CN110204614A

CN110204614A - Anti-human LAG-3 monoclonal antibody and its preparation method and application

Info

Publication number: CN110204614A
Application number: CN201910146172.2A
Authority: CN
Inventors: 郑勇; 吴琼; 李竞
Original assignee: Guangzhou Yuheng Biotechnology Co Ltd
Current assignee: Guangzhou Yuheng Biotechnology Co Ltd
Priority date: 2018-02-28
Filing date: 2019-02-27
Publication date: 2019-09-06
Anticipated expiration: 2039-02-27
Also published as: CN115057931A; CN110204614B

Abstract

Provide the novel complete human monoclonal antibodies in conjunction with people LAG-3.The hybridoma generation method using humanization rat is additionally provided, the nucleic acid molecules of anti-lag-3 antibody are encoded, for expressing the expression vector and host cell of anti-lag-3 antibody.Invention further provides the methods of Validation in vitro antibody function.Antibody of the invention provides the effective agent that kinds cancer is treated by adjusting human immunity function.

Description

Anti-human LAG-3 monoclonal antibody and its preparation method and application

Sequence table

The application includes sequence table, and entire contents are incorporated herein by reference.

Technical field

Present application relates generally to antibody.More specifically, this application involves combine the complete human monoclonal of people LAG-3 anti- Body, Its Preparation Method And Use.

Background technique

Lymphocyte activation gene 3 (CD223), also referred to as LAG-3 are a kind of I type transmembrane proteins, are immunoglobulins The member of superfamily (IgSF).

LAG-3 is the cell surface expressed on the T cell in activation, NK cell, B cell and plasmacytoid dendritic cellss Molecule, but it is not expressed on Resting T cells.LAG-3 and CD4 have about 20% amino acid sequence homology, but it is with more The major function ligand fibrin sample albumen 1 of high affinity combination MHC II class molecule and the LAG-3 independently of MHC-II (FGL1) class molecule, to provide the negative regulator to T cell receptor signal transduction.

The extracorporeal blocking enhancing T cell proliferation and cell factor of LAG-3 generates, and LAG-3 deficient mice is by super anti- Existing defects in the downward of former staphylococcal enterotoxin B, peptide or the infection induced t cell response of sendai virus.LAG-3 is being activated Natural Treg (nTreg) and induction CD4⁺FoxP3⁺It is expressed on Treg (iTreg) cell, wherein expression is higher than in work The effect CD4 of change⁺The expression observed in T cell.The repressor of Treg cell is eliminated in the blocking of LAG-3 on Treg cell Function, rather than Treg CD4⁺The ectopic expression of LAG-3 assigns inhibitory activity in T cell.Based on LAG-3 to chronic infection and cancer The immunoregulation effect of T cell function in disease, the mechanism of action of the prediction of LAG-3 monoclonal antibody specific are to inhibit tumour special The negative regulator of opposite effect T cell.

Only there are three types of potential antagonist antibodies to adjust LAG-3 function and antineoplastic immune in early clinic exploitation at present Response is to treat advanced solid tumor.These antibody are described in 20110150892 A1 of patent US, US 20170101472A1 and WO In 2015138920A1, hereinafter referred to as BMK1, BMK7 and BMK5.As described herein, BMK8 is the people of chimeric antibody BMK5 Source form.BMK1, BMK7 and BMK8 are used as reference-Ab in the context of this application.Therefore, there is still a need for having improves The effect of (such as high binding affinity, low across family reaction and good stability) anti-human LAG-3 antibody.In the application In, inventor produces a series of antibody and fully human antibodies for LAG-3 using humanization rat.The antibody of the application has There is high binding affinity, specifically binds people LAG-3 albumen without reacting across family, and effectively adjust immune response.

Summary of the invention

In the broadest sense, the present invention relates to provide new compound, method, composition and the system with improved antibody Product.Benefit provided by the invention is widely applicable for Antybody therapy and diagnostic field, and can with can be anti-with various targets The antibody combined use answered.The present invention provides the antibody in conjunction with people LAG-3, preferably completely human monoclonal antibodies.Also provide The method for generating hybridoma using humanization rat encodes the nucleic acid molecules of anti-lag-3 antibody, anti-for expressing anti-lag-3 The expression vector and host cell of body.Invention further provides the methods of Validation in vitro antibody function.Antibody of the invention Provide the effective agent that a variety of diseases are treated by adjusting human immunity function.

In some respects, the present invention includes isolated antibody or its antigen-binding portion thereof.

In some embodiments, isolated antibody or its antigen-binding portion thereof have one or more following properties:

(a) with 2 × 10^-10M or lower K_DIn conjunction with people LAG-3；

(b) inhibit the combination of LAG-3 and ajor histocompatibility (MHC) II class molecule；

(c) inhibit the combination of LAG-3 and fibrin sample albumen 1 (FGL1) ligand molecular；

(d) inhibit the combination of LAG-3 and LSECtin and/or galectin-3；

(e) combine people LAG-3 without reacting across family；Or

(f) there is no cross reactivity with people CD4.

In some embodiments, isolated antibody or its antigen-binding portion thereof include:

A) one or more heavy chain CDR (CDRH), it is selected from the following at least one: (i) with selected from SEQ ID NO:1 and CDRH1 shown in one of 7 sequence has the CDRH1 of at least 90% sequence identity；(ii) and selected from SEQ ID NO:2 and 8 One of sequence shown in CDRH2 there is the CDRH2 of at least 90% sequence identity；(iii) with selected from SEQ ID NO:3 and CDRH3 shown in one of 9 sequence has the CDRH3 of at least 90% sequence identity；

B) one or more light chain CDR (CDRL), it is selected from the following at least one: (i) with selected from SEQ ID NO:4 and CDRL1 shown in one of 10 sequence has the CDRL1 of at least 90% sequence identity；(ii) with selected from SEQ ID NO:5 and CDRL2 shown in one of 11 sequence has the CDRL2 of at least 90% sequence identity；(iii) and it is selected from SEQ ID NO:6 CDRL3 shown in one of sequence with 12 has the CDRL3 of at least 90% sequence identity；Or

C) A) one or more CDRH and B) one or more CDRL.

A) one or more (such as 1,2 or 3) heavy chain CDR (CDRH), it is selected from the following at least one: (i) be selected from SEQ There is the amino acid addition for being no more than 2 amino acid, missing with the amino acid sequence of the CDRH1 in the CDRH1 of ID NO:1 and 7 Or the CDRH1 of the difference replaced；(ii) CDRH2 selected from SEQ ID NO:2 and 8 or the amino acid sequence presence with the CDRH2 No more than the CDRH2 of the difference of the amino acid addition of 2 amino acid, missing or substitution；(iii) is selected from SEQ ID NO:3 and 9 CDRH3 or there is amino acid addition, missing or the difference replaced for being no more than 2 amino acid with the amino acid sequence of the CDRH3 Different CDRH3；

B) one or more (such as 1,2 or 3) light chain CDR (CDRL), it is selected from the following at least one: (i) be selected from SEQ There is the amino acid addition for being no more than 2 amino acid, missing with the amino acid sequence of the CDRL1 in the CDRL1 of ID NO:4 and 10 Or the CDRL1 of the difference replaced；(ii) CDRL2 selected from SEQ ID NO:5 and 11 or the amino acid sequence presence with the CDRL2 No more than the CDRL2 of the difference of the amino acid addition of 2 amino acid, missing or substitution；(iii) be selected from SEQ ID NO:6 and There is the amino acid addition for being no more than 2 amino acid, missing or substitution with the amino acid sequence of the CDRL3 in 12 CDRL3 The CDRL3 of difference；Or

C) A) one or more CDRH and B) one or more CDRL.

A) comprising the CDRH3 of SEQ ID NO:3 or 9；Or

B) there is at least 90% sequence identity with CDRH3 shown in one of the sequence selected from SEQ ID NO:3 and 9 CDRH3；Or

There is the amino acid addition for being no more than 2 amino acid, missing with the amino acid sequence of the CDRH3 of A) in C) CDRH3 Or the difference replaced,

And the antibody wherein separated or its antigen-binding portion thereof are with 2 × 10^-10M or lower K_DIn conjunction with people LAG-3.

(a) comprising the SEQ ID NO:1 or CDRH1 being made from it；

(b) comprising the SEQ ID NO:2 or CDRH2 being made from it；

(c) comprising the SEQ ID NO:3 or CDRH3 being made from it；

(d) comprising the SEQ ID NO:4 or CDRL1 being made from it；

(e) comprising the SEQ ID NO:5 or CDRL2 being made from it；With

(f) comprising the SEQ ID NO:6 or CDRL3 being made from it.

(a) comprising the SEQ ID NO:7 or CDRH1 being made from it；

(b) comprising the SEQ ID NO:8 or CDRH2 being made from it；

(c) comprising the SEQ ID NO:9 or CDRH3 being made from it；

(d) comprising the SEQ ID NO:10 or CDRL1 being made from it；

(e) comprising the SEQ ID NO:11 or CDRL2 being made from it；With

(f) comprising the SEQ ID NO:12 or CDRL3 being made from it.

(A) heavy chain variable region:

(i) comprising the amino acid sequence of SEQ ID NO:13；

(ii) comprising having the amino acid sequence of at least 85%, at least 90% or at least 95% identity with SEQ ID NO:13 Column；Or

(iii) comprising have with SEQ ID NO:13 compared with it is one or more (such as 1-10,1-5,1-3 is a, 1, 2,3,4 or 5) addition, missing and/or the substituted amino acid sequence of amino acid；And/or

(B) light chain variable region:

(i) comprising the amino acid sequence of SEQ ID NO:14；

(ii) comprising having the amino acid sequence of at least 85%, at least 90% or at least 95% identity with SEQ ID NO:14 Column；Or

(iii) comprising have with SEQ ID NO:14 compared with it is one or more (such as 1-10,1-5,1-3 is a, 1, 2,3,4 or 5) addition, missing and/or the substituted amino acid sequence of amino acid.

(A) heavy chain variable region:

(i) comprising the amino acid sequence of SEQ ID NO:15；

(ii) comprising having the amino acid sequence of at least 85%, at least 90% or at least 95% identity with SEQ ID NO:15 Column；Or

(iii) comprising have with SEQ ID NO:15 compared with it is one or more (such as 1-10,1-5,1-3 is a, 1, 2,3,4 or 5) addition, missing and/or the substituted amino acid sequence of amino acid；And/or

(B) light chain variable region:

(i) comprising the amino acid sequence of SEQ ID NO:16；

(ii) comprising having the amino acid sequence of at least 85%, at least 90% or at least 95% identity with SEQ ID NO:16 Column；Or

(iii) comprising have with SEQ ID NO:16 compared with it is one or more (such as 1-10,1-5,1-3 is a, 1, 2,3,4 or 5) addition, missing and/or the substituted amino acid sequence of amino acid.

In some respects, the present invention relates to isolated nucleic acid molecules, and it includes what coding separated as disclosed herein to resist The heavy chain variable region of body and/or the nucleic acid sequence of light chain variable region.

In some respects, the present invention relates to the nucleic acid comprising encoding antibody as disclosed herein or its antigen-binding portion thereof The expression vector of molecule.

In some respects, the present invention relates to the host cells comprising expression vector as disclosed herein.

In some respects, the present invention relates to pharmaceutical composition, it includes at least one antibody as disclosed herein or its Antigen-binding portion thereof and pharmaceutically acceptable carrier.

In some respects, the present invention relates to the methods for being used to prepare anti-lag-3 antibody or its antigen-binding portion thereof comprising Antibody or its antigen-binding portion thereof are expressed in host cell and from host cell separation antibody or antigen-binding portion thereof.

In some respects, the present invention relates to the methods for adjusting antigen-specific T cell response, including such as to subject's application Antibody disclosed herein or its antigen-binding portion thereof, so that the antigen-specific T cell response in subject is regulated.

In some respects, the present invention relates to the methods for the immune response for adjusting subject comprising such as to subject's application Antibody disclosed herein or its antigen-binding portion thereof, so that the immune response in subject is regulated.

In some respects, the present invention relates to for inhibiting or blocking side of the LAG-3 in conjunction with MHC II or FGL1 class molecule Method comprising make the MHC II class or FGL1 class molecule and antibody as disclosed herein or its antigen binding portion thereof.

In some respects, the present invention relates to for inhibiting or blocking LAG-3 and LSECtin and/or galectin-3 Combination method comprising make the LSECtin and/or galectin-3 and antibody as disclosed herein or it be anti- Former bound fraction contact.

In some respects, the present invention relates to for inhibiting the method for the growth of tumour cell in subject comprising to by Examination person applies antibody as disclosed herein or its antigen-binding portion thereof, so that the growth of tumour is suppressed in subject.

In certain aspects, the present invention relates to the methods for treating the virus infection in subject comprising to tested Person applies antibody as disclosed herein or its antigen-binding portion thereof, so that treating virus infection in subject.

In some respects, the present invention relates to the sides for treating or preventing such as cancer of the proliferative disorders in subject Method comprising apply a effective amount of antibody as disclosed herein or its antigen-binding portion thereof to subject.

In some respects, the present invention relates to antibody as disclosed herein or its antigen-binding portion thereof to prepare for treating Or the purposes in the drug of prevention proliferative disorders such as cancer.

In some respects, the present invention relates to antibody as disclosed herein or its antigen-binding portion thereof to prepare for diagnosing Purposes in the diagnosticum of proliferative disorders such as cancer.

In some respects, the present invention relates to antibody as disclosed herein or its antigen-binding portion thereof for treating or preventing Proliferative disorders such as cancer.

In some respects, the present invention relates to the kit for using antibody as disclosed herein or its antigen-binding portion thereof or Device and correlation technique and pharmaceutical composition as disclosed herein can be used for treating proliferative disorders such as cancer.For This, present invention preferably provides the product that can be used for treating such illness, it includes containing antibody as disclosed herein or it is anti- The container of former bound fraction and for being treated, being improved using antibody as disclosed herein or its antigen-binding portion thereof or in advance Anti- proliferative diseases or its progress or recurrence illustrate material.In selected embodiment, device and correlation technique will include The step of making at least one circulating tumor cell and antibody as disclosed herein or its antigen binding portion thereof.

The above content is a general introductions, therefore when necessary include the simplification, summary and omission of details；Therefore, this field skill Art personnel are it will be recognized that the being merely illustrative of property of general introduction, it is not intended to be limited in any way.Side as described herein The other aspects of method, composition and/or device and/or other themes, feature and advantage will become in introduction shown in this article Obviously.It provides and summarizes to simply introduce the concept of some selections, these concepts will be retouched further in the following detailed description It states.This general introduction is not intended to the key feature or essential characteristic for determining theme claimed, is not intended to and is used as determining wanted Seek the supplementary means of the range of the theme of protection.In addition, through the application reference all bibliography, patent and it is disclosed specially The content of benefit application is incorporated herein by reference in their entirety.

Brief description

Fig. 1 shows the combination of LAG-3 antibody and cell surface people LAG-3, is indicated by MFI (average fluorescent strength), and by BD FACSCanto II measurement.

Fig. 2 shows the blocking of the combination of the MHC-II expressed on LAG-3 albumen and Raji cell.

Fig. 3 shows the blocking of LAG-3 albumen and the combination of LSECtin.

Fig. 4 shows the blocking of the combination of LAG-3 albumen and galectin-3.

Fig. 5 shows the cross reactivity with machin LAG-3 measured by FACS.

Fig. 6 shows the cross reactivity with mouse LAG-3 measured by FACS.

Fig. 7 shows the cross reactivity with people CD4 measured by ELISA.

Fig. 8 A-E shows the epitope point storehouse (epitope binning) for reference-Ab BMK1, BMK7 and BMK5.

Fig. 9 A-B shows the result of epitope mapping.

Figure 10 shows effect of people's LAG-3 antibody in reporter-gene assays.

Figure 11 shows the influence of people LAG-3 antibody on human Allogeneic Mixed Lymphocyte reaction, is such as measured by ELISA And pass through IFN-γ level (ng/mL) Lai Fanying's.

Figure 12 shows the influence of people LAG-3 antibody on human Allogeneic Mixed Lymphocyte reaction, such as passes through³H- thymidine is mixed Enter measurement and is reflected by breeder reaction that the CPM (count per minute) with triplicate hole is indicated.

Figure 13 A-B shows by determining target cell lysis the result of the CDC test and ADCC test that carry out.

Figure 14 A-B shows that serum stability is tested as a result, MFI table that is such as being measured by FACS and passing through cell Show.

Detailed description of the invention

Although the present invention can be implemented in many different forms, disclosed herein is its for verifying the principle of the invention Specific illustrative embodiment.It is emphasized that the specific embodiment that the present invention is not limited to be illustrated.This Outside, any chapter title used herein is only used for organizational goal, is not interpreted to limit described theme.

Unless otherwise defined in this, the scientific and technical terms being otherwise employed in conjunction with the invention will have this field common The normally understood meaning of technical staff.In addition, unless the context otherwise requires, the term of singular should include plural form, The term of plural form should include singular.More specifically, being removed as used in the specification and the appended claims In addition non-context explicitly points out, otherwise singular " one ", "one" and "the" include plural referents.Thus, for example, mentioning And " a kind of protein " includes multiple proteins；Refer to that " cell " includes the mixture etc. of cell.In this application, unless It is otherwise noted, otherwise means "and/or" using "or".(such as " comprising " and " contain in addition, term "comprising" and other forms Have ") use be not limiting.In addition, the range provided in specification and appended include endpoint and breakpoint it Between all values.

In general, with cell and tissue culture described herein, molecular biology, immunology, microbiology, science of heredity and egg White matter and nucleic acid chemistry and the related term of hybridization and its technology are it is well known in the art that with common term.Unless another It is described, otherwise methods and techniques of the invention are carried out generally according to conventional method well known in the art, and such as in this specification In full text quote and discuss it is various general and referring more particularly to described in document carry out.See, for example, Abbas et al., Cellular and Molecular Immunology,6^thed.,W.B.Saunders Company(2010)；Sambrook J.&Russell D.Molecular Cloning:A Laboratory Manual,3rd ed.,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.(2000)；Ausubel et al., Short Protocols in Molecular Biology:A Compendium of Methods from Current Protocols in Molecular Biology,Wiley,John&Sons,Inc.(2002)；Harlow and Lane Using Antibodies:A Laboratory Manual,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y. (1998)；With Coligan et al., Short Protocols in Protein Science, Wiley, John&Sons, Inc. (2003).With analytical chemistry described herein, synthetic organic chemistry and drug and the related term of pharmaceutical chemistry and laboratory Program and technology are well known in the art and common terms.In addition, any chapter title used herein is only used for group Purpose is knitted, and is not interpreted to limit described theme.

Definition

For a better understanding of the present invention, the definition and explanation of relational language provide as follows.

As used herein, term " antibody " or " Ab " are typically referred to comprising by covalent disulfide bonds and noncovalent interaction The Y shape tetramer albumen of two heavy chains (H) and two light chains (L) polypeptide chain that keep together.The light chain of antibody can be divided into κ And lambda light chain.Heavy chain can be divided into μ, δ, γ, α and ε, they respectively by the isotype of antibody be defined as IgM, IgD, IgG, IgA and IgE.In light chain and heavy chain, variable region is connect by area " J " of about 12 or more amino acid with constant region, and heavy chain It also include area " D " of about 3 or more amino acid.Each heavy chain is by heavy chain variable region (VH) and heavy chain constant region (CH) group At.Heavy chain constant region is made of 3 structural domains (CH1, CH2 and CH3).Every light chain is by light chain variable region (VL) and chain constant Area (CL) composition.The area VH and VL can be further divided into the high change being spaced apart by the region (referred to as framework region (FR)) guarded relatively Area's (referred to as complementary determining region (CDR)).Each VH and VL is made of 3 CDR and 4 FR of following sequence: from N-terminal to C-terminal FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.The variable region (VH and VL) of each heavy chain/light chain pair is respectively formed antigen knot Coincidence point.Distribution of the amino acid in various regions or structural domain follows Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987and ) or Chothia&Lesk (1987) J.Mol.Biol.196:901-917 1991)；Chothia et al., (1989) Nature Definition in 342:878-883.Antibody can have different antibody isotypes, such as IgG (for example, IgG1, IgG2, IgG3 Or IgG4 hypotype), IgA1, IgA2, IgD, IgE or IgM antibody.

" antigen-binding portion thereof " or " antigen-binding fragment " of term antibody, can exchange in the context of this application It uses, refers to the polypeptide of the segment comprising full length antibody, remain the antigentic specificity knot with full length antibody specific binding The ability of conjunction and/or its antigen identical with full length antibody competitive binding.In general, referring to Fundamental (Paul, W., ed., the second edition, Raven Press, N.Y. (1989) pass through for all purposes by Immunology, Ch.7 It is incorporated herein by reference.The antigen-binding fragment of antibody can be cut by recombinant DNA technology or by the enzymatic or chemistry of complete antibody It cuts to generate.In some conditions, antigen-binding fragment includes Fab, Fab', F (ab')₂, Fd, Fv, dAb and complementary determining region (CDR) segment, single-chain antibody (such as scFv), chimeric antibody, double antibody and comprising being enough to assign polypeptid specificity antigen binding The polypeptide of at least part antibody of ability.The antigen-binding fragment of antibody can be from given antibody (for example, providing in this application Monoclonal anti-human LAG-3 antibody) by routine techniques well known by persons skilled in the art (for example, recombinant DNA technology or enzymatic Or chemical cleavage method) obtain, and specificity can be screened in a manner of identical with complete antibody.

As used herein, term " monoclonal antibody " or " mAb " refer to the antibody molecule preparation of unimolecule ingredient.Monoclonal Antibody shows the single binding specificity and affinity to defined epitope.

As used herein, term " human antibody " or " fully human antibodies " are intended to include the antibody with variable region, center Frame area and CDR region are derived from human germline immunoglobulin's sequence.In addition, if antibody contains constant region, constant region is also derived from Human germline immunoglobulin's sequence.Human antibody of the invention may include not by the amino of human germline immunoglobulin's sequential coding Sour residue (such as passing through external random or site-specific mutagenesis or the mutation introduced by internal somatic mutation).However, As used herein, term " human antibody " is not intended to including wherein from the germline of another mammalian species such as mouse CDR sequence has been grafted onto the antibody on people's Frame sequence.

As used herein, term " human monoclonal antibodies " refers to the antibody for showing single binding specificity, has wherein Frame and CDR region are derived from the variable region of human germline immunoglobulin's sequence.

Term " humanized antibody " is intended to refer to wherein from the CDR sequence of the germline of another mammalian species such as mouse Arrange the antibody being transplanted on people's Frame sequence.Additional framework region modification can be carried out in people's Frame sequence.

Term " chimeric antibody " as used herein refers to such antibody, and wherein variable region sequences come from a species simultaneously And constant-region sequences come from another species, such as wherein variable region sequences are anti-from people from mouse antibodies and constant-region sequences Body.

As used herein, term " LAG-3 " refers to lymphocyte activation gene -3.Term " LAG-3 " includes variant, of the same race Type, homologue, ortholog thing and collateral homologue.

As used herein, term " people LAG-3 " refers to human sequence LAG-3, such as with Genbank accession number NP_ The complete amino acid sequence of 002277 people LAG-3.People LAG-3 sequence can be with the people of Genbank accession number NP_002277 LAG-3 is different, such as has conservative mutation in non-conservative area, and LAG-3 has and Genbank accession number NP_ 002277 people LAG-3 substantially the same biological function.For example, the biological function of people LAG-3 is that have by the disclosure The biological function of epitope or people LAG-3 in the extracellular domain for the LAG-3 that the antibody specificity of content combines is and MHC II class or FGL1 class molecule combine.

As used herein, term " mouse LAG-3 " refers to mouse sequence LAG-3, such as with Genbank accession number NP_ The complete amino acid sequence of 032505 mouse LAG-3.

As used herein, term " machin LAG-3 " refers to machin sequence LAG-3, such as with Genbank accession number The complete amino acid sequence of the machin LAG-3 of XP_005570011.1.

As used herein, term " Ka " is intended to indicate that specific antibodies-antigen interactions association rate, and used herein Term " Kd " be intended to indicate that specific antibodies-antigen interactions dissociation rate.It is good that this field can be used in the Kd value of antibody The method established well determines.As used herein, term " K_D" it is intended to indicate that the dissociation of specific antibodies-antigen interactions is normal Number obtains from the ratio (that is, Kd/Ka) of Kd and Ka and is expressed as molar concentration (M).Determining the preferred method of antibody Kd is By using surface plasma body resonant vibration, it is preferable to use bio-sensor system such asSystem.

" high-affinity " of term IgG antibody as used herein refers to there is 1 × 10 for target antigen^-7M or lower, more It is preferred that 5 × 10^-8M or lower, even more preferably 1 × 10^-8M or lower, even more preferably 5 × 10^-9M or lower, and it is even more excellent Select 1 × 10^-9M or lower K_DAntibody.

Term " EC as used herein₅₀", also referred to as " medium effective concentration ", refers to and lured after specific exposure duration Lead the drug of 50% response between baseline and maximum value, the concentration of antibody or toxic agent.In the context of this application, EC₅₀Unit be " nM ".

" inhibit to combine " in this application or the ability of " competition same epitope " refers to antibody or the suppression of its antigen-binding fragment That makes two molecules (such as people LAG-3 and people's anti-lag-3 antibody) is bound to any detectable level.In certain embodiments In, the combination of two molecules can inhibit at least 50% by antibody or its antigen-binding fragment.In certain embodiments, this Inhibiting effect can be greater than 60%, be greater than 70%, be greater than 80% or greater than 90%.

As used herein, term " epitope " refers to immunoglobulin or the antigen part that antibody specificity combines." epitope " Also referred to as " antigenic determinant ".Epitope or antigenic determinant are usually by molecule such as amino acid, carbohydrate or carbohydrate side chain Chemically active surface group composition, and usually have specific three-dimensional structure and specific charge characteristic.For example, epitope is logical Often comprising at least 3,4,5,6,7,8,9,10,11,12,13,14 or 15 continuous or discontinuous ammonia in unique three-dimensional conformation Base acid, can be " linear " or " conformation " epitope.See, for example, Epitope Mapping Protocols in Methods in Molecular Biology,Vol.66,G.E.Morris,Ed.(1996).In linear epitope, protein and phase interaction Primary amino acid sequences with all interaction sites between molecule (such as antibody) along protein linearly exist.In conformation In epitope, interaction sites cross over the amino acid residue being separated from each other in protein.It is determined by those skilled in the art The competitiveness of the combination same epitope of known routine techniques detection, can be with screening antibodies.For example, can be at war with or intersect Competition research is to be contended with one other or the antibody of cross competition combination antigen (such as RSV fusion protein).In international monopoly Shen The high throughput method for obtaining the antibody for combining same epitope please be described in WO 03/48731, based on their intersection Competition.

As used herein, term " separation " refers to the state obtained by manual type from native state.If certain " separation " substance or component are naturally occurring, then may be because its naturally change perhaps substance with it is naturally isolated or The two haves both at the same time.For example, certain unsegregated polynucleotides or polypeptide are naturally present in some mobiles, it is natural from this The identical high-purity polynucleotides or polypeptide of state separation are referred to as isolated polynucleotides or polypeptide.Term " separation " was both It is not excluded for the artificial or synthetic of mixing, is also not excluded for not influencing other active foreign bodys of isolated substance.

As used herein, term " isolated antibody " be intended to refer to substantially free of with different antigentic specificities other are anti- The antibody of body is (for example, the isolated antibody of specific binding LAG-3 albumen removes LAG-3 albumen substantially free of specific binding The antibody of antigen in addition).However, the isolated antibody of specific binding people LAG-3 albumen such as comes from other to other antigens The LAG-3 albumen of species may have cross reactivity.In addition, isolated antibody can be substantially free of other cell materials And/or chemical substance.

As used herein, term " carrier " refer to can wherein be inserted into polynucleotides nucleic acid medium.When carrier is permitted When being permitted the expression of the protein for the polynucleotide encoding being inserted, which is known as expression vector.The carrier can be by turning Change, transduce or be transfected into host cell and express that the inhereditary material element carried in host cell.Carrier is this field skill Known to art personnel, including but not limited to plasmid, bacteriophage, clay, artificial chromosome such as yeast artificial chromosome (YAC), Bacterial artificial chromosome (BAC) or P1 are derivative artificial chromosome (PAC)；Bacteriophage such as λ bacteriophage or M13 bacteriophage and zoosis Poison.The animal virus that can be used as carrier includes but is not limited to retrovirus (including slow virus), adenovirus, adeno-associated virus, Herpesviral (such as herpes simplex virus), poxvirus, baculoviral, papillomavirus, papovavirus (such as SV40).Carrier can With comprising the multiple element for controlling expression, including but not limited to promoter sequence, transcriptional initiation sequence, enhancer sequence, Selection element and reporter.In addition, carrier may include replication orgin.

As used herein, term " host cell " refers to the cell that can import carrier, including but not limited to prokaryotic cell Such as Escherichia coli (E.coli) or bacillus subtilis (Bacillus subtilis), fungal cell such as yeast cells or aspergillus Belong to (Aspergillus), insect cell such as S2 drosophila cell or Sf9 and zooblast such as fibroblast, Chinese hamster ovary celI, COS cell, NSO cell, HeLa cell, bhk cell, HEK293 cell or people's cell.

As used herein, term " identity " refers to by comparing and comparing two or more polypeptides point that sequence determines Relationship between son or the sequence of two or more nucleic acid molecules." percentage identity " refer to compare in molecule amino acid or The percentage of identical residue between nucleotide, and calculated based on the size of the smallest molecule compared.For these calculating, compare In gap (if any) preferably addressed by specific mathematical model or computer program (i.e. " algorithm ").It can use It is included in Computational Molecular Biology in the method for the identity of the nucleic acid or polypeptide of calculating ratio pair, (Lesk,A.M.,ed.),1988,New York:Oxford University Press；Biocomputing Informatics and Genome Projects,(Smith,D.W.,ed.),1993,New York:Academic Press；Computer Analysis of Sequence Data,Part I,(Griffin,A.M.,and Griffin, H.G.,eds.),1994,New Jersey:Humana Press；von Heinje,G.,1987,Sequence Analysis in Molecular Biology,New York:Academic Press；Sequence Analysis Primer, (Gribskov,M.and Devereux,J.,eds.),1991,New York:M.Stockton Press；With Carillo etc. People, described in 1988, SIAMJ.Applied Math.48:1073 those.

As used herein, term " immunogenicity " refers to that specific antibody or sensitized lymphocyte are formed in stimulating organism body Ability.It refers not only to the activation of antigenic stimulus certain immune cells, proliferation and breaks up such as anti-finally to generate immunologic effector substance The property of body and sensitized lymphocyte also refers to that the specific immune response of antibody or sensitized T lymphocyte can be pierced with antigen It is formed in the immune system of organism after swashing organism.Immunogenicity is the most important characteristic of antigen.Whether antigen can be at Function induces the generation of immune response in host to depend on three factors: the property of antigen, the reactivity of host and immune means.

As used herein, term " transfection ", which refers to the process of, introduces eukaryocyte especially mammalian cell for nucleic acid. Scheme and technology for transfection include but is not limited to lipofection and chemically and physically method such as electroporation.Many rotaring dyeing technologies It is well known in the art and disclosed herein.See, for example, Graham et al., 1973, Virology 52:456； Sambrook et al., 2001, Molecular Cloning:A Laboratory Manual, ibid；Davis et al., 1986, Basic Methods in Molecular Biology,Elsevier；Chu et al,1981,Gene 13:197.In this hair In a bright specific embodiment, people's LAG-3 gene is transfected into 293F cell.

As used herein, term " hybridoma " and term " hybridoma cell line " may be used interchangeably.When referring to that term is " miscellaneous When friendship tumor " and term " hybridoma cell line ", they also include the subclone and progeny cell of hybridoma.

As used herein, term " SPR " or " surface plasma body resonant vibration " refer to and including allowing through detection biology The optical phenomena of real-time biospecific interaction is analyzed in the change of protein concentration in biosensor matrix, such as using BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).About retouching in detail State, referring to embodiment andEt al. U., (1993) Ann.Biol.Clin.51:19-26；U., etc. People (1991) Biotechniques 11:620-627；Johnsson, B., et al. (1995) J.Mol.Recognit.8:125- 131；And Johnnson, B., et al. (1991) Anal.Biochem.198:268-277.

As used herein, term " fluorescence-activated cell sorting " or " FACS " refer to the flow cytometry of specialized types.It It provides according to the scattering of the specific light of each cell and fluorescent characteristics, by the heterogeneous mixture of biological cell per next cell Method (FlowMetric. " the Sorting Out Fluorescence Activated being sorted in two or more containers Cell Sorting".2017-11-09).Instrument for carrying out FACS is known to the skilled in the art and can be right It is commercially available in the public.The example of this instrument includes the FACS of Becton Dickinson (Foster City, CA) Star Plus, FACScan and FACSort instrument, the Epics for coming from Coulter Epics Division (Hialeah, FL) The C and MoFlo for coming from Cytomation (Colorado Springs, Colorado).

As used herein, term " cytotoxicity of antibody dependent cellular mediation " or " ADCC " refer to wherein with it is certain thin Fc receptor (FcR) present on cellular toxicity cell (such as natural killer (NK) cell, neutrophil cell and macrophage) is tied The Ig of the secretion of conjunction enables these cytotoxic effect cells to specifically bind the target cell for carrying antigen and then uses cell The cytotoxic form of toxin kill target cell.Antibody " arms " cytotoxic cell, and be absolutely to need for this killing It wants.The main cell NK cell of ADCC is mediated only to express Fc γ RIII, and monocytes Fc γ RI, Fc γ RII and Fc γRIII.FcR expression on hematopoietic cell is summarised in Ravetch and Kinet, Annu.Rev.Immunol 9:457-92 (1991) in the table 3 of page 464.In order to assess the ADCC activity of molecules of interest, external ADCC measurement, such as beauty can be carried out Measurement described in state's patent No. 5,500,362 or 5,821,337.The effector cell that can be used for such measurement includes peripheral blood list Nucleus (PBMC) and natural kill (NK) cell.Alternatively or additionally, the ADCC activity of molecules of interest can be commented in vivo Estimate, such as is assessed in the animal model as disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

Term " complement-dependent cytotoxicity " or " CDC " refer to the cracking of target cell in the presence of complement.Classical complement The activation of approach by complement system the first component (C1q) and the antibody (subclass appropriate) in conjunction with its isogeneic in conjunction with and open It is dynamic.In order to assess complement activation, CDC measurement, such as Gazzano-Santoro et al., J.Immunol.Methods can be executed Described in 202:163 (1996).

Term " subject " includes anyone or non-human animal, preferably people.

As used herein, term " cancer " refer to cause medical conditions any tumour or Malignant cellular growth, proliferation or Shift the solid tumor mediated and non-physical knurl such as leukaemia.

Term " treatment " and " treatment " used in the case where treating the state of an illness herein relates generally to the treatment of human or animal And therapy, wherein some desired therapeutic effects are realized, for example, inhibit disease progression, including tempo decline, progress speed Degree is stagnated, and the state of an illness subsides, and the state of an illness improves and the state of an illness is cured.Further comprise the treatment as precautionary measures (preventing).For cancer Disease, " treatment " may refer to inhibition or slow down tumour or Malignant cellular growth, proliferation or transfer or its certain combination.For swollen Tumor, " treatment " include the development for removing all or part of tumour, inhibiting or slow down growth and metastasis of tumours, preventing or delaying tumour Or its certain combination.

As used herein, term " therapeutically effective amount " is related to reactive compound or the material comprising reactive compound, combination The amount of object or dosage form matches effective for generating with reasonable benefit/risk ratio when according to required therapeutic scheme application Certain required therapeutic effects.Specifically, " therapeutically effective amount " means that antibody or its antigen-binding portion thereof effectively treat people The amount or concentration of LAG-3 related disease or illness.

As used herein, " host cell " of the invention refers to the cell for introducing exogenous polynucleotide.

As used herein, term " pharmaceutically acceptable " refer to carrier, diluent, excipient and/or its salt chemistry and/ Or it is physically compatible with the other compositions in preparation and compatible in a physiologically with recipient.

As used herein, term " pharmaceutically acceptable carrier and/or excipient " refers in pharmacology and/or physiology Upper and subject and context of vaccine administration carrier and/or excipient, be well known in the present art (see, e.g., Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th Ed.Pennsylvania:Mack Publishing Company, 1995), and including but not limited to pH adjusting agent, surface are living Property agent, adjuvant and ionic strength reinforcing agent.For example, pH adjusting agent includes but is not limited to phosphate buffer；Surfactant packet It includes but is not limited to cation, anion or nonionic surfactant, such as Tween-80；Ionic strength reinforcing agent includes but not It is limited to sodium chloride.

As used herein, term " adjuvant " refers to nonspecific immunity strengthening agent, is delivered together with antigen to biology Body or while being delivered to organism in advance can be enhanced in organism to the immune response of antigen or change the class of immune response Type.There are a variety of adjuvants, including but not limited to aluminium adjuvant (such as aluminium hydroxide), Freund's adjuvant (such as Freund's complete adjuvant and Incomplete Freund's adjuvant), corynebacterium, lipopolysaccharides, cell factor etc..Freund's adjuvant is the most frequently used in current zoopery Adjuvant.Aluminum hydroxide adjuvant is more often available to clinical test.

Anti-lag-3 antibody

In the context of this application, " antibody " may include polyclonal antibody, monoclonal antibody, chimeric antibody, source of people Change and primatized antibody, CDR grafted antibody, human antibody, recombinates the antibody of generation, intracellular antibody, multi-specificity antibody, Bispecific antibody, univalent antibody, multivalent antibody, anti-idiotype, synthetic antibody, including its mutain and variant；And Its derivative (including Fc fusion protein and other modifications) and any other immunoreactivity molecule, if its show with The preferential combination or association of LAG-3 albumen.In addition, unless context dictates otherwise, otherwise the term further includes all categories Antibody (i.e. IgA, IgD, IgE, IgG and IgM) and all subclass (i.e. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2).One In a preferred embodiment, antibody is monoclonal antibody.In a more preferred embodiment, antibody is human monoclonal antibodies.

Various techniques known in the art can be used to generate in human antibody.A kind of technology is phage display, wherein (preferably people) antibody library is synthesized on bacteriophage, screens library with interested antigen or its antibody-binding fraction, and separate knot The bacteriophage for closing antigen, from wherein can be with adaptive immune reactivity segment.The method for being used to prepare and screening this library is this Field it is well known that and for generate phage display library kit it is commercially available (for example, Pharmacia weight Group phage antibody system, catalog number (Cat.No.) 27-9400-01；And Stratagene SurfZAPTM phage display kit, mesh Record number is 240612).It can be used for generating there are also other methods and reagent and screening antibodies display libraries be (see, for example, Barbas etc. People, Proc.Natl.Acad.Sci.USA 88:7978-7982 (1991)).

Human antibody can also be by introducing transgenic animals (such as wherein endogenous immune ball for human immunoglobulin gene's seat Protein gene partially or completely inactivates and has been introduced into the mouse of human immunoglobulin gene) in prepare.In attack, Observe that human antibody generates, in people various aspects observe closely similar, including gene rearrangement, assembly and antibody library. This method is for example described in United States Patent (USP) 5,545,807；5,545,806；5,569,825；5,625,126；5,633,425； 5,661,016 and about XenoMouse technology United States Patent (USP) 6,075,181 and 6,150,584；Lonberg and Huszar,Intern.Rev.Immunol.13:65-93(1995).Alternatively, the antibody of target antigen can be directed to by generation (such bone-marrow-derived lymphocyte can be from tumor disease or the individual that may be immunized in vitro for human B lymphocyte Middle acquisition) immortalization prepare human antibody.See, for example, Cole et al., Monoclonal Antibodies and Cancer Therapy,Alan R.Liss,p.77(1985)；Boerner et al., J.Immunol, 147 (l): 86-95 (1991)；With U.S.P.N.5,750,373。

Multiple technologies known in the art can be used to prepare in monoclonal antibody, including hybridoma technology, recombinant technique, Display technique of bacteriophage, transgenic animals (such as) or some combinations.It is, for example, possible to use hybridization Tumor and art-recognized biochemistry and genetic engineering technology produce monoclonal antibody, are such as described in detail in An, Zhigiang(ed.)Therapeutic Monoclonal Antibodies:From Bench to Clinic,John Wiley and Sons,1^sted.2009；Shire et.al.(eds.)Current Trends in Monoclonal Antibody Development and Manufacturing,Springer Science+Business Media LLC,1^st ed.2010；Harlow et al., Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory Press,2nd ed.1988；Hammerling, et al., in:Monoclonal Antibodies and T- Cell Hybridomas 563-681 (Elsevier, N.Y., 1981), every document are hereby incorporated by reference.It should Understand, can further change selected binding sequence, for example, with improve the affinity to target, make target binding sequence humanization, Improve its generation in cell culture, reduce its vivo immunogenicity, generate multi-specificity antibody etc., and include to change The antibody of target binding sequence be also antibody of the invention.In a preferred embodiment, it is made by using hybridoma Standby anti-human LAG-3 monoclonal antibody.

Generate the generation of the hybridoma of human monoclonal antibodies of the invention

In order to obtain the hybridoma for generating antibody of the present invention human monoclonal antibodies for example of the invention, it can separate to come from and exempt from The splenocyte and/or lymph node cells of epidemic disease mouse are simultaneously fused to suitable immortalized cell line, such as mouse myeloma is thin Born of the same parents system.The hybridoma that generation screening with regard to antigen-specific antibodies generates.The generation of hybridoma is well-known in the art 's.See, for example, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications,New York。

Generate the generation of the transfectoma of monoclonal antibody of the invention

Antibody of the invention can also in host cell transfectoma use example recombinant DNA skill as known in the art The combination (such as Morrison, S. (1985) Science 229:1202) of art and gene transfection method generates.In a reality It applies in scheme, the DNA of the coded portion or full-length light chains and heavy chain that are obtained by standard molecular biological technique is inserted into one Or in multiple expression vectors, so that the gene is operably connected to transcription and translation and adjusts sequence.In this context, art Language " being operably connected ", which is intended to mean that, is connected to antibody gene in carrier, so that carrying intracorporal transcription and translation control sequence Play the expectation function that they adjust antibody gene transcription and translation.

Term " regulating and controlling sequence " be intended to include the promoter of transcription or translation of control antibody chain gene, enhancer and other Expression control element (such as polyadenylation signal).These adjust sequence description in such as Goeddel (Gene Expression Technology.Methods in Enzymology 185,Academic Press,San Diego,CA (1990)) in.Exemplary regulating and controlling sequence for mammalian host cell expression includes instructing Gao Shui in mammalian cell The viral components of flat protein expression, such as from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (such as gland The promoter and/or enhancer of viral major late promoter (AdMLP) and polyomavirus, or nonviral regulatory can be used Sequence, such as ubiquitin promoter or beta-globin promoter；In addition, the controlling element being made of the sequence of separate sources, such as SRa Promoter systems, it includes the sequences of the long terminal repeats from 1 type of SV40 early promoter and human T cell leukemia virus It arranges (Takebe et al. (1988) MoI.Cell.Biol.8:466-472).Expression vector and expression control sequence be selected as with The expression host cell used is compatible.

Antibody light chain gene and antibody heavy chain gene can be inserted into identical or different expression vector.In some implementations In scheme, variable region is used for by inserting it into the encoded required heavy chain constant region of isotype and the table of constant region of light chain Up to the full length antibody gene for generating any antibody isotype in carrier, so that VH section, which is operably coupled to, carries intracorporal CH Section and VL section, which are operably coupled to, carries intracorporal CL section.Additionally or alternatively, recombinant expression carrier can encode rush The signal peptide secreted into antibody chain from host cell.Antibody chain gene can be cloned into carrier, so that signal peptide meets reading Frame it is connected to the amino terminal of antibody chain gene.Signal peptide can be immunoglobulin signal peptide or heterologous signal peptide (comes From the signal peptide of non-immunoglobulin protein).

Other than antibody chain gene and regulating and controlling sequence, recombinant expression carrier of the invention can also carry additional sequence Column, such as adjust sequence (such as replication orgin) and selectable marker gene that carrier replicates in host cell.Selected marker base The host cell of carrier has been imported (see, for example, U.S. Patent number 4,399,216 because facilitating selection；4,634,665 and 5, 179,017).For example, generally select marker gene assign carrier have been introduced into host cell therein to drug (such as G418, Hygromycin or methotrexate (MTX)) resistance.Selectable marker gene may include dihyrofolate reductase (DHFR) gene (for having Methotrexate (MTX) selection/amplification dhfr- host cell) and neo gene (being selected for G418).

In order to express light chain and heavy chain, it is thin that the expression vector of encoding heavy chain and light chain is transfected by host by standard technique In born of the same parents.The various forms of term " transfection " is intended to cover for exogenous DNA being introduced into protokaryon or eukaryotic host cell Various technologies, such as electroporation, calcium phosphate precipitation, DEAE- glucan transfection etc..Can protokaryon or eukaryotic host cell for example Expression antibody of the invention in mammalian host cell (it can assemble and secrete appropriate fold and immunocompetent antibody).

Mammalian host cell for expressing recombinant antibodies of the present invention includes with DHFR selected marker (for example, such as Described in R.J.Kaufman and P.A.Sharp (1982) J.MoI.Biol.159:601-621) China for being used together Hamster Qvary (Chinese hamster ovary celI) (is included in Urlaub and Chasin, (1980) Proc.Natl.Acad.Sci.USA 77:4216- Dhfr Chinese hamster ovary celI described in 4220), NSO myeloma cell, COS cell and SP2 cell.Particularly, in order to NSO marrow Tumor is used together, and another expression system is GS gene disclosed in WO 87/04462, WO 89/01036 and EP 338,841 Expression system.It is thin by culture host when the recombinant expression carrier of encoding antibody genes is imported mammalian host cell Born of the same parents are enough the period for allowing antibody to express in host cell or by the culture mediums of antibody-secreting to host cell growth Generate antibody.Antibody can be recycled from culture medium using Standard protein purification method.

Anti- LAG3 antibody with certain properties

Antibody of the invention is characterized in that the specific functional features or property of antibody.In some embodiments, it separates Antibody or its antigen-binding portion thereof have one or more following properties:

(a) with 2 × 10^-10M or lower K_DIn conjunction with people LAG-3；

(d) inhibit the combination of LAG-3 and LSECtin and/or galectin-3；Or

(e) combine people LAG-3 without reacting across family.

Antibody of the invention is with high-affinity combination people LAG-3.This can be used in the combination of antibody and LAG-3 of the invention One or more technologies for well establishing in field, such as ELISA are assessed.The binding specificity of antibody of the present invention can also lead to It crosses such as flow cytometry monitoring antibody and expresses the combination of the cell of LAG-3 albumen to determine.For example, antibody can pass through stream Formula cell art measures to test, and wherein the cell line of antibody and expression people LAG-3 have for example transfected on its cell surface Express the Chinese hamster ovary celI reaction of LAG-3.Other suitable cells for Flow Cytometry Assay include expressing natural LAG-3 AntiCD3 McAb-stimulation CD4⁺The T cell of activation.Additionally or alternatively, can in BIAcore binding assay test antibody knot It closes, including binding kinetics (such as Kd value).Other suitable binding analysis include elisa assay, such as use recombination LAG-3 Albumen.For example, antibody of the invention is with 5 × 10^-8M or lower K_DIn conjunction with people's LAG-3 albumen, with 2 × 10^-8M or lower K_D In conjunction with people's LAG-3 albumen, with 5 × 10^-9M or lower K_DIn conjunction with people's LAG-3 albumen, with 4 × 10^-9M or lower K_DIn conjunction with people LAG-3 albumen, with 3 × 10^-9M or lower K_DIn conjunction with people's LAG-3 albumen, with 2 × 10^-9M or lower K_DIn conjunction with people's LAG-3 egg It is white, with 1 × 10^-9M or lower K_DIn conjunction with people's LAG-3 albumen, with 5 × 10^-10M or lower K_DIn conjunction with people's LAG-3 albumen, or With 1 × 10^-10M or lower K_DIn conjunction with people's LAG-3 albumen.

The ability that antibody adjusts immune response (such as antigen-specific T cell response) can be for example, by antibody in antigen Specific T-cells response moderate stimulation generates the ability of interleukin 2 (IL-2) to indicate.In certain embodiments, this hair Bright antibody combination people LAG-3 and the ability for showing stimulator antigen specific T-cells response.Assessing antibody stimulates immune response energy The means of power may include that for example the ability of antibody inhibiting tumor growth or antibody stimulate itself in Replanting model mice in vivo The ability of immune response.

The antibody separated as disclosed herein or its antigen-binding portion thereof inhibit LAG-3 and ajor histocompatibility (MHC) combination of II class molecule, FGL1 class molecule, LSECtin and/or galectin-3.LAG-3 negative regulation T cell Signal transduction and function.The ligand of LAG-3 includes such as ajor histocompatibility (MHC) II class molecule, LSECtin and galactolipin Galectin-3.LAG-3 can be with MHC II class interaction of molecules (Baixeras et al. (1992) on cell surface J.Exp.Med.176:327-337；Huard et al. (1996) Eur.J.Immunol.26:1180-1186).It has been proposed that LAG-3 and being directly incorporated in for MHC II class molecule lower CD4⁺It works in the antigen dependent stimulation of T lymphocyte (Huard et al. (1994) Eur.J.Immunol.24:3216-3221).In the recent period, display equality people is tested by inside and outside into one Step card FGL1 is that the principal immune of LAG-3 inhibits ligand, proposes a new tumor immune escape access FGL1-LAG-3, Block FGL1-LAG-3 interaction that antitumor action (Cell.2019 Jan 10 can be enhanced；176(1-2):334- 347.e12.)。

Galectin-3 is 31kD agglutinin, by several mechanism regulatory T-cell responses, including Apoptosis, TCR crosslinking and TCR are lowered.Galectin-3 is in conjunction with LAG-3, and LAG-3 expression is situated between for galectin-3 The external CD8+T cell inhibition led is required.(Kouo et al. (2015) Cancer Immunol.Res.10.1158:2326- 6066).Have been displayed anti-LSECtin inhibit the growth of B16 melanoma cells (Xu et al. (2014) Cancer Res.74 (13): 3418-3428)。

Comprising having the anti-LAG3 antibody of the CDR of sequence identity with particular sequence

C) A) one or more CDRH and B) one or more CDRL.

Unless otherwise stated, can be according to one of numbering plan presented below by each CDR of Amino acid score dispensing: Kabat et al. (1991) Sequences of Proteins of Immunological Interest (5^th Ed.),US Dept.of Health and Human Services,PHS,NIH,NIH Publication no.91-3242；Chothia Et al., 1987, PMID:3681981；Chothia et al., 1989, PMID:2687698；MacCallum et al., 1996, PMID: 8876650；Or Dubel, Ed. (2007) Handbook of Therapeutic Antibodies, 3^rd Ed.,Wily-VCH Verlag GmbH and Co.。

Variable region and CDR in antibody sequence can be according to the general rules that this field has been developed (as described above, for example Kabat numbering system) or identified by comparing the database of sequence and known variable area.In Kontermann and Dubel, eds., Antibody Engineering, Springer, New York, NY, 2001 and Dinarello et al., It is retouched in Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000 The method for identifying these regions is stated.The exemplary database of antibody sequence is described in and available from www.bioinf.org.uk/ Website " Abysis " on abs is (by Department of Biochemistry&Molecular Biology University The A.C.Martin of College London, London, England are safeguarded) and the website VBASE2 www.vbase2.org, such as Retter et al., Nucl.Acids Res., described in 33 (Database issue): D671-D674 (2005).It is preferable to use Abysis database analysis sequence incorporates the sequence data from Kabat, IMGT and Protein Data Bank (PDB) and comes From the structured data of PDB, referring to the Protein Sequence and in the written book of Dr.Andrew C.R.Martin Structure Analysis of Antibody Variable Domains.In:Antibody Engineering Lab Manual(Ed.:Duebel,S.and Kontermann,R.,Springer-Verlag,Heidelberg,ISBN-13:978- 3540413547, can also be obtained on the bioinforg.uk/abs of website).Abysis database website further includes having developed use In the general rule for the CDR that identification can use according to the teaching of this article.Unless otherwise stated, as described herein all CDR is obtained all in accordance with the Abysis database website of Kabat.

The algorithm of E.Meyers and W.Miller can be used in percentage identity between two amino acid sequences (Comput.Appl.Biosci., 4:11-17 (1988)) determine, which has been incorporated into ALIGN program (version 2 .0), use PAM120 weight residue table, GAP LENGTH PENALTY 12, gap penalty 4.In addition, the percentage between two amino acid sequences Identity can be determining by the algorithm (J.Mol.Biol.48:444-453 (1970)) of Needleman and Wunsch, simultaneously Enter in the GAP program in GCG software package (can be obtained from http://www.gcg.com), using 62 matrix of Blossum or PAM250 matrix, gap weight are 16,14,12,10,8,6 or 4, Length Weight 1,2,3,4,5 or 6.

Additionally or alternatively, protein sequence of the invention can be further used as " search sequence " to execute and be directed to public affairs The search of database is altogether for example to identify correlated series.Altschul can be used in this search, et al. (1990) XBLAST program (version 2 .0) Lai Zhihang of J.MoI.Biol.215:403-10.BLAST albumen can be carried out with XBLAST program Matter search, score=50, word length=3, to obtain the amino acid sequence homologous with antibody molecule of the invention.In order to be used for Omparison purpose vacancy compares, and vacancy BLAST can be used, such as Altschul et al., (1997) Nucleic Acids Res.25 (17): described in 3389-3402.When using BLAST and gapped BLAST programs, can be used each program (for example, XBLAST and NBLAST) default parameters.Referring to www.ncbi.nlm.nih.gov.

In other embodiments, cdr amino acid sequence can with above-mentioned each sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% are identical.As illustrative example, antibody may include and be selected from SEQ CDRH1 shown in one of sequence of ID NO:1 and 7 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, the CDRH1 of 98% or 99% sequence identity.

Anti- LAG3 antibody comprising the CDR for adding, lacking or replacing with amino acid

A) one or more heavy chain CDR (CDRH), it is selected from the following at least one: (i) be selected from SEQ ID NO:1 and 7 CDRH1 or there is amino acid addition, missing or the difference replaced for being no more than 2 amino acid with the amino acid sequence of the CDRH1 Different CDRH1；(ii) it is selected from the CDRH2 of SEQ ID NO:2 and 8 or exists with the amino acid sequence of the CDRH2 no more than 2 ammonia The CDRH2 for the difference that the amino acid of base acid adds, lacks or replaces；(iii) be selected from SEQ ID NO:3 and 9 CDRH3 or with There is the CDRH3 of the amino acid addition, missing or the difference replaced that are no more than 2 amino acid in the amino acid sequence of the CDRH3；

B) one or more light chain CDR (CDRL), it is selected from the following at least one: (i) be selected from SEQ ID NO:4 and 10 CDRL1 or there is amino acid addition, missing or the difference replaced for being no more than 2 amino acid with the amino acid sequence of the CDRL1 Different CDRL1；(ii) it is selected from the CDRL2 of SEQ ID NO:5 and 11 or exists with the amino acid sequence of the CDRL2 no more than 2 The CDRL2 for the difference that the amino acid of amino acid adds, lacks or replaces；(iii) is selected from the CDRL3 of SEQ ID NO:6 and 12 Or there is the amino acid addition, the difference for lacking or replacing for being no more than 2 amino acid with the amino acid sequence of the CDRL3 CDRL3；Or

C) A) one or more CDRH and B) one or more CDRL.

Preferably, isolated antibody or the CDR of its antigen-binding portion thereof contain not more than 2 amino acid or not more than 1 The conservative substitution of amino acid.As used herein, term " conservative substitution " refers to and can not adversely influence or change comprising amino acid The amino acid substitution of the fundamental property of the protein/polypeptide of sequence.For example, conservative substitution can pass through standard known in the art Technology (such as mutagenesis of direct mutagenesis and PCR mediation) introduces.Conserved amino acid replaces to be had including wherein amino acid residue The substitution that another amino acid residue of similar side chain replaces, such as physics or intimate residue (such as with similar big Small, shape, charge, chemical property includes forming the ability etc. of covalent bond or hydrogen bond) to the substitution of corresponding amino acid residue.This The amino acid residue families with similar side chain have been defined in field.These families include the amino acid with basic side chain (such as lysine, arginine and histidine), the amino acid (such as aspartic acid and glutamic acid) with acid side-chain have not Electrically charged polar side chain amino acid (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, half Cystine, tryptophan), amino acid (such as alanine, valine, leucine, isoleucine, dried meat ammonia with non-polar sidechain Acid, phenylalanine, methionine), there is β-branched building block amino acid (such as threonine, valine, isoleucine) and have There is the amino acid (such as tyrosine, phenylalanine, tryptophan, histidine) of beta-branched side.Therefore, corresponding amino acid residue It is preferred that being replaced by another amino acid residue from identical side chain family.For identifying the method for conservative substitution at this It is well known in field (see, for example, Brummell et al., Biochem.32:1180-1187 (1993)；Kobayashi et al., Protein Eng.12(10):879-884(1999)；With Burks et al., Proc.Natl.Acad.Sci.USA 94:412- 417 (1997), are incorporated herein by reference).

Anti- LAG3 antibody comprising CDR

(a) comprising the CDRH1 of SEQ ID NO:1；

(b) comprising the CDRH2 of SEQ ID NO:2；

(c) comprising the CDRH3 of SEQ ID NO:3；

(d) comprising the CDRL1 of SEQ ID NO:4；

(e) comprising the CDRL2 of SEQ ID NO:5；With

(f) comprising the CDRL3 of SEQ ID NO:6.

In a particular embodiment, isolated antibody or its antigen-binding portion thereof include:

(a) CDRH1 being made of SEQ ID NO:1；

(b) CDRH2 being made of SEQ ID NO:2；

(c) CDRH3 being made of SEQ ID NO:3；

(d) CDRL1 being made of SEQ ID NO:4；

(e) CDRL2 being made of SEQ ID NO:5；With

(f) CDRL3 being made of SEQ ID NO:6.

(a) comprising the CDRH1 of SEQ ID NO:7；

(b) comprising the CDRH2 of SEQ ID NO:8；

(c) comprising the CDRH3 of SEQ ID NO:9；

(d) comprising the CDRL1 of SEQ ID NO:10；

(e) comprising the CDRL2 of SEQ ID NO:11；With

(f) comprising the CDRL3 of SEQ ID NO:12.

(a) CDRH1 being made of SEQ ID NO:7；

(b) CDRH2 being made of SEQ ID NO:8；

(c) CDRH3 being made of SEQ ID NO:9；

(d) CDRL1 being made of SEQ ID NO:10；

(e) CDRL2 being made of SEQ ID NO:11；With

(f) CDRL3 being made of SEQ ID NO:12.

Anti- LAG3 antibody comprising heavy chain variable region and light chain variable region

(A) heavy chain variable region:

(i) comprising the amino acid sequence of SEQ ID NO:13；

(iii) comprising having the one or more addition of amino acid, missing and/or substituted with SEQ ID NO:13 compared with Amino acid sequence；And/or

(B) light chain variable region:

(i) comprising the amino acid sequence of SEQ ID NO:14；

(ii) comprising having the amino acid sequence of at least 85%, at least 90% or at least 95% identity with SEQ ID NO:14 Column；

(iii) comprising having the one or more addition of amino acid, missing and/or substituted with SEQ ID NO:14 compared with Amino acid sequence.

(a) heavy chain variable region being made of the amino acid sequence of SEQ ID NO:13；And/or

(b) light chain variable region of the amino acid sequence comprising SEQ ID NO:14.

(A) heavy chain variable region:

(i) comprising the amino acid sequence of SEQ ID NO:15；

(iii) comprising having the one or more addition of amino acid, missing and/or substituted with SEQ ID NO:15 compared with Amino acid sequence；And/or

(B) light chain variable region:

(i) comprising the amino acid sequence of SEQ ID NO:16；

(iii) comprising having the one or more addition of amino acid, missing and/or substituted with SEQ ID NO:16 compared with Amino acid sequence.

(a) heavy chain variable region being made of the amino acid sequence of SEQ ID NO:15；And/or

(b) light chain variable region of the amino acid sequence comprising SEQ ID NO:16.

In other embodiments, the amino acid sequence of heavy chain variable region and/or light chain variable region can with it is above-mentioned each Sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% Or 99% is identical.As illustrative example, antibody may include can with the heavy chain of the amino acid sequence of SEQ ID NO:15 composition Become area have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, The heavy chain variable region of 98% or 99% sequence identity.

In some further embodiments, isolated antibody or its antigen-binding portion thereof may include heavy chain and/or The conservative substitution or modification of amino acid in the variable region of light chain.This field understands, can carry out certain not eliminating antigen In conjunction with conserved sequence modification.See, for example, Brummell et al. (1993) Biochem 32:1180-8；De Wildt et al. (1997)Prot.Eng.10:835-41；Komissarov et al. (1997) J.Biol.Chem.272:26864-26870；Hall Et al. (1992) J.Immunol.149:1605-12；Kelley and O'Connell(1993)Biochem.32:6862-35； Adib-Conquy et al. (1998) Int.Immunol.10:341-6 and Beers et al. (2000) Clin.Can.Res.6: 2835-43。

Terms used herein " conservative substitution " refer to amino acid substitution, can not adversely influence or change comprising amino The fundamental property of the protein/polypeptide of acid sequence.For example, conservative substitution can by standard technique known in the art (such as The mutagenesis that direct mutagenesis and PCR are mediated) it introduces.Conserved amino acid replaces including wherein amino acid residue by with similar side chain The substitution that another amino acid residue replaces, such as physics or intimate residue (such as with similar size, shape, electricity Lotus, chemical property include forming the ability etc. of covalent bond or hydrogen bond) to the substitution of corresponding amino acid residue.Determine this field Amino acid residue families of the justice with similar side chain.These families include have basic side chain amino acid (such as lysine, Arginine and histidine), the amino acid (such as aspartic acid and glutamic acid) with acid side-chain has uncharged polarity Amino acid (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, the color ammonia of side chain Acid), amino acid with non-polar sidechain (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, Methionine), there is β-branched building block amino acid (such as threonine, valine, isoleucine) and there is beta-branched side Amino acid (such as tyrosine, phenylalanine, tryptophan, histidine).Therefore, corresponding amino acid residue is preferably come from phase Another amino acid residue with side chain family replaces.Method for identifying that conservative replaces is known in the art (see, for example, Brummell et al., Biochem.32:1180-1187 (1993)；Kobayashi et al., Protein Eng.12(10):879-884(1999)；With Burks et al., Proc.Natl.Acad.Sci.USA 94:412-417 (1997), It is incorporated herein by reference).

Divide storehouse and epitope mapping

It will be further appreciated that disclosed antibody by with by selected target or its segment presentation discrete epitope or Immunogenic determinant association combines.In certain embodiments, epitope or Immunogenic determinant include the chemistry of molecule Active surface grouping, such as amino acid, carbohydrate side chain, phosphoryl or sulfonyl, and in certain embodiments, it can have spy Fixed three-dimensional structural feature and/or specific charge characteristics.Therefore, as used herein, term " epitope " include can with it is immune Globulin or T cell receptor specific binding or otherwise with any protein determinant of interaction of molecules.At certain In a little embodiments, when its target antigen in the preferential identification of protein of antibody and/or the complex mixture of macromolecular, antibody quilt Think specific binding (or immunologic specificity combines or reaction) antigen.In some embodiments, work as equilibrium dissociation constant (K_D) it is less than or equal to 10^-6M or be less than or equal to 10^-7When M, more preferably work as K_DLess than or equal to 10^-7When M, claim antibody and antigen Specific binding is equal to 10^-8M even more preferably works as K_DLess than or equal to 10^-9When M, antibody is considered molecule of the antigen binding.

The epitope (sometimes referred to as " linear " or " continuous " epitope) formed by continuous amino acid is usually in protein denaturation Retain, and folds the epitope to be formed by three-level and usually lost after protein denaturation.Under any circumstance, antibody epitope is usual It include at least three, more generally at least 5 or 8-10 amino acid in unique space conformation.

In this aspect, it will be understood that in certain embodiments, epitope can be with one of such as LAG-3 albumen Or multiple regions, structural domain or motif are combined or are located therein.Similarly, art recognized term " motif " will lead to according to it It is used with meaning, and should usually refer to the short conservative region of the protein of usual ten to 20 continuous amino acid residues.

Anyway, once it is determined that required epitope on antigen, it is possible to the antibody for being directed to the epitope is generated, such as It is immune with the peptide comprising epitope by using technology described in the present invention.Alternatively, in discovery procedure, the generation of antibody and table Sign can illustrate the information about the expectation epitope being located in specific domain or motif.It, can be competitive from these information The antibody of screening and the combination of same epitope.The method for realizing this point is to be at war with research to find that contending with one other property combines Antibody, i.e. antibody competition combination antigen.It is described in WO 03/48731 and point storehouse is carried out to antibody based on its cross competition High throughput method.Point storehouse or structural domain is horizontal or epitope mapping includes its of antibody competition or the antigen fragment expression on yeast He is well known in the present art method.

As used herein, term " point storehouse " refers to for being grouped or being divided to antibody based on antigen binding characteristics and competition The method of class.Although these technologies be for the antibody of the invention that defines and classify it is useful, these storehouses (bin) are not total Be directly in conjunction with epitope, and epitope combine this initial measurement can by this field and as described herein other Generally acknowledged method is further improved and confirmation.It is provided it is understood, however, that with the property tested distributing antibody to each storehouse It can indicate the information of the treatment potentiality of disclosed antibody.

More specifically, can determine selected reference by using method shown in known in the art and embodiment hereof Whether antibody (or its segment) combines identical epitope or in conjunction with the second test antibody cross competition (that is, in identical storehouse It is interior).

Other compatible epitope mapping techniques include Alanine scanning mutagenesis body, peptide trace (Reineke (2004) Methods Mol Biol 248:443-63) (being particularly incorporated herein by reference in their entirety) or peptide cleavage analysis.In addition, can To use the epitope excision of antigen, epitope is extracted and the methods of chemical modification (Tomer (2000) Protein Science 9: 487-496) (particularly it is incorporated herein by reference in their entirety).

Encode the nucleic acid molecules of antibody of the present invention

Standard molecular biological technique acquisition can be used in nucleic acid of the invention.For hybridoma expression antibody (for example, The hybridoma prepared as described further below by the transgenic mice for carrying human immunoglobulin gene), mark can be passed through Quasi- PCR amplification or cDNA clone technology obtain the cDNA of the light chain and heavy chain that encode the antibody prepared by hybridoma.For from The antibody (for example, using display technique of bacteriophage) that immunoglobulin gene libraries obtain, the nucleic acid for encoding this antibody can be with It is recycled from gene library.

By another DNA that the nucleic acid for encoding VH is operably coupled to encoding heavy chain constant (CH1, CH2 and CH3) The isolated nuclear transformation for encoding the area VH can be total length heavy chain gene by molecule.The sequence of people's weight chain constant area gene is in ability It is known (see, for example, Kabat etc. (1991), ibid) in domain, and can be obtained by standard PCR amplification comprising these The DNA fragmentation in region.Heavy chain constant region can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but more Preferably IgG1 or IgG4 constant region, most preferably IgG4 constant region.

By the way that the DNA for encoding VL to be operably coupled to another DNA molecular of coding constant region of light chain CL, can will encode The isolated nuclear transformation in the area VL is full-length light chains gene (and Fab light chain gene).The sequence of people's light chain constant region gene is (see, for example, Kabat etc., ibid) known in the art, and can be obtained by standard PCR amplification comprising these regions DNA fragmentation.In preferred embodiments, constant region of light chain can be κ or λ constant region.

Once obtain coding VH and VL section DNA fragmentation, can by the further operating of standard recombinant dna technology these DNA fragmentation, such as full length antibody chain gene, Fab fragment gene or scFv gene are converted by variable region gene.In these operations In, it encodes the DNA fragmentation of VL or VH and encodes another DNA fragmentation such as antibody constant region or flexible joint of another protein It is operably connected.Terminology used in this article " being operably connected " is intended to indicate that two DNA fragmentations are connected to two The amino acid sequence of a DNA fragmentation coding is maintained in frame.

Currently preferred nucleic acid molecules are that coding 1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L monoclonal is anti- The nucleic acid molecules of the VH and VL sequence of body.Encode the DNA of the VH sequence of 1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L Sequence is shown in SEQ ID NO:17 and 19.Encode the VL of 1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L The DNA sequence dna of sequence is shown in SEQ ID NO:18 and 20.In some embodiments, nucleic acid respectively with SEQ ID NO:17-20 have at least 80% (for example, at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%96%, 97%, 98% or sequence identity 99%).In some embodiments, the percentage source of identity From the degeneracy of genetic code, and the protein sequence encoded remains unchanged.

Pharmaceutical composition

The component of composition

Pharmaceutical composition can optionally contain one or more other active pharmaceutical ingredients, for example, another antibody or Drug.Pharmaceutical composition of the invention can also be with for example another immunostimulant, anticancer agent, antivirotic or vaccine combination Application, so that anti-lag-3 antibody enhances the immune response to vaccine.Pharmaceutically acceptable carrier may include for example pharmaceutically Acceptable liquid, gel or solid carrier, aqueous medium, non-aqueous medium, antimicrobial, isotonic agent, buffer, antioxygen Agent, anesthetic, suspension/dispersing agent, chelating agent, diluent, adjuvant, excipient or nontoxic auxiliary substance, it is known in the art Various components combination or more.

Suitable component may include such as antioxidant, filler, adhesive, disintegrating agent, buffer, preservative, profit Lubrication prescription, flavoring agent, thickener, colorant, emulsifier or stabilizer such as sugar and cyclodextrin.Suitable antioxidant may include for example Methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercapto glycerol, sulfydryl Acetic acid, sulfydryl D-sorbite, butyl methyl methyl phenyl ethers anisole, butylated hydroxytoluene and/or propylarsonic acid salt.As disclosed in the present invention , in the public containing the present invention of one or more antioxidants such as methionine comprising going back original antibody or its antigen-binding fragment In the antibody for the composition opened or the solvent of antigen-binding fragment, it can be oxidized.Redox can be prevented or reduced in conjunction with parent With the reduction of power, to enhance Antibody stability and extend the shelf life.Therefore, in some embodiments, the present invention provides Composition comprising one or more antibody or its antigen-binding fragment and one or more antioxidants such as methionine.This hair It is bright to further provide a variety of methods, wherein by antibody or its antigen-binding fragment and one or more antioxidants such as first sulphur ammonia Acid-mixed is closed.To which antibody or its antigen-binding fragment can be prevented from aoxidizing, to extend its shelf-life and/or increase activity.

Application, preparation and dosage

Pharmaceutical composition of the invention can be applied to subject in need, the approach packet in vivo by all means It includes but is not limited to take orally, intravenously, intra-arterial, subcutaneously, parenteral is intranasally, intramuscular, and encephalic is intracardiac, intra-ventricle, intratracheally, mouth Chamber, rectum is intradermal in peritonaeum, and part is percutaneous and intrathecal, or passes through implantation or sucking.The present composition can be configured to Solid, semisolid, liquid or gas form preparation；Including but not limited to tablet, capsule, pulvis, granule, ointment, Solution, suppository, enema, injection, inhalant and aerosol.It can choose properly according to expected application and therapeutic scheme Preparation and administration method.

Appropriate formulation for enteral administration includes hard or soft gelatine capsule, pill, tablet, including coated tablet, the wine made of broomcorn millet Agent, suspension, syrup or inhalant and its controlled release form.

Preparation suitable for parenteral administration (such as passing through injection) includes active constituent dissolution, is suspended in wherein or with it Aqueous or non-aqueous, isotonic, the apyrogeneity, sterile liquid (example of (for example, in liposome or other particles) that his mode provides Such as solution, suspension).In addition these liquid can contain other pharmaceutically acceptable ingredients, such as antioxidant, buffering Agent, preservative, stabilizer, bacteriostatic agent, suspending agent, thickener and blood (or other dependent bodies for making preparation and expected recipient Liquid) isotonic solute.The example of excipient includes such as water, alcohol, polyalcohol, glycerol, vegetable oil etc..Suitable for such preparation The example of isotonic vehicle includes sodium chloride injection, Ringer's solution or lactated ringer's injection.Similarly, particular dosage regimen (i.e. dosage, time and repetition) will depend on specific individual and individual medical history and such as pharmacokinetics (such as half-life period, Clearance rate etc.) experience consider.

Frequency of administration can be determined and be adjusted over the course for the treatment of, and the quantity based on reduction proliferation or tumorigenic cell, The reduction for maintaining this tumour cell reduces the proliferation of tumour cell or the development of Branch-delay.In some embodiments, it applies Dosage be can be adjusted or be reduced to control potential side effect and/or toxicity.Alternatively, therapeutic combination of the present invention is held Continuous continuous release formulations may be suitable.

It will be understood to those of skill in the art that suitable dosage can be different because of patient.It determines that optimal dose is usually directed to control Treat the balance of benefit level and any risk or harmful side effect.Selected dosage level will depend on many factors, including But it is not limited to the activity of specific compound, is applied, administration time, compound clearance rate, duration for the treatment of, other, which are combined, makes Drug, compound and/or material, the severity and species of illness, gender, age, weight, the state of an illness, one of patient As health status and pervious medical history.The amount and administration method of compound are finally determined by doctor, animal doctor or clinician, but are led to Often selection dosage with reach realize needed for effect site of action at local concentration, without will lead to substantive nocuousness or not Sharp side effect.

In general, antibody of the invention or its antigen-binding portion thereof can be applied with various ranges.These include every dosage about 5 μ g/kg weight is to about 100mg/kg weight；About 50 μ g/kg weight of every dosage is to about 5mg/kg weight；About 100 μ g/kg of every dosage Weight is to about 10mg/kg weight.Other ranges include about 100 μ g/kg weight of every dosage to about 20mg/kg weight and every dosage about 0.5mg/kg weight is to about 20mg/kg weight.In certain embodiments, dosage is at least about 100 μ g/kg weight, at least about 250 μ g/kg weight, at least about 750 μ g/kg weight, at least about 3mg/kg weight, at least about 5mg/kg weight, at least about 10mg/ Kg weight.

Anyway, antibody of the invention or its antigen-binding portion thereof are preferably applied in need tested as needed Person.Those skilled in the art can determine frequency of administration, such as year of the attending physician based on treated illness, treated subject The consideration of age, state of the illness, the general health of treated subject etc..

In certain preferred aspects, the therapeutic process for being related to antibody or its antigen-binding portion thereof of the invention will wrap It is contained in the selected drug products for the multi-dose applied in the time of several weeks or several months.More specifically, antibody of the invention or its Antigen-binding portion thereof can be daily, and every two days, every four days, weekly, every ten days, every two weeks, every three weeks, monthly, and every six weeks, every two A month, every ten weeks or every three months application.In this regard, it is to be understood that can based on patient respond and clinical practice come Change dosage or adjustment interval.

Can also be empirically determined in the individual for giving one or many applications disclosed therapeutic combination dosage and Scheme.For example, the therapeutic combination of individual increment dosage generated as described herein can be given.In selected embodiment, Dosage can the empirically determined respectively or side effect observed or toxicity gradually increase or reduce or mitigate.It is selected in order to assess The effect of composition selected, can track the marker of specified disease, illness or the state of an illness as previously described.For cancer, these packets It includes and tumor size is directly measured by palpation or visual observation, tumor size is measured by X-ray or other imaging techniques indirectly； The improvement assessed by the microexamination of direct tumor biopsy and tumor sample；Measurement is reflected according to methods described herein Fixed indirect tumor markers (such as PSA for prostate cancer) or oncogenicity antigen, the mitigation of pain or paralysis；With tumour Relevant speech, eyesight, breathing or the improvement of other disability；Appetite increases；Or the quality of life of the test measurement by receiving Raising or the extension of life cycle.It will be apparent to one skilled in the art that dosage will be according to individual, the type of the tumour state of an illness, tumour Whether the stage of the state of an illness, the tumour state of an illness have started the other positions being transferred in individual and the treatment used in the past and and have exercised Treatment and change.

COMPATIBLE FORMULATIONS for parenteral administration (such as intravenous injection) will be about 10 μ g/mL to about comprising concentration The antibody of 100mg/mL or its antigen-binding portion thereof.In certain selected embodiments, antibody or its antigen-binding portion thereof Concentration will include 20 μ g/mL, 40 μ g/mL, 60 μ g/mL, 80 μ g/mL, 100 μ g/mL, 200 μ g/mL, 300 μ g/ μ g/mL, 400 μ G/mL, 500 μ g/mL, 600 μ g/mL, 700 μ g/mL, 800 μ g/mL, 900 μ g/mL or 1mg/mL.In other preferred embodiment party In case, ADC concentration will include 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL, 6mg/mL, 8mg/mL, 10mg/mL, 12mg/mL, 14mg mL, 16mg/mL, 18mg/mL, 20mg/mL, 25mg/mL, 30mg/mL, 35mg/mL, 40mg/mL, 45mg/mL, 50mg/ ML, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL or 100mg/mL.

Application of the invention

Antibody, antibody compositions and method of the invention has many in vitro and in vivo purposes, the inspection including such as LAG-3 The immune response surveyed or pass through blocking LAG-3 enhancing.For example, these molecules can be applied to culture cell in vitro or in vitro, Or it is for example applied to people experimenter in vivo, to enhance the immunity in various situations.Immune response can be adjusted, such as be increased By force, it stimulates or raises.

Preferred subject includes the human patient for needing to enhance immune response.The method has especially suitable for treatment can Pass through the human patient of the illness of enhancing immune response (such as immune response of T cell mediation) treatment.In a specific implementation In scheme, the method is particularly suitable for interior therapeutic cancer.In order to realize immune antigentic specificity enhancing, can will resist LAG-3 antibody is applied together with interested antigen or antigen may be already present in subject to be treated and (such as take Subject with tumour or virus).When the antibody of LAG-3 is applied together with another medicament, the two can be in any order It applies or is administered simultaneously.

Invention further provides the presence for people LAG-3 antigen in test sample or measure people LAG-3 antigen The method of amount, being included under conditions of allowing antibody or part thereof to form compound between people LAG-3 makes sample and control sample The human monoclonal antibodies or its antigen binding portion thereof of product and specific binding people LAG-3.Then the formation of compound is detected, Wherein compared with control sample, the difference compound formation between sample shows that there are people's LAG-3 antigens in sample.In addition, this The anti-lag-3 antibody of invention can be used for through immunoaffinity purification come Purification of Human LAG-3.

Inhibit the combination of LAG-3 and MHC II class or FGL1 class molecule and stimulation anti-in view of anti-lag-3 antibody of the invention The ability of former specific T-cells response, the present invention also provides use antibody of the invention to stimulate, enhance or raise antigen-specific The in vitro and in vivo method of property t cell response.For example, the present invention provides the method for stimulator antigen specific T-cells response, packet It includes and applies antibody or its antigen-binding portion thereof of the invention to subject, thus stimulator antigen specific T-cells response.It can make Antigen-specific T cell response is measured with any suitable antigen-specific T cell response indicator.

The treatment of cancer

The non-limiting example of this suitable indicator be included in the presence of antibody increased T cell proliferation and/or Increased cell factor generates in the presence of antibody.In a preferred embodiment, stimulator antigen specific T-cells is white thin Born of the same parents' interleukin -2 generates.The present invention also provides the sides of the immune response (for example, antigen-specific T cell response) of stimulation subject Method, including applying antibody or its antigen-binding portion thereof of the invention to subject, so that stimulation immune response (such as antigen Specific T-cells response).In a preferred embodiment, the subject is subject and the stimulation for carrying cancer For the immune response of tumour.Antibody can enhance patient to the immune response of cancer cell to the cancer blocking of LAG-3.Anti-lag-3 Antibody can be used alone or with other immunogenic agents, standard cancer treatments agent or other antibody combined uses.

The example that the cancer of method treatment of the invention can be used includes osteocarcinoma, cancer of pancreas, cutaneum carcinoma, head and neck cancer, skin Skin or intraocular chromoma, uterine cancer, oophoroma, the carcinoma of the rectum, anal region cancer, gastric cancer, carcinoma of testis, carcinoma of fallopian tube, intrauterine Film cancer, cervical carcinoma, carcinoma of vagina, carcinoma of vulva, Hodgkin's disease, non-Hodgkin lymphoma, the cancer of the esophagus, carcinoma of small intestine, internal system cancer, Thyroid cancer, parathyroid carcinoma, adrenal, soft tissue sarcoma, carcinoma of urethra, carcinoma of penis, chronic or acute leukemia include anxious Property myelomatosis, chronic myelocytic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Er Tongshi Body tumor, lymphocytic lymphoma, bladder cancer, kidney or carcinoma of ureter, carcinoma of renal pelvis, central nervous system (CNS) cancer, primary CNS lymthoma, Tumor Angiongesis, spinal column axis tumour, brain stem glioma, pituitary adenoma, Kaposi sarcoma, epidermoid carcinoma, squama Shape cell cancer, t cell lymphoma, the cancer of environmental induction include by the combination of the cancer and the cancer of asbestos induction.

Antibody or its antigen-binding portion thereof can be applied in combination with chemotherapy or radiotherapy.

It is applied in combination with chemotherapy

Antibody or its antigen-binding portion thereof can be used with anticancer agent, cytotoxic agent or chemotherapeutic combination.

Term " anticancer agent " or " antiproliferative " mean any medicine that can be used for treating cell proliferative disorders such as cancer Agent, and including but not limited to cytotoxic agent, cytostatics, anti-angiogenic agent, radiotherapy and radiotherapy dose, target It is controlled to anticancer agent, BRM, therapeutic antibodies, cancer vaccine, cell factor, hormonotherapy, radiotherapy and anti-transfer agent with immune Treat agent.It should be understood that selecting in embodiment as described above, such anticancer agent may include conjugate and can be with Before administration in conjunction with disclosed site-specific antibodie.More specifically, in certain embodiments, by the anticancer of selection Agent is connected to the unpaired cysteine of engineered antibody to provide engineering conjugate as described herein.Therefore, such Engineering conjugate is clearly considered within the scope of the invention.In other embodiments, disclosed anticancer agent will be with Locus specificity conjugate comprising different therapeutic agents as described above is administered in combination.

As used herein, term " cytotoxic agent " refers to toxic to cell and reduces or inhibit cell function and/or draw Play the substance of cytoclasis.In certain embodiments, which is derived from the naturally occurring molecule of living organism.Cell toxicant Property agent example include but is not limited to bacterium (for example, diphtheria toxin, pseudomonad endotoxin and exotoxin, staphylococcus intestines poison Plain A), fungi (such as α-sarcine element, Restrictocin), plant (abrin, ricin (WA), modeccin, Viscin, pokeweed antiviral protein, saporin, gelonin, momoridin, trichosanthin, Barley Toxin, oil Paulownia (Aleurites fordii) albumen, China pink fibroin, Phytolacca mericana albumen (PAPI, PAPII and PAP- S), momordica charantia inhibitor, curcin, crotin, crystal soda grass inhibitor, gelonin, mitegellin, Aspergillus restrictus Element, phenomycin, neomycin and trichothecene) or animal (such as cytotoxicity RNA enzyme, such as extracellular pancreas RNA Enzyme；DNA enzymatic I, including its segment and/or variant) small molecule toxins or enzymatic activity toxin.

For the purposes of the present invention, " chemotherapeutant " includes the non-specific growth for reducing or inhibiting cancer cell, proliferation And/or the chemical compound (such as cytotoxic agent or cytostatics) of survival.These chemical reagent are raw generally directed to cell Intracellular processes needed for long or division, thus it is especially effective for the cancer cell of usual fast-growth and division.For example, Changchun New alkali makes microtubule depolymerization, so that cell be inhibited to enter mitosis.In general, chemotherapeutant may include inhibiting or being designed to use In any chemical agent for inhibiting cancer cell or being likely to become property or the cell of generation tumorigenesis offspring (such as TIC).These medicaments It is usually to be applied in combination, and it is usually most effective, for example, in the scheme of such as CHOP or FOLFIRI.

The anticancer agent that can be applied in combination with locus specificity construct of the invention is (as locus specificity conjugate Component or not conjugated state) example include but is not limited to alkylating agent, alkylsulfonate, aziridine, aziridine and methyl three Poly cyanamid, acetogenin (acetogenins), camptothecine, bryostatin, Cali's scholar's statin (callystatin), CC-1065, Crith support glad (cryptophycins), blocks meter Xing, Ai Liusu (eleutherobin), water ghost any of several broadleaf plants alkali, sand at dolastatin more Ke Diyin (sarcodictyin), Spongistatin (spongistatin), mustargen, antibiotic, Enediyne Antibiotic, Dynemicin, diphosphonate, ai sibo mycin, chromoprotein enediyne antibiotic chromophore, aclacinomycin class (aclacinomysins), D actinomycin D, Anthramycin, azaserine, bleomycin, act-C, Dan Karabin are pungent (carabicin), carminomycin, cardinophyllin, chromomycin class (chromomycinis), dactinomycin D, daunorubicin, hold in the palm Than star, 6- diazo -5- oxn-l-norieucin,Doxorubicin, epirubicin, according to rope ratio Star, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, Peplomycin, rich geomycin (potfiromycin), puromycin, triferricdoxorubicin, rodorubicin, streptonigrin, streptozotocin, tubercidin, black benzene U.S. department, Zinostatin, zorubicin；Anti- metabolin, Erlotinib, Wei Luofeini, gram azoles replace for Buddhist nun, Sorafenib, according to Shandong Such as not woods is sour for Buddhist nun, the miscellaneous Shandong amine of grace, folacin, purine analogue, androgen, anti-adrenaline, folic acid supplement (frolinic acid), aceglatone, aldophosphamideglycoside, amino-laevulic acid, eniluracil, amsacrine, bass Bush (bestrabucil), bisantrene, Edatrexate, defer's method bright (defofamine), demecolcine, diaziquone, Ai Funixin (elfornithine), Elliptinium Acetate, love wave like dragon, ethoglucid, gallium nitrate, hydroxycarbamide, lentinan, Lonidamine, beauty Smooth raw class compound (maytansinoids), mitoguazone, mitoxantrone, not pellet mole (mopidanmol), nit woods (nitraerine), Pentostatin, Phenamet, pirarubicin, Losoxantrone, podophyllic acid, 2- ethyl hydrazine, procarbazine,Polysaccharide compound (JHS Natural Products, Eugene, OR), razoxane；Rhizomycin；Sizofiran；Germanium spiral shell Amine；Acid is helped for slave；Triethyleneiminobenzoquinone；2,2', 2 "-trichlorotriethylamines；Trichothecenes (especially T-2 toxin, Wella library woods A (verracurin A), Roridine A and anguidin)；Urethane；Eldisine；Dacarbazine；Mannomustine；Dibromo Mannitol；Mitolactol；Pipobroman；Cassie support is glad (gacytosine)；Arabinoside (" Ara-C ")；Cyclophosphamide； Thiotepa；Taxanes；Chlorambucil (chloranbucil)；Gemcitabine；6-thioguanine； Mercaptopurine；Methotrexate；Platinum analogs；Vincaleukoblastinum；Platinum；Etoposide (VP-16)；Ifosfamide；Mitoxantrone；Changchun is new Alkali,Vinorelbine；Novantrone；Teniposide；Edatrexate；Daunorubicin；Aminopterin；Uncommon sieve It reaches；Ibandronate；Irinotecan (Camptosar, CPT-11)；Topoisomerase enzyme inhibitor RFS 2000；Difluoromethyl bird Propylhomoserin；Retinoid；Capecitabine；Combretastatin；Formyl tetrahydrofolic acid；Oxaliplatin；PKC- α, Raf, H-Ras, EGFR and The inhibitor (it reduces cell Proliferation) of VEGF-A and pharmaceutically acceptable salt, acid or the derivative of any of the above-described.This Also included in one definition is antihormone agent for adjusting or inhibiting to the hormonal action of tumour, such as antiestrogenic and selection Property estrogenic agents, inhibit adjust adrenal gland in estrogen generate aromatase enzyme aromatase inhibitor and anti-hero Hormone；And troxacitabine (1,3- dioxolane nucleosides analogue of cytosine)；Antisense oligonucleotides, ribozyme such as VEGF Expression inhibiting agent and HER2 expression inhibiting agent；Vaccine,rIL-2； 1 inhibitor of topoisomerase；rmRH；Vinorelbine and ai sibo mycin and any of the above-described Pharmaceutically acceptable salt, acid or derivative.

It is applied in combination with radiotherapy

The present invention also provides antibody or its antigen-binding portion thereof and radiotherapy (that is, for the part in tumour cell Any mechanism of induced DNA damage, such as gamma-irradiation, X-ray, UV- irradiation, microwave, electron emission etc.) combination.Also examine The conjoint therapy of the targeted delivery using radioactive isotope to tumour cell is considered, and disclosed conjugate can be with target To anticancer agent or other targeting means be used in combination.In general, radiotherapy is within about 1 week to about 2 weeks period with pulse Mode is applied.Radiotherapy can be to about 6 to 7 week of subject's application with head and neck cancer.Optionally, radiotherapy can be used as Single dose is applied as multiple sequential doses.

Diagnosis

The present invention provides for detecting, diagnosing or monitoring proliferative disorders in vitro and in vivo method and screening come from The cell of patient is to identify that tumour cell includes the method for tumorigenic cell.Such method includes identification for treating with cancer The individual of disease or the progress for monitoring cancer, including by patient or the sample obtained from patient (internal or external) with it is as described herein Antibody contact, and in test sample and combination or free target molecule combination antibody existence or non-existence or combine water It is flat.In some embodiments, antibody will include detectable label as described herein or reporter molecule.

In some embodiments, the combination of specific cells can indicate that sample may be thin containing tumorigenesis in antibody and sample Born of the same parents, to show to have the individual of cancer can effectively be treated with antibody as described herein.

It can analyze sample by many measure method, such as radioimmunoassay, enzyme immunoassay (such as ELISA), competition binding determination method, fluorescence immunoassay, immunoblot assay, Western blot analysis and fluidic cell Art measuring method.Compatible in-vivo diagnostic or diagnostic assay may include imaging well known in the art or monitoring technology, such as ability Magnetic resonance imaging known to field technique personnel, computerized tomography (such as cat scan), position emissron tomography (such as PET scan), radiography, ultrasonic wave etc..

Drug packages and kit

Additionally provide one or more containers comprising the antibody comprising one or more dosage or its antigen-binding portion thereof Drug packages and kit.In certain embodiments, unit dose is provided, wherein unit dose contains the combination of predetermined amount Object, the composition is including, for example, antibody or its antigen-binding portion thereof, with or without one or more other reagents.For Other embodiments, this unit dose are supplied with disposable pre-filled syringe with syringe.In other embodiments In, the composition for including in unit dose may include salt water, sucrose or the like；Buffer, such as phosphate；And/or match System is within the scope of stable and effective pH.Alternatively, in certain embodiments, conjugate composition can be used as freeze-dried powder and mention For that can be rebuild afterwards suitable liquid (such as sterile water or salting liquid) is added.In certain preferred aspects, group Closing object includes one or more substances for inhibiting protein aggregation, including but not limited to sucrose and arginine.On container or with appearance The conjugate composition of the associated any label instruction encapsulation of device is used for the tumor disease situation of therapeutic choice.

The present invention also provides the lists for generating locus specificity conjugate and optionally one or more anticancer agents The kit of dosage or multi-dose administration unit.The kit includes container and on container or label associated with container Or package insert.Suitable container includes such as bottle, bottle, syringe etc..Container can be formed of a variety of materials, such as glass Or plastics, and include the disclosed conjugation of pharmacy effective dose or the conjugate of unconjugated form.In other preferred embodiments In, container includes that (such as container can be intravenous solution bag or with can be pierced through by hypodermic needle to sterile access port The bottle of plug).Pharmaceutically acceptable system of such kit comprising engineering conjugate usually in suitable container Agent, and include one or more anticancer agents optionally in identical or different container.Kit can also contain its other medicine Acceptable preparation on, for diagnosing or being treated in combination.For example, in addition to antibody of the invention or its antigen-binding portion divide it Outside, such kit can contain any one or more of anticancer agent, such as chemotherapeutant or radiotherapy dose；It is anti-angiogenic Generating agent；Anti-transfer agent；Target anticancer agent；Cytotoxic agent；And/or other anticancer agents.

More specifically, kit can have the single container containing disclosed antibody or its antigen-binding portion thereof, Its with or without other component or they can have the different vessels for reagent needed for every kind.It is used in offer In the case where the combined therapy agent of conjugation, list can be pre-mixed in such a way that molar equivalent combines or a kind of component is more than another kind One solution.Alternatively, the conjugate of kit and any optional anticancer agent can be separately maintained in not before being applied to patient In same container.Kit can also be comprising for example pressing down for accommodating sterile pharmaceutically acceptable buffer or other diluents Bacterium water for injection (BWFI), phosphate buffered saline (PBS) (PBS), Ringer's solution and glucose solution second/third container dress It sets.

When the component of kit is provided with one or more liquid solutions, liquid solution is preferably aqueous solution, especially excellent Select aseptic aqueous solution or saline solution.However, the component of kit can be used as dry powder offer.When reagent or component are with dry powder shape It, can be by adding suitable solvent come reconstituted powders when formula provides.It is contemplated that solvent can also be provided in another container In.

As briefly described, the kit can also contain oriented patient's administration of antibodies or its antigen-binding portion thereof and any Select the tool of component, such as one or more needles, I.V. bags or syringe, or even eye drop device, pipette or other similar The affected areas of body can be injected by it or be introduced in animal body or be applied to preparation by device.Examination of the invention Agent box also typically includes the component of the device for accommodating bottle or the like and other deadends for commercial distribution, Such as injection or blow-moulding plastic containers, wherein placing and keeping required bottle and other devices.

Sequence table is summarized

The application is accompanied with the sequence table comprising many nucleic acid and amino acid sequence.Following table provides included sequence It summarizes.

Embodiment

The present invention being generally described herein will be more readily understood by reference to following embodiment, these embodiments be with What the mode of illustration provided, and it is not intended to be limited to the present invention.These embodiments are simultaneously intended to indicate that following experiment is complete Portion or the experiment only carried out.

Embodiment 1

The preparation of material

1. the generation of immunogene

Encoding human LAG-3 ECD (extracellular domain, ECD) with SEQ ID NO:22 is synthesized by Sangon Biotech Or the nucleic acid of the encoding full leng people LAG-3 with SEQ ID NO:24.The amino acid sequence of LAG-3ECD and the DNA sequence for encoding it Column are shown in SEQ ID NO:21 and 22, and the amino acid sequence of overall length LAG-3 and the DNA sequence dna for encoding it are shown in SEQ ID NO:23 and 24.LAG-3 genetic fragment is expanded from the nucleic acid of synthesis and is inserted into expression vector pcDNA3.3 (ThermoFisher) in.The LAG-3 genetic fragment of insertion is further confirmed by DNA sequencing.By the way that people's LAG-3 gene is turned It contaminates and obtains LAG-3ECD containing someone and various labels (including people Fc, mouse Fc and His in 293F cell (ThermoFisher) Label) fusion protein.By cell in 37 DEG C, 5%CO₂Under FreeStyle 293 express culture medium (ThermoFisher) Middle culture.After culture 5 days, protein purification will be used for from the supernatant for transiently transfecting cell culture harvest.Fusion protein is logical Cross nickel, albumin A and/or SEC column purification.By using Factor Xa protease (New England Biolabs) with cleavage site It cuts ECD-hFc fusion protein and generates untagged LAG-3ECD albumen.The protein of purifying is for being immunized, screening and table Sign.

2. the generation of reference-Ab

It is synthesized based on information disclosed in 20170101472 A1 of patent application US 20110150892 A1 and US anti-human (BMK1 and BMK7, wherein BMK1 is referred to as " 25F7 " and BMK7 in US to LAG-3 reference-Ab in 20110150892 A1 of US It is referred to as " H4sH15482P " in 20170101472 A1).Reference-Ab BMK8 is the humanization form of chimeric antibody BMK5, BMK5 is described in WO2015138920A1 and is known as " BAP050-chi ".BMK8 is referred to as in WO2015138920A1 "BAP050-hum01".As described in Section 1 above, the gene order of synthesis is integrated into plasmid pcDNA3.3.By plasmid wink When be transfected into 293F cell.By with it is identical described in Section 1 in a manner of culture cell.After culture 5 days, from transient transfection cell The supernatant of culture harvest is used for protein purification.Reference-Ab is purified from supernatant.

3. establishing stable cell line

Generate people, mouse and machin LAG-3 transfectional cell series.In brief, using 2000 turns of Lipofectamine Transfection reagent box is expressed with the pcDNA3.3 containing overall length people, mouse and machin LAG-3 carry respectively according to the scheme of manufacturer Body transfects Flp-In-293, Flp-In-CHO or 293F cell.Transfection 48-72 hour after, by the cell of transfection contain kill It cultivates for selection in the culture medium of piricularrin and tests LAG-3 expression.The thin of expression people LAG-3 is obtained by limiting dilution Born of the same parents system, the cell line for expressing machin LAG-3 and the cell line for expressing mouse LAG-3.

Embodiment 2

Antibody hybridoma generates

1. immune and cell fusion

It is handed over the people LAG-3ECD albumen of the 12.5 μ g hFc label in adjuvant and the mouse LAG-3 of 12.5 μ g His label For 24 week old of immunity inoculation OMT rat (with recombination immunoglobulin locus transgenic rat, such as US8,907, Described in 157B2), it generates framework region and CDR region is derived from the antibody of human germline immunoglobulin's sequence.Every two weeks to immune Rat extracting blood carries out serum collection, and the potency of anti-human LAG-3 in serum is measured by ELISA.Someone LAG- will be applied The plate of 3.ECD.hFc and diluted rat blood serum (then 1:100 first dilutes 3 times in 2%BSA) are incubated for 2 hours altogether. Use goat anti-rat-IgG-Fc-HRP as secondary antibody.It is developed the color by distributing the tmb substrate of 100 μ L, then with 100 μ L's 2N HCl is terminated.Absorbance is read in 450nM using microplate reader.Once antibody titer reaches sufficiently high, with the DPBS of no adjuvant In 40 μ g people LAG-3ECD albumen rat is finally reinforced.On the day of fusion, aseptically from immune rat Middle taking-up lymph node and spleen, and it is prepared into single cell suspension.Then by isolated cell and myeloma cell SP2/0 with 1:1 Ratio mixing.Electric cell fusion is carried out using 2000 Electro cell executor of BTX.Then by cell with 1 × 10⁴It is a The density of cells/well is seeded in 96 orifice plates, and in 37 DEG C, 5%CO₂Lower culture, until being ready for screening.

2. the preliminary screening of doma supernatant and confirmation screening

ELISA measuring method used as the first screening technique test the combination of doma supernatant and LAG-3 albumen.It will The plate of Section 1 of the present embodiment is coated with overnight in 4 DEG C of people LAG-3ECD.hFc with 1 μ g/mL.It, will be miscellaneous after closing and washing Tumor supernatant is handed over to be transferred to coated plate and be incubated at room temperature 1 hour.It is washed out plate, it is then anti-big with secondary antibody goat Mouse IgG HRP is incubated for 1 hour.After washing, tmb substrate is added, is reacted with 2M HCl color development stopping.It is read using microplate reader Absorbance at 450nm.

In order to confirm the natural combination for the conforma-tional LAG-3 molecule expressed on LAG-3 antibody and cell membrane, turn in LAG-3 Flow cytometry is carried out in the CHO-K1 cell line of dye.The CHO-K1 cell of people LAG-3 will be expressed with 1x10⁵A cell/ The density in hole is transferred in 96 hole U-shaped base plates.Then doma supernatant is transferred to plate and is incubated for 1 hour at 4 DEG C.With After 1 × PBS/1%BSA washing, secondary antibody goat anti-rat IgG Alexa647 is added and is incubated in the dark with cell at 4 DEG C 0.5 hour.It is washed out cell and is resuspended in 1 × PBS/1%BSA, then pass through flow cytometry.As negative right According to the parallel combination for carrying out antibody and parent's CHO-K1 cell line.Be combined as with the antibody of parent CHO-K1 cell line pair According to.

The blocking activity of antibody is used to screen as confirmatory to select potential antibody to hit.It is tested by facs analysis The ability of the combination of the cell line Raji of selected antibody blocking LAG-3 albumen and expression people MHC-II.By Raji cell with 1x10⁵ The density of a cells/well is transferred in 96 hole U-shaped base plates.The LAG-3 albumen of supernatant and mFc label is incubated for 30 at 4 DEG C Minute.Mixture is transferred in 96 orifice plates with Raji cell inoculation.The goat anti-mouse IgG antibody that secondary antibody PE is marked (to rat IgG Fc no cross reaction, Jackson Immunoresearch Lab) and cell are incubated in the dark at 4 DEG C 0.5 hour.It is washed out cell and is resuspended in 1 × PBS/1%BSA and passes through flow cytometry.

3. Hybridoma Subclones:

Once, then will by using Semi-solid cell culture based method by the specific binding of preliminary and confirmatory screening verification Positive hybridoma cell system is subcloned to obtain the anti-hLAG-3 antibody of monoclonal.In Semi-solid cell culture based method, for every kind Cell is diluted in semisolid Cloning Medium (STEMCELL Technologies) and is inoculated in 6 holes by hybridoma cell line In plate.Incubator (37 DEG C, 5%CO₂) middle culture cell 8-10 days, until monoclonal visible in semisolid culturemedium.It chooses It takes to clone and be transferred to 96 orifice plates and is in the HAT culture medium (hypoxanthine-aminopterin-thymidine medium) containing 10%FBS In.As described above, confirming positive colony by the combination ELISA and FACS for people LAG-3.

Embodiment 3

Hybridoma sequencing and fully human antibodies molecule construction

1. hybridoma is sequenced

Total serum IgE is separated from hybridoma using RNeasy Plus Mini kit (Qiagen), and such as table 1 and table The first chain cDNA is prepared shown in 2.As shown in Table 3 and Table 4, by using 3'- constant region degenerate primer and 5'- degenerate primer group (it is complementary with the upstream signal sequence code area of Ig variable sequence) expands antibody VH and VL gene from cDNA.Table 5 shows packet Include the reagent information including manufacturer.

PCR product (10 μ L) is connected in pMD18-T carrier, and 10 μ L connection products are transformed into Top10 competence In cell.The cell of conversion is layered on 2-YT+Cab plate and is incubated overnight at 37 DEG C.Positive colony is selected at random to be used for Shanghai Biosune Biotech Co., Ltd. are sequenced.

Table 1.cDNA amplified reaction (20 μ L)

Table 2.cDNA amplification reaction condition

	Step 1	Step 2	Step 3	Step 4
					Temperature (DEG C)	25	50	85	4
Time	10min	50min	5min	∞

Table 3.PCR reaction system (50 μ L)

Table 4.PCR reaction condition

5. reagent information of table

Two kinds of guide's antibody are respectively designated as " 1.53.3-uAb-IgG4k " and " 3.40.19-uAb-IgG4L ".

1.53.3-uAb-IgG4k CDR sequence measurement is as follows:

Description	SEQ ID NO.	Sequence information
			CDRH1	1	GGSFSGYYWS
CDRH2	2	EINHRGNTNYNPSLKS
			CDRH3	3	GEDYSDYDYYGDF
CDRL1	4	RASQSISSYLA
			CDRL2	5	AASNRAT
CDRL3	6	QQRSNWPLT

1.53.3-uAb-IgG4k the sequence of heavy chain and light chain variable region is as follows:

3.40.19-uAb-IgG4L CDR sequence it is as follows:

Description	SEQ ID NO.	Sequence information
			CDRH1	7	GDSISSTSYYWG
CDRH2	8	SFYYSGSTYYNPSLKS
			CDRH3	9	MQLWSYDVDV
CDRL1	10	TGTSSDVGGYDYVA
			CDRL2	11	DVSERPS
CDRL3	12	SSYTSTTTLVV

3.40.19-uAb-IgG4L the sequence of heavy chain and light chain variable region is as follows:

2. fully human antibodies molecule construction

VH and VL gene is expanded again using the cloning primer containing suitable restriction site, and is cloned into expression and is carried To generate corresponding chimeric antibody clone in body.

Embodiment 4

The combination of LAG-3 antibody and cell surface people LAG-3

The test antibody of various concentration, the positive and negative control are added in people LAG-3 transfection cell, phase is then passed through Combination of the secondary antibody detection antibody for the PE label answered on cell surface.Data are shown in Fig. 1, EC₅₀It is shown in table 6.

Table 6

Ab	EC₅₀(nM)
		1.53.3-uAb-IgG4k	0.43
3.40.19-uAb-IgG4L	0.13
		BMK1	0.32
BMK7	0.61
		BMK8	0.90

It was unexpectedly determined that as shown in Fig. 1 and table 6, EC of the 3.40.19-uAb-IgG4L in conjunction with cell surface LAG-3₅₀ (0.13) all three reference-Abs BMK1 (0.32), BMK7 (0.61) and BMK8 (0.90) are significantly lower than.In addition, 1.53.3- EC of the uAb-IgG4k in conjunction with cell surface LAG-3₅₀(0.43) it is far below the EC of BMK7 (0.61) and BMK8 (0.90)₅₀.This A bit the result shows that 1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L can effectively combine people LAG-3, and combine effect excellent In or be equivalent to reference-Ab.

Embodiment 5

The blocking of the combination of the MHC-II expressed on LAG-3 albumen and Raji cell

The serial dilution antibody in 1%BSA-PBS, and be incubated for 30 minutes at 4 DEG C with the LAG-3 albumen of mFc label.It will Mixture is transferred in 96 orifice plates with Raji cell inoculation.LAG-3 egg is detected using goat anti-mouse IgG Fc-PE antibody White and Raji cell combination.It is analyzed by hybridoma supematant assesse MFI and by FlowJo software (version 7.6.1).Data It is shown in Fig. 2, EC₅₀It is shown in table 7.

Table 7

Ab	EC₅₀(nM)
		1.53.3-uAb-IgG4k	0.80
3.40.19-uAb-IgG4L	0.67
		BMK1	0.76
BMK7	1.25
		BMK8	0.88

As shown in Fig. 2 and table 7, it was unexpectedly determined that the 3.40.19-uAb-IgG4L and MHC- expressed on Raji cell The EC of the combination of II₅₀(0.67) all three reference-Abs BMK1 (0.76), BMK7 (1.25) and BMK8 (0.88) are significantly lower than EC₅₀.In addition, the EC of the combination of 1.53.3-uAb-IgG4k and the MHC-II expressed on Raji cell₅₀(0.80) it is lower than The EC of BMK7 (1.25) and BMK8 (0.88)₅₀.These are the result shows that 1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L exist It is effective in terms of the combination of the MHC-II expressed in blocking and Raji cell, and barrier effect is better than or is equivalent to benchmark and resists Body.

Embodiment 6

The blocking of the combination of LAG-3 albumen and LSECtin and galectin-3

96 orifice plates are coated with the people LSECtin or galectin-3 of 0.5 μ g/mL respectively at 4 DEG C to stay overnight.Antibody exists In 1%BSA-PBS serial dilution and with mFc label LAG-3 albumen mix.After closing and washing, mixture is transferred to flat Plate is simultaneously incubated at room temperature 1 hour.It is washed out plate, is then incubated for 60 minutes with corresponding secondary antibody.After washing, TMB is added Substrate is reacted with 2M HCl color development stopping.The absorbance at 450nm is read using microplate reader.Data are shown in figures 3 and 4. EC₅₀It is shown in table 8.

Table 8

As shown in Fig. 3 and 4 and table 7,1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L can be blocked effectively The combination of LAG-3 and LSECtin or galectin-3, and blocking effect is better than or is equivalent to reference-Ab.

Embodiment 7

The test of Holonomic Dynamics binding affinity

The Holonomic Dynamics binding affinity tested by surface plasma body resonant vibration (SPR):

The affinity and binding kinetics that antibody is directed to people LAG-3 are characterized by using the SPR measurement of Biacore 8K. Goat anti-Human Fc is pre-fixed on sensor chip (CM5), and captures anti-lag-3 antibody when injecting chip.It will be various The people LAG-3 albumen and running buffer of concentration carry out the association phase of 300s with the flow velocity flows through sensor chip of 30 μ L/min, Then carry out the dissociation of 3600s.Association and dissociation curve negotiating are combined using the 1:1Langmuir of Biacore 8K assessment software Models fitting.Data are shown in table 9.

Table 9

Ab	ka(1/Ms)	kd(1/s)	KD(M)
				1.53.3-uAb-IgG4k	6.60E+05	3.33E-05	5.05E-11
3.40.19-uAb-IgG4L	1.05E+06	1.11E-05	1.06E-11
				BMK1	4.87E+05	3.34E-04	6.85E-10
BMK7	2.13E+05	1.06E-04	4.97E-10
				BMK8	8.46E+04	6.74E-06	7.97E-11

Pass through the LAG-3 antibody of fluorescence-activated cell sorting (FACS) test and the combination parent of cell surface LAG-3 molecule And power

The binding affinity of antibody on cell Surface L AG-3 is measured by facs analysis.The Flp- of people LAG-3 will be expressed In-293 cell is with 5 × 10⁵The density of a cell/mL is transferred in 96 hole U-shaped base plates.By the antibody of test in washing buffer In liquid (1 × PBS/1%BSA) serial dilution and at 4 DEG C with cell incubation 1 hour.Secondary antibody Goat anti-Human IgG Fc is added FITC (3.5 moles of FITC of every mole of IgG), and be incubated for 0.5 hour in the dark at 4 DEG C.Then cell washed once and lays equal stress on It is suspended from 1 × PBS/1%BSA, and passes through flow cytometry.Based on quantitative bead (QuantumTM MESF kit, Bangs Laboratories, Inc.) convert fluorescence intensity in molecule/cell of combination.Use Graphpad Prism 5 Calculate affinity.Data are shown in table 10.

Table 10

Ab	KD(M)
		1.53.3-uAb-IgG4k	1.60E-10
3.40.19-uAb-IgG4L	5.30E-11
		BMK1	2.70E-10
BMK7	5.80E-10
		BMK8	9.40E-10

As tested by SPR and FACS, by the sheet of 1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L expression The antibody of invention effectively combines people LAG-3, and is better than or is equivalent to reference-Ab in conjunction with effect.

Embodiment 8

Ortholog thing (across species) and homologue (across family) combine

With the cross reactivity of machin LAG-3 and mouse LAG-3

Pass through the cross reactivity of FACS measurement and machin and mouse LAG-3.The Flp-In-CHO for expressing mouse LAG-3 is thin Born of the same parents or machin LAG-3 expression 293F cell are with 1x10⁵The density of a cells/well is transferred in 96 hole U-shaped base plates.It will test Antibody serial dilution in washing buffer (1 × PBS/1%BSA) is simultaneously incubated for 1 hour at 4 DEG C with cell.With 1 × PBS/ After 1%BSA washing, apply corresponding secondary antibody, and be incubated for 1 hour in the dark at 4 DEG C with cell.It is washed out cell and is resuspended In 1 × PBS/1%BSA, then pass through flow cytometry.Data are shown in fig. 5 and fig..EC₅₀It is shown in table 11 In.

Table 11

Ab	EC₅₀(nM)
		1.53.3-uAb-IgG4k	4.01
3.40.19-uAb-IgG4L	3.92
		BMK1	86.0
BMK7	2.65
		BMK8	3.05

As shown in figure 5, LAG-3 antibody " 1.53.3-uAb-IgG4k " of the invention and " 3.40.19-uAb-IgG4L " with Cell surface machin LAG-3 is combined.As shown in fig. 6, LAG-3 antibody " 1.53.3-uAb-IgG4k " of the invention and " 3.40.19-uAb-IgG4L " be not in conjunction with cell surface mouse LAG-3.

With the cross reactivity of people CD4

Pass through the cross reactivity of ELISA measurement and people CD4.It is stayed overnight at 4 DEG C with the people CD4 coating plate of 1 μ g/mL.Envelope After closing and washing, plate is added in the LAG-3 antibody of 1 μ g/mL and is incubated at room temperature 1 hour.Be washed out plate, then with Corresponding secondary antibody is incubated for 45 minutes.After washing, tmb substrate is added, is reacted with 2M HCl color development stopping.It is read using microplate reader Absorbance at 450nm.Data are as shown in Figure 7.

These are the result shows that LAG-3 antibody " 1.53.3-uAb-IgG4k " of the invention and " 3.40.19-uAb-IgG4L " Do not combine human CD 4 protein.

Embodiment 9

Divide storehouse for the epitope of BMK1, BMK7 and BMK5

The combination epitope of LAG-3 antibody is subjected to a point storehouse for reference-Ab BMK1, BMK7 and BMK5 by facs analysis. By the biotinylation reference-Ab incubation 1 for being 1ug/mL in the Flp-In-293 cell of cell surface expression people LAG-3 and concentration Hour, the LAG-3 antibody of serial dilution is then added.Use Streptavidin-PE antibody (Jackson Immunoresearch Lab) combination of reference-Ab and cell is detected.Pass through hybridoma supematant assesse MFI and is analyzed by FlowJo.Data As shown in Fig. 8 A-E.

It was found that 1.53.3-uAb-IgG4k of the invention and BMK is competed, but 3.40.19-uAb-IgG4L is not competed with BMK. 1.53.3-uAb-IgG4k close epitope is shared with BMK1 and BMK7, but does not share epitope with BMK5.It was unexpectedly determined that 3.40.19-uAb-IgG4L there is the epitope different from all BMK1, BMK7 and BMK5.

Embodiment 10

Structural domain mapping and epitope mapping

1. structural domain is mapped

LAG-3 has the extracellular domain of 421aa (P30-L450), including four extracellular immunoglobulin superfamilies (IgSF) spline structure domain, i.e. structural domain 1 (" D1, " aa.37-167), structural domain 2 (" D2, " aa 168-252.), structural domain 3 (" D3, " aa.265-343) and structural domain 4 (" D4, " aa.348-419).By with corresponding mouse LAG-3 amino acid ( It in the context of the disclosure also referred to as " aa ") replaces following residues of people LAG-3 extracellular domain and constructs 10 kinds of variants.

(1) variant 1:xPro1.FL-x1: people LAG-3aa 168 to 419 is replaced by mouse corresponding part

(2) variant 2:xPro1.FL-x2: people LAG-3aa 37 to 167 and aa 265 to 419 is replaced by mouse corresponding part

(3) variant 3:xPro1.FL-x3: people LAG-3aa 37 to 252 and aa 348-419 is replaced by mouse corresponding part

(4) variant 4:xPro1.FL-x4: people LAG-3aa 37 to 343 is replaced by mouse corresponding part

(5) variant 5:xPro1.FL-x5: people LAG-3aa 265 to 419 is replaced by mouse corresponding part

(6) variant 6:xPro1.FL-x6: people LAG-3aa 37 to 167 and aa 348 to 419 is replaced by mouse corresponding part

(7) variant 7:xPro1.FL-x7: people LAG-3aa 37 to 252 is replaced by mouse corresponding part

(8) variant 8:xPro1.FL-x8: people LAG-3aa 168 to 343 is replaced by mouse corresponding part

(9) variant 9:xPro1.FL-x9: people LAG-3aa 348 to 419 is replaced by mouse corresponding part

(10) variant 10:xPro1.FL-x10: people LAG-3aa 37 to 167 is replaced by mouse corresponding part

This 10 kinds of variant clones into pcDNA3 carrier and are used for 293F cell transfecting.In short, using FreeStyle It is 1 × 10 that 293F cell is diluted to density by 293F culture medium⁶A cell/mL, and the aliquot in the hole 3mL/ is added to 24 In orifice plate.It is transfected using 293fectin reagent (Life Technologies).It is for transfecting every time, 3 μ g DNA are dilute It releases and subtracts in blood serum medium (Life Technologies) in 150 μ L Opti-MEM I, then with 6 μ L beforehand dilutions in 150 μ L Opti-MEM I subtracts the combination of the 293fectin reagent in blood serum medium.Before being added in culture, allow DNA/ Lipofectamine mixture stands 20 minutes at 25 DEG C.It is transfected after transfection by flow cytometry within 48 hours thin Born of the same parents.

Pass through flow cytometry antibody and chimeric LAG-3 variant or overall length people/mouse LAG-3 combination.In short, The 293F cell of 1 μ g/mL antibody LAG-3 chimeric with the expression of transfection is incubated for 1 hour at 4 DEG C, it is then anti-with 3 μ g/mL goats Human IgG Fc R-PE (Jackson) is incubated for 40 minutes at 4 DEG C.With flow cytometry analysis cell.

The binding ability of antibody 1.53.3-uAb-IgG4k and 3.40.19-uAb-hIgG4L and this 10 kinds of variants are detected, and And as the result is shown in the following table 12.

Combination MFI value of the table 12.LAG-3 antibody to 10 kinds of variants

Activity, two kinds of guide's antibody, that is, " 1.53.3-uAb-IgG4k " and " 3.40.19- are combined according to the FACS of antibody UAb-hIgG4L " is combined with structural domain 1 (i.e. aa.37-167).Therefore, structural domain 1 is carried out by Alanine scanning experiment (G37-Q167,131aa) further epitope mapping.

2. epitope mapping

Alanine scanning experiment is carried out to carry out epitope mapping to people LAG-3.By the alanine residue mutation on people LAG-3 For codon glycine, and every other residue (other than cysteine residues) is mutated into alanine codon.It is right In each residue of people LAG-3 extracellular domain (ECD), site amino acids displacement is carried out using two sequence PCR steps.It will compile The ECD structural domain 1 and structural domain 2 of code people LAG-3 and the pcDNA3.3-LAG-3-D12.mFc plasmid of C-terminal mFc- label are used as mould Plate, and one group of mutagenic primer is used for using QuikChange lightning multidigit point directed Mutagenesis Kit (Agilent Technologies, Palo Alto, CA) first step PCR.After being mutated chain synthesis reaction, Dpn I endonuclease is used Digest parental templates.In second step PCR, amplification comprising CMV promoter, LAG-3 extracellular domain 1 and 2 (D1 and D2), MFc- label and the polyadenylated linear DNA expression cassette of herpes simplex virus thymidine kinase (TK), and it is thin in Expi293 at 37 DEG C Transient expression (Life Technologies, Gaithersburg, MD) in born of the same parents, it is fixed by a-protein-HPLC and mFc-ELISA It is quantitative to measure kit (Bethyl, USA).

For ELISA binding assay, by antibody 1.53.3-uAb-IgG4k or 3.40.19-uAb-hIgG4L (2 μ g/mL) Coating is onboard.It interacts with comprising the supernatant of quantitative LAG-3 mutant or people's LAG-3-ECD.D12.mFc albumen Afterwards, the anti-mFc antibody (1:5000 of HRP conjugation is added；Bethyl, USA) as detection antibody.According to being averaged for control mutant Absorbance, by absorbance standard.After to combining variation multiple that other section (< 0.75) is arranged, what identification finally determined Epitope residues.The hot spot of antibody 1.53.3-uAb-IgG4k and 3.40.19-uAb-hIgG4L are shown in table 13 and table 14.

The hot spot of table 13.1.53.3-uAb-IgG4k antibody

The hot spot of table 14.3.40.19-uAb-IgG4L antibody

Due to not existing LAG-3 structure, glycoprotein structure (PDB:5FLU, sequence based on the connection of known myelin Column identity 18%), model the structure of LAG-3 (aa:31-431).Based on alanine scanning result, two kinds of antibody are identified Hot spot, and be shown in Fig. 9 A and Fig. 9 B.

Based on described results, it can be seen that the combination of 1.53.5-uAb-IgG4k antibody belongs to the W92 of outer ring (G70-Y99) Site, and the area 3.40.19-uAb-IgG4L antibody combination L134-P138.

Embodiment 11

Pass through the external function for the LAG-3 antibody that the measuring method based on cell is tested

Effect of people's LAG-3 antibody in reporter-gene assays

The Jurkat cell for expressing people LAG-3 and the IL-2 luciferase reporter gene with stable integration is deposited in SEE It is seeded in 96 orifice plates together with Raji cell lower.Test antibody is added in cell.By plate at 37 DEG C, 5%CO₂Under It is incubated overnight.After incubation, the luciferase substrate of reconstruct is added and luciferase intensity is measured by microplate spectrophotometer. Data are shown in Figure 10, EC₅₀It is shown in table 15.

Table 15

Ab	EC₅₀(nM)
		1.53.3-uAb-IgG4k	1.07
3.40.19-uAb-IgG4L	0.21
		BMK1	0.59
BMK7	2.65
		BMK8	65.3

As shown in Figure 10, LAG-3 antibody enhances the IL-2 approach of Jurkat in reporter measurement.In addition, such as table Shown in 15, the EC of 3.40.19-uAb-IgG4L in the measurement₅₀Significantly lower than all three reference-Ab.

The influence of people's LAG-3 antibody on human Allogeneic Mixed Lymphocyte reaction

Using Ficoll-Paque PLUS gradient centrifugation from healthy donors fresh separated human peripheral blood mononuclear cell (PBMC).Monocyte is separated using person monocytic cell's enrichment kit according to the explanation of manufacturer.Cell is being contained into GM- 5 to 7 days are cultivated in the culture medium of CSF and IL-4 with producing dendritic cell (DC).According to the scheme user CD4 of manufacturer⁺T is thin Born of the same parents' enrichment kit separates people CD4⁺T cell.By the CD4 of purifying⁺The Immature DC (iDC) of T cell and allogeneic and various dense The LAG-3 antibody of degree co-cultures in 96 orifice plates.At the 5th day, collects culture supernatants and carry out IFN-γ test and T cell increasing Grow test.People's IFN-γ is measured to by ELISA using matched antibody.By the plate capture antibody for being specific to people's IFN-γ (Pierce-M700A) pre-coated.Use the anti-IFN-γ antibody (Pierce-M701B) of biotin-conjugated as detection antibody. At last 16 hours, with the addition of 1 hole μ Ci/³H- thymidine.It is measured by scinticounting³H- thymidine incorporation, and by breeder reaction table It is shown as the CPM (count per minute) in triplicate hole.Data are shown in figs. 11 and 12.

As shown in figure 11, LAG-3 antibody " 1.53.3-uAb-IgG4k " of the invention and " 3.40.19-uAb-IgG4L " exist Enhance IFN-γ secretion in mixed lymphocyte reaction (MLP).In addition, as shown in figure 12, LAG-3 antibody " 1.53.3- of the invention UAb-IgG4k " and " 3.40.19-uAb-IgG4L " enhance T cell proliferation in mixed lymphocyte reaction (MLP).

Embodiment 12

ADCC and CDC test

In order to assess the ability of triggering Fc effector function, whether assessment anti-lag-3 antibody can be thin with mediate antibody dependence Cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activity that born of the same parents mediate.

ADCC test

The LAG-3 antibody of the Flp-In-293 cell for expressing people LAG-3 and various concentration is incubated in advance in 96 hole round bottom plates It educates 30 minutes；Then PBMC is added as effector, effector/target ratio is 50:1.Plate is maintained at 37 DEG C, 5%CO₂Under 4 hours.Target cell lysis is measured by the citotoxicity detection kit based on LDH.The suction at 492nm is read using microplate reader Luminosity.Use the cell line SK-Br-3 of Trastuzumab and expression HER2 as positive control.

CDC test

The Flp-In-293 of expression people LAG-3 as target and the LAG-3 antibody of various concentration are in 96 hole round bottom plates Middle mixing.People's complement is added with the final dilution of 1:50.Plate is maintained at 37 DEG C, 5%CO₂Lower 2 hours.Target cell lysis It is determined by CellTiter-Glo.Absorbance is read using microplate reader.Use the cell line Raji of Rituximab and expression CD20 As positive control.

Figure 13 A and 13B show the data of ADCC test and CDC test.Prove, by 1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L the LAG-3 antibody of the invention indicated does not mediate ADCC (Figure 13 A) and CDC (Figure 13 B) effect.

Embodiment 13

Serum stability test

Leading Ab is incubated in the human serum (serum content > 95%) of fresh separated at 37 DEG C.At the appointed time Then point is stored by the aliquot of the sample of serum processing from taking out in incubator and being rapidly frozen in liquid nitrogen at 80 DEG C Until preparing for testing.Sample quick-thawing immediately before stability test.By people LAG-3 transfection cell and various concentration Leading antibody be incubated for 1 hour at 4 DEG C.The combination of leading antibody and cell is detected using the Goat anti-Human IgG of PE label. The MFI of cell is measured by flow cytometer (BD FACSCanto II) and is analyzed by FlowJo.Data are shown in figure In 14A and 14B.

Prove the LAG-3 antibody of the invention represented by 1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L new Stablize in fresh human serum up to 14 days.

Those skilled in the art will be further appreciated that, in the case where without departing from its spirit or central feature, the present invention It can implement in other specific forms.Since foregoing description of the invention only discloses its exemplary implementation scheme, it should manage Solution, other variations are considered as within the scope of the invention.Therefore, the specific reality that the present invention is not limited to be described in detail here Apply scheme.On the contrary, should refer to appended claims to indicate the scope of the present invention and content.

Sequence table

<110>Guangzhou Yu Heng Biotechnology Co., Ltd

<120>anti-human LAG-3 monoclonal antibody and its preparation method and application

<130> IDC196003

<160> 28

<170> PatentIn version 3.5

<210> 1

<211> 10

<212> PRT

<213>artificial sequence

<220>

<223>CDRH1 of 1.53.3-uAb-IgG4k

<400> 1

Gly Gly Ser Phe Ser Gly Tyr Tyr Trp Ser

1 5 10

<210> 2

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<223>CDRH2 of 1.53.3-uAb-IgG4k

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Glu Ile Asn His Arg Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 3

<211> 13

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Gly Glu Asp Tyr Ser Asp Tyr Asp Tyr Tyr Gly Asp Phe

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Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Ala

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Ala Ala Ser Asn Arg Ala Thr

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<223>CDRL3 of 1.53.3-uAb-IgG4k

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Gln Gln Arg Ser Asn Trp Pro Leu Thr

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Gly Asp Ser Ile Ser Ser Thr Ser Tyr Tyr Trp Gly

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Ser Phe Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

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Met Gln Leu Trp Ser Tyr Asp Val Asp Val

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Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asp Tyr Val Ala

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Asp Val Ser Glu Arg Pro Ser

1 5

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<223>CDRL3 of 3.40.19-uAb-IgG4L

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Ser Ser Tyr Thr Ser Thr Thr Thr Leu Val Val

1 5 10

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<223>VH of 1.53.3-uAb-IgG4k

<400> 13

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Gly Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Met Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Arg Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Glu Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys Thr

85 90 95

Arg Gly Glu Asp Tyr Ser Asp Tyr Asp Tyr Tyr Gly Asp Phe Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

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<223>VL of 1.53.3-uAb-IgG4k

<400> 14

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

1 5 10 15

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

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Ile Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 15

<211> 120

<212> PRT

<213>artificial sequence

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<223>VH of 3.40.19-uAb-IgG4L

<400> 15

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Ser Ser Thr

20 25 30

Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Ser Phe Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Met Gln Leu Trp Ser Tyr Asp Val Asp Val Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

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<211> 111

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<223>VL of 3.40.19-uAb-IgG4L

<400> 16

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

1 5 10 15

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

20 25 30

Asp Tyr Val Ala Trp Tyr Gln Gln His Pro Gly Lys Val Pro Lys Leu

35 40 45

Met Ile Tyr Asp Val Ser Glu Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 17

<211> 363

<212> DNA

<213>artificial sequence

<220>

<223>VH of 1.53.3-uAb-IgG4k

<400> 17

caggtgcagc tacagcagtg gggcgcagga cttttgaagc cttcggagac cctgtccctc 60

acctgcggtg tctatggtgg gtccttcagt ggttactact ggagctggat ccgccagccc 120

ccagggatgg ggctggagtg gattggggaa atcaatcatc gtggaaacac caactacaac 180

ccgtccctca agagtcgcgt caccatatca gaagacacgt ccaagaacca gttctccctg 240

aggctgagct ctgtgaccgc cgcggacacg gctgtgtatt tctgtacgag aggagaggac 300

tatagtgact acgattacta tggggacttc tggggccagg gaaccctggt caccgtctcc 360

tca 363

<210> 18

<211> 321

<212> DNA

<213>artificial sequence

<220>

<223>VL of 1.53.3-uAb-IgG4k

<400> 18

gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctcaagggga aagagccacc 60

ctctcctgca gggccagtca gagtattagc agctacttag cctggtacca acagaaacct 120

ggccaggctc ccaggctcct catctatgct gcatccaaca gggccactgg catcccagcc 180

aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct 240

gaagattttg caatttatta ctgtcagcag cgtagcaact ggcctctcac tttcggcgga 300

gggaccaagg tggagatcaa a 321

<210> 19

<211> 360

<212> DNA

<213>artificial sequence

<220>

<223>VH of 3.40.19-uAb-IgG4L

<400> 19

cagctgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60

acctgcactg tctctggtga ctccatcagc agtactagtt actactgggg ctggatccgc 120

cagcccccag ggaaggggct ggagtggatt gggagtttct attatagtgg gagcacctac 180

tacaacccgt ccctcaagag tcgagtcacc atttccgtag acacgtccaa gaaccagttc 240

tccctgaagc tgaactctgt gaccgccgca gacacggctg tgtattactg tgcgaggatg 300

cagctatggt cgtacgatgt ggacgtctgg ggccaaggga ccacggtcac cgtctcctca 360

<210> 20

<211> 333

<212> DNA

<213>artificial sequence

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<223>VL of 3.40.19-uAb-IgG4L

<400> 20

cagtctgccc tgactcaacc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60

tcctgcactg gaaccagcag tgacgttggt gggtatgact atgtcgcctg gtaccaacaa 120

cacccaggca aagtccccaa actcatgatt tatgatgtca gtgagcggcc ctcaggggtt 180

tctaatcgct tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc 240

caggctgagg acgaggctga ttattactgc agctcatata caagcaccac cactctcgtt 300

gtgttcggcg gagggaccaa gctgtccgtc ctg 333

<210> 21

<211> 421

<212> PRT

<213>artificial sequence

<220>

<223>people LAG-3 ECD

<400> 21

Pro Val Val Trp Ala Gln Glu Gly Ala Pro Ala Gln Leu Pro Cys Ser

1 5 10 15

Pro Thr Ile Pro Leu Gln Asp Leu Ser Leu Leu Arg Arg Ala Gly Val

20 25 30

Thr Trp Gln His Gln Pro Asp Ser Gly Pro Pro Ala Ala Ala Pro Gly

35 40 45

His Pro Leu Ala Pro Gly Pro His Pro Ala Ala Pro Ser Ser Trp Gly

50 55 60

Pro Arg Pro Arg Arg Tyr Thr Val Leu Ser Val Gly Pro Gly Gly Leu

65 70 75 80

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

85 90 95

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

100 105 110

Ala Asp Ala Gly Glu Tyr Arg Ala Ala Val His Leu Arg Asp Arg Ala

115 120 125

Leu Ser Cys Arg Leu Arg Leu Arg Leu Gly Gln Ala Ser Met Thr Ala

130 135 140

Ser Pro Pro Gly Ser Leu Arg Ala Ser Asp Trp Val Ile Leu Asn Cys

145 150 155 160

Ser Phe Ser Arg Pro Asp Arg Pro Ala Ser Val His Trp Phe Arg Asn

165 170 175

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

180 185 190

Ala Glu Ser Phe Leu Phe Leu Pro Gln Val Ser Pro Met Asp Ser Gly

195 200 205

Pro Trp Gly Cys Ile Leu Thr Tyr Arg Asp Gly Phe Asn Val Ser Ile

210 215 220

Met Tyr Asn Leu Thr Val Leu Gly Leu Glu Pro Pro Thr Pro Leu Thr

225 230 235 240

Val Tyr Ala Gly Ala Gly Ser Arg Val Gly Leu Pro Cys Arg Leu Pro

245 250 255

Ala Gly Val Gly Thr Arg Ser Phe Leu Thr Ala Lys Trp Thr Pro Pro

260 265 270

Gly Gly Gly Pro Asp Leu Leu Val Thr Gly Asp Asn Gly Asp Phe Thr

275 280 285

Leu Arg Leu Glu Asp Val Ser Gln Ala Gln Ala Gly Thr Tyr Thr Cys

290 295 300

His Ile His Leu Gln Glu Gln Gln Leu Asn Ala Thr Val Thr Leu Ala

305 310 315 320

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

325 330 335

Lys Leu Leu Cys Glu Val Thr Pro Val Ser Gly Gln Glu Arg Phe Val

340 345 350

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

355 360 365

Leu Glu Ala Gln Glu Ala Gln Leu Leu Ser Gln Pro Trp Gln Cys Gln

370 375 380

Leu Tyr Gln Gly Glu Arg Leu Leu Gly Ala Ala Val Tyr Phe Thr Glu

385 390 395 400

Leu Ser Ser Pro Gly Ala Gln Arg Ser Gly Arg Ala Pro Gly Ala Leu

405 410 415

Pro Ala Gly His Leu

420

<210> 22

<211> 1263

<212> DNA

<213>artificial sequence

<220>

<223>people LAG-3 ECD

<400> 22

ccggtggtgt gggcccagga gggggctcct gcccagctcc cctgcagccc cacaatcccc 60

ctccaggatc tcagccttct gcgaagagca ggggtcactt ggcagcatca gccagacagt 120

ggcccgcccg ctgccgcccc cggccatccc ctggcccccg gccctcaccc ggcggcgccc 180

tcctcctggg ggcccaggcc ccgccgctac acggtgctga gcgtgggtcc cggaggcctg 240

cgcagcggga ggctgcccct gcagccccgc gtccagctgg atgagcgcgg ccggcagcgc 300

ggggacttct cgctatggct gcgcccagcc cggcgcgcgg acgccggcga gtaccgcgcc 360

gcggtgcacc tcagggaccg cgccctctcc tgccgcctcc gtctgcgcct gggccaggcc 420

tcgatgactg ccagcccccc aggatctctc agagcctccg actgggtcat tttgaactgc 480

tccttcagcc gccctgaccg cccagcctct gtgcattggt tccggaaccg gggccagggc 540

cgagtccctg tccgggagtc cccccatcac cacttagcgg aaagcttcct cttcctgccc 600

caagtcagcc ccatggactc tgggccctgg ggctgcatcc tcacctacag agatggcttc 660

aacgtctcca tcatgtataa cctcactgtt ctgggtctgg agcccccaac tcccttgaca 720

gtgtacgctg gagcaggttc cagggtgggg ctgccctgcc gcctgcctgc tggtgtgggg 780

acccggtctt tcctcactgc caagtggact cctcctgggg gaggccctga cctcctggtg 840

actggagaca atggcgactt tacccttcga ctagaggatg tgagccaggc ccaggctggg 900

acctacacct gccatatcca tctgcaggaa cagcagctca atgccactgt cacattggca 960

atcatcacag tgactcccaa atcctttggg tcacctggat ccctggggaa gctgctttgt 1020

gaggtgactc cagtatctgg acaagaacgc tttgtgtgga gctctctgga caccccatcc 1080

cagaggagtt tctcaggacc ttggctggag gcacaggagg cccagctcct ttcccagcct 1140

tggcaatgcc agctgtacca gggggagagg cttcttggag cagcagtgta cttcacagag 1200

ctgtctagcc caggtgccca acgctctggg agagccccag gtgccctccc agcaggccac 1260

ctc 1263

<210> 23

<211> 525

<212> PRT

<213>artificial sequence

<220>

<223>overall length people LAG-3

<400> 23

Met Trp Glu Ala Gln Phe Leu Gly Leu Leu Phe Leu Gln Pro Leu Trp

1 5 10 15

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

20 25 30

Trp Ala Gln Glu Gly Ala Pro Ala Gln Leu Pro Cys Ser Pro Thr Ile

35 40 45

Pro Leu Gln Asp Leu Ser Leu Leu Arg Arg Ala Gly Val Thr Trp Gln

50 55 60

His Gln Pro Asp Ser Gly Pro Pro Ala Ala Ala Pro Gly His Pro Leu

65 70 75 80

Ala Pro Gly Pro His Pro Ala Ala Pro Ser Ser Trp Gly Pro Arg Pro

85 90 95

Arg Arg Tyr Thr Val Leu Ser Val Gly Pro Gly Gly Leu Arg Ser Gly

100 105 110

Arg Leu Pro Leu Gln Pro Arg Val Gln Leu Asp Glu Arg Gly Arg Gln

115 120 125

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

130 135 140

Gly Glu Tyr Arg Ala Ala Val His Leu Arg Asp Arg Ala Leu Ser Cys

145 150 155 160

Arg Leu Arg Leu Arg Leu Gly Gln Ala Ser Met Thr Ala Ser Pro Pro

165 170 175

Gly Ser Leu Arg Ala Ser Asp Trp Val Ile Leu Asn Cys Ser Phe Ser

180 185 190

Arg Pro Asp Arg Pro Ala Ser Val His Trp Phe Arg Asn Arg Gly Gln

195 200 205

Gly Arg Val Pro Val Arg Glu Ser Pro His His His Leu Ala Glu Ser

210 215 220

Phe Leu Phe Leu Pro Gln Val Ser Pro Met Asp Ser Gly Pro Trp Gly

225 230 235 240

Cys Ile Leu Thr Tyr Arg Asp Gly Phe Asn Val Ser Ile Met Tyr Asn

245 250 255

Leu Thr Val Leu Gly Leu Glu Pro Pro Thr Pro Leu Thr Val Tyr Ala

260 265 270

Gly Ala Gly Ser Arg Val Gly Leu Pro Cys Arg Leu Pro Ala Gly Val

275 280 285

Gly Thr Arg Ser Phe Leu Thr Ala Lys Trp Thr Pro Pro Gly Gly Gly

290 295 300

Pro Asp Leu Leu Val Thr Gly Asp Asn Gly Asp Phe Thr Leu Arg Leu

305 310 315 320

Glu Asp Val Ser Gln Ala Gln Ala Gly Thr Tyr Thr Cys His Ile His

325 330 335

Leu Gln Glu Gln Gln Leu Asn Ala Thr Val Thr Leu Ala Ile Ile Thr

340 345 350

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

355 360 365

Cys Glu Val Thr Pro Val Ser Gly Gln Glu Arg Phe Val Trp Ser Ser

370 375 380

Leu Asp Thr Pro Ser Gln Arg Ser Phe Ser Gly Pro Trp Leu Glu Ala

385 390 395 400

Gln Glu Ala Gln Leu Leu Ser Gln Pro Trp Gln Cys Gln Leu Tyr Gln

405 410 415

Gly Glu Arg Leu Leu Gly Ala Ala Val Tyr Phe Thr Glu Leu Ser Ser

420 425 430

Pro Gly Ala Gln Arg Ser Gly Arg Ala Pro Gly Ala Leu Pro Ala Gly

435 440 445

His Leu Leu Leu Phe Leu Ile Leu Gly Val Leu Ser Leu Leu Leu Leu

450 455 460

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

465 470 475 480

Arg Arg Phe Ser Ala Leu Glu Gln Gly Ile His Pro Pro Gln Ala Gln

485 490 495

Ser Lys Ile Glu Glu Leu Glu Gln Glu Pro Glu Pro Glu Pro Glu Pro

500 505 510

Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu Gln Leu

515 520 525

<210> 24

<211> 1575

<212> DNA

<213>artificial sequence

<220>

<223>overall length people LAG-3

<400> 24

atgtgggagg ctcagttcct gggcttgctg tttctgcagc cgctttgggt ggctccagtg 60

aagcctctcc agccaggggc tgaggtcccg gtggtgtggg cccaggaggg ggctcctgcc 120

cagctcccct gcagccccac aatccccctc caggatctca gccttctgcg aagagcaggg 180

gtcacttggc agcatcagcc agacagtggc ccgcccgctg ccgcccccgg ccatcccctg 240

gcccccggcc ctcacccggc ggcgccctcc tcctgggggc ccaggccccg ccgctacacg 300

gtgctgagcg tgggtcccgg aggcctgcgc agcgggaggc tgcccctgca gccccgcgtc 360

cagctggatg agcgcggccg gcagcgcggg gacttctcgc tatggctgcg cccagcccgg 420

cgcgcggacg ccggcgagta ccgcgccgcg gtgcacctca gggaccgcgc cctctcctgc 480

cgcctccgtc tgcgcctggg ccaggcctcg atgactgcca gccccccagg atctctcaga 540

gcctccgact gggtcatttt gaactgctcc ttcagccgcc ctgaccgccc agcctctgtg 600

cattggttcc ggaaccgggg ccagggccga gtccctgtcc gggagtcccc ccatcaccac 660

ttagcggaaa gcttcctctt cctgccccaa gtcagcccca tggactctgg gccctggggc 720

tgcatcctca cctacagaga tggcttcaac gtctccatca tgtataacct cactgttctg 780

ggtctggagc ccccaactcc cttgacagtg tacgctggag caggttccag ggtggggctg 840

ccctgccgcc tgcctgctgg tgtggggacc cggtctttcc tcactgccaa gtggactcct 900

cctgggggag gccctgacct cctggtgact ggagacaatg gcgactttac ccttcgacta 960

gaggatgtga gccaggccca ggctgggacc tacacctgcc atatccatct gcaggaacag 1020

cagctcaatg ccactgtcac attggcaatc atcacagtga ctcccaaatc ctttgggtca 1080

cctggatccc tggggaagct gctttgtgag gtgactccag tatctggaca agaacgcttt 1140

gtgtggagct ctctggacac cccatcccag aggagtttct caggaccttg gctggaggca 1200

caggaggccc agctcctttc ccagccttgg caatgccagc tgtaccaggg ggagaggctt 1260

cttggagcag cagtgtactt cacagagctg tctagcccag gtgcccaacg ctctgggaga 1320

gccccaggtg ccctcccagc aggccacctc ctgctgtttc tcatccttgg tgtcctttct 1380

ctgctccttt tggtgactgg agcctttggc tttcaccttt ggagaagaca gtggcgacca 1440

agacgatttt ctgccttaga gcaagggatt caccctccgc aggctcagag caagatagag 1500

gagctggagc aagaaccgga gccggagccg gagccggaac cggagcccga gcccgagccc 1560

gagccggagc agctc 1575

<210> 25

<211> 521

<212> PRT

<213>artificial sequence

<220>

<223>overall length mouse LAG-3

<400> 25

Met Arg Glu Asp Leu Leu Leu Gly Phe Leu Leu Leu Gly Leu Leu Trp

1 5 10 15

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

20 25 30

Trp Ala Gln Glu Gly Ala Pro Val His Leu Pro Cys Ser Leu Lys Ser

35 40 45

Pro Asn Leu Asp Pro Asn Phe Leu Arg Arg Gly Gly Val Ile Trp Gln

50 55 60

His Gln Pro Asp Ser Gly Gln Pro Thr Pro Ile Pro Ala Leu Asp Leu

65 70 75 80

His Gln Gly Met Pro Ser Pro Arg Gln Pro Ala Pro Gly Arg Tyr Thr

85 90 95

Val Leu Ser Val Ala Pro Gly Gly Leu Arg Ser Gly Arg Gln Pro Leu

100 105 110

His Pro His Val Gln Leu Glu Glu Arg Gly Leu Gln Arg Gly Asp Phe

115 120 125

Ser Leu Trp Leu Arg Pro Ala Leu Arg Thr Asp Ala Gly Glu Tyr His

130 135 140

Ala Thr Val Arg Leu Pro Asn Arg Ala Leu Ser Cys Ser Leu Arg Leu

145 150 155 160

Arg Val Gly Gln Ala Ser Met Ile Ala Ser Pro Ser Gly Val Leu Lys

165 170 175

Leu Ser Asp Trp Val Leu Leu Asn Cys Ser Phe Ser Arg Pro Asp Arg

180 185 190

Pro Val Ser Val His Trp Phe Gln Gly Gln Asn Arg Val Pro Val Tyr

195 200 205

Asn Ser Pro Arg His Phe Leu Ala Glu Thr Phe Leu Leu Leu Pro Gln

210 215 220

Val Ser Pro Leu Asp Ser Gly Thr Trp Gly Cys Val Leu Thr Tyr Arg

225 230 235 240

Asp Gly Phe Asn Val Ser Ile Thr Tyr Asn Leu Lys Val Leu Gly Leu

245 250 255

Glu Pro Val Ala Pro Leu Thr Val Tyr Ala Ala Glu Gly Ser Arg Val

260 265 270

Glu Leu Pro Cys His Leu Pro Pro Gly Val Gly Thr Pro Ser Leu Leu

275 280 285

Ile Ala Lys Trp Thr Pro Pro Gly Gly Gly Pro Glu Leu Pro Val Ala

290 295 300

Gly Lys Ser Gly Asn Phe Thr Leu His Leu Glu Ala Val Gly Leu Ala

305 310 315 320

Gln Ala Gly Thr Tyr Thr Cys Ser Ile His Leu Gln Gly Gln Gln Leu

325 330 335

Asn Ala Thr Val Thr Leu Ala Val Ile Thr Val Thr Pro Lys Ser Phe

340 345 350

Gly Leu Pro Gly Ser Arg Gly Lys Leu Leu Cys Glu Val Thr Pro Ala

355 360 365

Ser Gly Lys Glu Arg Phe Val Trp Arg Pro Leu Asn Asn Leu Ser Arg

370 375 380

Ser Cys Pro Gly Pro Val Leu Glu Ile Gln Glu Ala Arg Leu Leu Ala

385 390 395 400

Glu Arg Trp Gln Cys Gln Leu Tyr Glu Gly Gln Arg Leu Leu Gly Ala

405 410 415

Thr Val Tyr Ala Ala Glu Ser Ser Ser Gly Ala His Ser Ala Arg Arg

420 425 430

Ile Ser Gly Asp Leu Lys Gly Gly His Leu Val Leu Val Leu Ile Leu

435 440 445

Gly Ala Leu Ser Leu Phe Leu Leu Val Ala Gly Ala Phe Gly Phe His

450 455 460

Trp Trp Arg Lys Gln Leu Leu Leu Arg Arg Phe Ser Ala Leu Glu His

465 470 475 480

Gly Ile Gln Pro Phe Pro Ala Gln Arg Lys Ile Glu Glu Leu Glu Arg

485 490 495

Glu Leu Glu Thr Glu Met Gly Gln Glu Pro Glu Pro Glu Pro Glu Pro

500 505 510

Gln Leu Glu Pro Glu Pro Arg Gln Leu

515 520

<210> 26

<211> 1563

<212> DNA

<213>artificial sequence

<220>

<223>overall length mouse LAG-3

<400> 26

atgagggagg acctgctcct tggctttttg cttctgggac tgctttggga agctccagtt 60

gtgtcttcag ggcctgggaa agagctcccc gtggtgtggg cccaggaggg agctcccgtc 120

catcttccct gcagcctcaa atcccccaac ctggatccta actttctacg aagaggaggg 180

gttatctggc aacatcaacc agacagtggc caacccactc ccatcccggc ccttgacctt 240

caccagggga tgccctcgcc tagacaaccc gcacccggtc gctacacggt gctgagcgtg 300

gctccaggag gcctgcgcag cgggaggcag cccctgcatc cccacgtgca gctggaggag 360

cgcggcctcc agcgcgggga cttctctctg tggttgcgcc cagctctgcg caccgatgcg 420

ggcgagtacc acgccaccgt gcgcctcccg aaccgcgccc tctcctgcag tctccgcctg 480

cgcgtcggcc aggcctcgat gattgctagt ccctcaggag tcctcaagct gtctgattgg 540

gtccttttga actgctcctt cagccgtcct gaccgcccag tctctgtgca ctggttccag 600

ggccagaacc gagtgcctgt ctacaactca ccgcgtcatt ttttagctga aactttcctg 660

ttactgcccc aagtcagccc cctggactct gggacctggg gctgtgtcct cacctacaga 720

gatggcttca atgtctccat cacgtacaac ctcaaggttc tgggtctgga gcccgtagcc 780

cctctgacag tgtacgctgc tgaaggttct agggtggagc tgccctgtca tttgccccca 840

ggagtgggga ccccttcttt gctcattgcc aagtggactc ctcctggagg aggtcctgag 900

ctccccgtgg ctggaaagag tggcaatttt acccttcacc ttgaggctgt gggtctggca 960

caggctggga cctacacctg tagcatccat ctgcagggac agcagctcaa tgccactgtc 1020

acgttggcgg tcatcacagt gactcccaaa tccttcgggt tacctggctc ccgggggaag 1080

ctgttgtgtg aggtaacccc ggcatctgga aaggaaagat ttgtgtggcg tcccctgaac 1140

aatctgtcca ggagttgccc gggccctgtg ctggagattc aggaggccag gctccttgct 1200

gagcgatggc agtgtcagct gtacgagggc cagaggcttc ttggagcgac agtgtacgcc 1260

gcagagtcta gctcaggcgc ccacagtgct aggagaatct caggtgacct taaaggaggc 1320

catctcgttc tcgttctcat ccttggtgcc ctctccctgt tccttttggt ggccggggcc 1380

tttggctttc actggtggag aaaacagttg ctactgagaa gattttctgc cttagaacat 1440

gggattcagc catttccggc tcagaggaag atagaggagc tggagcgaga actggagacg 1500

gagatgggac aggagccgga gcccgagccg gagccacagc tggagccaga gcccaggcag 1560

ctc 1563

<210> 27

<211> 533

<212> PRT

<213>artificial sequence

<220>

<223>overall length machin LAG-3

<220>

<221> misc_feature

<222> (74)..(74)

<223>Xaa can be any naturally occurring amino acid

<400> 27

Met Trp Glu Ala Gln Phe Leu Gly Leu Leu Phe Leu Gln Pro Leu Trp

1 5 10 15

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

20 25 30

Trp Ala Gln Glu Gly Ala Pro Ala Gln Leu Pro Cys Ser Pro Thr Ile

35 40 45

Pro Leu Gln Asp Leu Ser Leu Leu Arg Arg Ala Gly Val Thr Trp Gln

50 55 60

His Gln Pro Asp Ser Gly Pro Pro Ala Xaa Ala Pro Gly His Pro Pro

65 70 75 80

Val Pro Gly His Arg Pro Ala Ala Pro Tyr Ser Trp Gly Pro Arg Pro

85 90 95

Arg Arg Tyr Thr Val Leu Ser Val Gly Pro Gly Gly Leu Arg Ser Gly

100 105 110

Arg Leu Pro Leu Gln Pro Arg Val Gln Leu Asp Glu Arg Gly Arg Gln

115 120 125

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

130 135 140

Gly Glu Tyr Arg Ala Thr Val His Leu Arg Asp Arg Ala Leu Ser Cys

145 150 155 160

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

165 170 175

Gly Ser Leu Arg Thr Ser Asp Trp Val Ile Leu Asn Cys Ser Phe Ser

180 185 190

Arg Pro Asp Arg Pro Ala Ser Val His Trp Phe Arg Ser Arg Gly Gln

195 200 205

Gly Arg Val Pro Val Gln Gly Ser Pro His His His Leu Ala Glu Ser

210 215 220

Phe Leu Phe Leu Pro His Val Gly Pro Met Asp Ser Gly Leu Trp Gly

225 230 235 240

Cys Ile Leu Thr Tyr Arg Asp Gly Phe Asn Val Ser Ile Met Tyr Asn

245 250 255

Leu Thr Val Leu Gly Leu Glu Pro Ala Thr Pro Leu Thr Val Tyr Ala

260 265 270

Gly Ala Gly Ser Arg Val Glu Leu Pro Cys Arg Leu Pro Pro Ala Val

275 280 285

Gly Thr Gln Ser Phe Leu Thr Ala Lys Trp Ala Pro Pro Gly Gly Gly

290 295 300

Pro Asp Leu Leu Val Ala Gly Asp Asn Gly Asp Phe Thr Leu Arg Leu

305 310 315 320

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

325 330 335

Leu Gln Gly Gln Gln Leu Asn Ala Thr Val Thr Leu Ala Ile Ile Thr

340 345 350

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

355 360 365

Cys Glu Val Thr Pro Ala Ser Gly Gln Glu His Phe Val Trp Ser Pro

370 375 380

Leu Asn Thr Pro Ser Gln Arg Ser Phe Ser Gly Pro Trp Leu Glu Ala

385 390 395 400

Gln Glu Ala Gln Leu Leu Ser Gln Pro Trp Gln Cys Gln Leu His Gln

405 410 415

Gly Glu Arg Leu Leu Gly Ala Ala Val Tyr Phe Thr Glu Leu Ser Ser

420 425 430

Pro Gly Ala Gln Arg Ser Gly Arg Ala Pro Gly Ala Leu Arg Ala Gly

435 440 445

His Leu Pro Leu Phe Leu Ile Leu Gly Val Leu Phe Leu Leu Leu Leu

450 455 460

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

465 470 475 480

Arg Arg Phe Ser Ala Leu Glu Gln Gly Ile His Pro Pro Gln Ala Gln

485 490 495

Ser Lys Ile Glu Glu Leu Glu Gln Glu Pro Glu Leu Glu Pro Glu Pro

500 505 510

Glu Leu Glu Arg Glu Leu Gly Pro Glu Pro Glu Pro Gly Pro Glu Pro

515 520 525

Glu Pro Glu Gln Leu

530

<210> 28

<211> 1599

<212> DNA

<213>artificial sequence

<220>

<223>overall length machin LAG-3

<220>

<221> misc_feature

<222> (219)..(220)

<223>n is a, c, g, or t

<400> 28

atgtgggagg ctcagttcct gggcttgctg tttctgcagc cgctctgggt ggctccagtg 60

aagcctcccc agccaggggc tgagatctcg gtggtgtggg cccaggaggg ggctcctgcc 120

cagctcccct gcagccccac aatccccctc caggatctca gccttctgcg aagagcaggg 180

gtcacttggc agcatcaacc agacagtggc ccgcccgcnn ccgcccccgg ccaccccccg 240

gtccccggcc atcgcccggc ggcgccctac tcttgggggc ccaggccccg ccgctacacg 300

gtgctgagcg tgggtcctgg aggcctgcgc agcgggaggc tgcccctgca gccccgcgtc 360

cagctggatg agcgcggccg gcagcgcggg gacttctcgc tgtggctgcg cccagcccgg 420

cgcgcggacg ccggcgagta ccgcgccacg gtgcacctca gggaccgcgc cctctcctgc 480

cgccttcgtc tgcgcgtggg ccaggcctcg atgactgcca gccccccagg gtctctcagg 540

acctctgact gggtcatttt gaactgctcc ttcagccgcc ctgaccgccc agcctctgtg 600

cattggttcc ggagccgtgg ccagggccga gtccctgtcc aggggtcccc ccatcaccac 660

ttagcggaaa gcttcctctt cctgccccat gtcggcccca tggactctgg gctctggggc 720

tgcatcctca cctacagaga tggcttcaat gtctccatca tgtataacct cactgttctg 780

ggtctggagc ccgcaactcc cttgacagtg tacgctggag caggttccag ggtggagctg 840

ccctgccgcc tgcctcctgc tgtggggacc cagtctttcc ttactgccaa gtgggctcct 900

cctgggggag gccctgacct cctggtggct ggagacaatg gcgactttac ccttcgacta 960

gaggatgtaa gccaggccca ggctgggacc tacatctgcc atatccgtct acagggacag 1020

cagctcaatg ccactgtcac attggcaatc atcacagtga ctcccaaatc ctttgggtca 1080

cctggctccc tggggaagct gctttgtgag gtgactccag catctggaca agaacacttt 1140

gtgtggagcc ccctgaacac cccatcccag aggagtttct caggaccatg gctggaggcc 1200

caggaagccc agctcctttc ccagccttgg caatgccagc tgcaccaggg ggagaggctt 1260

cttggagcag cagtatactt cacagaactg tctagcccag gtgcacaacg ctctgggaga 1320

gccccagggg ccctccgagc aggccacctc ccgctgtttc tcatccttgg tgtccttttt 1380

ctgctccttt tggtgactgg agcctttggc tttcaccttt ggagaagaca gtggcgacca 1440

agaagatttt ctgccttaga gcaagggatt caccctccgc aggctcagag caagatagag 1500

gagctcgagc aagaaccgga gctggaacca gagccggagc tggagcgcga gctggggccg 1560

gagcccgagc cggggcctga gcccgagccg gagcagctc 1599

Claims

1. isolated antibody or its antigen-binding portion thereof, wherein the isolated antibody or its antigen-binding portion thereof include:

A) one or more heavy chain CDR (CDRH), it is selected from the following at least one:

(i) there is at least 90% sequence identity with CDRH1 shown in one of the sequence selected from SEQ ID NO:1 and 7 CDRH1；

(ii) there is at least 90% sequence identity with CDRH2 shown in one of the sequence selected from SEQ ID NO:2 and 8 CDRH2；With

(iii) there is at least 90% sequence identity with CDRH3 shown in one of the sequence selected from SEQ ID NO:3 and 9 CDRH3；

B) one or more light chain CDR (CDRL), it is selected from the following at least one:

(i) there is at least 90% sequence identity with CDRL1 shown in one of the sequence selected from SEQ ID NO:4 and 10 CDRL1；

(ii) there is at least 90% sequence identity with CDRL2 shown in one of the sequence selected from SEQ ID NO:5 and 11 CDRL2；With

(iii) there is at least 90% sequence identity with CDRL3 shown in one of the sequence selected from SEQ ID NO:6 and 12 CDRL3；Or

C) A) one or more CDRH and B) one or more CDRL.

2. the isolated antibody or its antigen-binding portion thereof of claim 1, wherein the isolated antibody or its antigen-binding portion Subpackage contains:

(i) CDRH1 selected from SEQ ID NO:1 and 7 or exist with the amino acid sequence of the CDRH1 and be no more than 2 amino acid The CDRH1 of the difference of amino acid addition, missing or substitution；

(ii) CDRH2 selected from SEQ ID NO:2 and 8 or exist with the amino acid sequence of the CDRH2 and be no more than 2 amino acid The CDRH2 of the difference of amino acid addition, missing or substitution；With

(iii) it is selected from the CDRH3 of SEQ ID NO:3 and 9 or exists with the amino acid sequence of the CDRH3 no more than 2 amino acid Amino acid addition, missing or replace difference CDRH3；

(i) CDRL1 selected from SEQ ID NO:4 and 10 or exist with the amino acid sequence of the CDRL1 and be no more than 2 amino acid The CDRL1 of the difference of amino acid addition, missing or substitution；

(ii) it is selected from the CDRL2 of SEQ ID NO:5 and 11 or exists with the amino acid sequence of the CDRL2 no more than 2 amino acid Amino acid addition, missing or replace difference CDRL2；With

(iii) it is selected from the CDRL3 of SEQ ID NO:6 and 12 or exists with the amino acid sequence of the CDRL3 no more than 2 amino acid Amino acid addition, missing or replace difference CDRL3；Or

C) A) one or more CDRH and B) one or more CDRL.

3. the isolated antibody or its antigen-binding portion thereof of claim 1, wherein the isolated antibody or its antigen-binding portion Subpackage contains:

(a) comprising the SEQ ID NO:1 or CDRH1 being made from it；

(b) comprising the SEQ ID NO:2 or CDRH2 being made from it；

(c) comprising the SEQ ID NO:3 or CDRH3 being made from it；

(d) comprising the SEQ ID NO:4 or CDRL1 being made from it；

(e) comprising the SEQ ID NO:5 or CDRL2 being made from it；With

(f) comprising the SEQ ID NO:6 or CDRL3 being made from it.

4. the isolated antibody or its antigen-binding portion thereof of claim 1, wherein the isolated antibody or its antigen-binding portion Subpackage contains:

(a) comprising the SEQ ID NO:7 or CDRH1 being made from it；

(b) comprising the SEQ ID NO:8 or CDRH2 being made from it；

(c) comprising the SEQ ID NO:9 or CDRH3 being made from it；

(d) comprising the SEQ ID NO:10 or CDRL1 being made from it；

(e) comprising the SEQ ID NO:11 or CDRL2 being made from it；With

(f) comprising the SEQ ID NO:12 or CDRL3 being made from it.

5. the isolated antibody or its antigen-binding portion thereof of claim 1, wherein the isolated antibody or its antigen-binding portion Subpackage contains:

(A) heavy chain variable region:

(i) comprising the amino acid sequence of SEQ ID NO:13；

(ii) comprising having the amino acid sequence of at least 85%, 90% or 95% identity with SEQ ID NO:13；Or

(iii) comprising having addition, missing and/or the substituted amino of one or more amino acid compared with SEQ ID NO:13 Acid sequence；And/or

(B) light chain variable region:

(i) comprising the amino acid sequence of SEQ ID NO:14；

(ii) comprising having the amino acid sequence of at least 85%, 90% or 95% identity with SEQ ID NO:14；Or

(iii) comprising having addition, missing and/or the substituted amino of one or more amino acid compared with SEQ ID NO:14 Acid sequence.

6. the isolated antibody or its antigen-binding portion thereof of claim 1, wherein the isolated antibody or its antigen-binding portion Subpackage contains:

(A) heavy chain variable region:

(i) comprising the amino acid sequence of SEQ ID NO:15；

(ii) comprising having the amino acid sequence of at least 85%, 90% or 95% identity with SEQ ID NO:15；Or

(iii) comprising having addition, missing and/or the substituted amino of one or more amino acid compared with SEQ ID NO:15 Acid sequence；And/or

(B) light chain variable region:

(i) comprising the amino acid sequence of SEQ ID NO:16；

(ii) comprising having the amino acid sequence of at least 85%, 90% or 95% identity with SEQ ID NO:16；Or

(iii) comprising having addition, missing and/or the substituted amino of one or more amino acid compared with SEQ ID NO:16 Acid sequence.

7. the isolated antibody or its antigen-binding portion thereof of any one of claim 1-6 have one or more following property Matter:

(a) with 2 × 10^-10M or lower K_DIn conjunction with people LAG-3；

(d) inhibit the combination of LAG-3 and LSECtin and/or galectin-3；

(e) combine people LAG-3 without reacting across family；Or

(f) there is no cross reactivity with people CD4.

8. the isolated antibody or its antigen-binding portion thereof of any one of claim 1-7, wherein the antibody is that monoclonal is anti- Body, such as complete human monoclonal antibodies, such as the complete human monoclonal antibodies generated by transgene mammal, the transgenosis Mammal is preferably transgenic rat, more preferably with the transgenic rat of recombination immunoglobulin locus.

9. a kind of isolated nucleic acid molecules, it includes the weights of the antibody of separation defined in any one of coding claim 1-8 The nucleic acid sequence of chain variable region and/or light chain variable region, for example, nucleic acid sequence shown in SEQ ID NOs:17-20.

10. a kind of expression vector, it includes the nucleic acid molecules of claim 9.

11. a kind of host cell, it includes the expression vectors of claim 10.

12. a kind of pharmaceutical composition, it includes at least one antibody as defined in any one of claim 1-8 or its antigens Bound fraction and pharmaceutically acceptable carrier.

13. a kind of method for preparing antibody defined in any one of claim 1-8 or its antigen-binding portion thereof comprising with Lower step:

Antibody or its antigen binding defined in any one of claim 1-8 are expressed in the host cell of claim 11 Part；With

From host cell separation antibody or its antigen-binding portion thereof.

14. a kind of antigen-specific T cell response adjusted in subject or the method for adjusting the immune response in subject, Including applying antibody or its antigen-binding portion thereof defined in any one of claim 1-8 to subject, so that in subject Antigen-specific T cell response or immune response be regulated.

15. a kind of inhibition or blocking LAG-3 and MHC II class molecule, FGL1 class molecule, LSECtin and/or galactose agglutinin- The method of 3 combination comprising make the MHC II class molecule, FGL1 class molecule, LSECtin and/or galectin-3 With antibody or its antigen binding portion thereof defined in any one of claim 1-8.

16. a kind of method for inhibiting the growth of tumour cell in subject comprising apply in claim 1-8 and appoint to subject One defined antibody or its antigen-binding portion thereof, so that the growth of tumour is suppressed in subject.

17. a method of treat or prevent the proliferative disorders such as cancer in subject comprising Xiang Suoshu subject applies With the antibody or its antigen-binding portion thereof of any one of a effective amount of claim 1-8 definition.

18. antibody defined in any one of claim 1-8 or its antigen-binding portion thereof increase in preparation for treating or preventing Grow the purposes in the drug of venereal disease disease such as cancer.

19. antibody defined in any one of claim 1-8 or its antigen-binding portion thereof are in preparation for diagnosing proliferative diseases Purposes in the diagnosticum of disease such as cancer.

20. antibody defined in any one of claim 1-8 or its antigen-binding portion thereof, are used to treat or prevent proliferative Illness such as cancer.

21. antibody defined in any one of claim 1-8 or its antigen-binding portion thereof, are used to diagnose proliferative disorders example Such as cancer.

22. the kit for treating or diagnosing proliferative disorders such as cancer, it includes contain at least one such as claim The container of antibody or its antigen-binding portion thereof defined in any one of 1-8.