in the pharmaceutical composition in multidose container, its bag Include phenols or cresol, mercurial, benzyl alcohol, chlorobutanol, methyl and propyl para-hydroxybenzoate, thiophene hydrargyrum spread, Neotran Ammonium and chlorine Benzethonium.Applicable adjuvant may include, for example, water, salt, glucose, glycerol or ethanol.Applicable non-toxic auxiliary substances May include, for example, emulsifying agent, pH, stabilizer, solubilizing agent, or Sodium Acetate Trihydrate, sorbitan monolaurate, The material of Emulphor FM or cyclodextrin etc.

Described pharmaceutical composition can be liquid solution, suspension, Emulsion, pill, capsule, tablet, extended release preparation Or powder.Oral formulations can include the Mannitol of standard vector such as pharmaceutical grade, Lactose, starch, magnesium stearate, polyvinyl pyrrole Alkanone, saccharin sodium, cellulose, magnesium carbonate etc..

In some embodiments, described pharmaceutical composition is formulated to injectable compositionss.Injectable medicine group Compound can be prepared in the form of any routine, for example, liquid flux, suspending agent, emulsifying agent or be applied to generation liquid flux, outstanding Floating agent or the solid form of emulsifying agent.Ejection preparation may include used aseptic and/or pyrogen-free solution, using front existing and solvent In conjunction with aseptic drying soluble substance, such as lyophilized powder, include subcutaneous, injection sterile suspensions i.e., using front existing with Jie The insoluble product of aseptic drying that matter combines, 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 and carries pin Syringe in.This area is known, and the preparation of all drug administration by injection should be aseptic apyrogeneity.

In some embodiments, suitable by antibody disclosed in the present application or its Fab are dissolved in certain Aseptic freeze-dried powder can be prepared in solvent.Described solvent can contain a kind of restructuring solution improving powder or being obtained by powder Stability, or improve powder or other pharmacology components of restructuring solution.Applicable adjuvant includes, but are not limited to, water, glucose, Minashi sugar alcohol, Fructose, corn syrup, xylitol, glycerol, glucose, sucrose or other applicable materials.Solvent can contain buffering Liquid, such as citrate buffer, sodium phosphate or kaliumphosphate buffer or buffer known to other this technologies skilled person, in one kind In embodiment, the pH of buffer is neutrality.Carry out described dissolving is carried out subsequent under standard conditions known in the art Filtration sterilization, then lyophilizing is obtained preferable preparation.In one embodiment, the solvent subpackage of gained is frozen to tubule Dry.Every tubule can accommodate described anti-PD-L1 antibody or its Fab or a combination thereof of single dose or multidose Thing.Charge weight in every tubule can be slightly higher than (such as 10% is excessive) needed for each dosage or needed for multidose, thus Ensure sampling accurately and be administered accurately.Lyophilized powder can store under suitable condition, such as arrives room temperature scope at about 4 DEG C.

With water for injection by molten for the lyophilizing grain weight preparation obtaining for drug administration by injection.In one embodiment, can will freeze It is molten that dry powder adds to weight in aseptic apirogen water or other applicable liquid carrier.Accurate amount is determined by the therapy selecting, can root Determine according to empirical value.

Additionally provide Therapeutic Method, apply including by the antibody described herein of therapeutically effective amount or its Fab With to needing its experimenter, thus treating or preventing the situation related to PD-L1 or disease.On the other hand, additionally provide The method of the situation of experimenter that treatment can benefit from the immune response raising, controls including applying to described its experimenter of needs Treat antibody described herein or its Fab of effective dose.

The treatment effective dose of antibody provided herein or its Fab depends on well known in the art many Kind of factor, such as body weight, the age, passing medical history, now with potentiality for the treatment of, the health status of object and cross infection, allergy, super Quick and side effect, and the degree of route of administration and tumor development.One skilled in the art (such as doctor or veterinary) can basis These or other condition or requirement reduce in proportion or raise dosage.

In some embodiments, the antibody that the present invention provides or its Fab can in treatment effective dose about Between 0.01mg/kg to about 100mg/kg administration (for example, about 0.01mg/kg, about 0.5mg/kg, about 1mg/kg, about 2mg/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 is with about 50mg/kg or less dosed administration, in some embodiments, dosage is 10mg/ Kg or less, 5mg/kg or less, 1mg/kg or less, 0.5mg/kg or less or 0.1mg/kg or less.Certain given dose Can in multiple doses at intervals, for example once a day, twice daily or more, monthly twice or more, weekly, every two weeks one Secondary, every once three weeks, monthly or every two months or more moon once.In some embodiments, dosage can be with treatment Process changes.For example, in some embodiments, initial dosages are high than subsequent dose dosage.In some embodiments In, dosage reaction according to administration object in treatment process is adjusted.

Dosage regimen can reach peak optimization reaction (as therapeutic response) by adjustment.For example, can carry out single dose administration or The dosed administration of a period of time point multiple separations.

Antibody disclosed in the present invention and Fab can be administered by administering mode well known in the art, for example, note Penetrate administration (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 related to PD-L1 and disease can be Ia disease or disease.In some embodiments, institute State the situation related to PD-L1 and disease include tumor and cancer, such as nonsmall-cell lung cancer, small cell lung cancer, renal cell carcinoma, Colorectal carcinoma, ovarian cancer, breast carcinoma, pancreatic cancer, gastric cancer, bladder cancer, the esophageal carcinoma, mesothelioma, melanoma, incidence cancer, first Shape adenocarcinoma, sarcoma, carcinoma of prostate, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphoma, myeloma, gill fungus sample meat Bud swells (mycoses fungoids), merkel's cellses cancer and other malignant hematologic disease, such as Classical Hodgkin Lymphoma (CHL), Primary Mediastinal large B cell lymphoid tumor, T cell/histiocytic richness B cell lymphoma, the positive and negative PTLD of EBV Diffusivity large B cell lymphoid tumor (DLBCL) related with EBV, plasmablast lymphoma, lymphoma extranodal NK/Tcell, nasopharynx Cancer lymphoma primary effusion related with HHV8, Hodgkin lymphoma, central nervous system (CNS) tumor, such as constitutional CNS lymphoma, ridge axle tumor, brain stem glioma.In some embodiments, described tumor and cancer are metastatic, Especially express the metastatic tumo(u)r of PD-L1.In some embodiments, the described situation related to PD-L1 and disease include Autoimmune disease, such as systemic lupus erythematosus (sle) (SLE), psoriasises, systemic sclerosiss, Autoimmune Diabetes.At certain In a little embodiments, the described situation related to PD-L1 and disease include chronic viral infection, such as hepatitis B, the third type liver Inflammation, herpesviruss, Epstein-Barr virus, HIV (human immunodeficiency virus), cytomegaloviruses, herpes simplex virus I-type, herpes simplex virus 2 types, human papillomaviruss, the virus infection of adenoviruss, the related herpesviruss epidemic diseases of card ripple cc sarcoma, thin circovirus virus (Torquetenovirus), JC virus or BK virus etc..

Using method

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

In some embodiments, this application provides treating the side of the situation related to PD-L1 or disease in individuality Method, including the PD-L1 antibody described herein applying therapeutically effective amount or its Fab.In some embodiments In, described individuality is accredited as with the disease that PD-L1 antagonist may be responded or situation.

In target biological tissue, the presence situation of PD-L1 and level can indicate that the individuality in described biological sample source is No PD-L1 antagonist may be responded.PD- can determined using multiple methods in described individual biological sample to be measured The presence situation of L1 or level.For example, it is possible to described biological sample to be measured is exposed to anti-PD-L1 antibody or its antigen binding Fragment, its PD-L1 protein binding with expression simultaneously detects the PD-L1 albumen of expression.Alternatively, it is possible to use such as qPCR, reversion Record PCR, microarray, SAGE, FISH etc. detect PD-L1 in nucleic acid expression level.In some embodiments, described testing sample From cancerous cell or tissue, or the immunocyte entering tumor.In some embodiments, in described biological sample to be measured PD-L1 there is a possibility that or level rise expression response.Term " rise " used in this application refers to resist with using identical In the reference sample that health check-up is surveyed, PD-L1 protein level is compared, and is being treated using antibody described herein or its Fab In test sample product detection PD-L1 protein level total increase be no less than 10%, 15%, 20%, 25%, 30%, 35%, 40%th, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or more.Described reference sample can be from health or The control sample obtaining no in the individuality of disease, or from the individuality in testing sample source the health that obtains or no disease sample Product.For example, described reference sample can be near testing sample (as tumor) or adjacent no disease sample.

Antibody disclosed by the invention and Fab can be administered alone or with one or more other treatment means or Agents in combination is administered.For example, antibody disclosed by the invention and Fab can be performed the operation (such as with chemotherapy, radiotherapy, treatment of cancer Tumorectomy), the therapy of complication that leads to of one or more Antiemetics or other chemotherapy or any other be used for cancer Therapeutic substance or the therapeutic substance of any disease being mediated by PD-L1 be combined.In some such embodiments, this When the antibody of disclosure of the invention and Fab and one or more therapeutic substance combination, can control with described one or more Treat material to be administered simultaneously, in some such embodiments, described antibody and Fab can be used as same medicines A part for compositions is administered simultaneously.But, do not need together with the antibody of other treatment material " combination " and antigen conjugates When administration or be administered in same compositionss with this therapeutic substance.The implication " being combined " in the present invention is additionally included in another treatment Before or after material, the antibody of administration and antigen conjugates are also considered as and this therapeutic substance " combination ", even if described antibody Or its Fab is administered by different modes of administration with second material.In the conceived case, open with the present invention Antibody or other treatment material associated with its Fab can refer to the side of the product description of this other treatment material Method medication, 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 abilities Method known to domain.

In some embodiments, described therapeutic substance can induce or strengthen the immunoreation for cancer.For example, swell Tumor vaccine can be used for inducing the immunne response to some tumors or cancer.Cytokine therapy can be used for improving and resists tumor Former to immune submission.The example of cytokine therapy includes but is not limited to interferon such as interferon-ALPHA, β and γ, and colony pierces The sharp factor such as macrophage CSF, granular leukocyte macrophage CSF and granulocyte-CSF, interleukin such as IL-1, IL-1 α, IL-2, IL- 3rd, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 and IL-12, tumor necrosis factor such as TNF-α and TNF- β.Can also be using the reagent of inactivation immunosuppressant target, such as TGF-β inhibitor, IL-10 inhibitor and FasL inhibitor. Another group reagent includes activating those reagent of the immune response for tumor or cancerous cell, for example, improves t cell activation (as T Cell co-stimulatory molecules agonist such as CTLA-4, ICOS and OX-40), and improve Dendritic Cell Function and antigen and pass Those being in.

The application further provides monitoring treatment reaction or progression of disease in the experimenter with PD-L1 antagonist for treating Method, including with herein described anti-PD-L1 antibody or its Fab from described individual biology sample to be measured Presence situation or the level of PD-L1 is determined in product.In some embodiments, methods described further includes biology sample to be measured PD-L1 level in product is compared with the PD-L1 level in the comparable sample obtaining from same individuality before this, is wherein surveying In examination biological sample, the increase of PD-L1 level reduces or is slowed or stopped, and shows that positive therapeutic response or controlled disease are entered Exhibition.Described can be same type of sample with testing sample than sample, but it is before the treatment or in treatment initial stage Obtain from same individual.

Following examples 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, materials It is not limited to the present invention with method, and be simply specific embodiment to be described within the scope of the invention.This area is ripe Practice technical staff and can develop equivalent compositionss, material and method without creative and without departing from the scope of the invention.Should Understand, can still be included in the scope of the present invention in multiple changes that the method for the present invention is made.Inventor is intended to this The variation of sample is included within the scope of the invention.

Embodiment 1：The generation of antibody hybridoma

1.1 it is immune：The female OMT rat of～8 week old is (available from Open Monoclonal Technology, Inc., Paro Many, the U.S. difficult to understand) it is injected at the human PD-L 1 ECD protein sensitization in 10 μ g TiterMax via foot pad, subsequently it is used in phosphoric acid within every 3 days PD-L1ECD albumen in alumina gel adjuvant excites through foot pad until being suitable for merging.Pass through ELISA or FACS detection every two weeks anti- PD-L1 antibody serum titer.

1.2 cell fusion：First 3 days in fusion, animal receives human PD-L 1 ECD albumen in the PBS of 10 μ g through lumbar injection Last exciting.Merging the same day, harvesting lymph-node cell and prepare single cell suspension.By obtain lymphocyte with Myeloma cell (P3) is mixed with suitable ratio.Cell mixture is washed and with 2.0x10 in ECF solution⁶cells/mL Density resuspended.Carry out cell fusion using BTX 2000 electro' asion instrument.

1.3 first times and second doma supernatant screening：After 37 DEG C of cultures 7-14 days, by a part of hybridoma Clear liquid analyzes detection by Mirrorball.Briefly, doma supernatant is diluted 5 times with 1XPBS.Expression PD-L1's CHO-K1 cell is mixed with the antibody of secondary fluorescence labelling and DraQ5.The cell adding 20 μ L in each hole of 384 orifice plates mixes The doma supernatant sample of the dilution of compound and 20 μ L at room temperature lucifuge are incubated at least 2 hours until preparingIt is analyzed on high sensitivity microwell plate cell instrument.By using expressing the CHO-K1 cell of PD-L1 through FACS Checking positive cell.With doma supernatant sample to cell dyeing, the subsequently goat anti mouse IgG Fc with being connected with FITC Carry out two anti-bindings.With corresponding parental cells system as negative control.FACSCanto II using BD Biosciences And FlowJo version software is analyzed to the cell combining.

1.4 sub-clone：Carry out Ya Ke using being verified as the positive hybridoma cell line combining with PD-L1 express cell Grand.Briefly, for every strain of hybridoma system, it is diluted to every 200 μ L5 or 1 by cell counting and in Cloning Medium Individual cell.Carry out bed board with 200 μ L/ holes in 96 orifice plates.Plate is placed in 37 DEG C, 5%CO₂Incubation is up to subsequent analysis.

1.5 homotype tests：With the goat anti-rat IgG1 in 50 μ L/ holes, IgG2a, IgG2b, IgG3, IgA and IgM antibody Elisa plate is coated respectively with 1 μ g/mL.After closure, the doma supernatant sample of 50 μ L is added every hole, under room temperature, be incubated 2 Hour.Goat anti-rat kappa light chain-HRP is used as detection antibody.Using tmb substrate incubation 10min colour developing, use 2M HCl terminating reaction.Read plate at 450nm in ELISA microplate reader.

1.6 Binding experiment based on cell：For inspection human antibody and the binding activity of target, human PD-L 1 will be expressed CHO-K1 or maturation dendritic cell (mDC) with human antibody combine, the subsequently goat anti-human IgG with being connected with FITC Two anti-bindings that Fc combines.Corresponding parental cells system is used as negative control.Using BD Biosciences's FACSCanto II and FlowJo version software is analyzed to the cell combining.

For arrogant mouse hybridoma for human PD-L 1 antibodies transfected total length human PD-L 1 CHO thin Born of the same parents, the two resistive connection merging facs analysis that subsequently the goat anti-rat IgG Fc with being connected with FITC is combined.As shown in figure 1, it is anti- Body 1.4.1,1.14.4,1.20.15 and 1.46.11 are with the EC of about 1nM₅₀The specifically PD-L1 knot with expression on Chinese hamster ovary celI Close.

Embodiment 2：Change Fc part and purification

Antibody in 293F cell culture supernatant uses protein A affinity chromatography column purification.

Embodiment 3：The sign of human antibody

The competitive test that 3.1FACS measures：In order to check whether human antibody can block the knot of PD-L1 and PD-1 Close, the CHO-K1 cell of expression human PD-L 1 is incubated 1 hour with the human antibody of variable concentrations at 4 DEG C.Will be unconjugated Antibody elution, then adds the people PD-1 of mice Fc- labelling in cell.The goat anti mouse IgG detection being connected using FITC People PD-1 and the combination of the cell of expression PD-L1, subsequently use the FACSCanto II and FlowJo of BD Biosciences Version software is analyzed.

By the human antibody of Chinese hamster ovary celI and the variable concentrations of expression human PD-L 1 (1.4.1,1.14.4,1.20.15 and 1.46.11) or control antibodies incubation, subsequently the people PD-1 of mice Fc- labelling is added in cell.The goat being connected using FITC Anti-mouse IgG detects the combination of people PD-1 and the cell of expression PD-L1, subsequently passes through facs analysis.As shown in Fig. 2 being needed The combination of the full people source PD-L1 antibody blocking PD-1 the surveying and PD-L1 of expression on the Chinese hamster ovary celI of conversion, and 1.4.1, 1.14.4,1.20.15 and 1.46.11 shows the IC50 value of about 10nM.

The affinity test that 3.2 surface plasma body resonant vibrations (SPR) measure：ProteOn XPR36 is used by SPR method (Bio-Rad) antagonist and the affinity of PD-L1 and binding kineticses are characterized.Protein A/Protein (Sigma) is even by amine Connection is fixed on (Bio-Rad) on GLM sensing chip.Make the antibody flows through sensor chip of purification and captured by protein A.By chip Rotate 90 ° and use electrophoresis buffer solution (1XPBS/0.01%Tween20, Bio-Rad) up to baseline stability.Make 5 concentration Human PD-L 1 and electrophoretic buffer described antibody flow unit is flowed through with flow velocity 100 μ L/min, be first with reference to mutually flow 240s, Subsequently dissociation phase 600s.The H of pH 1.7 is used after each run₃PO₄Regenerate described chip.Using ProteOn software will in conjunction with and Dissociation curve matching is to 1:1 Langmiur combination model.

As shown in figure 5, by using surface plasma body resonant vibration detection full people source PD-L1 antibody to restructuring human PD-L 1 Affinity be from 4.78E-10 to 2.26E-10mol/L.

The affinity test that 3.3 FACS measure：By using expressing the CHO-K1 cell of human PD-L 1, through facs analysis, Carry out measuring with the antibodies affinity of cell surface PD-L1.Test antibodies elution buffer is with 1 to 2 multiple series Dilution (1XPBS/1%BSA) is simultaneously incubated 1 hour at 4 DEG C.Add two anti-goat anti-human IgG Fc FITC (Jackson Immunoresearch Lab) and at 4 DEG C lucifuge be incubated 1 hour.Subsequently washed once cell and in 1XPBS/1%BSA Resuspended, using flow cytometry (BD) analysis.Based on quantitative beads QuantumTM MESF Kit (Bangs Laboratories, Inc.), fluorescence intensity will be converted into related to molecule/cell.Calculated using Graphpad Prism5 K_D.

3.4 external functional examinations：(include cytokine production and thin in order to estimate the response of human antibody regulatory T-cell Born of the same parents breed) ability, carried out three below experiment.

3.4.1 allogeneic MLR：Using person monocytic cell's enrichment kit, separated single from healthy donors according to explanation Nucleuss.By cell culture 5-7 days to be divided into dendritic cell (DC).Before using 18 to 24 hours, 1 μ g/mL LPS is added Enter induction DC in cell culture ripe.

Using people CD4⁺T cell enrichment kit, separates CD4 according to description⁺T cell, is subsequently being with or without Quan Renyuan In the case of anti-PD-L1 antibody or control antibodies, stimulated using ripe or jejune allochthonous DC.Exist respectively Measure IL-2 and IFN γ level in culture supernatant with ELISA within 3rd day and the 5th day.By [³H] mensure T is thin for thymidine incorporation Born of the same parents breed.

As shown in figure 9, all full people sources PD-L1 antibody (1.4.1,1.14.4,1.20.15 and 1.46.11) to be measured with Dose-dependent mode increased the secretion of IL-2.As shown in figure 8, all full people source PD-L1 antibody to be measured (1.4.1, 1.14.4,1.20.15 and 1.46.11) secretion of IFN γ is increased in dose-dependent mode.As shown in Figure 10, all to be measured Full people source PD-L1 antibody (1.4.1,1.14.4,1.20.15 and 1.46.11) all can increase concentration dependant T cell propagation.

3.4.2 self antigen specific immune response：PBMC and mononuclear cell is separated from identical donor.In CMV PBMC is cultivated in the presence of pp65 peptide and low dosage IL2 (20U/ml).Pass through to cultivate mononuclear cell production according to preceding method simultaneously DC.After 5 days, add DC with pp65 peptide pulsed, subsequently under the conditions of human antibody or control antibodies are presence or absence of, will DC adds to CD4⁺T cell.Measure IL-2 and IFN γ level in culture supernatant at the 3rd day with ELISA.CMVpp65 is special Property CD4⁺The propagation of T cell pass through [³H] thymidine incorporation mensure.

As shown in fig. 6, being improve by full people source PD-L1 antibody (1.4.1,1.14.4,1.20.15 and 1.46.11) The generation of IFN γ in specific T-cells response.Fig. 7 shows that full people source PD-L1 antibody increased using the load of CMV pp65 peptide Autologous DC concentration dependant CMV⁺-CD4⁺T cell is bred.

3.4.3 Treg inhibition test：Regulatory T cells (Treg) are critical immune regulon in maintaining self tolerance Play an important role.CD4⁺CD25⁺Treg is related to tumor, and this is due to finding that in multiple cancer patient Treg quantity increases Plus, and related to poor prognosis.In order to directly estimate work in suppression Treg suppression function for the anti human PD-L 1 human antibody With, under the conditions of human antibody or control antibodies are presence or absence of, comparing the function of Treg.In short, CD4⁺CD25⁺ Tregs and CD4⁺CD25^-T cell is separated by MACS.Presence or absence of in the human antibody of variable concentrations or control antibodies Under the conditions of, compare the function of Treg.In short, CD4⁺CD25⁺Tregs and CD4⁺CD25^-T cell is separated by MACS, with of the same race Allosome mDC co-cultures CD4⁺CD25⁺Tregs and CD4⁺CD25^-T cell (Treg:Teff ratio is 1:1).Will be without antibody or same Type antibody is as negative control.Measure generation and the T cell propagation of cytokine with preceding method.

As shown in figure 11, PD-L1 antibody 1.20.15 has abolished the suppression function of Treg, and has recovered reaction-ive T cell Propagation and the secretion of IFN γ.

Cytotoxicity (CDC) test of the cell-mediated cytotoxicity (ADCC) of 3.5 antibody-dependant and Complement Dependent：By It is expressed in various kinds of cell type in human PD-L 1, in order to will be to healthy PD-L1 in health and tumor cell⁺Being not intended to of cell Toxicity be reduced to minimum, the anti-PD-L1 human antibody demonstrating selection does not have ADCC and CDC function.

3.5.1 ADCC：By the human antibody of target cell (mDC) and variable concentrations in 96 orifice plates preincubate 30min, Subsequently with the ratio 50 of effector/target:1 PBMC (effector) adding IL-1 activation.By described plate in 37 DEG C, 5%CO₂Incubate Educate in device and be incubated 6 hours.Measure the cracking of target cell by citotoxicity detection kit (Roche).Using Molecular Devices SpectraMax M5e micropore board detector measures optical density (OD).Comparison hAb (IgG1) and comparison hAb (IgG4) point Not as positive and negative control.

Figure 12 shows, PBMC the originating and will express Gao Shui as NK cell (NK) cell of the IL-2- activation being used The mDC of the PD-L1 of flat cell surface as target cell, full people source PD-L1 antibody (1.4.1,1.14.4,1.20.15 and 1.46.11) do not mediate ADCC.

3.5.2 CDC：Full people by target cell (mDC), the complement (Quidel-A112) of dilution and variable concentrations Source antibody mixes in 96 orifice plates.By described plate in 37 DEG C, 5%CO₂It is incubated 4 hours in couveuse.Using CellTiter glo (Promega-G7573) target cell lysis are measured.Rituximab (Roche) and human B cell lymphoma cell line Raji (CD20 Positive) as positive control.As shown in figure 13, full people source PD-L1 antibody does not mediate CDC.

3.6 classification being determined by FACS (Binning) tests：In order to check whether human antibody is had with reference-Ab There are identical epitope classes, will be little in 4 DEG C of incubations 1 with the human antibody of variable concentrations for the CHO-K1 cell of expression human PD-L 1 When.Eluting is not associated with antibody, adds the control antibodies of biotin labeling in cell.The streptomycin detection being connected using PE is biological The control antibodies of plain labelling and the combination of PD-L1 express cell, subsequently use the FACSCanto II of BD Biosciences And FlowJo version software is analyzed.

The result of class test shows that full people source PD-L1 antibody (i.e. 1.4.1,1.14.4,1.20.15 and 1.46.11) exists Combination epi-position on the PD-L1 of people source is different from known PD-L1 antibody (i.e. reference-Ab).

3.7 across species combination mensurations：Antibody is measured by ELISA to the cross reaction of machin and Muridae PD-L1.Will The PD-L1 of people, machin and mice is coated on ELISA flat board respectively.After closing, human antibody is added in plate and in room The lower incubation of temperature at least 2 hours.Combination with goat anti-human's IgG Fc-HRP detection antibody and coated antigen.Using tmb substrate Incubation 10min colour developing, with 2M HCl terminating reaction.Using read plate at 450nm in Molecular Device M5e microplate reader.

As shown in figure 4, ELISA is test result indicate that full people source PD-L1 and machin PD-L1 to be measured is with dose-dependent Form combines.However, test antibodies (1.4.1,1.14.4,1.20.15 and 1.46.11) are not combined (data with Muridae PD-L1 Do not show).

3.8 across the family combination mensuration of FACS：For detecting across the family binding activity of human antibody, use human antibody In conjunction with the cell line of expression PD-L2, subsequently carry out two anti-bindings with the goat anti-human IgG Fc being connected with FITC.PD-L1 expresses Cell is as positive control.Corresponding blast cell system is as negative control.FACSCanto II using BD Biosciences And FlowJo version software is analyzed to the cell combining.

PD-L1 antibody using full people source dyes to the Chinese hamster ovary celI having transfected PD-L1 or PD-L2, and is divided with FACS Analysis.As shown in figure 3, the PD-L1 antibody specificity of Quan Renyuan combines PD-L1, but be not combined with the PD-L2 of PD-1 ligand family.

Embodiment 4：The Epitope Identification of full people source PD-L1 antibody

In order to determine the epi-position of antibody 1.14.4 described herein, carry out the third ammonia for hPD-L1 using 1.14.4 Acid scanning mutating experiment (alanine scanning experiment) and the recruitment evaluation of antibodies.

Alanine residue in hPD-L1 is mutated into codon glycine, and every other residue mutations are alanine Codon.For each residue of hPD-L1 ectodomain (ECD), entered using two step consecutive PCRs (sequential PCR) The point of row aminoacid replaces.By the coding ECD of human PD-L 1 and the pcDNA3.3-hPD-1_ECD.His matter of C-terminal His- label Grain, as template, is used a set of mutagenic primer as first step PCR, and uses QuikChangeLighting multiple spot rite-directed mutagenesises Test kit (Agilent technologies, Palo Alto, CA).Disappeared after mutation combinations become reaction using Dpn I restriction endonuclease Change parent template.In second step PCR, linear DNA expression cassette comprises CMV promoter, PD-L1 ectodomain (ECD), His- Label and the Polyadenylation of herpes simplex virus thymidine kinase (TK), described linear DNA expression cassette is expanded and Transient expression (Life Technologies, Gaithersburg, MD) in HEK293F cell.

Monoclonal antibody 1.14.4 is coated and is onboard used for ELISA combination mensuration.With comprise quantitative PD-L1 mutation Or after the supernatant reaction of people/mice PD-L1_ECD.His albumen (Sino Biological, China), by HRP be coupled anti- His antibody (1:5000；Rockland Immunochemicals, Pottstown, PA) add as detection antibody.According to right Meansigma methodss according to mutation carry out benchmark to absorbance.To changing with reference to multiple (<0.55) after carrying out extra critical value setting, The final epitope residues determining of identification.

Carry out the binding activity (Figure 14) that antibody 1.14.4 is directed to people and mice PD-L1.Find that antibody 1.14.4 combines Human PD-L 1 (Figure 14 A), but (Figure 14 B) is not combined with mice PD-L1.

List the impact of the hPD-L1 point mutation antagonist combination that 131 points replace in table 2.In hPD-L1 crystal structure On (PDB code 3RRQ and 4ZQK), the position of all these residues is checked, show some aminoacid (e.g.Gly159, Tyr160, Pro161) unlikely directly contact with any antibody.The combination reduction observed is most-likely due to alanine takes The unstable or or even structure collapses of the hPD-L1 structure after generation.To changing with reference to multiple (<0.55) carry out other critical After value sets, the final epitope residues determining are listed in table 3.They are 6 positions for 1.14.4.

All data in table 3 are mapped preferably to carry out visualization and to compare (figure on the crystal structure of hPD-L1 15).

The impact that table 2.PD-L1 point mutation antagonist combines

1.14.4

^aIn conjunction with multiple change be with respect to some silences alanine replace combination for.

The potential epi-position of table 3. identification

Marginal value:Multiple changes<0.55

As shown in figure 15, the described focus residue that responsible hPD-L1 combines all concentrates on C chain, CC ' ring and F chain (figure 15).The crystal structure of hPD-1/hPD-L1 complex is verified to the position of residue (PDB code 4ZQK,) show Illustrate that these residues are predominantly located at A, C, F and G chain.The epi-position of antibody 1.14.4 is mainly contributed by the residue on C chain, and these are residual Base is directly overlapping with hPD-1 and hPD-L1 interaction sites, the mechanism indicating with reference to hPD-L1 and blocking hPD-1.

Embodiment 5：The internal inhibitory action of full people source PD-L1 antibodies on tumor growth

For assessing the inhibitory action to tumour growth for the human antibody hPD-L1, by MC38-B7H1 tumor cell 5 × 10⁵ Individual/0.1mL is inoculated in that male B-hPD-1 humanization right side of mice fore flank flank is subcutaneous, and corotation connects 42 animals.Treat tumour growth Arrive about 100mm³Shi Jinhang packet administration, every group 7, totally 5 groups, respectively：Solvent control group, BMK6 antibody control group (referring to Description in WO2011066389A1), 1.14.4 antibody 3mg/kg group, 1.14.4 antibody 10mg/kg group and 1.14.4 antibody 30mg/kg group.All groups of route of administration are lumbar injection, every two days 1 time, successive administration 6 times, and administration is further continued for after terminating observing 2 weeks.Measurement gross tumor volume and body weight 2 times weekly, the change of record Mouse Weight and gross tumor volume and the relation of administration time.Real At the end of testing, calculate treatment group and solvent control group gross tumor volume ratio (T/C) and inhibition rate of tumor growth (TGI) and count Credit is analysed.Application Graphpad Prism 5 software carries out T-test inspection, carries out statistical analysis to gross tumor volume.P<0.05 Think there is significant difference.

Using slide gauge, gross tumor volume is measured 2 times a week, measured major diameter and the minor axis of tumor, its volume is calculated Formula is：Gross tumor volume=0.5 × major diameter × minor axis².Calculate tumor appreciation rate T/C (%)=treatment group according to measurement result to swell Tumor volume/negative control group gross tumor volume x100%.Tumor control rate TGI (%)=[1- (Ti-T0)/(Vi-V0)] x100

(Ti:Treatment group is in the administration gross tumor volume average of i-th day, T0:Treatment group is equal in the administration gross tumor volume of the 0th day Value；Vi:Matched group is in the administration gross tumor volume average of i-th day, V0:Matched group is in the administration gross tumor volume average of the 0th day).

The full people source PD-L1 antibody 1.14.4 of table 4. transplants the suppression of hPD-1 humanization mice to MC38-B7H1 Mus source colon cancer Tumor acts on

Note:^a.Mean ± standard error；^b.Compare with matched group

In whole experiment process, each group experimental animals weight average has no obvious and reduces (table 4 and Figure 16), shows to tested Compound well-tolerated.Administration terminates rear (after tumor cell inoculation 25 days) for 19 days, and solvent control group tumor volume growth arrives 2359mm³, compared with matched group, gross tumor volume all has obvious reduction (gross tumor volume average to the basic, normal, high dosage group of 1.14.4 It is respectively 949mm³, 1416mm³And 1115mm³), three dosage all show that (tumor suppression ratio is respectively significant antitumor action 62.8%, 42.0% and 55.4%) (table 4 and Figure 17).Control antibodies BMK6 also produce obvious antitumor action (gross tumor volume Average is 1241mm³, tumor control rate is 49.7%).Result shows, 1.14.4 antibody has a significant antitumor action, low, Middle and high dosage group is to the suppression ratio of tumor all more than 40%.

Although the disclosure has specifically illustrated and has entered with reference to specific embodiment (some of them are preferred embodiment) Go description, 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 Interior, the change on various forms and in details can be carried out.

Claims

1. a kind of detached antibody or its Fab, it includes the heavy CDR sequences being selected from the group：SEQ ID NO:1、 3rd, 5,13,15,17,25,27,29,37,39 and 41.

2. antibody according to claim 1 or its Fab, it further includes the light chain CDR sequence being selected from the group Row：SEQ ID NO:7th, 9,11,19,21,23,31,33 and 35.

3. the antibody according to aforementioned any one claim or its Fab, it includes the heavy chain being selected from the group Variable region：

4. the antibody according to aforementioned any one claim or its Fab, it includes the light chain being selected from the group Variable region：

5. the antibody according to aforementioned any one claim or its Fab, it includes：

A) weight chain variable district, it includes SEQ ID NO:1、SEQ ID NO:3 and/or SEQ ID NO:5；And light chain variable district, It includes SEQ ID NO:7、SEQ ID NO:9 and/or SEQ ID NO:11；

B) weight chain variable district, it includes SEQ ID NO:13、SEQ ID NO:15 and/or SEQ ID NO:17；And light chain variable Area, it includes SEQ ID NO:19、SEQ ID NO:21 and/or SEQ ID NO:23；

C) weight chain variable district, it includes SEQ ID NO:25、SEQ ID NO:27 and/or SEQ ID NO:29；And light chain variable Area, it includes SEQ ID NO:31、SEQ ID NO:33 and/or SEQ ID NO:35；Or

D) weight chain variable district, it includes SEQ ID NO:37、SEQ ID NO:39 and/or SEQ ID NO:41；And light chain variable Area, it includes SEQ ID NO:19、SEQ ID NO:21 and/or SEQ ID NO:23.

6. the antibody according to aforementioned any one claim or its Fab, it includes the heavy chain being selected from the group Variable region：SEQ ID NO:43、SEQ ID NO:47、SEQ ID NO:51 and SEQ ID NO:55.

7. the antibody according to aforementioned any one claim or its Fab, it includes the light chain being selected from the group Variable region：SEQ ID NO:45、SEQ ID NO:49 and SEQ ID NO:53.

8. the antibody according to aforementioned any one claim or its Fab, it includes：

9. the detached antibody according to aforementioned any one claim or its Fab, it can be to be less than 10^-8The Kd value of M is specifically combined with human PD-L 1, and described Kd value is measured by plasma resonance combined techniqueses.

10. the antibody according to aforementioned any one claim or its Fab, its with less than 10nM, or not EC more than 1nM₅₀Be combined with monkey PD-L1, and/or be not combined with mice PD-L1.

11. antibody according to aforementioned any one claim or its Fab, it can be with less than 100nM IC50 block the combination of people or monkey PD-L1 and its receptor.

12. antibody according to aforementioned any one claim or its Fab, it is not substantially tied with PD-L2 Close.

13. antibody according to aforementioned any one claim or its Fab, its do not mediate ADCC or CDC or Both of which does not mediate.

14. antibody according to aforementioned any one claim or its Fab, it is that full people's resource monoclonal resists Body.

15. antibody according to claim 14 or its Fab, wherein said full human monoclonal antibody by turn Genetic rat produces.

A kind of 16. antibody or its Fab, it is resisted with the antibody according to aforementioned any one claim or its Former binding fragment competes identical epi-position.

17. antibody according to aforementioned any one claim or its Fab, its can block human PD-L 1 with Its receptor binding, and at least one of following activity is therefore provided：

A) in CD4⁺The generation of IL-2 is induced in T cell；

B) in CD4⁺The generation of IFN γ is induced in T cell；

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

D) reverse T reg suppression function.

18. antibody according to aforementioned any one claim or its Fab, 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.

19. antibody according to aforementioned any one claim or its Fab, it further includes immune ball Albumen constant region.

20. antibody according to aforementioned any one claim or its Fab, it further includes conjugate.

A kind of 21. detached polynucleotide, it encodes antibody or its antigen according to any one in claim 1-19 Binding fragment.

A kind of 22. carriers, it includes detached polynucleotide according to claim 21.

A kind of 23. host cells, it includes carrier according to claim 22.

A kind of 24. antibody expressed according to any one in claim 1-19 or the method for its Fab, its According to claim 23 including cultivating under conditions of expressing detached polynucleotide according to claim 21 Host cell.

A kind of 25. test kits, it includes antibody or its Fab according to any one in claim 1-20.

A kind of 26. detection people or presence of monkey PD-L1 or the methods of level in biological sample, including by described biological sample with Antibody according to any one in claim 1-20 or the contact of its Fab, and determine people in described sample Or the presence of monkey PD-L1 or level.

A kind of 27. methods of the individuality differentiating with the disease that PD-L1 antagonist may be responded or situation, it includes：With root According to the antibody described in any one in claim 1-20 or its Fab from described individual biology sample to be measured Presence or the level of PD-L1 is determined, the presence of PD-L1 or level raise and represent response wherein in described biological sample in product Probability.

28. methods according to claim 27, it further includes to described individual administration therapeutically effective amount according to power Profit requires antibody or its Fab described in any one in 1-20.

A kind of 29. methods of monitoring treatment reaction or progression of disease in the experimenter with PD-L1 antagonist for treating, it includes using Anti- PD-L1 antibody according to any one in claim 1-20 or its Fab are from described individuality Presence or the level of PD-L1 is determined in biological sample to be measured.

A kind of 30. pharmaceutical compositions, including the antibody according to any one in claim 1-20 or its antigen binding fragment Section and one or more pharmaceutically acceptable carrier.

A kind of 31. methods treating the situation of experimenter that can benefit from the immune response raising, including to described experimenter Apply the antibody according to any one in claim 1-20 or its Fab of therapeutically effective amount.

32. method according to claim 31, wherein said experimenter has the PD-L1 expression of rise.

33. antibody according to any one in claim 1-20 or its Fab can be from for treatment in preparation Purposes in the medicine of situation benefiting in the immune response raising.

34. purposes according to claim 33, wherein said situation is cancer or chronic viral infection.

35. antibody according to claim 16 or its Fab, wherein said epi-position includes following PD-L1 amino At least one of sour residue：E58, E60, D61, K62, N63 and R113.