CN110386981A - Anti- GITR antibody and application thereof - Google Patents

Abstract

Composition the present invention relates to the novel antibodies of specific binding GITR and antibody fragment and containing the antibody or antibody fragment.In addition, the present invention relates to the nucleic acid of encoding said antibody or its antibody fragment and including its host cell and associated uses.In addition, the present invention relates to the treatment of these antibody and antibody fragment and diagnostic uses.

Description

Anti- GITR antibody and application thereof

The present invention relates to the novel antibodies of specific binding GITR and antibody fragment and contain the antibody or antibody piece The composition of section.In addition, the present invention relates to the nucleic acid of encoding said antibody or its antibody fragment and comprising its host cell, with And associated uses.In addition, the present invention relates to the treatment of these antibody and antibody fragment and diagnostic uses.

Background technique

Tumor Necrosis Factor Receptors (the glucocorticoid induced tumor of glucocorticoid inducible Necrosis factor receptor, GITR), also referred to as CD357 and GITR-D are the mouse in Doxorubicin processing earliest (Nocentin, the PNAS.1997 being found in T cell；It 94:6216-6221), is Tumor Necrosis Factor Receptors (tumor Necrosis factor receptor, TNFR) superfamily the 18th member (TNFRSF18), other members include CD40, CD27,4-1BB, OX40 etc..GITR can activate the NF-kappa in downstream by its cognate ligand, GITR ligand (GITRL) activation B signal access.

GITR is in Resting T cells with low expression level；After T cell activation, significant up-regulation is expressed.GITR is in modulability High-level constitutive expression in T cell (Treg), and when these cells are activated, expression is dialled further up (Nocentini And Riccardi, E.J.Immunol.2005,35:1016-1022).

GITR ligand (GITRL) mainly antigen presenting cell (APC, including macrophage, B cell, Dendritic Cells and Endothelial cell) on express.The combination of GITR triggers GITR signal transduction, stimulating effect on GITRL and response T cell on APC Property t cell activation and the activity for inhibiting Treg cell.GITR pairing effect T cell and regulatory T cells have several works as a result, With, comprising: costimulation and activating effect T cell, inhibition of reduction Treg cell pairing effect T cell etc. (Nocentini, Eur.J.Immunol.2007,37:1165-1169)。

These effects mean that the activation of GITR signal path can lead to the enhancing of immune response.Therefore, it can activate The substance of GITR signal path can activate immune response, and then increase that body is antitumor and the ability of infection etc., simultaneously The high expression in Treg cell in tumor microenvironment of GITR molecule, therefore GITR molecule is removed using the ADCC activity of GITR antibody Highly expressed Treg cell can further enhance anti-tumor activity.

The prior art have studied a variety of GITR antibody (referring to CN103951753A, CN105829343A, CN106459203A, WO2016196792A1, WO2017068186A9 etc.).Such antibody is the agonist of GITR (that is, being Activated form antibody), it can induce or enhance GITR signal transduction, need to enhance a variety of GITR related diseases of immune response in treatment It or in illness is effective.It is affine compared with the combination of itself and antigen since such anti-GITR antibody is activated form antibody Power, the height of Activation Activity be its play activity (such as.Immune-enhancing activity) more crucial index.

Therefore, it is necessary to develop with better Activation Activity, more preferable ADCC effect, and there is better curative effect, such as The antibody of antitumor action.

Summary of the invention

This invention therefore provides the antibody and its antigen-binding fragment of a kind of new combination GITR.

In some embodiments, anti-GITR antibody of the invention has following one or more characteristics:

(i) with high-affinity in conjunction with GITR；

(ii) it is effectively combined with the GITR of cell surface；

(iii) there is agonist activity, such as can effectively activate NF-kappaB signal path；

(iv) effectively T cell (such as cd4 t cell) is activated；

(v) ADCC effect can effectively be mediated；

(vi) there is better anti-tumor activity, such as can reduce the gross tumor volume in subject, at the same do not influence by The weight of examination person；

(vii) it can preferably inhibit tumor promotion with anti-PD-1 antibody combination, such as can reduce swollen in subject Knurl product, while subject's weight is not influenced.

In some embodiments, the present invention provides the antibody or its antigen-binding fragment that combine GITR, comprising such as SEQ 3 heavy chain CDR (HCDR) of sequence shown in ID NO:17,18,19 or 20, and/or such as SEQ ID NO:21,22,23 or 24 Shown in sequence 3 light chain CDR (LCDR).

In some embodiments, the present invention provides the nucleic acid for encoding antibody of the present invention or its segment, include the core The carrier of acid, the host cell comprising the carrier.

In some embodiments, the present invention provides the methods for preparing antibody of the present invention or its segment.

In some embodiments, the present invention provides the immunoconjugates comprising antibody of the present invention, pharmaceutical composition and Combination product.

The present invention also provides ADCC or activation NF-kappaB signal path are mediated in subject using antibody of the present invention Method, and prevention or treating cancer or the method for infection.

The invention further relates to the methods for detecting GITR in the sample.

The present invention is further illustrated in following drawings and specific embodiments.However, these attached drawings and specific implementation Scheme should not be considered limiting the scope of the present invention, and the change that those skilled in the art are readily apparent that is included within the present invention Spirit and appended claims protection scope in.

Detailed description of the invention

Fig. 1 shows Flow Cytometry Assay chimeric antibody and expresses the CHO-S cell strain (CHO-hGITR) of people GITR Binding ability.

Fig. 2 shows Flow Cytometry Assay humanized antibody and expresses the CHO-S cell strain (CHO-hGITR) of people GITR Binding ability.

Fig. 3 shows Flow Cytometry Assay humanized antibody and expresses the CHO-S cell strain (CHO- of machin GITR CynoGITR binding ability).

Fig. 4 shows MOA method measurement humanized antibody activation Hela-GITR-NF-Kappa B luciferace cell strain Ability.

Fig. 5 shows humanized antibody activation cd4 t cell release IL-2 ability detection.

Fig. 6 shows humanized antibody activation cd4 t cell release IFN-γ ability detection.

Fig. 7 shows that MOA method measures humanized antibody ADCC activity.

Fig. 8 shows Composition analyzed in HZ37G5 molecule body.

Fig. 9 shows Composition analyzed in HZ22F4 molecule body.

Figure 10 shows that mouse weight detects.

Detailed description of the invention

I. it defines

Before the present invention is described in detail below, it should be understood that the present invention is not limited to particular methodologies described herein, scheme And reagent, because these can change.It will also be understood that term used herein is not only for describing specific embodiment, and not Intention limits the scope of the invention, and can only be limited by the appended claims.Unless otherwise defined, used herein all Technical and scientific term has the same meaning with the common understanding of those of ordinary skill in fields of the present invention.

In order to explain this specification, will use it is defined below, as long as and it is appropriate, term used in the singular can also To include plural number, and vice versa.The term as used herein is appreciated that merely to describing specific embodiment, and It is not intended to be restrictive.

Term " about " meant when being used in combination with digital numerical value cover with smaller than designation number numerical value 5% lower limit and Digital numerical value in the range of bigger than designation number numerical value 5% upper limit.

As used herein, term "and/or" means two of any one of option or option.

As used herein, term "comprising" or " comprising " mean to include the element, integer or step, but are not excluded for Any other element, integer or step.Herein, when using term "comprising" or " comprising ", unless otherwise specified, otherwise It is also covered by the situation combined by element, integer or the step addressed.For example, when the antibody for referring to some particular sequence of "comprising" When variable region, it is also intended to the antibody variable region covered and be made of the particular sequence.

As used herein, term " GITR " refers to " the TNF related gene and/or polypeptide of glucocorticoid inducible ", at this In field also referred to as TNF receptor superfamily 18 (TNFRSF18), refer to from any vertebrate origin, including mammal is such as Any natural GITR of primate (such as people, machin) and rodent (such as mouse and rat), unless otherwise indicated.The art Language is covered " overall length ", unprocessed GITR and because of the processing in cell caused by any type of GITR.The term also covers The natural generation variant of GITR, such as splice variant or allelic variant.People's GITR amino acid sequence can be found in GenBank registration number Q9Y5U5.The amino acid sequence of one specific people's GITR polypeptide is found in SEQ ID NO:41.One specific machin The amino acid sequence of GITR polypeptide is shown in SEQ ID NO:42.

The term as used herein " anti-GITR antibody ", " anti-GITR ", " GITR antibody " refer to " in conjunction with the antibody of GITR " Such antibody, the antibody can combine (people or machin) GITR albumen or its segment so that described with enough affinities Antibody may be used as the diagnosticum and/or therapeutic agent in targeting (people or machin) GITR.In one embodiment, anti-GITR Antibody is lower than pact of the antibody in conjunction with (people or machin) GITR with the protein bound degree of non-(people or machin) GITR 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80% or about 90% or more, it is such as example logical Cross radioimmunoassay (RIA) or bio-light interferometry or MSD measuring method measurement.

" antibody fragment " refers to the molecule different from complete antibody, it includes a part of complete antibody and combines complete antibody In conjunction with antigen.The example of antibody fragment includes but is not limited to Fv, Fab, Fab ', Fab '-SH, F (ab ') 2；Double antibody；Line Property antibody；Single-chain antibody (such as scFv)；Single domain antibody；Bivalent or bispecific antibody or its segment；Camelidae antibodies； With the bispecific antibody or multi-specificity antibody formed by antibody fragment.

As used herein, term " epitope " refers to the portion in antigen (for example, GITR) with the interaction of antibody molecule specificity Point.

Refer to such antibody with reference antibody " in conjunction with identical or overlapping epitope antibody ", is blocked in competition assay 50% or more the reference antibody and the combination of its antigen, conversely, reference antibody blocks 50% or more in competition assay The antibody and its antigen combination.

Refer to such antibody with the antibody of reference antibody competitive binding its antigen, block 50% in competition assay, 60%, 70%, 80%, 90% or 95% or more the reference antibody and the combination of its antigen.Conversely, reference antibody is competing Strive the combination of the antibody and its antigen of 50%, 60%, 70%, 80%, 90% or 95% or more blocking in measurement.Numerous types Competitive binding assay can be used for determining whether a kind of antibody competes with another kind, these measurements for example: solid phase directly or Radiommunoassay (RIA), the direct or indirect enzyme immunoassay (EIA) of solid phase (EIA), sandwich competition measurement are connect (see, for example, Stahli Deng 1983, Methods in Enzymology 9:242-253).

Inhibit (such as Reverse transcriptase) reference antibody and the antibody of the combination of its antigen to refer to such antibody, inhibits 50%, 60%, 70%, 80%, 90% or 95% or more the reference antibody and the combination of its antigen.Conversely, referring to anti- Body inhibits the combination of 50%, 60%, 70%, 80%, 90% or 95% or more antibody and its antigen.Antibody and its antigen In conjunction with can be measured with affinity (such as equilibrium dissociation constant).The method for measuring affinity is known in the art.

Show that the same or similar binding affinity and/or the antibody of specificity refer to such antibody with reference antibody, It can have the binding affinity of at least 50%, 60%, 70%, 80%, 90% or 95% of reference antibody or more and/or spy It is anisotropic.This can be measured by the method for any measurement binding affinity known in the art and/or specificity.

" complementary determining region " or " CDR region " or " CDR " are that height becomes and is formed in sequence in constant region for immunoglobulin sequence The ring (" hypermutation ring ") determined in structure and/or the region containing antigen contact residue (" antigen contact point ").CDR is mainly responsible for In conjunction with epitope.The CDR of heavy chain and light chain is commonly referred to as CDR1, CDR2 and CDR3, the serial number since the end N-.Position It is referred to as HCDR1, HCDR2 and HCDR3 in the CDR in heavy chain of antibody variable domains, and is located at antibody light chain variable domains Interior CDR is referred to as LCDR1, LCDR2 and LCDR3.In a given light chain variable region or heavy chain variable amino acid sequence In, any or combinations thereof of many well known antibody CDR delegation systems can be used in the precise amino acid sequences boundary of each CDR It determines, the delegation system includes for example: the topological Chothia (Chothia of three-dimensional structure and CDR ring based on antibody Et al. (1989) Nature 342:877-883, Al-Lazikani et al., " Standard conformations for the canonical structures of immunoglobulins”,Journal of Molecular Biology,273, 927-948 (1997)), Kabat (Kabat et al., Sequences of the Proteins of based on antibody sequence changeability Immunological Interest, the 4th edition, U.S.Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), international ImMunoGeneTics database (IMGT) is (on the world wide web (www On imgt.cines.fr/), and based on neighbour's propagation clustering (affinity propagation using a large amount of crystal structures Clustering North CDR definition).In one embodiment, the CDR of antibody of the present invention determines side by AbM rule Boundary, such as shown in table 1.

It is to be noted, however, that the boundary of the CDR of the variable region of the same antibody based on different delegation system acquisitions may Difference.The CDR sequence of the i.e. different undefined same antibody variable regions of delegation system is different.Therefore, it is being related to using When the specific CDR sequence that the present invention defines limits antibody, the range of the antibody also covers such antibody, variable region sequence Column include the specific CDR sequence, but due to applying different schemes (such as different delegation system rule or group Close) and the boundary CDR that causes it to be claimed with specifically the boundary CDR is different defined in the present invention.

Antibody with not homospecificity (that is, for different binding sites of not synantigen) has different CDR.So And although CDR is different between antibody and antibody, the amino acid position of only limited quantity directly participates in CDR Antigen binding.Using Kabat, Chothia, in AbM, Contact and North method at least two, can determine minimum weight Folded region, to provide " the minimum bonding unit " for being used for antigen binding.Minimum bonding unit can be a sub-portion of CDR Point.As those skilled in the art be illustrated, by the structure and protein folding of antibody, can determine CDR sequence rest part Residue.Therefore, present invention also contemplates that the variant of any CDR given in this article.For example, in the variant of a CDR, most brief summary The amino acid residue for closing unit can remain unchanged, and remaining the CDR residue defined according to Kabat or Chothia can be protected Keep amino acid residue substitution.

Term " PD-1 axis binding antagonists " refers to following molecule, inhibit PD-1 axis combination spouse with it is one or more Its combination spouse interaction, so that removal is originated from the T cell dysfunction-one of the signal transduction in PD-1 signaling axis Item is the result is that restore or enhance T cell function (such as being proliferated, cell factor generates, target cell killing).As used in this article, PD-1 axis binding antagonists include PD-1 binding antagonists (such as anti-PD-1 antibody), PD-L1 binding antagonists (such as anti-PD- L1 antibody) and PD-L2 binding antagonists (such as anti-PD-L2 antibody).

Term " PD-1 binding antagonists " refers to following molecule, reduces, and blocks, and inhibits, and eliminates or interference is originated from PD-1 And it is one or more it combination spouse (such as PD-L1, PD-L2) interaction signal transduction.In some embodiments, PD-1 binding antagonists be inhibit PD-1 combine it is one or more it combination spouse molecule.In a particular aspects, PD-1 Binding antagonists inhibit PD-1 combination PD-L1 and/or PD-L2.For example, PD-1 binding antagonists include reducing, block, inhibit, Eliminate or interfere the anti-PD-1 antibody for being originated from the signal transduction that PD-1 and PD-L1 and/or PD-L2 interact, antigen binding Segment, immunoadhesin, fusion protein, oligopeptides and other molecules.In one embodiment, PD-1 binding antagonists reduce by Or the negative costimulatory signal mediated via the cell cortex protein expressed in T lymphocyte (mediates signal to pass via PD-1 Lead) so that dysfunction T cell less dysfunction (such as the effector response of enhancing to antigen recognizing).In In some embodiments, PD-1 binding antagonists are anti-PD-1 antibody.In a specific embodiment, PD-1 binding antagonists It is MDX-1106 disclosed in WO2015/095423 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab) or AMP-224.In one embodiment, anti-PD-1 antibody is " Antibody C " (WO2017/ 133540)。

Term " PD-L1 binding antagonists " refers to following molecule, reduces, and blocks, and inhibits, and eliminates or interference is originated from PD- L1 and it is one or more it combination spouse (such as PD-1, B7-1) interaction signal transduction.In some embodiments, PD-L1 binding antagonists are to inhibit PD-L1 in conjunction with the molecule of its combination spouse.In a particular aspects, PD-L1 combination antagonism Agent inhibits PD-L1 combination PD-1 and/or B7-1.In some embodiments, PD-L1 binding antagonists include reducing, and are blocked, suppression System, eliminate or interference be originated from PD-L1 and it is one or more it combination spouse (such as PD-1, B7-1) interaction signal turn The anti-PD-L1 antibody led, antigen-binding fragment, immunoadhesin, fusion protein, oligopeptides and other molecules.Implement at one In scheme, PD-L1 binding antagonists are reduced by or via negative being total to of mediating of the cell cortex protein expressed in T lymphocyte Stimulus signal (mediates signal transduction via PD-L1), so that dysfunction T cell less dysfunction (such as increase By force to the effector response of antigen recognizing).In some embodiments, PD-L1 binding antagonists are anti-PD-L1 antibody.One A specific aspect, anti-PD-L1 antibody are YW243.55.S70, MDX-1105, MPDL3280A disclosed in WO2015/095423 Or MEDI4736.

Term " PD-L2 binding antagonists " refers to following molecule, reduces, and blocks, and inhibits, and eliminates or interference is originated from PD- L2 and it is one or more it combination spouse (such as PD-1) interaction signal transduction.In some embodiments, PD-L2 Binding antagonists be inhibit PD-L2 combine it is one or more it combination spouse molecule.In a particular aspects, PD-L2 knot It closes antagonist and inhibits PD-L2 combination PD-1.In some embodiments, PD-L2 antagonist includes reducing, and is blocked, and is inhibited, and is eliminated Or interference be originated from PD-L2 and it is one or more it combination spouse (such as PD-1) interact signal transduction anti-PD-L2 Antibody, antigen-binding fragment, immunoadhesin, fusion protein, oligopeptides and other molecules.In one embodiment, PD-L2 Binding antagonists reduce the negative costimulatory signal (warp mediated by or via the cell cortex protein expressed in T lymphocyte Signal transduction is mediated by PD-L2) so that dysfunction T cell less dysfunction (such as enhancing is to antigen recognizing Effector response).In some embodiments, PD-L2 binding antagonists are immunoadhesins.

" cytotoxicity of antibody dependent cellular mediation " or " ADCC ", which refer to, is wherein integrated to certain cytotoxic cell (examples Such as NK cell, neutrophil cell and macrophage) present on S-IgA on Fc receptor (FcR) make this A little cytotoxic effect cells can specifically bind the target cell for carrying antigen, then kill the thin of target cell with cytotoxin Cellular toxicity form.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 γ RIII.The 464th page table 3 of Ravetch and Kinet, Annu.Rev.Immunol.9:457-92 (1991) summarizes FcR expression on hematopoietic cell.For the ADCC activity of purpose of appraisals molecule, external ADCC measuring method, such as U.S. can be carried out Documented by patent No.5,500,362 or 5,821,337 or United States Patent (USP) No.6,737,056 (Presta).It can be used for this The effector cell of class measuring method includes PBMC and NK cell.Or can purpose of appraisals molecule in vivo ADCC activity, Such as in animal model, disclosed in such as Clynes et al., PNAS (USA) 95:652-656 (1998).Implement herein A kind of illustrative measuring method for assessing ADCC activity is provided in example.

Term " cytotoxic agent " finger used for this invention inhibits or prevents cell function and/or cause cell death or broken Bad substance.

" chemotherapeutics " includes useful chemical compound in treating cancer.The example of chemotherapeutics is referring to WO2015/ Those of disclosed in 031667, including but not limited to antitumor agent, including alkylating agent；Antimetabolite；Natural products；Antibiosis Element；Enzyme；Miscellaneous agents；Hormone and antagonist；Antiestrogenic；Antiandrogen；And Nonsteroidal anti-androgens etc..

Term " small-molecule drug " refers to the organic compound that can adjust bioprocess of low molecular weight.

" the functional area Fc " possesses " effector functions " in the area native sequences Fc.Illustrative " effector functions " include C1q is combined；CDC；Fc receptor combines；ADCC；Phagocytosis；Cell surface receptor (such as B-cell receptor；BCR) lower etc..This Class effector functions generally require the area Fc to combine with binding structural domain (such as antibody variable domains), and many measure can be used Method is assessed, such as those of disclosed herein.

For term " area Fc " herein for defining the C-terminal region of heavy chain immunoglobulin, the region includes at least one Partial constant region.The term includes the area native sequences Fc and the area variant Fc.In certain embodiments, the area human IgG heavy chain Fc The carbonyl end of heavy chain is extended to from Cys226 or Pro230.However, the C-terminal lysine (Lys447) in the area Fc may exist or can To be not present.Unless otherwise stated, the number of the area Fc or the amino acid residue in constant region be according to EU numbering system, also by Referred to as EU index, such as in Kabat, Sequences of Proteins of Immunological Interest, 5th Institute in Ed.Public Health Service, National Institutes of Health, Bethesda, MD, 1991 It states.

" antibody of IgG form " refers to IgG form belonging to the heavy chain constant region of antibody.The antibody of all same types Heavy chain constant region is all identical, the heavy chain constant region difference between the antibody of different shaped.For example, the antibody of IgG1 form refers to Its heavy chain constant region Ig structural domain is the Ig structural domain of IgG1.

Term " therapeutic agent " as described herein, which is covered, is preventing or is treating tumour (such as cancer) and infecting (such as chronic sense Dye) in effective any substance, including chemotherapeutics, cytotoxic agent, vaccine, other antibody, anti-infection activity agent, small molecule medicine Object or immunomodulator.

Term " effective quantity " refers to the such amount or dosage of antibody or segment or conjugate or composition of the invention, with After single or multidose application patient, desired effect is generated in needing the patient treated or prevented.Effective quantity can be by making It is readily determined for the attending physician of those skilled in the art by considering following many factors: the object of such as mammal Kind；Its size, age and general health；The disease specific being related to；The degree or seriousness of disease；The response of individual patient； The antibody specific of application；Administration mode；Apply the bioavailability characteristics of preparation；The dosage regimen of selection；With any with treatment The use of method.

The period that " therapeutically effective amount " refers to the dosage with needs and be continuously needed, treatment results needed for effectively realizing Amount.The therapeutically effective amount of antibody or antibody fragment or its conjugate or composition can be according to many factors such as morbid state, a Age, gender and the weight and antibody or antibody moiety of body excite in individual the required ability reacted and are changed.Treatment is effective Amount is also such a amount, and wherein any toxic or illeffects of antibody or antibody fragment or its conjugate or composition is not And treatment beneficial effect.Relative to untreated object, " therapeutically effective amount " preferably inhibits mensurable parameter (such as tumour is raw Long rate, gross tumor volume etc.) at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60% or 70% and still more preferably at least about 80% or 90%.It can be evaluated in the animal model system of effect in indication human tumour Compound inhibits the ability of mensurable parameter (for example, cancer).

The period that " prevention effective dose " refers to the dosage with needs and be continuously needed, prevention result needed for effectively realizing Amount.It is used in object before disease earlier stage or in disease earlier stage generally, due to preventative dosage, therefore prevention has Effect amount will be less than therapeutically effective amount.

Term " host cell ", " host cell line " and " host cell cultures " convertibly using and refer to and wherein draw The cell for entering exogenous nucleic acid, the offspring including this cell.Host cell includes " transformant " and " cell of conversion " comprising The cell of primary transformants and offspring derived therefrom, the number without considering passage.Offspring may be with parent on nucleic acid content Cell is not exactly the same, but may include mutation.Herein include in the cell initially converted screen or select have The Mutant progeny of identical function or biological activity.

" human antibody " refers to that the antibody with such amino acid sequence, the amino acid sequence correspond to the ammonia of following antibody Base acid sequence, the antibody generate or derive from inhuman source by people or people's cell, utilizes human antibody library or other human antibodies Coded sequence.This definition of human antibody clearly excludes the humanized antibody comprising non-human antigen-binding residues.

" humanization " antibody refers to embedding comprising the amino acid residue from inhuman CDR and the amino acid residue from people FR Close antibody.In some embodiments, humanized antibody will include at least one, usually two essentially all of varistructures Domain, wherein all or substantially all CDR (for example, CDR) corresponds to those of non-human antibody, and all or substantially institute Some FR correspond to those of human antibody.Humanized antibody optionally may include at least part of antibody from human antibody Constant region." humanization form " of antibody (such as non-human antibody) refers to the antibody that humanization has been carried out.

Feature is usually that cell grows the life not adjusted in term " cancer " and " carcinous " direction or description mammal Manage illness.

Term " tumour " refers to all neoplastic (neoplastic) cell growths and proliferation, and either pernicious is still benign And (pre-cancerous) and cancerous cells and tissue before all cancers.Term " cancer ", " carcinous ", " cell Proliferation venereal disease Disease ", " proliferative disorders " and " tumour " are not mutually exclusive when mentioning herein.

Term " infectious diseases " refers to the disease that pathogen causes, including such as virus infection, bacterium infection, fungi sense Dye or protozoan such as parasitic infection.

Term " chronic infection " refers to such infection, wherein infectious agent (for example, pathogen such as virus, bacterium, it is primary move Object such as helminth, fungi or the like) in the host of infection induction of immune response, but not yet such as in acute sense It is the same during dye to be removed or eliminated from host.Chronic infection can be duration, latency or slow.Although Acute infection (such as influenza) is usually solved by immune system within a few days or weeks, the level that the infection of duration can be relatively low Periods of months, several years, many decades or all one's life (for example, hepatitis B).In contrast, the infection of latency is characterized in long-term Asymptomatic activity, punctuated by the hyperinfection of a period of time to increase sharply and raised pathogen levels (such as it is simple Bleb).Finally, slowly infect be characterized in disease symptoms gradually and increase continuously, such as long-term incubation period is then facing Bed symptom is that extended and progress clinical process starts after occurring.

" immunoconjugates " are conjugated with one or more of the other substance (including but not limited to cytotoxic agent or label) Antibody.

Term as used herein " label ", which refers to, is directly or indirectly conjugated or is fused to reagent (such as polynucleotides spy Needle or antibody) and the detection of the reagent that promotes it to be conjugated or merge compound or composition.Label can be in itself can (for example, labelled with radioisotope or the fluorescent marker) of detection or detectable bottom can be catalyzed in the case where enzymatic labelling The chemical modification of compounds or composition.Term is intended to cover by the way that detectable substance to be coupled to (that is, physical connection) to spy Needle or antibody come direct label probe or antibody and by reacting with another reagent directly marked come indirect labelling probe Or antibody.The example of indirect labelling is included the detection of the Primary antibodies carried out using the secondary antibody of fluorescent marker and had biological The end mark of the DNA probe of element, makes it possible to the streptavidin of fluorescent marker and detects.

" individual " or " subject " includes mammal.Mammal includes but is not limited to, domestic animal (for example, ox, Sheep, cat, dog and horse), primate (for example, people and non-human primate such as monkey), rabbit and rodent (for example, Mouse and rat).In some embodiments, individual or subject are people.

" separation " antibody is such antibody, is separated with the component of its natural surroundings.In some embodiments In, by antibody purification to more than 95% or 99% purity, such as example, by electrophoresis (for example, SDS-PAGE, isoelectric focusing (IEF), Capillary Electrophoresis) or chromatography (for example, ion exchange or reversed-phase HPLC) determination.For the method for assessing antibody purity Summary, see, e.g., Flatman etc., J.Chromatogr.B848:79-87 (2007).

" the anti-GITR antibody of coding of separation or the nucleic acid of its segment " refers to one or more nucleic acid molecules, encoding antibody Heavy chain or light chain (or its segment), including such nucleic acid molecules in single carrier or separated carrier, and are present in Such nucleic acid molecules at one or more positions in host cell.

The following calculating for carrying out sequence identity between sequence.

For the homogeneity percentage for determining two amino acid sequences or two nucleic acid sequences, the sequence is compared for best It compares compared with purpose (for example, can be for optimal comparison and in one or both of the first and second amino acid sequences or nucleic acid sequence Middle introducing vacancy can abandon nonhomologous sequence to be omparison purpose).In a preferred embodiment, to be omparison purpose, The length of the reference sequences compared is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more Preferably at least 70%, 80%, 90%, 100% reference sequences length.Then relatively in orresponding amino acid position or nucleotide Amino acid residue or nucleotide at position.When the position in First ray is by the identical amino of corresponding position in the second sequence When sour residue or nucleotide occupy, then the molecule is identical at this position.

It can use mathematical algorithm and realize that the sequence between two sequences compares calculating with homogeneity percentage.It is excellent at one It selects in embodiment, uses the Needlema and Wunsch ((1970) in the GAP program for being integrated to GCG software package J.Mol.Biol.48:444-453) algorithm (can get in http://www.gcg.com), using 62 matrix of Blossum or PAM250 matrix and gap weight 16,14,12,10,8,6 or 4 and Length Weight 1,2,3,4,5 or 6, determine two amino acid sequences Homogeneity percentage between column.In still another preferred embodiment, using the GAP program (In in GCG software package Http:// www.gcg.com can get), use NWSgapdna.CMP matrix and gap weight 40,50,60,70 or 80 and length Weight 1,2,3,4,5 or 6 is spent, determines the homogeneity percentage between two nucleotide sequences.Particularly preferred parameter sets (and The parameter sets that otherwise should be used unless otherwise stated) it is empty using gap penalty 12, gap extension penalties 4 and frameshit 62 rating matrix of Blossum of position point penalty 5.

PAM120 weighted residual table, GAP LENGTH PENALTY 12, gap penalty 4 can also be used), using having been incorporated into E.Meyers the and W.Miller algorithm of ALIGN program (2.0 editions), ((1989) CABIOS, 4:11-17) determine two amino acid Homogeneity percentage between sequence or nucleotide sequence.

Additionally or alternately, nucleic acid sequence as described herein and protein sequence can be further used as " inquiry Sequence " is to execute retrieval for public database, for example to identify other family member's sequences or correlated series.

Term " pharmaceutic adjuvant " refers to the diluent applied together with active material, adjuvant, and (such as Freund's adjuvant is (completely and not Completely)), excipient, carrier or stabilizer etc..

Term " pharmaceutical composition " refers to such composition, living with the biology for the active constituent for allowing to be included in Property effective form exist, and do not include there is the other of unacceptable toxicity to the subject of applying said compositions Ingredient.

Term " combination product " refers to a kind of fixed Combination of dosage unit form or non-fixed combinations or applies for combining Partial kit, two of them or more therapeutic agent can be separated independently in the same time or in the time interval Application especially allows combined partner to show cooperation, for example, when synergistic effect in these time intervals.Term " fixed Combination " Refer to antibody and combined partner of the present invention (such as other therapeutic agents, such as anti-PD1 antibody, anti-PDL1 antibody or anti-PDL2 antibody) It is administered simultaneously in the form of single entities or dosage in patient.Term " non-fixed combinations " means antibody and combined partner of the present invention (such as other therapeutic agents, such as anti-PD1 antibody, anti-PDL1 antibody or anti-PDL2 antibody) as separated entity simultaneously, parallel Or it is successively applied to patient, without specific time restriction, wherein such application provides two kinds of compounds of patient's body Treat effective level.The latter is also applied for cocktail therapy, such as applies three or more therapeutic agents.It is preferred real at one It applies in scheme, pharmaceutical composition is non-fixed combinations.

Term " combination treatment ", which refers to, applies two or more therapeutic agents to treat cancer or sense as described in the present disclosure Dye.This application includes that these therapeutic agents are co-administered in a substantially simultaneous manner, such as with the activity with fixed proportion The single capsule of ingredient.Alternatively, this application includes for each active constituent a variety of or in separated container (such as piece Agent, capsule, powder and liquid) in co-administration.Powder and/or liquid can reconstruct or be diluted to before administration required agent Amount.In addition, this application further includes using each type in a sequential manner with the roughly the same time or in different times Therapeutic agent.In any case, therapeutic scheme will provide pharmaceutical composition and treat having in illness or symptom as described herein Benefit effect.

When for this paper, " treatment " refer to slow down, interrupt, block, alleviate, stop, reducing or reverse already present symptom, The progress or seriousness of illness, the patient's condition or disease.

When for this paper, " prevention " includes the generation or development of the symptom to disease or illness or specified disease or illness Inhibit.In some embodiments, the subject with cancer family's medical history is the candidate of prevention scheme.In general, in cancer Background in, term " prevention " refer to cancer symptom or symptom occur before, especially in the subject with risk of cancer Medicament administration before middle generation.

Term " anti-infection activity agent " is included in specificity under application concentration and dosing interval and suppresses or eliminates microorganism life It grows but to the nonlethal any molecule of host, the microorganism such as virus, bacterium, fungi or protozoan, such as helminth. When for this paper, term anti-infection activity agent includes antibiotic, antibacterial agent, antivirotic, antifungal agent and antiprotozoal. In In a specific aspect, anti-infection activity agent is nontoxic to host in application concentration and dosing interval.

Antibacterial anti-infection activity agent or antibacterial agent can widely be classified as the (that is, directly killing) or antibacterial of sterilization (that is, preventing division).The anti-infection activity agent of antibacterial can further be resorted to as narrow spectrum antibacterial agent (that is, only influencing group bacterium Hypotype, for example, Gram-negative etc.) or broad spectrum antimicrobial agent (that is, influencing broad variety).

Term " antivirotic " includes any substance for suppressing or eliminating viral growth, causing a disease and/or surviving.

Term " antifungal agent " includes any substance for suppressing or eliminating fungi growth, causing a disease and/or surviving.

Term " antiprotozoal " includes suppressing or eliminating protozoan organisms (such as helminth) growth, morbidity And/or any substance of survival.

Term " carrier " is the nucleic acid molecules for referring to be proliferated another coupled nucleic acid as used herein. The term includes the carrier as self-replicating nucleic acid structure and is integrated in the genome for having been incorporated into its host cell Carrier.Some carriers can instruct the expression that the nucleic acid being connected can be operated with it.Such carrier is referred to herein as " table Up to carrier ".

" subject/Patient Sample A " refers to the set of the cell or fluid that obtain from patient or subject.Tissue or cell sample The source of product can be solid tissue, as from fresh, freezing and/or preservation organ or tissue's sample or biopsy samples Or puncture sample；Blood or any blood constitutent；Body fluid, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites) or Gap liquid；Gestation or the cell of development any time from subject.Tissue sample be possibly comprised in nature it is natural not with Organize the compound that mixes, such as preservative, anti-coagulants, buffer, fixative, nutrients, antibiotic, etc..Tumor sample Example herein include but is not limited to tumor biopsy, fine needle aspirate, bronchial perfusate, liquor pleurae (hydrothorax), sputum, Urine, specimens from pri, the tumour cell in circulation, serum, blood plasma, the plasma proteins in circulation, ascites, derived from tumour or The primary cell culture or cell line of tumor-like properties are shown, and the tumor sample saved, such as formalin are fixed , the tumor sample of paraffin embedding or the tumor sample of freezing.

II. antibody

In some embodiments, anti-GITR antibody of the invention or its segment are with high-affinity combination GITR (such as people GITR or machin GITR), for example, with following equilibrium dissociation constant (K_D) in conjunction with GITR, the K_DIt is excellent less than about 10nM Selection of land, less than or equal to about 7nM, more preferably less or equal to about 6nM, more preferably less or equal to about 5nM, 4.9nM, 4.8nM, 4.7nM, 4.6nM, 4.5nM, 4nM or 3nM, most preferably, the K_DLess than or equal to about 2.5nM, 2.4nM,2.3nM,2.2nM,2.1nM.In some embodiments, anti-GITR antibody of the invention is with 1nM-6nM, preferably The K of 1.5nM-5nM, 1.7nM-5nM, 1.8nM-5nM, 1.9nM-5nM_DIn conjunction with GITR.In some embodiments, GITR is people GITR.In some embodiments, antibody binding affinity is using (such as the affine survey of Fortebio of bio-light interferometry Amount) measurement.

In some embodiments, antibody of the invention or its segment combine the cell of expression GITR, for example, to be less than or Equal to the EC50 of about 2nM, 1.9nM, 1.5nM, 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM.Some In embodiment, the combination is measured with flow cytometry (such as FACS).In some embodiments, the cell of GITR is expressed For the Chinese hamster ovary celI (such as CHO-S cell) for expressing GITR.In some embodiments, GITR is people GITR or machin GITR.

In some embodiments, antibody of the invention or its segment in conjunction with the GITR molecule with cell surface and swash The NF-kappaB signal path in the downstream GITR living.In some embodiments, antibody of the invention or its segment activation capability are excellent In known anti-GITR antibody, such as patent application US20130183321A1 (GITR, INC. (Cambridge, MA, US)) The GITR antibody of report.In one embodiment, it is known that anti-GITR antibody molecule light chain and sequence of heavy chain exist It is respectively SEQ ID NO:44 and SEQ ID NO:54 in US20130183321A1.In some embodiments, of the invention GITR antibody molecule compares control antibodies molecule, significantly can effectively activate NF kappa B signal access.In some embodiment party In case, the activation is detected by luciferase.

In some embodiments, antibody of the present invention or its segment raising effector T cell function, such as activation effect T are thin Born of the same parents.In some embodiments, antibody of the invention or its segment are capable of the proliferation of enhancement effect T cell.In some embodiment party In case, antibody of the invention or its segment improve interferon (such as IFN-γ) secretion/expression.In some embodiments, originally The antibody of invention or its segment improve interleukins (such as IL-2) secretion/expression.In some embodiments, T cell is Cd4 t cell.

In some embodiments, GITR antibody of the invention or its segment can activate regulatory T-cell (such as Treg are thin Born of the same parents) in GITR signal path, and then by mediate ADCC effect eliminate cell.Effector T cell itself is on the one hand released as a result, Inhibition signal, enhance the activity of cd4 t cell and/or cd8 t cell, eliminate regulatory T-cell pairing effect T cell on the other hand Inhibiting effect, thus farthest activation effect T cell, enhancing is directed to the immune response of tumour cell.Some In embodiment, the ADCC activity of antibody is detected by detection NF-AT signal activation.In some embodiments, the NF- The activation of AT signal is embodied by the expression of fluorescent reporter gene.In some embodiments, GITR antibody molecule phase of the invention Than known anti-GITR antibody molecule, NF-AT signal path significantly can be effectively activated.In some embodiments, of the invention GITR antibody molecule compare known anti-GITR antibody molecule, significantly can effectively mediate ADCC effect.In some embodiment party In case, GITR antibody molecule of the invention compares known anti-GITR antibody molecule, significantly can effectively suppress or eliminate and adjust T Cell.In some embodiments, effector T cell of the invention is cd4 t cell.In some embodiments, it is known that it is anti- GITR antibody molecule is reported in such as patent application US20130183321A1 (GITR, INC. (Cambridge, MA, US)) Anti- GITR antibody.In one embodiment, it is known that anti-GITR antibody molecule light chain and sequence of heavy chain exist It is respectively SEQ ID NO:44 and SEQ ID NO:54 in US20130183321A1.

In some embodiments, antibody of the invention or its segment (optionally with therapeutic modality and/or other treatments Agent, such as the combination of PD-1 axis binding antagonists) it can prevent or treat tumour.In some embodiments, antibody of the invention Or its segment can be used in inhibiting or reducing tumour growth (such as reducing gross tumor volume).In some embodiments, of the invention Antibody or its segment can also be used to maintain tumor patient weight.In some embodiments, antibody of the invention or its piece Section can inhibit or reduce tumour growth (example with therapeutic modality and/or other therapeutic agents, such as the combination of PD1- axis binding antagonists Such as reduce gross tumor volume).In some embodiments, antibody of the invention or its segment can be with therapeutic modalities and/or other Therapeutic agent, such as the combination of PD1- axis binding antagonists maintain tumor patient weight.In some embodiments, antibody of the invention Or its segment (optionally being combined with therapeutic modality and/or other therapeutic agents, such as PD-1 axis binding antagonists) be able to suppress or Reduce tumour growth (such as reducing gross tumor volume), while not influencing tumor patient weight.Preferably, tumour is swollen for gastrointestinal tract Tumor.Preferably, tumour is cancer, such as gastrointestinal cancer, such as gastric cancer, the carcinoma of the rectum, colon cancer, colorectal cancer etc..One In a little embodiments, antibody of the invention or its segment (optionally combining with PD-1 axis binding antagonists) can prevent or treat Infection, such as chronic infection, including bacterium infection, fungal infection, virus infection, protozoal infections etc..

In some embodiments, anti-GITR antibody of the invention or its antigen-binding fragment include heavy chain variable region (VH), wherein the VH includes

(i) three complementarity determining regions (CDR) contained in the VH of any antibody listed by table B, or

(ii) sequence relative to (i), altogether comprising at least one and no more than 5,4,3,2 or 1 on three CDR regions The sequence of a amino acid change (preferred amino acid displacement, preferably conservative substitution).

In some embodiments, anti-GITR antibody of the invention or its antigen-binding fragment include light chain variable region (VL), wherein the VL includes:

(i) three complementarity determining regions (CDR) contained in the VL of any antibody listed by table B；Or

(ii) sequence relative to (i), altogether comprising at least one and no more than 5,4,3,2 or 1 on three CDR regions The sequence of a amino acid change (preferred amino acid displacement, preferably conservative substitution).

In some embodiments, anti-GITR antibody of the invention or its antigen-binding fragment include heavy chain variable region VH and Light chain variable region VL, wherein

(a) VH includes

(i) three complementarity determining regions (CDR) contained in the VH of any antibody listed by table B, or

(ii) sequence relative to (i), altogether comprising at least one and no more than 5,4,3,2 or 1 on three CDR regions The sequence of a amino acid change (preferred amino acid displacement, preferably conservative substitution)；And/or

(b) VL includes:

(i) three complementarity determining regions (CDR) contained in the VL of any antibody listed by table B；Or

(ii) sequence relative to (i), altogether comprising at least one and no more than 5,4,3,2 or 1 on three CDR regions The sequence of a amino acid change (preferred amino acid displacement, preferably conservative substitution).

In preferred embodiments, VH includes to be selected from amino acid sequence shown in SEQ ID NO:17,18,19 or 20, Or it is made of the amino acid sequence.

In preferred embodiments, VL includes to be selected from amino acid sequence shown in SEQ ID NO:21,22,23 or 24, Or it is made of the amino acid sequence.

In preferred embodiments, the anti-GITR antibody of the present invention or its antigen-binding fragment include

(i) 3 complementary determining region HCDR of the heavy chain variable region as shown in SEQ ID NO:17 or 19, and such as SEQ ID 3 complementary determining region LCDR of light chain variable region shown in NO:21 or 23, or

(ii) 3 complementary determining region HCDR of the heavy chain variable region as shown in SEQ ID NO:18 or 20, and such as SEQ 3 complementary determining region LCDR of light chain variable region shown in ID NO:22 or 24.

In some embodiments, anti-GITR antibody of the invention or its antigen-binding fragment include heavy chain variable region (VH) And/or light chain variable region (VL), wherein

(i) VH includes complementarity determining region (CDR) HCDR1, HCDR2 and HCDR3, and wherein HCDR1 includes to be selected from SEQ The amino acid sequence of ID NO:1,2,3 or 4, or be made of the amino acid sequence or HCDR1 includes and selected from SEQ ID Compared to having one, two or three to change, (preferred amino acid displacement, preferably guards and sets the amino acid sequence of NO:1,2,3 or 4 Change) amino acid sequence；HCDR2 includes the amino acid sequence selected from SEQ ID NO:5 or 6, or by the amino acid sequence group At or HCDR2 include with selected from SEQ ID NO:5 or 6 amino acid sequence compare have one, two or three change The amino acid sequence of (preferred amino acid displacement, preferably conservative substitution)；HCDR3 includes the amino acid selected from SEQ ID NO:7 or 8 Sequence or by the amino acid sequence form or HCDR3 include with selected from SEQ ID NO:7 or 8 amino acid sequence compared with Change the amino acid sequence of (preferred amino acid displacement, preferably conservative substitution) with one, two or three；

And/or

(ii) wherein the VL includes complementarity determining region (CDR) LCDR1, LCDR2 and LCDR3, and wherein LCDR1 includes choosing It is formed from the amino acid sequence of SEQ ID NO:9 or 10 or by the amino acid sequence or LCDR1 includes and is selected from SEQ ID The amino acid sequence of NO:9 or 10 is compared, and there is one, two or three to change (preferred amino acid displacement, preferably conservative substitution) Amino acid sequence；LCDR2 includes to be selected from the amino acid sequence of SEQ ID NO:11,12,13 or 14 or by the amino acid sequence Column composition or LCDR2 include to have one, two compared with the amino acid sequence selected from SEQ ID NO:11,12,13 or 14 Or the amino acid sequence of three changes (preferred amino acid displacement, preferably conservative substitution)；LCDR3 includes to be selected from SEQ ID NO:15 16 amino acid sequence or be made of the amino acid sequence or LCDR3 includes and selected from SEQ ID NO:15 or 16 Amino acid sequence compares the amino acid sequence that there is one, two or three to change (preferred amino acid displacement, preferably conservative substitution) Column.

In preferred embodiments, the present invention provides anti-GITR antibody or its antigen-binding fragment, and it includes heavy chains can Become area (VH) and light chain variable region (VL), wherein

(a) VH includes

(i) combination of HCDR1, HCDR2 and HCDR3 shown in Table A；Or

(ii) variant of the HCDR combination of (i), the variant include at least one on three CDR regions and do not surpass altogether Cross 5,4,3,2 or 1 amino acid changes (preferred amino acid displacement, preferably conservative substitution)；

And/or

(ii) VL includes

(i) combination of LCDR1, LCDR2 and LCDR3 shown in Table A；Or

(ii) variant of the LCDR combination of (i), the variant include at least one on three CDR regions and do not surpass altogether Cross 5,4,3,2 or 1 amino acid changes (preferred amino acid displacement, preferably conservative substitution).

In preferred embodiments, the present invention provides anti-GITR antibody or its antigen-binding fragment, and it includes heavy chains can Become area (VH) and light chain variable region (VL), wherein the VH comprising complementarity determining region (CDR) HCDR1, HCDR2 and HCDR3 simultaneously And the VL includes (CDR) LCDR1, LCDR2 and LCDR3, wherein the antibody or its antigen-binding fragment are included Shown in the combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 such as following table (Table A):

Table A: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 in antibody of the present invention or its antigen-binding fragment Example combinations

In some embodiments, anti-GITR antibody of the invention or its antigen-binding fragment include heavy chain variable region VH And/or light chain variable region VL, wherein

(a) heavy chain variable region VH

(i) comprising with selected from SEQ ID NO:17,18,19 or 20 amino acid sequence have at least 90%, 91%, 92%, it the amino acid sequence of 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or is made from it；Or

(ii) comprising being selected from the amino acid sequence of SEQ ID NO:17,18,19 or 20 or being made from it；Or

(iii) comprising having one or more (excellent compared with the amino acid sequence selected from SEQ ID NO:17,18,19 or 20 Choosing be no more than 10, more preferably no more than 5,4,3,2,1) amino acid change (preferred amino acid displacement, more preferable amino acid Conservative substitution) amino acid sequence, it is preferable that the amino acid change does not occur in CDR region；

And/or

(b) light chain variable region VL

(i) comprising with selected from SEQ ID NO:21,22,23 or 24 amino acid sequence have at least 90%, 91%, 92%, it the amino acid sequence of 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or is made from it；

(ii) comprising being selected from the amino acid sequence of SEQ ID NO:21,22,23 or 24 or being made from it；Or

(iii) comprising having one or more (excellent compared with the amino acid sequence selected from SEQ ID NO:21,22,23 or 24 Choosing be no more than 10, more preferably no more than 5,4,3,2,1) amino acid change (preferred amino acid displacement, more preferable amino acid Conservative substitution) amino acid sequence, it is preferable that the amino acid change does not occur in CDR region.

In preferred embodiments, the present invention provides anti-GITR antibody or its antigen-binding fragment, and it includes heavy chains can Become area (VH) and light chain variable region (VL), wherein heavy chain variable region VH that the antibody or its antigen-binding fragment are included and gently Shown in the combination of chain variable region VL such as following table (table B):

Table B: the exemplary group of heavy chain variable region VH and light chain variable region VL in antibody of the present invention or its antigen-binding fragment It closes

In some embodiments, anti-GITR antibody of the invention or its antigen-binding fragment include heavy chain and/or light chain, Wherein

(a) heavy chain

(i) comprising with selected from SEQ ID NO:25,26,27 or 28 amino acid sequence have at least 85%, 90%, 91%, it the amino acid sequence of 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or is made from it；

(ii) comprising being selected from the amino acid sequence of SEQ ID NO:25,26,27 or 28 or being made from it；Or

(iii) comprising having one or more (excellent compared with the amino acid sequence selected from SEQ ID NO:25,26,27 or 28 Choosing be no more than 20 or 10, more preferably no more than 5,4,3,2,1) amino acid change (preferred amino acid displacement, more preferably Conservative aminoacid substitutions) amino acid sequence, it is preferable that the amino acid change does not occur in the CDR region of heavy chain, more preferably Ground, the amino acid change do not occur in heavy chain variable region；

And/or

(b) light chain

(i) comprising with selected from SEQ ID NO:29,30,31 or 32 amino acid sequence have at least 85%, 90%, 91%, it the amino acid sequence of 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or is made from it；

(ii) comprising being selected from the amino acid sequence of SEQ ID NO:29,30,31 or 32 or being made from it；Or

(iii) comprising having one or more (excellent compared with the amino acid sequence selected from SEQ ID NO:29,30,31 or 32 Choosing be no more than 20 or 10, more preferably no more than 5,4,3,2,1) amino acid change (preferred amino acid displacement, more preferably Conservative aminoacid substitutions) amino acid sequence, it is preferable that the amino acid change does not occur in the CDR region of light chain, more preferably Ground, the amino acid change do not occur in light chain variable region.

In preferred embodiments, the present invention provides anti-GITR antibody or its antigen-binding fragment, it includes heavy chain and Light chain, wherein shown in the combination of heavy chain and light chain that the antibody or its antigen-binding fragment are included such as following table (table C):

Table C: the example combinations of heavy chain and light chain in antibody of the present invention or its antigen-binding fragment

In some embodiments, the anti-GITR antibody of the present invention or the heavy chain and/or light chain of its segment also include signal peptide Sequence, such as METDTLLLWVLLLWVPGSTG (SEQ ID NO:43).

In one embodiment of the invention, amino acid change as described herein include amino acid displacement, insertion or Missing.Preferably, amino acid change as described herein is amino acid replacement, preferably conservative substitution.

In preferred embodiments, the region outside CDR occurs for amino acid change of the present invention (such as in FR In).It is highly preferred that the region outside heavy chain variable region and/or outside light chain variable region occurs for amino acid change of the present invention.

In some embodiments, it is replaced into preservative replacement.Conservative substitution refers to an amino acid through in the same category Another amino acid replacement, such as an acidic amino acid replaces through another acidic amino acid, and a basic amino acid is through another Basic amino acid substitution or a neutral amino acid are replaced through another neutral amino acid.Illustrative displacement is as shown in following table D:

Table D

In certain embodiments, the CDR region in antibody occurs for displacement.In general, the variant obtained is relative to parental antibody (for example, increased affinity) has modification (for example, improvement) and/or will have parental antibody in terms of certain biological characteristics The certain biological characteristics substantially retained.Exemplary permutation variant is affinity maturation antibody.

In certain embodiments, antibody presented herein is changed to increase or decrease antibody through glycosylated journey Degree.Addition or missing to the glycosylation site of antibody can generate or remove one or more sugar by changing amino acid sequence Base site and conveniently realize.When antibody includes the area Fc, thus it is possible to vary be attached to its carbohydrate.In some applications, it removes It goes the modification of undesired glycosylation site can be useful, such as removes fucose module to improve antibody dependent cellular Property cytotoxicity (ADCC) function (referring to Shield etc. (2002) JBC277:26733).In other applications, half can be carried out Lactose glycosidation is modified to modify complement-dependent cytotoxicity (CDC).

In certain embodiments, can herein provided antibody the area Fc in introducing one or more amino acid repair Decorations, generate Fc region variants with this, to enhance the validity of such as antibodies for treating cancer or cell proliferation disorders.Fc region variants May include comprising amino acid modification (such as displacement) at one or more amino acid positions people Fc region sequence (such as human IgG l, The area IgG2, IgG3 or IgG4Fc).About the exemplified in U.S. patents number 7 of Fc variant, 332,581, U.S. Patent number 6,737, 056, U.S. Patent number 6,737,056；WO 2004/056312 and Shields et al., J.Biol.Chem.9 (2): 6591- 6604 (2001), U.S. Patent number 6,194,551, WO 99/51642 and Idusogie et al. J.Immunol.164:4178- 4184 (2000), U.S. Patent number 7,371,826, Duncan&Winter, Nature322:738-40 (1988)；United States Patent (USP) Numbers 5,648,260；U.S. Patent number 5,624,821；With WO 94/29351.

In some embodiments it may be necessary to the antibody engineered through cysteine is generated, such as " thio MAb ", Wherein one or more residues of antibody are replaced through cysteine residues.Can be as, such as U.S. Patent number 7, described in 521,541 Ground generates cysteine engineered antibody.

In certain embodiments, antibody presented herein can be further modified to containing as is generally known in the art and Other non-protein portions obtained easily.It is suitble to the part of antibody derivatization to include, but are not limited to water-soluble polymer. The non-limiting example of water-soluble polymer includes, but are not limited to polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxylic Methylcellulose, glucan, polyvinyl alcohol, polyvinylpyrrolidone, poly- 1,3- dioxane, tri- alkane of poly- 1,3,6-, ethylene/Malaysia Acid anhydride copolymer, polyaminoacid (homopolymer or random copolymer) and glucan or poly- (n- vinyl pyrrolidone) poly- second two Alcohol, propropylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymer, oxyethylated polyols (such as glycerol), polyethylene Alcohol and its mixture.

In some embodiments, anti-GITR antibody of the invention or its antigen-binding fragment have following one or more Characteristic:

(i) display is with any antibody listed by table 3 to the same or similar binding affinity of GITR and/or specificity；

(ii) inhibit the combination of any antibody and GITR listed by (for example, Reverse transcriptase) table 3；

(iii) with table 3 shown in identical or overlapping epitope in conjunction with any antibody；

(iv) with table 3 shown in GITR in conjunction with any antibody competition；

(v) one or more biological characteristics with any antibody molecule listed by table 3.

In some embodiments, anti-GITR antibody of the invention be the antibody of IgG1 form or the antibody of IgG2 form or The antibody of IgG4 form.

In some embodiments, anti-GITR antibody is monoclonal antibody.

In some embodiments, anti-GITR antibody is humanization.Distinct methods for making antibody humanization are skills Known to art personnel, as summarized by Almagro&Fransson, content is completely incorporated herein (Almagro JC by mentioning stating With Fransson J (2008) Frontiers inBioscience13:1619-1633).

In some embodiments, anti-GITR antibody is human antibody.Various technologies as known in the art can be used to make Standby human antibody.Human antibody general description is in van Dijk and van de Winkel, Curr.Opin.Pharmacol 5:368-74 (2001) and Lonberg, Curr.Opin.Immunol 20:450-459 (2008).

In some embodiments, anti-GITR antibody is chimeric antibody.

In some embodiments, the Frame sequence of at least part of anti-GITR antibody is human consensus framework sequence.One In a embodiment, anti-GITR antibody of the invention also covers its antibody fragment, is preferably chosen from antibody fragment below: Fab, Fab ', Fab '-SH, Fv, single-chain antibody (such as scFv) or (Fab ')₂, single domain antibody, double antibody (dAb) or linear anti- Body.

In certain embodiments, anti-GITR antibody molecule is in bispecific or multi-specificity antibody molecular forms.In In one embodiment, bi-specific antibody molecule has the first binding specificity for GITR and is directed to PD-1 or PD-L1 Or the second binding specificity of PD-L2.In one embodiment, bi-specific antibody molecule is in conjunction with GITR and PD-1.In In another embodiment, bi-specific antibody molecule is in conjunction with GITR and PD-L1.In yet another embodiment, double special Property antibody molecule is in conjunction with GITR and PD-L2.It is special that multi-specificity antibody molecule can have any combination for previous molecular Anisotropic combination.Multi-specificity antibody molecule for example can be three-specific antibody molecule, and it includes the first knots for being directed to GITR Close second and third binding specificity of specificity and the molecule for one or more of: PD-1, PD-L1 or PD-L2.

II. nucleic acid of the invention and the host cell comprising it

On the one hand, the present invention provides the coding anti-GITR antibody of any of the above or the nucleic acid of its segment.Implement at one In scheme, the carrier comprising the nucleic acid is provided.In one embodiment, carrier is expression vector.In an embodiment In, the host cell comprising the nucleic acid or the carrier is provided.In one embodiment, host cell is eukaryon.In In another embodiment, host cell is selected from yeast cells, mammalian cell (such as Chinese hamster ovary celI or 293 cells) or suitable It is used to prepare antibody or other cells of its antigen-binding fragment.In another embodiment, host cell is protokaryon.

On the one hand, the present invention provides the coding anti-GITR antibody of any of the above or the nucleic acid of its segment.The nucleic acid can With the nucleic acid of the amino acid sequence of the light chain variable region comprising encoding antibody and/or heavy chain variable region, or include encoding antibody The nucleic acid of the amino acid sequence of light chain and/or heavy chain.The nucleic acid sequence of illustrative encoding antibody heavy variable region includes and choosing Have at least 80% from the nucleic acid sequences of SEQ ID NO:33,34,35 or 36,85%, 90%, 91%, 92%, 93%, 94%, 95%, the nucleic acid sequence of 96%, 97%, 98% or 99% identity, or comprising selected from SEQ ID NO:33,34,35 or 36 nucleic acid sequence.The nucleic acid sequence of illustrative encoding antibody light variable region includes and is selected from SEQ ID NO:37,38,39 Or 40 nucleic acid sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% Or 99% identity nucleic acid sequence, or include be selected from SEQ ID NO:37,38,39 or 40 nucleic acid sequence.

In one embodiment, one or more carriers comprising the nucleic acid are provided.In one embodiment, it carries Body is expression vector, such as carrier for expression of eukaryon.Carrier includes but is not limited to virus, plasmid, clay, λ bacteriophage or yeast people Work chromosome (YAC).In one embodiment, carrier is pTT5 carrier.

In one embodiment, the host cell comprising the carrier is provided.For cloning or expressing encoding antibody The appropriate host cell of carrier includes protokaryon or eukaryotic described herein.For example, antibody can generate in bacterium, especially work as When not needing glycosylation and Fc effector function.For the expression of antibody fragment and polypeptide in bacterium, see, for example, the U.S. is special Benefit number 5,648,237,5,789,199 and 5,840,523, sees also Charlton, Methods in Molecular Biology, Volume 248 (B.K.C.Lo, editor, Humana Press, Totowa, NJ, 2003), describes antibody fragment and exists by the 245-254 pages Expression in Escherichia coli).After expression, antibody can be separated from the bacterial cell paste in solvable fraction, and can be with It is further purified.

In one embodiment, host cell is eukaryon.In another embodiment, host cell is selected from yeast Cell, mammalian cell or other cells suitable for preparing antibody or its antigen-binding fragment.For example, eukaryotic microorganisms are all If filamentous fungi or yeast are the suitable clones or expressive host of the carrier about encoding antibody.For example, glycosylation approach is The fungi and yeasts strain for carrying out " humanization " causes to generate the antibody with partially or completely people's glycosylation pattern.Referring to Gerngross, Nat.Biotech.22:1409-1414 (2004) and Li et al., Nat.Biotech.24:210-215 (2006). Host cell suitable for expressing glycosylated antibodies is also derived from multicellular organisms (invertebrate and vertebrate).It can also be with Vertebrate cells are used as host.It is, for example, possible to use be modified to be suitable for the mammal cell line of suspension growth.Have It is the monkey kidney CV1 system (COS-7) converted with SV40 with other examples of mammalian host cell line；Human embryo kidney (HEK) system (293HEK Or 293F or 293 cells, such as described in such as Graham, J.Gen Virol.36:59 (1977)) etc..It is other useful Mammalian host cell line include Chinese hamster ovary (CHO) cell, including DHFR-CHO cell (Urlaub etc., Proc.Natl.Acad.Sci.USA 77:216 (1980), CHO-S cell etc.；And myeloma cell line such as Y0, NS0 and Sp2/0.Summary about the suitable certain mammalian host cell lines for generating antibody is shown in such as Yazaki and Wu, Methods In Molecular Biology, roll up 248 (B.K.C.Lo, ed., Humana Press, Totowa, NJ), the 255-268 pages (2003)。

In one embodiment, present invention offer prepares anti-GITR antibody or its segment (preferred antigen-binding fragment) Method, wherein the method includes in the core for being suitable for expressing encoding said antibody or its segment (preferred antigen-binding fragment) The host cell is cultivated under conditions of acid, and is optionally separated the antibody or its segment (preferably antigen binding fragment Section).In some embodiment, the method also includes (preferably anti-from the anti-GITR antibody of host cell recycling or its segment Former binding fragment).

In one embodiment, the method for preparing anti-GITR antibody is provided, wherein the method includes being suitble to resist Under conditions of body surface reaches, culture comprising encoding said antibody nucleic acid host cell, supply as mentioned above, and optionally from The host cell (or host cell culture medium) recycles the antibody.Anti- GITR antibody is generated in order to recombinate, separation coding is anti- The nucleic acid of body (such as antibody as described above), and one or more carriers are inserted into, it is used in host cell into one Step clone and/or expression.The routine protocols separation easy to use of such nucleic acid and sequencing (such as by using can be with encoding antibody The oligonucleotide probe that the gene specific of heavy chain and light chain combines carries out).

III. measuring method

It can be screened by many measure method as known in the art to anti-GITR Identification of the antibodies provided herein, or Characterize its physical/chemical properties and/or biological activity.On the one hand, to its antigen-binding activity of antibody test of the invention, example Such as by known method such as ELISA, western blot is waited to carry out.Means known in the art can be used to measure pair The combination of GITR, there is disclosed herein exemplary methods.In some embodiments, using bio-light interferometry (such as Measurement that Fortebio is affine) or MSD measuring method.

On the other hand, competition assay can be used to identify with any anti-GITR antibody competition disclosed herein to GITR Combination antibody.In certain embodiments, such competitive antibody combines and any anti-GITR antibody disclosed herein Combined epitope is identical or the epitope (such as linear or comformational epitope) of overlapping.For positioning the detailed example of the combined epitope of antibody Exemplary method is shown in Morris (1996) " Epitope Mapping Protocols ", Methods in Molecular Biology vol.66(Humana Press,Totowa,NJ)。

The present invention also provides for identifying the measuring method of the anti-GITR antibody with biological activity.Biological activity can To include for example improving signal transduction that GITR mediates and (such as improving in conjunction with GITR (such as in conjunction with people and/or machin GITR) NFkappa-B signal path), cut down the cell (such as Treg cell) of expression GITR by ADCC, enhances T effector cell function (such as CD4 effector T cell) (such as the generation of the cell factor by improving effector T cell (such as interferon such as IFN-γ or white Cytokine such as IL2)).It also provides in vivo and/or in vitro with the antibody of such biological activity.

In certain embodiments, to the such biological activity of antibody test of the invention.

Methods known in the art can be used to measure T cell activation.For example, thin by being discharged after T cell activation Intracellular cytokine, such as the level of interferon (such as IFN-γ) or interleukins (such as IL2) measure.This field can also be used Well known method measures GITR signal transduction (such as NF-kappaB signal path), to measure the activation of T cell.One In a embodiment, the expression people GITR and reporter gene (NF- comprising being fused to reporter gene (such as β luciferase) is generated Kappa B promoter) transgenic cell.Adding anti-GITR antibody to cell causes NF-kappa B transcription to increase, this use It is detected for the measuring method (such as luciferase reporter gene measuring method) of reporter gene.

Methods known in the art can be used to measure the ADCC effect of antibody.Such as pass through NF-AT signal activation.In In one embodiment, obtaining ADCC effector cell, (NF-AT comprising being fused to reporter gene (such as β luciferase) starts Son) transgenic cell.The cell of pairing effect cell and high expression GITR are co-cultured, while being added anti-GITR antibody and caused Effector cell NF-AT transcription increases, this uses the measuring method (such as luciferase reporter gene measuring method) for being directed to reporter gene To detect.

Include natural expression GITR or engineered for the cell that any of above vitro assay uses and expresses GITR cell System.Such cell includes the T cell of natural expression GITR, the memory T cell after Treg cell and activation.Such cell further includes It expresses GITR and not expresses the cell line of the coding GITR DNA transfection of GITR under normal circumstances.

It is understood that it is any to carry out to be able to use the anti-GITR antibody of immunoconjugates replace or supplement of the invention Said determination method.

It is understood that being able to use anti-GITR antibody and other activating agent to carry out any of above measuring method.

IV. immunoconjugates

In some embodiments, the present invention provides immunoconjugates, and it includes any anti-GITR provided herein Antibody and other materials, such as cytotoxic agent.In some embodiments, other materials such as therapeutic agent, such as cytotoxicity Agent or immunosuppressor or chemotherapeutics.Cytotoxic agent includes the harmful medicament of any pair of cell.It is suitable for forming immunoconjugates The example of the cytotoxic agent (such as chemotherapeutics) of object is as known in the art.For example, cytotoxic agent includes but is not limited to: Radioactive isotope；Growth inhibitor；Enzyme and its segment such as hydrolase nucleic acid；Antibiotic；Toxin such as small molecule toxins or bacterium, The enzymatic activity toxin of fungi, plant or animal origin, including its segment and/or variant；With it is known various antitumor or anti- Cancer agent.

In some embodiments, the immunoconjugates are for preventing or treating tumour, such as gastroenteric tumor.One In a little embodiments, tumour is cancer, such as gastrointestinal cancer, such as gastric cancer, the carcinoma of the rectum, colon cancer, colorectal cancer etc..In In some embodiments, the immunoconjugates are for preventing or treating infection, such as chronic infection, such as bacterium infection, disease Malicious infection, fungal infection, protozoal infections etc..

V. pharmaceutical composition and pharmaceutical preparation

In some embodiments, the present invention is provided comprising any anti-GITR antibody as described herein or its segment (preferably Its antigen-binding fragment of ground) or its immunoconjugates composition, preferably composition be pharmaceutical composition.In an embodiment party In case, the composition also includes pharmaceutic adjuvant.In one embodiment, composition, for example, pharmaceutical composition, comprising this The anti-GITR antibody or its segment or its immunoconjugates of invention and one or more other therapeutic agents (such as it is chemotherapeutics, thin Cellular toxicity agent, vaccine, other antibody, anti-infection activity agent, small-molecule drug or immunomodulator, preferably PD-1 axis combine short of money Anti-agent, such as anti-PD-1 antibody, anti-PD-L1 antibody or anti-PD-L2 antibody) combination.

In some embodiments, the composition is for preventing or treating tumour, such as gastroenteric tumor.In some realities It applies in scheme, tumour is cancer, such as gastrointestinal cancer, such as gastric cancer, the carcinoma of the rectum, colon cancer, colorectal cancer etc..Some In embodiment, the composition is for preventing or treat infection, such as chronic infection, such as bacterium infection, virus infection, very Bacterium infection, protozoal infections etc..

The invention also includes composition (including pharmaceutical composition or drugs comprising anti-GITR antibody or its immunoconjugates Preparation) and the polynucleotides comprising encoding anti-GITR antibody composition (including pharmaceutical composition or pharmaceutical preparation).Certain In embodiment, composition include one or more combination GITR antibody its segment or it is one or more encode it is a kind of or more Kind is in conjunction with the antibody of GITR or the polynucleotides of its segment.These compositions can also include suitable pharmaceutic adjuvant, such as ability Known pharmaceutical carrier, pharmaceutical excipient in domain, including buffer.

Being suitable for the invention pharmaceutical carrier can be sterile liquid, Ru Shui and oil, including those with petroleum, animal, What plant or synthesis originated from, such as peanut oil, soybean oil, mineral oil, sesame oil.When intravenously application pharmaceutical composition, water It is preferred carrier.Saline solution and aqueous dextrose and glycerite can also be used as liquid-carrier, are especially used for Injectable solution.Suitable pharmaceutical excipient include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, Silica gel, odium stearate, glyceryl monostearate, talcum, sodium chloride, the skimmed milk of drying, glycerol, propylene, glycol, water, ethyl alcohol Deng.Using and application thereof for excipient, also referring to " Handbook of PharmaceuticalExcipients ", the 5th Version, R.C.Rowe, P.J.Seskey and S.C.Owen, PharmaceuticalPress, London, Chicago.If desired Words, the composition can also contain a small amount of wetting agent or emulsifier or pH buffer.These compositions can use molten The form of liquid, suspension, emulsion, tablet, pill, capsule, powder, sustained release preparaton etc..Oral preparaton can wrap Containing standard vector, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin.

It can be by by anti-GITR antibody of the invention with the desired purity and one or more optional pharmaceutic adjuvants (Remington ' s Pharmaceutical Sciences, the 16th edition, Osol, A. compile (1980)) mixing includes this to prepare The pharmaceutical preparation of anti-GITR antibody described in text, preferably in the form of lyophilized preparation or aqueous solution.

Illustrative lyophilized antibodies preparation is described in U.S. Patent number 6,267,958.Aqueous antibody formulation includes that the U.S. is special Those of described in benefit number 6,171,586 and WO2006/044908, latter preparation includes histidine-acetate buffer.

Pharmaceutical composition or preparation of the invention can also include one or more other active constituents, the active constituent Be needed for treated specific adaptations card preferably have can not adversely influence those of mutual complementary activity activity at Point.For example, it is desirable that also provide other anticancer active constituents, for example, it is chemotherapeutics, cytotoxic agent, vaccine, other antibody, anti- Infection activity agent, small-molecule drug or immunomodulator, such as PD-1 axis binding antagonists (such as anti-PD-1 antibody or anti-PD- L1 antibody or anti-PD-L2 antibody) etc..The active constituent exists effectively to measure suitably combination for purpose purposes.

Extended release preparation can be prepared.The suitable example of extended release preparation includes solid hydrophobic polymers antibody-containing Semi-permeable matrix, the matrix is in formed article, such as film or microencapsulated form.

About the other components of pharmaceutical preparation/pharmaceutical composition comprising antibody of the present invention, referring also to WO2015/ Disclosed in 031667 or WO2015/187835 etc. those.

VI. combination product

In some embodiments, the present invention also provides combination products, and it includes antibody of the invention or its antigen knots Close segment or its immunoconjugates and one or more other therapeutic agents (such as chemotherapeutics, other antibody, cytotoxicity Agent, vaccine, anti-infection activity agent, small-molecule drug or immunomodulator etc.).In some embodiments, other antibody are for example Anti- PD-1 antibody or anti-PD-L1 antibody or anti-PD-L2 antibody.

In some embodiments, the combination product is for preventing or treating tumour, such as gastroenteric tumor.Some In embodiment, tumour is cancer, such as gastrointestinal cancer, such as gastric cancer, the carcinoma of the rectum, colon cancer, colorectal cancer etc..One In a little embodiments, the combination product is for preventing or treating infection, such as chronic infection, such as bacterium infection, virus sense Dye, fungal infection, protozoal infections etc..

VII. purposes

One aspect of the present invention provides the method that NF-KappaB signal path is activated in subject, including to subject Apply antibody or its antigen-binding fragment, immunoconjugates, pharmaceutical composition or the combination of a effective amount of anti-GITR of the invention Product.

Further aspect of the present invention provides the raising effector T cell function in subject, such as the side of activation effect T cell Method, antibody from a effective amount of anti-GITR of the invention to subject or its antigen-binding fragment, immunoconjugates, medicine including applying Compositions or combination product.In some embodiments, the function is effector T cell proliferation enhancing.In some embodiment party In case, the activation T cell shows as interferon or interleukins secretion/expression increases.In some embodiments, white thin Born of the same parents' interleukin is IL2.In some embodiments, interferon is IFN-γ.

Further aspect of the present invention is supplied to mediates ADCC and the method for eliminating regulatory T-cell in subject, including to Subject applies the antibody or its antigen-binding fragment, immunoconjugates, pharmaceutical composition of a effective amount of anti-GITR of the invention Or combination product.In some embodiments, the regulatory T-cell is Treg cell.

One aspect of the present invention additionally provides the method prevented in subject or treat tumour, and the method includes to described Subject applies a effective amount of any anti-GITR antibody as described herein or its segment, immunoconjugates, pharmaceutical composition or group Close product.

One aspect of the present invention additionally provides the method prevented in subject or treat infection, and the method includes to described Subject applies a effective amount of any anti-GITR antibody as described herein or its segment, immunoconjugates, pharmaceutical composition or group Close product.

The present invention also provides the sides that the prevention in subject or treatment and Treg are proliferated related other symptom or the patient's condition Method, the method includes applying a effective amount of any anti-GITR antibody as described herein or its segment to the subject, be immunized Conjugate, pharmaceutical composition or combination product.

Subject can be mammal, for example, primate, it is preferable that advanced primate, for example, the mankind are (for example, suffer from There are disease described herein or the patient with the risk with disease described herein).In one embodiment, subject suffers from Disease described herein (for example, tumour or infectious diseases as described herein) or with the risk for suffering from disease described herein. In certain embodiments, subject receives or had received other treatments, such as chemotherapeutic treatment and/or radiotherapy.It is standby Under selection of land or combination, subject immunocompromised host or risk with the immunocompromised host due to infection due to infection.

In some embodiments, tumour as described herein, such as cancer, including but not limited to solid tumor, hematology cancer, Soft tissue neoplasm and metastasis (metastases).In some embodiments, it is as described herein for treatment cancer include but breast cancer, Lung cancer, oophoroma, prostate cancer, colon and rectum carcinoma, colorectal cancer, cervical carcinoma, the cancer of the brain, cutaneum carcinoma, liver cancer, cancer of pancreas Or the solid tumors such as gastric cancer and leukemia such as leukaemia and lymthoma.In some embodiments, cancer is gastrointestinal cancer, Such as gastric cancer, the carcinoma of the rectum, colon cancer, colorectal cancer etc..

Antibody molecule as described herein can be used to realize to metastatic carcinoma (for example, the metastatic of expression GITR or PD1 Cancer) treatment.

In one embodiment, tumour is to express the cancer of raised levels of GITR and/or PD1.

In some embodiments, cancer as described herein is colon cancer and its metastatic cancer.

In some embodiments, the infection is acute or chronic.In some embodiments, the chronic sense Dye is infection, latent infection or the slowly infection of duration.In some embodiments, the chronic infection is by selected from thin Caused by bacterium, virus, fungi and protozoic pathogen.

In other respects, the present invention provides anti-GITR antibody or its segment and is producing or preparing the purposes in drug, described Drug is for treating the related disease or illness being mentioned above.

In some embodiments, antibody of the invention or antibody fragment or immunoconjugates or composition or product can prolong The breaking-out of slow illness and/or symptom relevant to illness.

In some embodiments, prevention as described herein or treatment method further include to the subject or individual combination Antibody molecule disclosed herein or pharmaceutical composition or immunoconjugates and one or more other therapies are applied, such as are controlled Treatment mode and/or other therapeutic agents.

In some embodiments, therapeutic modality includes surgical operation (such as tumorectomy)；Radiotherapy (for example, External beam therapy, it is related to wherein designing the three-dimensional potential theory of irradiation area), local irradiation (for example, be directed toward pre-selection The irradiation of target or organ) or focusing illumination) etc..

In some embodiments, therapeutic agent is selected from chemotherapeutics, cytotoxic agent, vaccine, other antibody, anti-infection activity Agent, small-molecule drug or immunomodulator.

Illustrative vaccine includes but is not limited to cancer vaccine.Vaccine can be the vaccine based on DNA, the epidemic disease based on RNA Seedling or vaccine based on viral transduction.Cancer vaccine can be preventative or therapeutic.

Illustrative anti-infection activity agent includes but is not limited to antivirotic, antifungal agent, antiprotozoal, antibacterial Agent, such as nucleoside analog Zidovudine (AST), Ganciclovir, phosphonic acid or cidovir, as described above.

Illustrative other antibody include but is not limited to anti-PD-1 antibody, anti-PDL1 antibody or anti-PDL2 antibody.This field Known a variety of anti-PD-1 antibody, anti-PDL1 antibody or anti-PDL2 antibody, see, for example, WO2007/005874, WO2009/ 101611, WO2009/114335, WO2010/027827 and WO2011/066342 etc..

In some embodiments, antibody combination described herein can be applied respectively, for example, as individual antibody It applies respectively, or application when (such as bispecific or three-specific antibody molecule) connection.

More WO2015/ can be may refer to the therapy or therapeutic agent of anti-GITR antibody or its fragment combination 031667。

Such combination treatment cover combined administration (such as two or more therapeutic agents be included in same preparaton or separate Preparaton in) and separate administration, in this case, can before applying other therapeutic agent and/or medicament, meanwhile, and/ Or the application of antibody of the invention occurs later.In one embodiment, the application of anti-GITR antibody and other therapeutic agent Application occurs in about one month, or in about one, two or three week, or in about 1,2,3,4,5 or 6 day each other.

The therapeutically effective amount of pharmaceutical composition containing anti-GITR antibody to be employed will be for example depending on treatment background and mesh Mark.It will be appreciated by those skilled in the art that the suitable dosage level for treatment will partly change according to following factor: delivering Molecule, the targeted indication used, weight, body surface area or the organ size and/or feelings of administration method and patient Condition (age and general health status).In certain embodiments, the titratable dosage of clinician and change administration method with Obtain optimum therapeuticing effect.

Administration frequency will depend on the pharmacokinetic parameter of the specific anti-GITR antibody in preparation used.In general, facing Bed doctor applies the dosage that composition obtains desired effect until reaching.Therefore antibody of the invention can be applied with single dose, Or within a certain period of time with dosage two or more times (it may include the required molecule of identical or different amount) application, Huo Zhetong Cross implanted device or the application of conduit continuous infusion.Dosage appropriate can be determined by using dose response data appropriate.At certain It, can be in extended time limit to patient's administration of antibodies in a little embodiments.In certain embodiments, antibody is every two Week, monthly, the every two moon, every three months, four months every, every five months or every six months are administered.

The administration method of pharmaceutical composition be according to known method, for example, orally, by intravenous injection, in peritonaeum, brain Interior (in essence), the ventricles of the brain are interior, in intramuscular, intraocular, intra-arterial, portal vein or intralesional routes；By sustained release system or pass through Implanted device.In certain embodiments, composition can be applied by bolus injection or by continuous infusion or by implanted device With.

Composition can also be via the another kind material appropriate of molecule needed for implantation film, sponge or absorption thereon or capsule encapsulating Material is applied topically.In certain embodiments, when using implanted device, described device can be implanted to any suitable group Knit or organ in, and molecule needed for being delivered via diffusion, the bolus of time controlled released or continuous administration.

It is understood that it is any to carry out to be able to use the anti-GITR antibody of immunoconjugates replace or supplement of the invention Treatment.

VIII. the method and composition for diagnosing and detecting

In certain embodiments, any anti-GITR antibody or its antigen-binding fragment provided herein can be used for examining Survey the presence of GITR in the biological sample.When term " detection " is used in this article, including quantitative or qualitative detection, it is illustrative to examine It is compound that survey method can be related to immunohistochemistry, immunocytochemistry, flow cytometry (for example, FACS), antibody molecule Magnetic bead, ELISA measuring method, PCR- technology (for example, RT-PCR).In certain embodiments, biological sample is blood, serum or life Other fluid samples in object source.In certain embodiments, biological sample includes cell or tissue.In some embodiments In, biological sample comes from hyperproliferative or cancerous lesions.

In one embodiment, it provides for diagnosing or the anti-GITR antibody of detection method.In another aspect, it mentions For the existing method of detection GITR in the biological sample.In certain embodiments, method includes detection GITR albumen in life Presence in object sample.In certain embodiments, GITR is people GITR.In certain embodiments, the method includes will Biological sample contacts under conditions of allowing anti-GITR antibody in conjunction with GITR with anti-GITR antibody as described herein, and detects Whether compound is formed between anti-GITR antibody and GITR.The formation of compound indicates that there are GITR.This method can be body Outer or vivo approaches.In one embodiment, the treatment that anti-GITR antibody be used to select to be suitble to utilize anti-GITR antibody Subject, such as wherein GITR is the biomarker for selecting the subject.

In one embodiment, antibody diagnosis cancer or tumour of the present invention, such as evaluation (for example, monitoring) can be used The treatment of disease (for example, cancer or tumour) described herein or progress in object, its diagnosis and/or by stages.In certain embodiment party In case, the anti-GITR antibody of label is provided.Label include but is not limited to the label being detected directly or part (such as fluorescent marker, Chromophore label, electron dense label, chemiluminescent labeling and radioactive label), and the part being indirectly detected, such as enzyme or Ligand, for example, passing through enzymatic reaction or interaction of molecules.Exemplary indicia includes but is not limited to radioactive isotope³²P、¹⁴C、¹²⁵I、³H and¹³¹I, fluorogen such as Rare Earth Chelate or fluorescein and its derivative, rhodamine and its derivative, dansyl (dansyl), umbelliferone (umbelliferone), luciferase (luceriferase), for example, firefly luciferase and thin Bacteriofluorescein enzyme (U.S. Patent number 4,737,456), fluorescein, 2,3- dihydro phthalazine diketones, horseradish peroxidase (HR), alkali Acid phosphatase, beta galactosidase, glucoamylase, lyase, Carbohydrate oxidase, for example, glucose oxidase, gala glycosyloxy Change enzyme and glucose-6-phosphate dehydrogenase (G6PD), Heterocyclic oxidases such as uricase and xanthine oxidase, and utilizes hydrogen peroxide The enzyme of oxidation dye precursors such as HR, lactoperoxidase or microperoxisome (microperoxidase), biotin/affine Element, spin labeling, bacteriophage labels, stable free radical, etc..

In some embodiments of any invention provided herein, sample is obtained before with anti-GITR Antybody therapy ?.In some embodiments, sample is obtained before with Medication for Cancer.In some embodiments, sample It is to be obtained after cancer is transferred.In some embodiments, sample be formalin fix, paraffin coating (FFPE) 's.In some embodiments, sample is biopsy (such as core biopsy), specimens from pri (such as sample from operation excision), Or fine needle aspirate.

In some embodiments, before treatment, for example, certain before initial treatment or after treatment interval is controlled GITR is detected before treating.

In some embodiments, a kind of method for treating tumour or infection is provided, which comprises to subject (for example, sample) (for example, Samples subjects comprising cancer cell) examine the presence of GITR, thus determine GITR value, by GITR Value compared with control value, and if GITR value be greater than control value, to subject apply therapeutically effective amount it is optional with it is a kind of Or the anti-GITR antibody (for example, anti-GITR antibody as described herein) of various other therapy combinations, thus treat tumour or infection.

Exemplary anti-GITR antibody IX of the invention

In terms of these and other of the invention and embodiment is in attached drawing (followed by brief description) and hair below It is described and is illustrated in following embodiment in bright detailed description.In above and entire the application it is discussed any or all Feature can combine in the various embodiments of the present invention.Following embodiment further illustrates the present invention, it should be understood, however, that real It applies example and illustrates and not to the mode limited to describe, and those skilled in the art can carry out a variety of modifications.

Embodiment

The preparation of 1. hybridoma of embodiment

Hybridoma technology is to keep the main feature of the two simultaneously by merging two kinds of cells.Both cells are respectively The mouse boosting cell and murine myeloma cell being immunized through antigen.(B lymph is thin for the mouse boosting cell being immunized by specific antigen Born of the same parents) the antibody-secreting function of being mainly characterized by it, but cannot continuously cultivate in vitro, murine myeloma cell can then cultivated Under the conditions of infinitely divide, be proliferated, that is, there is so-called immortality.Under the action of Selective agar medium, only B cell and myeloma is thin The hybrid cell of born of the same parents' fusion could have the ability of lasting culture, and formation is provided simultaneously with antibody-secreting function and keeps cell immortality The cell clone of two kinds of features of property.This experiment is by people GITR protein immunization mouse, then obtains the splenocyte and myeloma of mouse Cell fusion obtains the hybridoma that can express positive antibody.

Hybridoma fusion

Experimental animal and immunologic information

Electro' asion ware prepares: thoroughly impregnating electro' asion ware with 70% ethyl alcohol, and dries up in super-clean bench spare.

Separating Morr. cell: mouse is put to death in neck dislocation, with 75% alcohol disinfecting body surface 5min, is put into immediately small in super-clean bench In mouse dissection plate, left lateral position, with No. 7 syringe needle fixing limbs.Spleen is taken out in sterile opening abdominal cavity, with basal medium (configuration Method such as following table) washing, and carefully remove the connective tissue around adhered to.Spleen is then transferred to another and fills basic training In the plate for supporting base.Push down spleen with elbow syringe needle, with small pinhead on spleen jack, and squeezed with tweezers, fill splenocyte Divide release, splenocyte suspension is made.Cell suspension is washed one time after 70 μM of cell screen clothes filter with 30ml basal medium, 1200rpm is centrifuged 6min.

Splitting erythrocyte: cell is resuspended with 10ml RBC lysis buffer (GIBCO) in removal supernatant.Then it adds 20ml RBC lysis buffer.1100rpm is centrifuged 6min after suspension stands 5min.It is resuspended after removing supernatant with 10ml basal medium Cell, then adds 30ml basal medium, and 1100rpm is centrifuged 6min.After removing supernatant, cell is resuspended in the training of the basis 20ml It supports in base and counts.

Electro' asion: murine myeloma cell SP2/0 cell (ATCC) is resuspended with 20ml basal medium and counts.By SP2/ 0 and splenocyte mixed with the ratio of 1:2~1:1,1000rpm is centrifuged 6min.Mixed cell is resuspended in after removal supernatant 10ml is merged in buffer (BTXpress).15ml fusion buffer is added, 1000rpm is centrifuged 5min, removes supernatant.It repeats After above-mentioned steps one time, with appropriate fusion buffer gravity treatment cell, adjustment cell mixing density to 1 × 10⁷A cell/ml.Electricity The parameter setting of fusion instrument is as follows.2ml cell suspension is added in each electro' asion ware and carries out electro' asion.

Condition	Mouse(SP2/0-ECF-F)
		Alignment:	60v,30sec
Membrane breaking:	1500V,30μs,3X
		Post-fusion pulse:	60V,3sec

Bed board after electro' asion: cell is stored at room temperature 5min in electro' asion ware.Cell is transferred in centrifuge tube, with screening Culture medium (configuration method such as following table) diluting cells are to 1~2 × 10⁴A cell/ml.100 μ l cells are added in every hole in 96 orifice plates Suspension.7th day replacement screening and culturing medium after fusion.It cultivates the 10th day and is screened after (or more long, according to cell growth state). The hybridoma of the anti-GITR antibody of expression specificity is filtered out by FACS (C6 (BD Biosciences)) detection.

Positive hybridoma cell subclone

Subcloning steps: preparing one piece of 96 orifice plate, and 200 μ l basis culture as described above is added in the 2nd to the 8th every hole of column Base.Cell suspension is made in the cell in the positive hole that above-mentioned fusion filters out and the 1st column are added.1st column cell suspension is taken 100 The 2nd column are added in μ l, take 100 μ l that next column is added after mixing well.It repeats the above steps, until last column volume becomes 300 μ l；96 orifice plate 15min are stood, microscopically observation counts.Take the corresponding volume of 100 cells that 20ml base as described above is added In basal culture medium, and bed board is mixed, every 200 μ l of hole.Microscopically observation after a week judges and marks monoclonal hole, to be measured Choose positive hole.

Cell cryopreservation: observation cell state waits cells well-grown, and when vigor > 90%, 1000rpm is centrifuged 5min, removal Supernatant.Cell is resuspended to 1 × 10 with frozen stock solution (45.5%FBS, 44.5%RPMI-1640,10%DMSO)⁷A cell/ml, point It is filled to cryopreservation tube, is put into program temperature reduction box, -80 DEG C freeze.

The production and purifying of 2. chimeric antibody of embodiment

The present invention utilizes Protocols in Molecular Biology, obtains the antibody sequence in anti-GITR positive hybridoma cell, and utilize It constructs human mouse chimeric antibody.

Hybridoma sequencing

RNA extracting: fresh cells, 300g are centrifuged 5min, remove supernatant, 500 μ l LY buffers are added in precipitating (Biomiga) it (20 μ l β mercaptoethanols are added using preceding every 1ml), mixes to clarification.It is added in DNA removal pipe, 13000rpm is centrifuged 2min, and collection flows through liquid.100% ethyl alcohol is added in liquid to flowing through in 1/2 ratio, mixes 5 times to clarifying. Clear solution is added in RNA collecting pipe, 13000rpm is centrifuged 1min and removes liquid, and 500 μ l RB are added (RecoveryBuffer recycles buffer) (Takara), 13000rpm are centrifuged 30s, add 500 μ l RNA washing buffers (Biomiga) (ethyl alcohol is added before using), is centrifuged 30s, after coming again the above process, after being centrifuged thoroughly volatilization removal ethyl alcohol, 30 μ l DEPC water are added in column to collecting, 12000g is centrifuged 2min, collects eluent.Measure RNA concentration.

It is obtained using PrimeScript II 1st Strand cDNA Synthesis Kit (Takara) reverse transcription CDNA:

It is as follows to configure reaction system I:

After 65 DEG C of incubation 5min, cooled on ice is set rapidly.Following reverse transcription system is added into reaction system I, total amount is 20 μ l:

Carry out reverse transcription translation by following condition after slowly mixing: then 42 DEG C of 60min → 95 DEG C 5min are put cold on ice But, cDNA is obtained.

CDNA is connected into carrier T:

PCR expands heavy chain and light chain variable region respectively, and PCR reaction system is as follows:

PCR reaction condition is as follows:

The PCR product for taking the above-mentioned PCR reaction of 4.5 μ l to obtain, is added 0.5 μ l pMD20-T carrier (Clontech), 5 μ LLigation Mighty Mix (Takara), mixes gently, and in 37 DEG C of reaction 2h, obtains connection product.

Heavy chain variable region (VH) primer (Primer Mix 1) of the anti-GITR antibody of 6. mouse of table

Light chain variable region (VL) primer (Primer Mix 2) of the anti-GITR antibody of 7. mouse of table:

Transformed cells:

- 80 DEG C of taking-up TOP10 competent cells (TIANGEN Biotech (Beijing) Co., Ltd.), melt on ice, take above-mentioned The 5 μ l of connection product of acquisition is added in the TOP10 competent cell of thawing, is incubated for 30min after mixing on ice.42 DEG C of heat shocks Rapid cooled on ice 2min after 90s adds 900 μ l LB culture mediums (the raw limited public affairs of work bioengineering (Shanghai) share into EP pipe Department), 37 DEG C, 220rpm shaking table culture 1h.3000g is centrifuged 2min, absorbs 800 μ l supernatants, with remaining culture medium by thallus weight On the plate for hanging and being coated on amicillin resistance.In 37 DEG C of overnight incubations, cloning and sequencing is chosen.

Construct chimeric antibody

The anti-area GITR antibody VH and VL of mouse that hybridoma generates in the embodiment 1 that PCR amplification has been sequenced

PCR system is as follows:

* VH is expanded, using Primer Mix 1, VL is expanded, using Primer Mix 2；

^ is the correct pMD20-T plasmid of above-mentioned sequencing

Gel extraction pcr amplification product.

Homologous recombination reaction:

Homologous recombination system is as follows:

37 DEG C of reaction 30min obtain recombinant products.Recombinant products convert TOP10 competent cell, and picking monoclonal is surveyed Sequence selects the clone comprising the correct plasmid of direction of insertion as positive colony, saves positive colony.

The expression and purifying of chimeric antibody

The plasmid comprising anti-GITR antibody is extracted from the positive colony obtained above.

According to required transfection volume pass on 293F cell (Invitrogen), the day before transfection by cell density adjust to 1.5×10⁶A cell/ml.Transfection same day cell density is about 3 × 10⁶A cell/ml.Take the F17 culture medium of final volume 1/10 Plasmid appropriate is added as transfection buffer in (Gibco, A13835-01), mixes.Add suitable polyethyleneimine (PEI) (Polysciences, 23966) into plasmid (ratio of plasmid and PEI are 1:3 in 293F cell), is incubated at room temperature after mixing 10min obtains DNA/PEI mixture.After cell is resuspended with DNA/PEI mixture, 36.5 DEG C, 8% CO₂.It adds and turns afterwards for 24 hours The FEED (Sigma) for contaminating volume 2%, in 36.5 DEG C, 120rpm, 8% CO₂Under the conditions of cultivate.Continuous culture to the 6th day or When cell viability≤60%, collects cell conditioned medium and purified.

Use 0.5M NaOH to handle overnight on the gravity column that uses of purifying, vial etc. cleaned with distilled water after at 180 DEG C Dry roasting 4h, obtains purification column.The culture medium 4500rpm of collection is centrifuged 30min before purification, discards cell.Supernatant is used again The filter of 0.22 μ l filters.Every pipe loads 1ml Protein A, and uses 10ml combination buffer (sodium phosphate 20mM.NaCl150mM, PH7.0) balance.15ml combination buffer rebalancing is used after purification column is added in filtered supernatant. Add 5ml elution buffer (citric acid+sodium citrate 0.1M, PH3.5), collect eluent, 80 μ l are added in the eluent of every 1ml Tris-HCl.The antibody ultrafiltration concentration of collection is exchanged in PBS (Gibco, 70011-044), and detectable concentration.

CDR, light chain variable region and the heavy chain variable region for 2 chimeric antibodies (CH22F4 and CH37G5) that the present invention obtains, The amino acid sequence and sequence number of light chain and heavy chain refer to table 1-3.

Control antibodies used in the present invention be patent application US20130183321A1 (GITR, INC. (Cambridge, MA, US the GITR antibody reported in)), light chain and sequence of heavy chain be respectively in US20130183321A1 SEQ ID NO:44 and SEQ ID NO:54, is hereinafter referred to as TRX518.

3 biomembrane thin-layers interference technology of embodiment measures the binding kinetics of chimeric antibody and antigen of the invention

The equilibrium solution of antibody combination people GITR of the present invention is measured using biomembrane thin-layers interference determination techniques (ForteBio) From constant (KD).ForteBio affinity determination is according to existing method (Estep, P et al., High throughput solution Basedmeasurement of antibody-antigen affinity and epitope Binning.MAbs, 2013.5 (2): the 270-8 pages) it carries out.

Experiment starts preceding half an hour, according to sample size, takes appropriate number of AMQ (Pall, 1506091) (for sample Product examine survey) or AHQ (Pall, 1502051) (for positive control detect) sensor be soaked in SD buffer (PBS 1 ×, BSA0.1%, Tween-20 0.05%) in.

Take 100 μ l SD buffer, antibody (CH22F4, CH37G5 and TRX518), antigen (including people GITR and food crab Monkey GITR, from Acrobiosystems buy) be added separately to 96 hole black polystyrenes partly measure microwell plate (Greiner, 675076) in.According to sample position fabric swatch, sensor position is selected.It is as follows that parameter is arranged in instrument: operating procedure: Baseline, Loading~1nm, Baseline, Association and Dissociation；Each step runing time depends on sample knot It closes and dissociation speed, revolving speed 400rpm, temperature is 30 DEG C.Use ForteBio analysis software K_DValue.

In the experiment described in the above measuring method, the affinity of antibody CH22F4, CH37G5 are as shown in table 8:

Table 8.ForteBio detects the affinity constant (equilibrium dissociation constant) that antigen-antibody combines

In the above test, the K of chimeric antibody CH22F4, CH37G5_DValue respectively 1.96E-09M, 2.04E-09M, with The TRX518 of control group is compared, and the antibody in this research has more preferably K_DValue.

The Binding experiment of the CHO-S cell of 4 chimeric antibody of embodiment and overexpression people GITR

The chimeric antibody of the invention of this research and utilization flow cytomery gradient dilution and surface are overexpressed people The combination situation of the CHO-S stable cell line of GITR.

By the cDNA clone of encoding human GITR (SEQ ID NO:41) in pCHO1.0 carrier (Invitrogen), it will obtain Plasmid transfection to CHO-S cell (Invitrogen, ExpiCHOTMExpression System Kit, article No.: A29133), the CHO-S cell (CHO-hGITR) for being overexpressed people GITR is generated.

By CHO-hGITR cell count, and it is diluted to 2 × 10⁶100 μ l/ are added into 96 orifice plate of U-shaped bottom by a cell/ml Hole.400g is centrifuged 5min, removes cell culture medium.By sample (being chimeric antibody CH22F4, CH37G5, and TRX518 respectively) U is added in (antibody dilution process are as follows: highest antibody concentration is 400nM, and three times are diluted in PBS, tests 12 concentration in total) Simultaneously cell is resuspended in template, and 100 holes μ l/ stand 30min on ice.400g is centrifuged 5min and removes supernatant, and PBS is washed cell 1 time.400g It is centrifuged 5min and removes PBS, the secondary antibody (SoutherBiotech of the PE label of the 100 anti-human Fc of μ l is added in every hole；2040-09)(1: 200 are diluted in PBS), it is protected from light is incubated for 30min on ice.400g is centrifuged 5min and removes supernatant, and PBS is washed cell 1 time.With 80 μ l Cell, FACS detection is resuspended in 1 × PBS.

In the experiment described in the above measuring method, the combination situation of antibody CH22F4, CH37G5 and CHO-hGITR cell is such as Shown in Fig. 1.

In the above test, antibody CH22F4, CH37G5 combine the people's GITR molecule being overexpressed on CHO-S cell, EC50 is respectively 0.4568nM, 0.9069nM, and compared with TRX518, binding ability is similar.

The humanization of 5 chimeric antibody of embodiment

The chimeric antibody that embodiment 2 is obtained carries out humanization.And humanization is carried out by following steps:

1. determining CDR ring structure；

2. finding immediate homologous sequence in human germ line sequences' database for each region V/J of heavy chain and light chain；

3. the back mutation of screening and the most matched ethnic group system of heavy chain light chain and minimum flow；

4. constructing the CDR region of chimeric antibody to the skeleton area of people；

5. using sequence and structure feature, the amino acid position for playing in skeleton area and maintaining CDR function is determined；

6. carrying out back mutation being determined as important sequence location (back to input amino acid classes)；

7. optimizing the amino acid in risk site.

CDR, light chain variable region and the weight chain variable for 2 humanized antibodies (HZ22F4 and HZ37G5) that the present invention obtains The amino acid sequence of area, light chain and heavy chain refers to table 1-3 as described above.

The binding kinetics of embodiment 6ForteBio measurement humanized antibody and antigen

Equilibrium dissociation constant (the K of humanized antibody combination people GITR of the invention is measured using ForteBio measuring method_D)。 ForteBio affinity determination method with embodiment 3, exception be using antibody be humanized antibody HZ22F4 and HZ37G5.In In experiment described in the above measuring method, the affinity of antibody HZ22F4 and HZ37G5 are as shown in table 9:

Table 9.ForteBio detects the affinity constant that antigen-antibody combines

In the above test, the K of humanized antibody HZ22F4, HZ37G5 as described herein_DValue respectively 4.58E-09M, 4.83E-09M, compared with control antibodies, the humanized antibody in this research has more preferably K_DValue.

The Binding experiment of the CHO-S cell of 7 humanized antibody of embodiment and overexpression people GITR

The humanized antibody of the invention and CHO-hGITR cell of this research and utilization flow cytomery gradient dilution Combination situation.Test method with embodiment 4, exception be using antibody be humanized antibody HZ22F4 and HZ37G5.In conjunction with Situation is as shown in Figure 2.

In the above test, the people GITR being overexpressed on humanized antibody HZ22F4, HZ37G5 combination CHO-S cell, EC50 is respectively 0.5492nM, 1.974nM, compared with TRX518, has similar or more preferably EC50 numerical value, i.e., similar or more excellent Binding ability.

The Binding experiment of the cell of 8 humanized antibody of embodiment and overexpression machin GITR

The humanized antibody of the invention of this research and utilization flow cytomery gradient dilution and surface, which are overexpressed, eats The combination situation of the CHO-S stable cell line of crab monkey GITR.

It will encode on the cDNA clone to pCHO1.0 carrier (Invitrogen) of machin GITR (SEQ ID NO:42), By plasmid transfection to CHO-S cell (Invitrogen, ExpiCHOTMExpression System Kit, article No.: A29133), Generate the CHO-S cell (CHO-cynoGITR) for being overexpressed machin GITR.Rest part test method is the same as embodiment 4, exception Be using antibody be humanized antibody HZ22F4 and HZ37G5, it is as shown in Figure 3 in conjunction with situation.

In the above test, the machin that is overexpressed on humanized antibody HZ22F4, HZ37G5 combination CHO-S cell GITR, EC50 are respectively 0.3533nM, 0.9931nM, compared with TRX518, have similar or more preferably binding ability.

The biological activity of embodiment 9MOA method detection antibody

NF-kappa B signal in downstream can be activated in conjunction with cell surface GITR molecule for the activity antibody of GITR Access.This research uses the Hela-GITR-NF kappa B luciferase's (hereinafter referred to as Hela-GITR) of internal build Cell strain is detected, the expression response by detecting fluorescent reporter gene goes out the activation situation of NF-kappa B signal, to detect The activation of antibody of the present invention.

The building of Hela-GITR-NF kappa B luciferase cell strain

By the cDNA clone of encoding human GITR (sequence is shown in SEQ ID NO:41) to pCHO1.0 carrier (Invitrogen) On, by the plasmid of acquisition and NF-kappaB luciferace reporter plasmid (Promega) cotransfection to Hela cell (ATCC, article No.: CCL-2TM) generates and is overexpressed people GITR simultaneous with NF-kappaB luciferace Reporter System Hela cell (Hela-GITR).

The Hela-GITR cell of logarithmic growth phase, abandons culture supernatant, and PBS (Gibco) washes a cell.It is added appropriate Trypsin (Gibco) is in 37 DEG C, 5%CO₂Digest 2min.The DMEM culture medium containing 10%FBS of 4 times of Trypsin volumes is added (ATCC), metastatic cells to 50ml centrifuge tube and count, 400g, are centrifuged 5min.It is added DMEM culture medium (ATCC), cell is resuspended To 1 × 10⁵A cell/mL.96 hole white tissue culture plates (Nunclon), 50 holes μ l/ are added in cell.Every hole is added simultaneously 50 μ l samples (humanized antibody HZ22F4, HZ37G5 prepared by the present invention) and positive control TRX518), (antibody dilution process Are as follows: highest antibody concentration is 80nM, and three times are diluted in measurement buffer (2%FBS DMEM culture medium), tests 10 in total A concentration).In 37 DEG C/5%CO₂It is cultivated 6 hours in incubator.

Detection: in advance melting Bio-GloTM buffer (promega), is added Bio-GloTM substrate (promega), mixes It is even, obtain Bio-GloTM reagent.Bio-GloTM reagent, 100 μ l/ are added into the cell for having cultivated 6 hours as described above Hole.It reads immediately.

In the above test, experimental result is as shown in figure 4, antibody HZ22F4, HZ37G5 can effectively activate NF KappaB signal path, EC50 are respectively 0.3394nM, 0.3208nM, compared with TRX518 (1.139nM), are had significant more excellent Activation capability.

The experiment of 10 CD4T active cell of embodiment

Antibody and the cd4 t cell activated are incubated for by this research jointly, pass through the phase of IL-2 in detection architecture and IFN-γ To expression quantity, to reflect different antibodies to the activation of cd4 t cell.

PBMC separation: taking contributor new blood 50ml, add 2.5 times of PBS, be gently added to FiColl (Thermo), Divide 4 pipes, 400g is centrifuged 30min, lifting speed 7, deceleration 0.Centrifugation terminates, and gently takes out centrifuge tube, draws middle white Into PBS, PBS is washed 2 times cloud cell mass.

CD4⁺T cell separation: the PBMC cell separated as described above is taken, according to EasySep Human CD4⁺T CellEnrichment Kit (stem cell) specification separation and concentration CD4⁺T cell, and (formula is shown in using T cell culture medium Following table) it is resuspended.

CD4⁺T cell activation: the CD4 for taking above-mentioned separation to obtain⁺T cell, and be with T cell culture medium adjustment cell density 1*10⁶/ ml, according to cell: Dynabeads Human T-Activator CD3/CD28 is added in beads (1:1) (Invitrogen), in 37 DEG C, 5%CO₂It is cultivated one week in incubator.

T cell activation experiment: taking 96 hole flat underside (Nunclon) of Nunc, and the diluted 0.25 μ g/ of PBS is added in 100 holes μ l/ The anti-human CD3Clone UCHT1 of Purified NA/LE Mouse (BD Biosciences) of ml, 37 DEG C are coated with 2 hours, removal Coating buffer.By the CD4 of above-mentioned activation one week⁺T cell removes beads, is cleaned twice with T cell culture medium, and by cell density It is adjusted to 1*10⁶/ ml, every 100 μ l cell suspension of pore volume are added in the flat underside of above-mentioned 96 hole, while of the invention resist is added 100 μ l of body (HZ22F4, HZ37G5) and TRX518 (antibody dilution process are as follows: highest antibody concentration is 400nM, three times dilution In T cell culture medium, 10 concentration are tested in total) and the anti-human CD28Clone CD28 of Purified NA/LE Mouse (BD Biosciences) (final concentration of 2 μ g/ml), cultivates 3 and 5 days, respectively with Human IL-2Kit 1000Test and Human IFN gamma 1000test kit (being purchased from cisbio) detect IL-2 and IFN-γ relative expression quantity (with DeltaF% meter).

Experimental result is as shown in table 10 and 11 and Fig. 5,6.Antibody of the invention can effectively activate CD4 in vitro⁺T is thin Born of the same parents.Wherein the data unit in table is DeltaF% average value.

The relative expression quantity (DeltaF% average value) of table 10.IL2

The relative expression quantity (DeltaF% average value) of table 11.IFN- γ

11 ADCC effect experiment of embodiment

Can be in conjunction with the highly expressed GITR molecule in the surface Treg for the IgG1 subclass antibodies of GITR, and then mediate ADCC Effect removes Treg.This research is (following simple using the Jurkat-ADCCNF-AT luciferase effector cell strain of Promega Claim ADCC effector cell) as detection cell strain and as the CHO-hGITR cell (as described above) of target cell and of the invention Antibody is incubated for altogether, and the expression response by detecting fluorescent reporter gene goes out the activation situation of NF-AT signal, to detect antibody ADCC activity.

Supernatant is removed in ADCC effector cell's (illustrating to cultivate according to Promega company) of logarithmic growth phase, centrifugation, and PBS washes 2 Time, it is 2*10 that adjustment cell density, which is resuspended, with detection culture medium (1640 culture mediums (Gibco) of 5%low IgG serum)⁷/ ml, The CHO-hGITR target cell for preparing and being incubated for as described above is taken, supernatant is removed in centrifugation, and adjustment cell density is resuspended with detection culture medium For 2*10⁶Above-mentioned cell is mixed in 1:1 ratio, and is added to 96 hole white tissue culture plates (Nunclon), 50 μ l/ by/ml Hole.50 μ l samples (humanized antibody HZ22F4, HZ37G5 prepared by the present invention) and positive control antibodies are added in every hole simultaneously (TRX518), (antibody dilution process are as follows: the final concentration of 66.66nM of highest antibody, three times are diluted in detection culture medium, in total Test 12 concentration).In 37 DEG C/5%CO₂It is cultivated 12 hours in incubator.

Detection: in advance melting Bio-GloTM buffer (promega), is added Bio-GloTM substrate (promega), mixes It is even, obtain Bio-GloTM reagent.Bio-GloTM reagent, 100 μ l/ are added into the cell for having cultivated 12 hours as described above Hole.It reads immediately.

In the above test, experimental result is as shown in fig. 7, antibody HZ22F4, HZ37G5 can be activated effectively under ADCC The NF-AT signal path of trip, EC50 are respectively 0.02958nM, 0.04056nM, compared with TRX518, are had significantly more preferably EC50 numerical value.

The antitumor pharmacodynamic test of embodiment 12

This experiment measures the antitumor work of GITR antibody of the invention using MC38 cell inoculation hGITR transgenic mice With.

People's GITR transgenic mice:

The people GITR transgenic mice (about 5 weeks big) of female C57Bl/6 background has purchased from hundred Olympic Competition figure experimental animal technologies Limit company.Mouse is tamed 7 days after arrival, then begins to study.

Cell:

Mouse MC38 cell is purchased from Nanjing milky way biological medicine Co., Ltd, and carries out routine passage in strict accordance with specification Culture is used for subsequent experiment in vivo.Cell is collected by centrifugation, it is 5 × 10 that cell is resuspended in sterile PBS and adjusts cell density⁶ A/ml.It took 0.2ml cell suspension to inoculate into people's GITR transgenic mice right abdomen region at the 0th day to establish MC38-hGITR bearing mouse model.

Administration:

Tumor cell inoculation detects each mouse tumor volume after 6 days, pick out knurl product in 87.4mm³~228.4mm³Model For mouse in enclosing by knurl product average packet (every group of 5 mouse) grouping, dosage and mode are as shown in table 12, wherein AntibodyC is the monoclonal antibody " Antibody C " (WO2017/133540) of anti-PD-1.Respectively after inoculation the 6th, 10, 13, it is administered within 17 days, monitors each group mouse tumor volume during administration and changes of weight, monitoring frequency are 2 times/week, it is continuous to supervise It surveys 5 weeks.Measurement weight and gross tumor volume before each administration, the 24th day calculating Relative tumor inhibiting rate (TGI%) after inoculation, meter It is as follows to calculate formula: TGI%=100%* (h-IgG control group gross tumor volume-treatment group tumors volume)/(h-IgG control group tumour Pre-neoplastic volume is administered in volume-h-IgG control group), wherein h-IgG control group administration pre-neoplastic bulk averaged value is 106mm³。 Gross tumor volume measurement: maximum long axis (L) and maximum wide axis (W) using vernier caliper measurement tumour, gross tumor volume is by following public Formula calculates: V=L × W²/2.Weight is measured using electronic balance.

12. experimental design table of table

* it was administered every 3 or 4 days, totally 4 times.

^h-IgG is purchased from Equitech-Bio, article No.: SLH56-0001.

Tumor control rate result such as Fig. 8,9 and table 13 shown in: the 24th day after inoculation, HZ37G5 and the mono- medicine of HZ22F4 were aobvious Show good tumor control rate, be respectively as follows: 53% and 50%, better than the 36% of TRX518 group.HZ37G5 and HZ22F4 and anti-PD- 1 antibody A ntibody C drug combination, tumor inhibitory effect are significantly better than that Antibody C, HZ37G5, HZ22F4, and The mono- medicine group of TRX518.Wherein HZ37G5 and Antibody C drug combination group and HZ22F4 and Antibody C drug combination 5 mouse of group have 4 cases of complete remission.We detect mouse weight simultaneously, the results are shown in Figure 10 Mice Body Weight is without significant difference.Therefore, the antibodies on tumor for GITR molecule of the invention has apparent inhibitory effect, and when described anti- Body and the antibody combined medication of PD-1 are more significant to the inhibitory effect of tumour.

The 24th day tumor control rate of table 13.

Claims (23)

1. the antibody or its antigen-binding fragment of GITR is combined, it includes

(i) 3 complementary determining region HCDR of the heavy chain variable region as shown in SEQ ID NO:17 or 19, and such as SEQ ID NO: 3 complementary determining region LCDR of light chain variable region shown in 21 or 23, or

(ii) 3 complementary determining region HCDR of the heavy chain variable region as shown in SEQ ID NO:18 or 20, and such as SEQ ID 3 complementary determining region LCDR of light chain variable region shown in NO:22 or 24.

2. combining the antibody or its antigen-binding fragment of GITR, it includes 3 complementary determining region HCDR of heavy chain variable region, and 3 complementary determining region LCDR of light chain variable region, wherein

(i) HCDR1 includes amino acid sequence shown in SEQ ID NO:1 or 3 or 4, and HCDR2 includes shown in SEQ ID NO:5 Amino acid sequence, HCDR3 include amino acid sequence shown in SEQ ID NO:7, and LCDR1 includes ammonia shown in SEQ ID NO:9 Base acid sequence, LCDR2 includes amino acid sequence shown in SEQ ID NO:11,13 or 14, and LCDR3 includes SEQ ID NO:15 Shown in amino acid sequence；Or

(ii) HCDR1 includes amino acid sequence shown in SEQ ID NO:2, and HCDR2 includes amino acid shown in SEQ ID NO:6 Sequence, HCDR3 include amino acid sequence shown in SEQ ID NO:8, and LCDR1 includes amino acid sequence shown in SEQ ID NO:10 Column, LCDR2 includes amino acid sequence shown in SEQ ID NO:12, and LCDR3 includes amino acid shown in SEQ ID NO:16 Sequence.

3. combining the antibody or its antigen-binding fragment of GITR, it includes heavy chain variable region and/or light chain variable regions, wherein

The heavy chain variable region includes:

(i) three complementarity determining regions (HCDR) contained in the VH of any antibody listed by table B；

(ii) combination of HCDR1, HCDR2 and HCDR3 shown in Table A；

(iii) HCDR of (i) or (ii) combination variant, the variant on three CDR regions altogether comprising at least one and No more than 5,4,3,2 or 1 amino acid changes (preferred amino acid displacement, preferably conservative substitution)；Or

(iv) HCDR1, HCDR2 and HCDR3, wherein HCDR1 includes the amino acid sequence selected from SEQ ID NO:1,2,3 or 4, or It is made of the amino acid sequence or HCDR1 includes to have compared with the amino acid sequence selected from SEQ ID NO:1,2,3 or 4 There is one, two or three to change the amino acid sequence of (preferred amino acid displacement, preferably conservative substitution)；HCDR2 includes to be selected from The amino acid sequence of SEQ ID NO:5 or 6, or be made of the amino acid sequence or HCDR2 includes and selected from SEQ ID The amino acid sequence of NO:5 or 6 is compared, and there is one, two or three to change (preferred amino acid displacement, preferably conservative substitution) Amino acid sequence；HCDR3 is made of comprising the amino acid sequence selected from SEQ ID NO:7 or 8 or the amino acid sequence, or HCDR3 includes compared with the amino acid sequence selected from SEQ ID NO:7 or 8 there is one, two or three to change (preferably amino Acid displacement, preferably conservative substitution) amino acid sequence；

And/or

The light chain variable region includes:

(i) three complementarity determining regions (LCDR) contained in the VL of any antibody listed by table B；

(ii) combination of LCDR1, LCDR2 and LCDR3 shown in Table A；

(iii) LCDR of (i) or (ii) combination variant, the variant on three CDR regions altogether comprising at least one and No more than 5,4,3,2 or 1 amino acid changes (preferred amino acid displacement, preferably conservative substitution)；Or

(iv) LCDR1, LCDR2 and LCDR3, wherein LCDR1 includes to be selected from the amino acid sequence of SEQ ID NO:9 or 10 or by institute State amino acid sequence composition or LCDR1 include with selected from SEQ ID NO:9 or 10 amino acid sequence compare with one, Two or three change the amino acid sequence of (preferred amino acid displacement, preferably conservative substitution)；LCDR2 includes to be selected from SEQ ID The amino acid sequence of NO:11,12,13 or 14 is made of or LCDR2 includes and selected from SEQ ID the amino acid sequence Compared to having one, two or three to change, (preferred amino acid displacement, is preferably protected the amino acid sequence of NO:11,12,13 or 14 Keep displacement) amino acid sequence；LCDR3 includes to be selected from the amino acid sequence of SEQ ID NO:15 or 16 or by the amino acid sequence Column composition or LCDR3 include to have one, two or three compared with the amino acid sequence selected from SEQ ID NO:15 or 16 Change the amino acid sequence of (preferred amino acid displacement, preferably conservative substitution).

4. the antibody of any one of claims 1 to 3 or its antigen-binding fragment, it includes

(i) comprising having the amino acid sequence of at least 90% sequence identity with amino acid sequence shown in SEQ ID NO:17 or 19 The heavy chain variable region of column, and/or comprising having at least 90% sequence same with amino acid sequence shown in SEQ ID NO:21 or 23 The light chain variable region of the amino acid sequence of one property, or

(ii) comprising having the amino acid of at least 90% sequence identity with amino acid sequence shown in SEQ ID NO:18 or 20 The heavy chain variable region of sequence, and/or comprising there is at least 90% sequence with amino acid sequence shown in SEQ ID NO:22 or 24 The light chain variable region of the amino acid sequence of identity, or

(iii) heavy chain variable region comprising amino acid sequence shown in SEQ ID NO:17 or 19, and/or include SEQ ID NO: The light chain variable region of amino acid sequence shown in 21 or 23, or

(iv) heavy chain variable region comprising amino acid sequence shown in SEQ ID NO:18 or 20, and/or include SEQ ID NO: The light chain variable region of amino acid sequence shown in 22 or 24.

5. the antibody of any one of claims 1 to 4 or its antigen-binding fragment, it includes

(i) comprising having the amino acid sequence of at least 90% sequence identity with amino acid sequence shown in SEQ ID NO:25 or 27 The heavy chain of column, and/or comprising there is at least 90% sequence identity with amino acid sequence shown in SEQ ID NO:29 or 31 The light chain of amino acid sequence, or

(ii) comprising having the amino acid of at least 90% sequence identity with amino acid sequence shown in SEQ ID NO:26 or 28 The heavy chain of sequence, and/or comprising there is at least 90% sequence identity with amino acid sequence shown in SEQ ID NO:30 or 32 Amino acid sequence light chain, or

(iii) heavy chain comprising amino acid sequence shown in SEQ ID NO:25 or 27, and/or include SEQ ID NO:29 or 31 Shown in amino acid sequence light chain, or

(iv) heavy chain comprising amino acid sequence shown in SEQ ID NO:26 or 28, and/or include SEQ ID NO:30 or 32 Shown in amino acid sequence light chain.

6. the antibody or its antigen-binding fragment of GITR is combined, with following one or more characteristics:

(i) display is with any antibody listed by table 3 to the same or similar binding affinity of GITR and/or specificity；

(ii) inhibit the combination of any antibody and GITR listed by (for example, Reverse transcriptase) table 3；

(iii) with table 3 shown in identical or overlapping epitope in conjunction with any antibody；

(iv) with table 3 shown in GITR in conjunction with any antibody competition；

(v) one or more biological characteristics with any antibody molecule listed by table 3.

7. the antibody or its antigen-binding fragment of the combination GITR of any one of claims 1 to 6 have with next or more A characteristic:

(i) with high-affinity combination GITR, such as people GITR；

(ii) there is agonist activity, such as can effectively activate NF-kappaB signal path；

(iii) ADCC effect can effectively be mediated；

(iv) T cell, such as cd4 t cell can be activated；

(v) there is better anti-tumor activity, such as can reduce the gross tumor volume in subject, while not influencing subject's Weight；

(vi) it can preferably inhibit tumor promotion with anti-PD-1 antibody combination, such as can reduce the tumour body in subject Product, while subject's weight is not influenced.

8. the antibody or its antigen-binding fragment of the combination GITR of any one of claims 1 to 7, wherein the antibody is IgG1 The antibody or antigen-binding fragment of form or IgG2 form or IgG4 form.

9. the antibody or its antigen-binding fragment of the combination GITR of any one of claims 1 to 8, wherein the antibody is Dan Ke Grand antibody.

10. the antibody or its antigen-binding fragment of the combination GITR of any one of claims 1 to 9, wherein the antibody is people The antibody or human antibody or chimeric antibody in source.

11. the antibody of any one of claims 1 to 10 or its antigen-binding fragment, wherein the antigen-binding fragment is to be selected from Antibody fragment below: Fab, Fab ', Fab '-SH, Fv, single-chain antibody (such as scFv) or (Fab ')₂, single domain antibody, Double antibody (dAb) or linear antibodies.

12. the antibody of any one of claims 1 to 11 or its antigen-binding fragment, wherein the antibody is bispecific or more Specific antibody molecules, it is preferable that bi-specific antibody molecule is in conjunction with GITR and PD-1, PD-L1 or PD-L2.

13. isolated nucleic acid encodes the antibody or its antigen binding fragment of the combination GITR of any one of claims 1 to 12 Section.

14. the carrier of the nucleic acid comprising claim 13, the preferably described carrier is expression vector.

15. the host cell of the carrier of nucleic acid or claim 14 comprising claim 13, it is preferable that the host cell is It is protokaryon or eukaryon, it is more preferably selected from yeast cells, mammalian cell (such as 293 cells or Chinese hamster ovary celI) or is applicable in In the other cells for preparing antibody or its antigen-binding fragment.

16. preparation is in conjunction with the method for the antibody or its antigen-binding fragment of GITR, the method includes encoding power suitable for expression Benefit requires to cultivate claim under conditions of any one of 1 to 12 antibody of combination GITR or the nucleic acid of its antigen-binding fragment 15 host cell is optionally separated the antibody or its antigen-binding fragment, and optionally the method also includes from the place Chief cell recycles the antibody or its antigen-binding fragment of the combination GITR.

17. immunoconjugates, it includes the antibody of the combination GITR of any one of claims 1 to 12 or its antigen-binding fragments And other materials, such as cytotoxic agent.

18. pharmaceutical composition, it includes the antibody of the combination GITR of any one of claims 1 to 12 or its antigen-binding fragments Or the immunoconjugates of claim 17, and optionally one or more other therapeutic agents, such as anti-PD-1 antibody, anti-PD- L1 antibody or anti-PD-L2 antibody, and optionally pharmaceutic adjuvant.

19. combination product, it includes the antibody of the combination GITR of any one of claims 1 to 12 or its antigen-binding fragment or The immunoconjugates of claim 17 and one or more other therapeutic agents, for example, chemotherapeutics, cytotoxic agent, vaccine, its Its antibody, anti-infection activity agent, small-molecule drug or immunomodulator, preferably PD-1 axis binding antagonists, such as anti-PD-1 Antibody, anti-PD-L1 antibody or anti-PD-L2 antibody.

20. it is thin to mediate ADCC or activation effect T cell or activation NF-kappaB signal path or elimination to adjust T in subject The method of born of the same parents' (such as Treg cell), the method includes applying in a effective amount of claim 1 to 12 to appoint to the subject The immunoconjugates or claim 18 of the antibody or its antigen-binding fragment or claim 17 of one combination GITR The combination product of pharmaceutical composition or claim 19.

21. prevention or treatment subject's tumour or the method for infection, a effective amount of the method includes applying to the subject The antibody of the combination GITR of any one of claims 1 to 12 or the immunoconjugates of its antigen-binding fragment or claim 17 The combination product of the pharmaceutical composition or claim 19 of object or claim 18, it is preferable that the tumour is cancer, such as Gastrointestinal cancer, such as gastric cancer, the carcinoma of the rectum, colon cancer, colorectal cancer etc.；Preferably, it is described infection be such as bacterium infection, Virus infection, fungal infection or protozoal infections, the preferably described infection is chronic infection.

22. method described in claim 21 further includes one or more therapies being administered in combination to the subject, such as control Treatment mode and/or other therapeutic agents, it is preferable that therapeutic modality includes operative treatment and/or radiotherapy and/or other treatments Agent is selected from chemotherapeutics, cytotoxic agent, vaccine, other antibody, anti-infection activity agent, small-molecule drug or immunomodulator, excellent Selection of land PD-1 axis binding antagonists, such as anti-PD-1 antibody or anti-PD-L1 antibody or anti-PD-L2 antibody.

23. the method for GITR in test sample, the method includes

(a) sample is contacted with the antibody of any combination GITR of any one of claims 1 to 12 or its antigen-binding fragment； With

(b) detection combines the formation of the compound between the antibody or its antigen-binding fragment and GITR of GITR；Optionally, in conjunction with The antibody of GITR is detectably labeled.