CN111918878B

CN111918878B - anti-GITR antibodies and uses thereof

Info

Publication number: CN111918878B
Application number: CN201980022627.8A
Authority: CN
Inventors: 翟天航; 付凤根; 黄威峰; 刘军建
Original assignee: Innovent Biologics Suzhou Co Ltd
Current assignee: Innovent Biologics Suzhou Co Ltd
Priority date: 2018-04-20
Filing date: 2019-04-18
Publication date: 2022-08-23
Anticipated expiration: 2039-04-18
Also published as: TW201946658A; CN111918878A; TWI743469B; WO2019201301A1; CN110386981A

Abstract

The present invention relates to novel antibodies and antibody fragments that specifically bind GITR and compositions containing the same. Furthermore, the invention relates to nucleic acids encoding said antibodies or antibody fragments thereof and host cells comprising the same, as well as related uses. Furthermore, the invention relates to therapeutic and diagnostic uses of these antibodies and antibody fragments.

Description

anti-GITR antibodies and uses thereof

Background

Glucocorticoid-induced tumor necrosis factor receptors (GITRs), also known as CD357 and GITR-D, were first found in T-cells of doxorubicin-treated mice (Nocentin, PNAS. 1997; 94: 6216-. GITR is activated by its cognate ligand, GITR ligand (GITRL), which activates the downstream NF-kappa B signaling pathway.

GITR is expressed at low levels in resting T cells; expression is significantly upregulated after T cell activation. GITR is constitutively expressed at high levels in regulatory T cells (Tregs) and expression is further upregulated when these cells are activated (Nocentini and Riccaradi, E.J.Immunol.2005, 35: 1016-1022).

GITR ligand (GITRL) is predominantly expressed on antigen presenting cells (APC, including macrophages, B cells, dendritic cells and endothelial cells). The binding of GITRL on APC to GITR on responder T cells triggers GITR signaling, stimulates effector T cell activation and suppresses Treg cell activity. Thus, GITR has several effects on effector T cells and regulatory T cells, including: co-stimulating and activating effector T cells, reducing the suppression of effector T cells by Treg cells, etc. (Nocentini, Eur.J.Immunol.2007, 37: 1165-1169).

These effects imply that activation of the GITR signaling pathway can lead to an enhancement of the immune response. Therefore, the substance capable of activating the GITR signal channel can activate the immune response of the organism, further increase the capacity of resisting tumor and infection of the organism and the like, and meanwhile, the GITR molecules are highly expressed in Treg cells in a tumor microenvironment, so that the tumor killing activity can be further enhanced by clearing the Treg cells highly expressed by the GITR molecules by utilizing the ADCC activity of the GITR antibody.

The prior art has investigated a variety of antibodies to GITR (see CN103951753A, CN105829343A, CN106459203A, WO2016196792a1, WO2017068186a9, etc.). Such antibodies are agonists of GITR (i.e., are activating antibodies), can induce or enhance GITR signaling, and are effective in treating a variety of GITR-related diseases or disorders where enhanced immune responses are desired. Since such anti-GITR antibodies are activating antibodies, the level of their activating activity relative to their binding affinity to the antigen is a more critical indicator of their activity (e.g., immunopotentiating activity).

Therefore, there is a need to develop antibodies with better activation activity, better ADCC effect, and better therapeutic effect, such as anti-tumor effect.

Disclosure of Invention

The present invention thus provides a novel antibody that binds GITR, as well as antigen-binding fragments thereof.

In some embodiments, the anti-GITR antibodies of the invention have one or more of the following properties:

(i) binds to GITR with high affinity;

(ii) binds efficiently to GITR on the cell surface;

(iii) have agonist activity, e.g., are capable of effectively activating the NF-kappaB signaling pathway;

(iv) effective activation of T cells (e.g., CD4T cells);

(v) can effectively mediate ADCC effect;

(vi) have better anti-tumor activity, for example, can reduce the tumor volume in a subject, and simultaneously does not affect the body weight of the subject;

(vii) in combination with an anti-PD-1 antibody, better inhibition of tumor activity, e.g., reduction of tumor volume in a subject, while not affecting the subject's body weight, can be achieved.

In some embodiments, the invention provides an antibody or antigen-binding fragment thereof that binds GITR comprising an amino acid sequence as set forth in SEQ ID NO: 17. 18, 19 or 20, and/or a sequence as set forth in SEQ ID NO: 21. 22, 23 or 24 (c) and 3 light chain cdrs (lcdr).

In some embodiments, the invention provides nucleic acids encoding the antibodies or fragments thereof of the invention, vectors comprising the nucleic acids, host cells comprising the vectors.

In some embodiments, the invention provides methods of making the antibodies of the invention or fragments thereof.

In some embodiments, the invention provides immunoconjugates, pharmaceutical compositions and combinations comprising the antibodies of the invention.

The invention also provides methods of using the antibodies of the invention to mediate ADCC or activate the NF-kappaB signaling pathway in a subject, as well as methods of preventing or treating cancer or infection.

The invention also relates to methods of detecting GITR in a sample.

The invention is further illustrated in the following figures and detailed description. However, these drawings and specific embodiments should not be construed as limiting the scope of the invention, and modifications readily ascertainable by those skilled in the art would be included within the spirit of the invention and the scope of the appended claims.

Drawings

FIG. 1 shows flow cytometry for the binding ability of chimeric antibodies to CHO-S cell line expressing human GITR (CHO-hGITR).

FIG. 2 shows flow cytometry to determine the binding ability of humanized antibodies to CHO-S cell strain expressing human GITR (CHO-hGITR).

FIG. 3 shows flow cytometry for the binding ability of humanized antibodies to CHO-S cell line expressing cynomolgus monkey GITR (CHO-cynoGITR).

FIG. 4 shows the MOA method for determining the ability of the humanized antibody to activate the Hela-GITR-NF-Kappa B luciferace cell line.

FIG. 5 shows the measurement of the ability of the humanized antibody to activate CD4T cells to release IL-2.

FIG. 6 shows the measurement of IFN-. gamma.releasing ability of humanized antibody-activated CD4T cells.

FIG. 7 shows the determination of ADCC activity of humanized antibody by MOA method.

FIG. 8 shows the in vivo efficacy test of HZ37G5 molecule.

FIG. 9 shows the in vivo efficacy assay of HZ22F4 molecules.

Figure 10 shows the mouse weight measurements.

Detailed Description

I. Definition of

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodologies, protocols, and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

For the purpose of interpreting this specification, the following definitions will be used, and terms used in the singular may also include the plural and vice versa, as appropriate. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

The term "about," when used in conjunction with a numerical value, is intended to encompass a numerical value within a range having a lower limit that is 5% less than the stated numerical value and an upper limit that is 5% greater than the stated numerical value.

As used herein, the term "and/or" means either one of the selectable items or both of the selectable items.

As used herein, the terms "comprises" or "comprising" are intended to be inclusive of the stated elements, integers, or steps, but not to exclude any other elements, integers, or steps. When the term "comprising" or "comprises" is used herein, unless otherwise indicated, the combination of the recited elements, integers or steps is also encompassed. For example, when referring to an antibody variable region "comprising" a particular sequence, it is also intended to encompass antibody variable regions consisting of that particular sequence.

As used herein, the term "GITR" refers to "glucocorticoid-induced TNF-related genes and/or polypeptides," also known in the art as TNF receptor superfamily 18(TNFRSF18), and refers to any native GITR from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length," unprocessed GITR as well as any form of GITR that results from processing in a cell. The term also encompasses naturally occurring variants of GITR, such as splice variants or allelic variants. The amino acid sequence of human GITR can be found in GenBank accession No. Q9Y5U 5. One specific amino acid sequence of a human GITR polypeptide can be found in SEQ ID NO: 41. A specific cynomolgus GITR polypeptide has an amino acid sequence shown in SEQ ID NO: 42.

the term "anti-GITR antibody", "anti-GITR", "GITR antibody" or "antibody that binds GITR" as used herein refers to an antibody that is capable of binding (human or cynomolgus monkey) GITR protein or fragment thereof with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent in targeting (human or cynomolgus monkey) GITR. In one embodiment, the anti-GITR antibody binds to a non- (human or cynomolgus) GITR protein to a lesser extent than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% or more of the binding of the antibody to (human or cynomolgus) GITR as measured, for example, by Radioimmunoassay (RIA) or biophotonic interferometry or MSD assay.

An "antibody fragment" refers to a molecule distinct from an intact antibody that comprises a portion of an intact antibody and binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab ', Fab ' -SH, F (ab ') 2; a diabody; a linear antibody; single chain antibodies (e.g., scFv); a single domain antibody; a bivalent or bispecific antibody or fragment thereof; camelid antibodies; and bispecific or multispecific antibodies formed from antibody fragments.

As used herein, the term "epitope" refers to the portion of an antigen (e.g., GITR) that specifically interacts with an antibody molecule.

An "antibody that binds to the same or an overlapping epitope" as a reference antibody refers to an antibody that blocks binding of more than 50% of the reference antibody to its antigen in a competition assay, and conversely, a reference antibody blocks binding of more than 50% of the antibody to its antigen in a competition assay.

An antibody that competes with a reference antibody for binding to its antigen refers to an antibody that blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the reference antibody's binding to its antigen in a competition assay. Conversely, a reference antibody blocks binding of more than 50%, 60%, 70%, 80%, 90%, or 95% of the antibody to its antigen in a competition assay. Numerous types of competitive binding assays can be used to determine whether one antibody competes with another, such as: solid phase direct or indirect Radioimmunoassay (RIA), solid phase direct or indirect Enzyme Immunoassay (EIA), sandwich competition assays (see, e.g., Stahli et al, 1983, Methods in Enzymology 9: 242-.

An antibody that inhibits (e.g., competitively inhibits) the binding of a reference antibody to its antigen refers to an antibody that inhibits more than 50%, 60%, 70%, 80%, 90%, or 95% of the binding of the reference antibody to its antigen. In contrast, a reference antibody inhibits 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the antibody to its antigen. Binding of an antibody to its antigen can be measured by affinity (e.g., equilibrium dissociation constant). Methods for determining affinity are known in the art.

An antibody that exhibits the same or similar binding affinity and/or specificity as a reference antibody refers to an antibody that is capable of having a binding affinity and/or specificity that is at least 50%, 60%, 70%, 80%, 90%, or 95% or more of the reference antibody. This can be determined by any method known in the art for determining binding affinity and/or specificity.

"complementarity determining regions" or "CDR regions" or "CDRs" are regions of antibody variable domains that are mutated in sequence and form structurally defined loops ("hypervariable loops") and/or contain antigen-contacting residues ("antigen-contacting points"). The CDRs are primarily responsible for binding to an epitope of the antigen. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2 and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2 and LCDR 3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or combination of a number of well-known antibody CDR assignment systems, including, for example: chothia (Chothia et Al (1989) Nature 342: 877- & 883, Al-Lazikani et Al, "Standard constraints for the structural characterization of Immunological constructs", Journal of Molecular Biology, 273, 927- & 948(1997)), based on antibody sequence variations Kabat (Kabat et Al, Sequences of Proteins of Immunological constructs, 4 th edition, U.S. Department of Health and Human Services, National instruments of Health (1987)), AbM (fundamental of Molecular), activity compatibility, London, Molecular Biology (IMGT), and the bulk of the clustering of CDs (International patent publication).

For example, according to different CDR determination schemes, the residues of each CDR are as follows.

CDRs may also be determined based on the same Kabat numbering position as a reference CDR sequence (e.g., any one of the exemplary CDRs of the invention).

Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the ways described above.

Unless otherwise indicated, in the present invention, when referring to residue positions in the variable region of an antibody (including heavy chain variable region residues and light chain variable region residues), reference is made to the numbering positions according to the Kabat numbering system (Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

In one embodiment, the CDRs of an antibody of the invention are bounded by AbM rules, e.g., as shown in table 1.

However, it should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may differ. I.e., the CDR sequences of the same antibody variable region defined under different assignment systems differ. Thus, where reference is made to an antibody defined with a particular CDR sequence as defined herein, the scope of the antibody also encompasses an antibody whose variable region sequences comprise the particular CDR sequence but whose claimed CDR boundaries differ from the particular CDR boundaries as defined herein due to the application of different protocols (e.g., different assignment system rules or combinations).

Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. However, although CDRs vary from antibody to antibody, only a limited number of amino acid positions within a CDR are directly involved in antigen binding. Using at least two of Kabat, Chothia, AbM, Contact, and North methods, the region of minimum overlap can be determined, thereby providing a "minimum binding unit" for antigen binding. The minimum binding unit may be a sub-portion of the CDR. As will be appreciated by those skilled in the art, the residues in the remainder of the CDR sequences can be determined by the structure and protein folding of the antibody. Thus, the present invention also contemplates variants of any of the CDRs given herein. For example, in a variant of one CDR, the amino acid residue of the smallest binding unit may remain unchanged, while the remaining CDR residues according to Kabat or Chothia definition may be replaced by conserved amino acid residues.

The term "PD-1 axis binding antagonist" refers to a molecule that inhibits the interaction of the PD-1 axis binding partner with one or more of its binding partners, thereby removing T cell dysfunction resulting from signaling on the PD-1 signaling axis-one outcome is restoration or enhancement of T cell function (e.g., proliferation, cytokine production, target cell killing). As used herein, PD-1 axis binding antagonists include PD-1 binding antagonists (e.g., anti-PD-1 antibodies), PD-L1 binding antagonists (e.g., anti-PD-L1 antibodies), and PD-L2 binding antagonists (e.g., anti-PD-L2 antibodies).

The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners (such as PD-L1, PD-L2). In some embodiments, the PD-1 binding antagonist is a molecule that inhibits binding of PD-1 to one or more of its binding partners. In a particular aspect, the PD-1 binding antagonist inhibits PD-1 from binding to PD-L1 and/or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signal transduction resulting from the interaction of PD-1 with PD-L1 and/or PD-L2. In one embodiment, the PD-1 binding antagonist reduces a negative co-stimulatory signal (mediated signaling via PD-1) mediated by or via a cell surface protein expressed on a T lymphocyte, thereby rendering a dysfunctional T cell less dysfunctional (e.g., enhancing effector response to antigen recognition). In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In a specific embodiment, the PD-1 binding antagonist is MDX-1106(nivolumab), MK-3475(pembrolizumab), CT-011(pidilizumab), or AMP-224 as disclosed in WO 2015/095423. In one embodiment, the anti-PD-1 Antibody is "Antibody C" (WO 2017/133540).

The term "PD-L1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners (such as PD-1, B7-1). In some embodiments, the PD-L1 binding antagonist is a molecule that inhibits binding of PD-L1 to its binding partner. In a particular aspect, the PD-L1 binding antagonist inhibits PD-L1 from binding to PD-1 and/or B7-1. In some embodiments, PD-L1 binding antagonists include anti-PD-L1 antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners (such as PD-1, B7-1). In one embodiment, the PD-L1 binding antagonist reduces negative co-stimulatory signals mediated by or via cell surface proteins expressed on T lymphocytes (signal transduction mediated via PD-L1), thereby rendering dysfunctional T cells less dysfunctional (e.g., enhancing effector response to antigen recognition). In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In a particular aspect, the anti-PD-L1 antibody is yw243.55.s70, MDX-1105, MPDL3280A, or MEDI4736 disclosed in WO 2015/095423.

The term "PD-L2 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with signal transduction resulting from the interaction of PD-L2 with one or more of its binding partners (such as PD-1). In some embodiments, the PD-L2 binding antagonist is a molecule that inhibits binding of PD-L2 to one or more of its binding partners. In a particular aspect, the PD-L2 binding antagonist inhibits PD-L2 from binding to PD-1. In some embodiments, PD-L2 antagonists include anti-PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signal transduction resulting from the interaction of PD-L2 with one or more of its binding partners (such as PD-1). In one embodiment, the PD-L2 binding antagonist reduces negative co-stimulatory signals mediated by or via cell surface proteins expressed on T lymphocytes (signaling is mediated via PD-L2), thereby rendering dysfunctional T cells less dysfunctional (e.g., enhancing effector response to antigen recognition). In some embodiments, the PD-L2 binding antagonist is an immunoadhesin.

"antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cytotoxic form in which secreted immunoglobulins that bind to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) enable these cytotoxic effector cells to specifically bind to antigen-bearing target cells, followed by killing of the target cells with cytotoxins. The major cells mediating ADCC NK cells express Fc γ RIII only, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. Ravatch and Kinet, annu.rev.immunol.9: 457-92(1991) 464 Page table 3 summarizes FcR expression on hematopoietic cells. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay may be performed, such as described in U.S. Pat. No.5,500,362 or 5,821,337 or U.S. Pat. No.6,737,056 (Presta). Effector cells useful in such assays include PBMC and NK cells. Alternatively/additionally, ADCC activity of a molecule of interest may be assessed in vivo, for example in animal models, such as Clynes et al, pnas (usa) 95: 652-656 (1998). An exemplary assay for assessing ADCC activity is provided in the examples herein.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cell function and/or causes cell death or destruction.

"chemotherapeutic agents" include chemical compounds useful in the treatment of cancer. Examples of chemotherapeutic agents are disclosed in WO2015/031667, including but not limited to antineoplastic agents, including alkylating agents; an antimetabolite; a natural product; (ii) an antibiotic; an enzyme; a miscellaneous agent; hormones and antagonists; an antiestrogen; an antiandrogen; and non-steroidal antiandrogens and the like.

The term "small molecule drug" refers to low molecular weight organic compounds capable of modulating biological processes.

A "functional Fc region" possesses the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors; BCR), and the like. Such effector functions generally require that the Fc region be associated with a binding domain (e.g., an antibody variable domain) and can be evaluated using a variety of assays, such as those disclosed herein.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which region comprises at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In certain embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carbonyl end of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise indicated, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, which is also referred to as the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. public Health Service, National Institutes of Health, Bethesda, MD, 1991.

An "IgG-form antibody" refers to the IgG form to which the heavy chain constant region of an antibody belongs. The heavy chain constant regions are the same for all antibodies of the same type, and differ between antibodies of different types. For example, an antibody in the form of IgG1 refers to a heavy chain constant region, Ig, domain that is the Ig domain of IgG 1.

The term "therapeutic agent" as used herein encompasses any substance that is effective in preventing or treating tumors (e.g., cancer) and infections (e.g., chronic infections), including chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies, anti-infective agents, small molecule drugs, or immunomodulators.

The term "effective amount" refers to an amount or dose of an antibody or fragment or conjugate or composition of the invention which, upon administration to a patient in a single or multiple dose, produces the desired effect in the patient in need of treatment or prevention. An effective amount can be readily determined by the attending physician, as one skilled in the art, by considering a number of factors: species such as mammals; its size, age and general health; the specific diseases involved; the degree or severity of the disease; the response of the individual patient; the specific antibody administered; a mode of administration; bioavailability characteristics of the administered formulation; a selected dosing regimen; and the use of any concomitant therapies.

"therapeutically effective amount" means an amount effective, at dosages and for periods of time as required, to achieve the desired therapeutic result. The therapeutically effective amount of the antibody or antibody fragment, or conjugate or composition thereof, may vary depending on factors such as the disease state, the age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or deleterious effects of the antibody or antibody fragment or conjugate or composition thereof are less than the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 50%, 60%, or 70%, and still more preferably by at least about 80% or 90%, relative to an untreated subject. The ability of a compound to inhibit a measurable parameter (e.g., cancer) can be evaluated in an animal model system predictive of efficacy in human tumors.

A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time as required, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in a subject prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid is introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include primarily transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny screened or selected for the same function or biological activity in the originally transformed cell.

"human antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell or derived from a non-human source using a human antibody repertoire or other human antibody coding sequence. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues.

"humanized" antibodies refer to chimeric antibodies comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In some embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. "humanized forms" of antibodies (e.g., non-human antibodies) refer to antibodies that have been humanized.

The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth.

The term "tumor" refers to all neoplastic (neoplastic) cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein.

The term "infectious disease" refers to a disease caused by a pathogen, including, for example, viral infections, bacterial infections, fungal infections, or protozoan infections such as parasitic infections.

The term "chronic infection" refers to an infection in which an infectious agent (e.g., a pathogen such as a virus, bacterium, protozoan such as a parasite, fungus, or the like) has induced an immune response in the infected host, but has not been cleared or eliminated from the host as in the course of an acute infection. Chronic infection can be persistent, latent, or slow. Although acute infections (such as influenza) are usually resolved by the immune system within days or weeks, persistent infections can persist at relatively low levels for months, years, decades, or a lifetime (e.g., hepatitis b). In contrast, latent infections are characterized by long-term asymptomatic activity, interrupted by a period of rapidly increasing high-grade infection and elevated levels of pathogens (e.g., herpes simplex). Finally, slow infection is characterized by a gradual and continuous increase in disease symptoms, such as a long latency period, followed by the onset of a prolonged and progressive clinical course after the appearance of clinical symptoms.

An "immunoconjugate" is an antibody conjugated to one or more other substances, including but not limited to cytotoxic agents or labels.

The term "label" as used herein refers to a compound or composition that is conjugated or fused, directly or indirectly, to an agent (such as a polynucleotide probe or antibody) and facilitates detection of the agent to which it is conjugated or fused. The label may be detectable by itself (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze chemical alteration of a detectable substrate compound or composition. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody as well as indirect labeling of the probe or antibody by reaction with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.

An "individual" or "subject" includes a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

An "isolated" antibody is one that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al, j.chromatogr.b848: 79-87(2007).

An "isolated nucleic acid encoding an anti-GITR antibody or fragment thereof" refers to one or more nucleic acid molecules encoding an antibody heavy or light chain (or fragment thereof), including such nucleic acid molecules in a single vector or separate vectors, as well as such nucleic acid molecules present at one or more locations in a host cell.

The calculation of sequence identity between sequences is performed as follows.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences can be discarded for comparison purposes). In a preferred embodiment, the length of the aligned reference sequences is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence for comparison purposes. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.

Sequence comparisons between two sequences and calculation of percent identity can be accomplished using mathematical algorithms. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needlema and Wunsch ((1970) J.mol.biol.48: 444-. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http:// www.gcg.com), using the NWSgapdna. CMP matrix and GAP weights 40, 50, 60, 70 or 80 and

length weights

1, 2, 3, 4,5 or 6. A particularly preferred set of parameters (and one that should be used unless otherwise specified) is the Blossum 62 scoring matrix using a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

PAM120 weighted residue table, gap length penalty 12, gap penalty 4) can also be used, using the e.meyers and w.miller algorithms that have been incorporated into the ALIGN program (version 2.0) ((1989) cabaos, 4: 11-17) determining the percentage identity between two amino acid sequences or nucleotide sequences.

Additionally or alternatively, the nucleic acid sequences and protein sequences described herein may be further used as "query sequences" to perform searches against public databases, for example, to identify other family member sequences or related sequences.

The term "pharmaceutical excipient" refers to diluents, adjuvants (e.g., freund's adjuvant (complete and incomplete)), excipients, carriers, stabilizers or the like, which are administered with the active substance.

The term "pharmaceutical composition" refers to a composition that is present in a form that allows for the biological activity of the active ingredients contained therein to be effective, and that does not contain additional ingredients that have unacceptable toxicity to the subject to which the composition is administered.

The term "combination product" refers to a kit of parts for combined administration or a fixed combination or a non-fixed combination or for combined administration in the form of a dosage unit, wherein two or more therapeutic agents may be administered separately at the same time or within time intervals, especially when these time intervals allow the combination partners to exhibit a synergistic, e.g. synergistic, effect. The term "fixed combination" means that the antibody of the invention and a combination partner (e.g. other therapeutic agent, such as an anti-PD 1 antibody, an anti-PDL 1 antibody or an anti-PDL 2 antibody) are administered to a patient simultaneously, in the form of a single entity or dose. The term "non-fixed combination" means that the antibody of the invention and the combination partner (e.g. the other therapeutic agent, e.g. the anti-PD 1 antibody, the anti-PDL 1 antibody or the anti-PDL 2 antibody) are administered to the patient as separate entities simultaneously, concurrently or sequentially, without specific time constraints, wherein such administration provides therapeutically effective levels of both compounds in the patient. The latter also applies to cocktail therapies, such as the administration of three or more therapeutic agents. In a preferred embodiment, the pharmaceutical combination is a non-fixed combination.

The term "combination therapy" refers to the administration of two or more therapeutic agents to treat cancer or infection as described in the present disclosure. Such administration includes co-administering the therapeutic agents in a substantially simultaneous manner, for example, in a single capsule having a fixed ratio of active ingredients. Alternatively, such administration includes co-administration of the active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). The powder and/or liquid may be reconstituted or diluted to the desired dosage prior to administration. In addition, such administration also includes the use of each type of therapeutic agent at approximately the same time or in a sequential manner at different times. In either case, the treatment regimen will provide a beneficial effect of the drug combination in treating the disorders or conditions described herein.

As used herein, "treating" refers to slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

As used herein, "prevention" includes inhibition of the occurrence or development of a disease or disorder or a symptom of a particular disease or disorder. In some embodiments, subjects with a family history of cancer are candidates for a prophylactic regimen. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in a subject at risk for cancer.

The term "anti-infective active agent" includes any molecule that specifically inhibits or eliminates the growth of a microorganism, such as a virus, bacterium, fungus, or protozoan, e.g., a parasite, at the administered concentration and dosing interval, but is not lethal to the host. As used herein, the term anti-infective active agent includes antibiotics, antibacterials, antivirals, antifungals, and antiprotozoals. In a particular aspect, the anti-infective active agent is non-toxic to the host at the administration concentration and dosing interval.

Anti-infective actives or antimicrobials that are antibacterial may be broadly classified as either bactericidal (i.e., direct killing) or bacteriostatic (i.e., preventing division). Antimicrobial anti-infective actives may be further sub-classified as either narrow spectrum antimicrobials (i.e., affecting only a small subset of bacteria, e.g., gram negative, etc.) or broad spectrum antimicrobials (i.e., affecting a wide variety).

The term "antiviral agent" includes any substance that inhibits or eliminates viral growth, pathogenesis and/or survival.

The term "antifungal agent" includes any substance that inhibits or eliminates fungal growth, disease, and/or survival.

The term "antiprotozoal agent" includes any substance that inhibits or eliminates the growth, pathogenesis and/or survival of a protozoan organism (e.g., a parasite).

The term "vector" when used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors which are self-replicating nucleic acid structures as well as vectors which are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors".

"subject/patient sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample may be a solid tissue, like from a fresh, frozen and/or preserved organ or tissue sample or biopsy sample or punch sample; blood or any blood component; body fluids such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from a subject at any time of pregnancy or development. Tissue samples may contain compounds that are not naturally intermixed with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. Examples of tumor samples herein include, but are not limited to, tumor biopsies, fine needle aspirates, bronchial lavage, pleural fluid (pleural fluid), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples, such as formalin-fixed, paraffin-embedded tumor samples or frozen tumor samples.

Antibodies

In some embodiments, the anti-GITR antibodies or fragments thereof of the invention bind GITR (e.g., human GITR or cynomolgus GITR) with high affinity, e.g., with the following equilibrium dissociation constant (K) _D ) Binding to GITR, said K _D Less than about 10nM, preferably less than or equal to about 7nM, more preferably less than or equal to about 7nMAbout 6nM, more preferably less than or equal to about 5nM, 4.9nM, 4.8nM, 4.7nM, 4.6nM, 4.5nM, 4nM, or 3nM, and most preferably the K _D Less than or equal to about 2.5nM, 2.4nM, 2.3nM, 2.2nM, 2.1 nM. In some embodiments, the anti-GITR antibodies of the invention have a K of 1nM to 6nM, preferably 1.5nM to 5nM, 1.7nM to 5nM, 1.8nM to 5nM, 1.9nM to 5nM _D Binds GITR. In some embodiments, the GITR is human GITR. In some embodiments, antibody binding affinity is determined using a biological light interferometry (e.g., Fortebio affinity measurement).

In some embodiments, an antibody or fragment thereof of the invention binds to a GITR-expressing cell, e.g., with an EC50 of less than or equal to about 2nM, 1.9nM, 1.5nM, 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4 nM. In some embodiments, the binding is determined by flow cytometry (e.g., FACS). In some embodiments, the GITR-expressing cells are GITR-expressing CHO cells (e.g., CHO-S cells). In some embodiments, the GITR is human GITR or cynomolgus GITR.

In some embodiments, the antibodies or fragments thereof of the invention activate the NF-kappaB signaling pathway downstream of GITR by binding to GITR molecules on the cell surface. In some embodiments, the antibodies or fragments thereof of the invention have superior activation capacity over known anti-GITR antibodies, such as those reported in patent application US20130183321a1(GITR, INC. (Cambridge, MA, US)). In one embodiment, the light and heavy chain sequences of known anti-GITR antibody molecules are SEQ ID NOs: 44 and SEQ ID NO: 54. in some embodiments, GITR antibody molecules of the invention are capable of significantly and effectively activating the NF kappa B signaling pathway compared to control antibody molecules. In some embodiments, the activation is obtained by luciferase assay.

In some embodiments, an antibody or fragment thereof of the invention increases effector T cell function, e.g., activates effector T cells. In some embodiments, the antibodies or fragments thereof of the invention are capable of enhancing the proliferation of effector T cells. In some embodiments, an antibody or fragment thereof of the invention increases interferon (e.g., IFN- γ) secretion/expression. In some embodiments, an antibody or fragment thereof of the invention increases interleukin (e.g., IL-2) secretion/expression. In some embodiments, the T cell is a CD4T cell.

In some embodiments, the GITR antibodies or fragments thereof of the present invention are capable of activating GITR signaling pathways in regulatory T cells (e.g., Treg cells), thereby depleting the cells by mediating ADCC effects. Therefore, on one hand, the inhibition signal of effector T cells is relieved, the activity of CD4T cells and/or CD8T cells is enhanced, and on the other hand, the inhibition effect of regulatory T cells on the effector T cells is relieved, so that the effector T cells are activated to the maximum extent, and the immune response reaction aiming at tumor cells is enhanced. In some embodiments, the ADCC activity of the antibody is detected by detecting NF-AT signal activation. In some embodiments, the activation of the NF-AT signal is represented by the expression of a fluorescent reporter. In some embodiments, GITR antibody molecules of the invention are capable of significantly effective activation of NF-AT signaling pathways as compared to known anti-GITR antibody molecules. In some embodiments, GITR antibody molecules of the invention are capable of significantly effectively mediating ADCC effects as compared to known anti-GITR antibody molecules. In some embodiments, GITR antibody molecules of the invention are capable of significantly effective inhibition or elimination of regulatory T cells as compared to known anti-GITR antibody molecules. In some embodiments, the effector T cells of the invention are CD4T cells. In some embodiments, the known anti-GITR antibody molecule is an anti-GITR antibody, for example, as reported in patent application US20130183321a1(GITR, INC. (Cambridge, MA, US)). In one embodiment, the light and heavy chain sequences of known anti-GITR antibody molecules are in US20130183321a1 as SEQ ID NOs: 44 and SEQ ID NO: 54.

in some embodiments, the antibodies or fragments thereof of the invention (optionally in combination with a therapeutic modality and/or other therapeutic agent, e.g., a PD-1 axis binding antagonist) are capable of preventing or treating a tumor. In some embodiments, the antibodies or fragments thereof of the invention can be used to inhibit or reduce tumor growth (e.g., reduce tumor volume). In some embodiments, the antibodies or fragments thereof of the invention can also be used to maintain tumor patient body weight. In some embodiments, the antibodies or fragments thereof of the invention are capable of inhibiting or reducing tumor growth (e.g., reducing tumor volume) in combination with a therapeutic modality and/or other therapeutic agent, such as a PD 1-axis binding antagonist. In some embodiments, the antibodies or fragments thereof of the invention are capable of maintaining tumor patient body weight in combination with a therapeutic modality and/or other therapeutic agent, such as a PD 1-axis binding antagonist. In some embodiments, an antibody or fragment thereof of the invention (optionally in combination with a therapeutic modality and/or other therapeutic agent, e.g., a PD-1 axis binding antagonist) is capable of inhibiting or reducing tumor growth (e.g., reducing tumor volume) while not affecting tumor patient body weight. Preferably, the neoplasm is a gastrointestinal tumor. Preferably, the tumor is a cancer, such as a cancer of the gastrointestinal tract, e.g., gastric, rectal, colon, colorectal, etc. In some embodiments, the antibodies or fragments thereof of the invention (optionally in combination with a PD-1 axis binding antagonist) are capable of preventing or treating an infection, e.g., a chronic infection, including a bacterial infection, a fungal infection, a viral infection, a protozoan infection, and the like.

In some embodiments, the anti-GITR antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH), wherein the VH comprises

(i) Three Complementarity Determining Regions (CDRs) contained in the VH of any one of the antibodies listed in Table B, or

(ii) (ii) sequences which collectively comprise at least one and no more than 5, 4, 3, 2 or 1 amino acid alterations (preferably amino acid substitutions, preferably conservative substitutions) in the three CDR regions relative to the sequence of (i).

In some embodiments, the anti-GITR antibody or antigen-binding fragment thereof of the present invention comprises a light chain variable region (VL), wherein the VL comprises:

(i) three Complementarity Determining Regions (CDRs) contained in the VL of any one of the antibodies listed in table B; or

In some embodiments, the anti-GITR antibodies or antigen-binding fragments thereof of the invention comprise a heavy chain variable region VH and a light chain variable region VL, wherein

(a) Said VH comprises

(ii) (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid alterations (preferably amino acid substitutions, preferably conservative substitutions) in total on the three CDR regions relative to the sequence of (i); and/or

(b) The VL comprises:

In a preferred embodiment, the VH comprises a sequence selected from SEQ ID NO: 17. 18, 19 or 20, or consists of said amino acid sequence.

In a preferred embodiment, the VL comprises an amino acid sequence selected from SEQ ID NOs: 21. 22, 23 or 24, or consists of said amino acid sequence.

In a preferred embodiment, the anti-GITR antibodies or antigen-binding fragments thereof of the present invention comprise

(i) As shown in SEQ ID NO:17 or 19, and 3 complementarity determining regions HCDR of the heavy chain variable region set forth in SEQ ID NO:21 or 23, or 3 complementarity determining regions LCDR of the light chain variable region, or

(ii) As shown in SEQ ID NO:18 or 20, and 3 complementarity determining regions HCDR of the heavy chain variable region set forth in SEQ ID NO:22 or 24, and 3 complementarity determining regions, LCDRs, of the light chain variable region.

In some embodiments, the anti-GITR antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein

(i) The VH comprises Complementarity Determining Regions (CDRs) HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises a sequence selected from SEQ ID NO: 1. 2, 3 or 4, or consists of said amino acid sequence, or HCDR1 comprising a sequence identical to or consisting of SEQ ID NO: 1. 2, 3 or 4 with one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of the polypeptide; HCDR2 comprises an amino acid sequence selected from SEQ ID NO:5 or 6, or consists of said amino acid sequence, or HCDR2 comprising a sequence identical to or consisting of SEQ ID NO:5 or 6 with one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence; HCDR3 comprises a sequence selected from SEQ ID NO:7 or 8, or HCDR3 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:7 or 8 with one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence;

and/or

(ii) Wherein the VL comprises Complementarity Determining Regions (CDRs) LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 or 10, or consists of said amino acid sequence, or LCDR1 comprising a nucleotide sequence identical to or selected from the group consisting of SEQ ID NO:9 or 10 with one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of the polypeptide; LCDR2 comprising a nucleotide sequence selected from SEQ ID NO: 11. 12, 13 or 14 or LCDR2 comprising or consisting of an amino acid sequence selected from SEQ ID NO: 11. 12, 13 or 14 with one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence; LCDR3 comprising a nucleotide sequence selected from SEQ ID NO:15 or 16 or consists of said amino acid sequence, or LCDR3 comprising a nucleotide sequence identical to or consisting of an amino acid sequence selected from SEQ ID NO:15 or 16 with one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence.

In a preferred embodiment, the present invention provides an anti-GITR antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein

(a) Said VH comprises

(i) The combinations of HCDR1, HCDR2, and HCDR3 shown in table a; or

(ii) (ii) a variant of the HCDR combination of (i) which comprises in total at least one and no more than 5, 4, 3, 2 or 1 amino acid alterations (preferably amino acid substitutions, preferably conservative substitutions) in the three CDR regions;

and/or

(ii) Said VL comprising

(i) The combination of LCDR1, LCDR2 and LCDR3 shown in table a; or

(ii) (ii) a variant of the LCDR combination of (i) which comprises in total at least one and no more than 5, 4, 3, 2 or 1 amino acid alterations (preferably amino acid substitutions, preferably conservative substitutions) in said three CDR regions.

In a preferred embodiment, the invention provides an anti-GITR antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising Complementarity Determining Regions (CDRs) HCDR1, HCDR2 and HCDR3 and/or a light chain variable region (VL) Comprising (CDRs) LCDR1, LCDR2 and LCDR3, wherein the antibody or antigen-binding fragment thereof comprises a combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in the following table (table a):

table a: exemplary combinations of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 in an antibody or antigen-binding fragment thereof of the invention

In some embodiments, the anti-GITR antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region VH and/or a light chain variable region VL, wherein,

(a) heavy chain variable region VH

(i) Comprising a nucleotide sequence substantially identical to a sequence selected from SEQ ID NO: 17. 18, 19 or 20, or an amino acid sequence consisting of or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity; or alternatively

(ii) Comprises a sequence selected from SEQ ID NO: 17. 18, 19 or 20 or consists thereof; or alternatively

(iii) Comprising a nucleotide sequence substantially identical to a sequence selected from SEQ ID NO: 17. 18, 19 or 20 (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to an amino acid sequence having 1 or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions), preferably the amino acid changes do not occur in the CDR regions;

and/or

(b) Light chain variable region VL

(i) Comprising a nucleotide sequence substantially identical to a sequence selected from SEQ ID NO: 21. 22, 23 or 24 has or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity;

(ii) comprising a nucleic acid sequence selected from SEQ ID NO: 21. 22, 23 or 24 or consists thereof; or

(iii) Comprising a nucleotide sequence substantially identical to a sequence selected from SEQ ID NO: 21. 22, 23 or 24 (preferably amino acid substitutions, more preferably amino acid conservative substitutions) with 1 or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions), preferably the amino acid changes do not occur in the CDR regions.

In a preferred embodiment, the present invention provides an anti-GITR antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the antibody or antigen-binding fragment thereof comprises a combination of a heavy chain variable region VH and a light chain variable region VL as set forth in the following table (table B):

table B: exemplary combinations of heavy chain variable region VH and light chain variable region VL in an antibody or antigen binding fragment thereof of the invention

In some embodiments, the anti-GITR antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain and/or a light chain, wherein

(a) Heavy chain

(i) Comprising a nucleotide sequence substantially identical to a sequence selected from SEQ ID NO: 25. 26, 27 or 28, or an amino acid sequence consisting of or having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity;

(ii) comprising a nucleic acid sequence selected from SEQ ID NO: 25. 26, 27 or 28 or consists thereof; or alternatively

(iii) Comprising a nucleotide sequence substantially identical to a sequence selected from SEQ ID NO: 25. 26, 27 or 28 (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions), preferably the amino acid changes do not occur in the CDR regions of the heavy chain, more preferably the amino acid changes do not occur in the heavy chain variable region;

and/or

(b) Light chains

(i) Comprising a nucleotide sequence substantially identical to a sequence selected from SEQ ID NO: 29. 30, 31 or 32, or an amino acid sequence consisting of or having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity;

(ii) comprises a sequence selected from SEQ ID NO: 29. 30, 31 or 32 or consists thereof; or

(iii) Comprising a nucleotide sequence substantially identical to a sequence selected from SEQ ID NO: 29. 30, 31 or 32 (preferably, no more than 20 or 10, more preferably, no more than 5, 4, 3, 2, 1) amino acid changes (preferably, amino acid substitutions, more preferably, amino acid conservative substitutions), preferably, the amino acid changes do not occur in the CDR regions of the light chain, more preferably, the amino acid changes do not occur in the light chain variable region.

In a preferred embodiment, the present invention provides an anti-GITR antibody, or antigen-binding fragment thereof, comprising a heavy chain and a light chain, wherein the antibody, or antigen-binding fragment thereof, comprises a combination of heavy and light chains as set forth in the following table (table C):

table C: exemplary combinations of heavy and light chains in an antibody or antigen-binding fragment thereof of the invention

In some embodiments, the heavy and/or light chain of an anti-GITR antibody or fragment thereof of the invention further comprises a signal peptide sequence, e.g., METDTLLLWVLLLWVPGSTG (SEQ ID NO: 43).

In one embodiment of the invention, the amino acid alterations described herein comprise amino acid substitutions, insertions or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

In a preferred embodiment, the amino acid changes described herein occur in regions outside the CDRs (e.g., in the FRs). More preferably, the amino acid changes of the invention occur in regions outside the heavy chain variable region and/or outside the light chain variable region.

In some embodiments, the substitution is a conservative substitution. Conservative substitutions are those where one amino acid is substituted with another within the same class, for example, where one acidic amino acid is substituted with another acidic amino acid, one basic amino acid is substituted with another basic amino acid, or one neutral amino acid is substituted with another neutral amino acid. Exemplary substitutions are shown in table D below:

table D

Original residues	Exemplary permutations	Preferred conservative amino acid substitutions
			Ala(A)	Val、Leu、Ile	Val
Arg(R)	Lys、Gln、Asn	Lys
			Asn(N)	Gln、His、Asp、Lys、Arg	Gln
Asp(D)	Glu、Asn	Glu
			Cys(C)	Ser、Ala	Ser
Gln(Q)	Asn、Glu	Asn
			Glu(E)	Asp、Gln	Asp
Gly(G)	Ala	Ala
			His(H)	Asn、Gln、Lys、Arg	Arg
Ile(I)	Leu, Val, Met, Ala, Phe, norleucine	Leu
			Leu(L)	Norleucine, Ile, Val, Met, Ala, Phe	Ile
Lys(K)	Arg、Gln、Asn	Arg
			Met(M)	Leu、Phe、Ile	Leu
Phe(F)	Trp、Leu、Val、Ile、Ala、Tyr	Tyr
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Val、Ser	Ser
Trp(W)	Tyr、Phe	Tyr
			Tyr(Y)	Trp、Phe、Thr、Ser	Phe
Val(V)	Ile, Leu, Met, Phe, Ala, norleucine	Leu

In certain embodiments, the substitutions occur in the CDR regions of the antibody. Typically, the obtained variant will have a modification (e.g., an improvement) in certain biological properties (e.g., increased affinity) relative to the parent antibody and/or will have certain biological properties of the parent antibody that are substantially retained. Exemplary substitutional variants are affinity matured antibodies.

In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites of an antibody can be conveniently achieved by altering the amino acid sequence so as to create or remove one or more glycosylation sites. When the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. In some applications, modifications that remove unwanted glycosylation sites may be useful, for example, to remove fucose moieties to enhance antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al (2002) JBC 277: 26733). In other applications, galactosylation modifications may be made to modify Complement Dependent Cytotoxicity (CDC).

In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein to produce a variant Fc region, in order to enhance the effectiveness of the antibody in treating, for example, cancer or a cell proliferative disease. Fc region variants may include human Fc region sequences (e.g., human IgGl, IgG2, IgG3, or IgG4Fc regions) that include amino acid modifications (e.g., substitutions) at one or more amino acid positions. For examples of Fc variants see U.S. Pat. nos. 7,332,581, U.S. Pat. No.6,737,056; WO 2004/056312 and Shields et al, j.biol.chem.9 (2): 6591 and 6604(2001), U.S. Pat. No.6,194,551, WO 99/51642 and Idusogene et al J.Immunol.164: 4178-: 738-40 (1988); U.S. Pat. nos. 5,648,260; U.S. Pat. nos. 5,624,821; and WO 94/29351.

In certain embodiments, it may be desirable to generate cysteine engineered antibodies, such as "thio mabs," in which one or more residues of the antibody are replaced with a cysteine residue. Cysteine engineered antibodies can be produced as described, for example, in U.S. patent No. 7,521,541.

In certain embodiments, the antibodies provided herein can be further modified to contain other non-protein moieties known and readily available in the art. Suitable antibody-derived moieties include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyaminoacids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

In some embodiments, the anti-GITR antibodies or antigen-binding fragments thereof of the present invention have one or more of the following properties:

(i) exhibits the same or similar binding affinity and/or specificity for GITR as any of the antibodies listed in table 3;

(ii) inhibiting (e.g., competitively inhibiting) the binding of any of the antibodies listed in table 3 to GITR;

(iii) binds to the same or an overlapping epitope as any one of the antibodies shown in table 3;

(iv) competes for binding to GITR with any one of the antibodies shown in table 3;

(v) having one or more of the biological properties of any of the antibody molecules listed in table 3.

In some embodiments, the anti-GITR antibody of the invention is an antibody of the IgG1 format or an antibody of the IgG2 format or an antibody of the IgG4 format.

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

In some embodiments, the anti-GITR antibody is humanized. Different methods for humanizing antibodies are known to the skilled artisan, as reviewed by Almagro & Fransson, the contents of which are incorporated herein by reference in their entirety (Almagro JC and Fransson J (2008) Frontiers in bioscience 13: 1619-1633).

In some embodiments, the anti-GITR antibody is a human antibody. Human antibodies can be made using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, curr. 368-74(2001) and Lonberg, curr. opin. immunol 20: 450-459(2008).

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

In some embodiments, at least a portion of the framework sequence of the anti-GITR antibody is a human consensus framework sequence. In one embodiment, the anti-GITR antibodies of the invention also encompass antibody fragments thereof, preferably antibody fragments selected from the group consisting of: fab, Fab '-SH, Fv, single chain antibodies (e.g., scFv) or (Fab') ₂ A single domain antibody, a diabody (dAb) or a linear antibody.

In certain embodiments, the anti-GITR antibody molecule is in the form of a bispecific or multispecific antibody molecule. In one embodiment, the bispecific antibody molecule has a first binding specificity for GITR and a second binding specificity for PD-1 or PD-L1 or PD-L2. In one embodiment, the bispecific antibody molecule binds to GITR and PD-1. In another embodiment, the bispecific antibody molecule binds to GITR and PD-L1. In yet another embodiment, the bispecific antibody molecule binds to GITR and PD-L2. Multispecific antibody molecules may have any combination of binding specificities for the foregoing molecules. The multispecific antibody molecule may, for example, be a trispecific antibody molecule comprising a first binding specificity for GITR and second and third binding specificities for molecules of one or more of: PD-1, PD-L1 or PD-L2.

Nucleic acids of the invention and host cells comprising the same

In one aspect, the invention provides a nucleic acid encoding any of the above anti-GITR antibodies or fragments thereof. In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell comprising said nucleic acid or said vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell (e.g., a CHO cell or 293 cell), or other cell suitable for production of an antibody or antigen-binding fragment thereof. In another embodiment, the host cell is prokaryotic.

In one aspect, the invention provides a nucleic acid encoding any of the above anti-GITR antibodies or fragments thereof. The nucleic acid may comprise a nucleic acid encoding an amino acid sequence of a light chain variable region and/or a heavy chain variable region of an antibody, or a nucleic acid encoding an amino acid sequence of a light chain and/or a heavy chain of an antibody. An exemplary nucleic acid sequence encoding an antibody heavy chain variable region comprises a nucleotide sequence substantially identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 33. 34, 35 or 36, or a nucleic acid sequence comprising at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a nucleic acid sequence selected from SEQ ID NOs: 33. 34, 35 or 36. An exemplary nucleic acid sequence encoding an antibody light chain variable region comprises a nucleotide sequence identical to a sequence selected from the group consisting of SEQ ID NOs: 37. 38, 39 or 40, or a nucleic acid sequence comprising a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a nucleic acid sequence selected from SEQ ID NOs: 37. 38, 39 or 40.

In one embodiment, one or more vectors comprising the nucleic acid are provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or Yeast Artificial Chromosomes (YACs). In one embodiment, the vector is the pTT5 vector.

In one embodiment, a host cell comprising the vector is provided. Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199 and 5,840,523, and also Charlton, Methods in Molecular Biology, Vol.248 (B.K.C.Lo, eds., Humana Press, Totowa, NJ, 2003), page 245-. After expression, the antibody can be isolated from the bacterial cell paste in the soluble fraction and can be further purified.

In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell, or other cell suitable for the production of an antibody or antigen-binding fragment thereof. For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors. For example, fungal and yeast strains in which the glycosylation pathway has been "humanized" result in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, nat. biotech.22: 1409-: 210-215(2006). Host cells suitable for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, mammalian cell lines engineered to be suitable for growth in suspension may be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney lines (293HEK or 293F or 293 cells, as described, for example, in Graham et al, J.Gen Viro 1.36: 59 (1977)), and the like. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al, Proc. Natl. Acad. Sci. USA 77: 216(1980), CHO-S cells, etc., and myeloma cell lines such as Y0, NS0, and Sp 2/0. for reviews of certain mammalian host cell lines suitable for antibody production see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol.248 (B.K.C.Lo, ed., Humana Press, Totowwa, NJ), pp.255-268 (2003).

In one embodiment, the invention provides a method of making an anti-GITR antibody or fragment thereof (preferably an antigen-binding fragment), wherein the method comprises culturing the host cell under conditions suitable for expression of a nucleic acid encoding the antibody or fragment thereof (preferably an antigen-binding fragment), and optionally isolating the antibody or fragment thereof (preferably an antigen-binding fragment). In a certain embodiment, the method further comprises recovering the anti-GITR antibody or fragment thereof (preferably an antigen-binding fragment) from the host cell.

In one embodiment, a method of making an anti-GITR antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium). For recombinant production of anti-GITR antibodies, nucleic acids encoding the antibodies (e.g., the antibodies described above) are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids are readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of an antibody).

Assay method

The anti-GITR antibodies provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activities by a variety of assays known in the art. In one aspect, antibodies of the invention are tested for antigen binding activity, for example, by known methods such as ELISA, Western blot, and the like. Binding to GITR can be determined using methods known in the art, exemplary methods are disclosed herein. In some embodiments, a biophotonic interferometry (e.g., Fortebio affinity measurement) or MSD assay is used.

In another aspect, a competition assay can be used to identify antibodies that compete for binding to GITR with any of the anti-GITR antibodies disclosed herein. In certain embodiments, such competitive antibodies bind to the same or overlapping epitopes (e.g., linear or conformational epitopes) as any of the anti-GITR antibodies disclosed herein. A detailed exemplary method for locating epitopes bound by antibodies is described in Morris (1996) "Epitope Mapping Protocols", Methods in Molecular Biology vol.66(Humana Press, Totowa, NJ).

The invention also provides assays for identifying anti-GITR antibodies that are biologically active. Biological activities may include, for example, binding to GITR (e.g., binding to human and/or cynomolgus GITR), increasing GITR-mediated signal transduction (e.g., increasing NFkappa-B signaling pathway), depleting GITR-expressing cells (e.g., Treg cells) by ADCC, enhancing T effector cell function (e.g., CD4 effector T cells) (e.g., by increasing cytokine production by effector T cells (e.g., interferons such as IFN- γ or interleukins such as IL 2)). Also provided are antibodies having such biological activity in vivo and/or in vitro.

In certain embodiments, antibodies of the invention are tested for such biological activity.

T cell activation can be determined using methods known in the art. For example, by the level of cytokines, e.g., interferons (e.g., IFN-. gamma.) or interleukins (e.g., IL2), released upon T cell activation. Activation of T cells can also be measured using methods well known in the art to measure GITR signaling (e.g., NF-kappaB signaling pathway). In one embodiment, transgenic cells are generated that express human GITR and a reporter gene comprising the NF-kappa B promoter fused to a reporter gene (e.g., beta luciferase). Addition of anti-GITR antibody to the cells resulted in increased NF-kappa B transcription, which was detected using an assay directed to a reporter gene (e.g., luciferase reporter assay).

ADCC effects of antibodies can be determined using methods known in the art. For example by NF-AT signals. In one embodiment, transgenic cells are obtained that have ADCC effector cells comprising the NF-AT promoter fused to a reporter gene (e.g. beta luciferase). Co-culture of effector cells with high GITR-expressing cells, with the addition of anti-GITR antibody, results in increased NF-AT transcription of the effector cells, which is detected using an assay for a reporter gene (e.g., luciferase reporter assay).

Cells for use in any of the above in vitro assays include cell lines that naturally express GITR or are engineered to express GITR. Such cells include T cells that naturally express GITR, Treg cells, and activated memory T cells. Such cells also include cell lines transfected with GITR-expressing and GITR-encoding DNA that does not normally express GITR.

It will be appreciated that any of the above assays can be performed using the immunoconjugates of the invention in place of or in addition to anti-GITR antibodies.

It will be appreciated that any of the above assays can be performed using anti-GITR antibodies and other active agents.

Immunoconjugates

In some embodiments, the invention provides immunoconjugates comprising any of the anti-GITR antibodies provided herein and an additional substance, such as a cytotoxic agent. In some embodiments, the other agent is, for example, a therapeutic agent, such as a cytotoxic agent or an immunosuppressive or chemotherapeutic agent. Cytotoxic agents include any agent that is harmful to cells. Examples of cytotoxic agents (e.g., chemotherapeutic agents) suitable for forming immunoconjugates are known in the art. For example, cytotoxic agents include, but are not limited to: a radioisotope; a growth inhibitor; enzymes and fragments thereof such as nucleic acid hydrolases; (ii) an antibiotic; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various known antitumor or anticancer agents.

In some embodiments, the immunoconjugate is for use in the prevention or treatment of a tumor, e.g., a gastrointestinal tumor. In some embodiments, the tumor is a cancer, e.g., a gastrointestinal cancer, e.g., gastric cancer, rectal cancer, colon cancer, colorectal cancer, and the like. In some embodiments, the immunoconjugate is used for the prevention or treatment of infection, e.g., chronic infection, e.g., bacterial infection, viral infection, fungal infection, protozoan infection, and the like.

Pharmaceutical compositions and pharmaceutical formulations

In some embodiments, the invention provides a composition comprising any of the anti-GITR antibodies or fragments thereof (preferably antigen-binding fragments thereof) or immunoconjugates thereof described herein, preferably the composition is a pharmaceutical composition. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, a composition, e.g., a pharmaceutical composition, comprises a combination of an anti-GITR antibody, or fragment thereof, or immunoconjugate thereof, of the invention, and one or more other therapeutic agents (e.g., a chemotherapeutic agent, a cytotoxic agent, a vaccine, other antibodies, an anti-infective active agent, a small molecule drug, or an immunomodulator, preferably a PD-1 axis binding antagonist, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody).

In some embodiments, the composition is for preventing or treating a tumor, for example a gastrointestinal tumor. In some embodiments, the tumor is a cancer, e.g., a cancer of the gastrointestinal tract, e.g., gastric cancer, rectal cancer, colon cancer, colorectal cancer, and the like. In some embodiments, the compositions are used to prevent or treat an infection, e.g., a chronic infection, such as a bacterial infection, a viral infection, a fungal infection, a protozoal infection, and the like.

The invention also includes compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising an anti-GITR antibody or immunoconjugate thereof, and compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising a polynucleotide encoding an anti-GITR antibody. In certain embodiments, the compositions comprise one or more antibodies or fragments thereof that bind GITR or one or more polynucleotides encoding one or more antibodies or fragments thereof that bind GITR. These compositions may also contain suitable pharmaceutical excipients such as pharmaceutically acceptable carriers, excipients, including buffers, as are known in the art.

Pharmaceutical carriers suitable for use in the present invention can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. For the use of excipients and their use, see also "Handbook of pharmaceutical excipients", fifth edition, r.c. rowe, p.j.seskey and s.c. owen, pharmaceutical press, London, Chicago. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin.

Pharmaceutical formulations comprising the anti-GITR antibodies described herein can be prepared by mixing the anti-GITR antibodies of the invention with the desired purity with one or more optional Pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16 th edition, Osol, a. eds. (1980)), preferably in the form of a lyophilized formulation or an aqueous solution.

Exemplary lyophilized antibody formulations are described in U.S. Pat. No.6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No.6,171,586 and WO2006/044908, the latter formulation including histidine-acetate buffer.

The pharmaceutical compositions or formulations of the present invention may also contain one or more other active ingredients that are required for the particular indication being treated, preferably those active ingredients that have complementary activities that do not adversely affect each other. For example, it may be desirable to also provide other anti-cancer active ingredients, such as chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies, anti-infective active agents, small molecule drugs, or immunomodulators, such as PD-1 axis binding antagonists (e.g., anti-PD-1 antibodies or anti-PD-L1 antibodies or anti-PD-L2 antibodies), and the like. The active ingredients are suitably present in combination in an amount effective for the intended use.

Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

For further components of pharmaceutical formulations/pharmaceutical compositions comprising the antibodies of the invention, see also those disclosed in WO2015/031667 or WO2015/187835 etc.

VI. combination product

In some embodiments, the invention also provides a combination product comprising an antibody or antigen-binding fragment thereof of the invention, or an immunoconjugate thereof, and one or more additional therapeutic agents (e.g., chemotherapeutic agents, additional antibodies, cytotoxic agents, vaccines, anti-infective active agents, small molecule drugs, or immunomodulators, and the like). In some embodiments, the other antibody is, for example, an anti-PD-1 antibody or an anti-PD-L1 antibody or an anti-PD-L2 antibody.

In some embodiments, the combination product is for use in the prevention or treatment of a neoplasm, for example a neoplasm of the gastrointestinal tract. In some embodiments, the tumor is a cancer, e.g., a gastrointestinal cancer, e.g., gastric cancer, rectal cancer, colon cancer, colorectal cancer, and the like. In some embodiments, the combination product is used to prevent or treat an infection, e.g., a chronic infection, such as a bacterial infection, a viral infection, a fungal infection, a protozoan infection, and the like.

Use of

In one aspect, the invention provides a method of activating the NF-KappaB signaling pathway in a subject, comprising administering to the subject an effective amount of an anti-GITR antibody or antigen-binding fragment thereof, immunoconjugate, pharmaceutical composition, or combination product of the invention.

In a further aspect, the invention provides a method of increasing effector T cell function, e.g., activating effector T cells, in a subject, comprising administering to the subject an effective amount of an anti-GITR antibody, or antigen-binding fragment thereof, immunoconjugate, pharmaceutical composition, or combination product of the invention. In some embodiments, the function is enhanced proliferation of effector T cells. In some embodiments, the activated T cell exhibits increased secretion/expression of an interferon or interleukin. In some embodiments, the interleukin is IL 2. In some embodiments, the interferon is IFN- γ.

In yet another aspect, the invention provides a method of mediating ADCC to eliminate regulatory T cells in a subject, comprising administering to the subject an effective amount of an anti-GITR antibody, or antigen-binding fragment thereof, immunoconjugate, pharmaceutical composition or combination product of the invention. In some embodiments, the regulatory T cells are Treg cells.

In one aspect, the invention also provides a method of preventing or treating a tumor in a subject, the method comprising administering to the subject an effective amount of any of the anti-GITR antibodies or fragments thereof, immunoconjugates, pharmaceutical compositions or combination products described herein.

In one aspect, the invention also provides a method of preventing or treating an infection in a subject, the method comprising administering to the subject an effective amount of any of the anti-GITR antibodies or fragments thereof, immunoconjugates, pharmaceutical compositions or combination products described herein.

The invention also provides a method of preventing or treating other conditions or disorders associated with Treg proliferation in a subject, the method comprising administering to the subject an effective amount of any of the anti-GITR antibodies or fragments thereof, immunoconjugates, pharmaceutical compositions or combination products described herein.

The subject can be a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., a patient having or at risk of having a disease as described herein). In one embodiment, the subject has or is at risk of having a disease described herein (e.g., a tumor or infectious disease as described herein). In certain embodiments, the subject receives or has received other treatment, such as chemotherapy treatment and/or radiation therapy. Alternatively or in combination, the subject is or is at risk of being immunocompromised due to the infection.

In some embodiments, a tumor, e.g., a cancer, described herein includes, but is not limited to, a solid tumor, a hematologic cancer, a soft tissue tumor, and a metastatic lesion. In some embodiments, the cancer for treatment described herein includes, but is not limited to, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, rectal cancer, colorectal cancer, cervical cancer, brain cancer, skin cancer, liver cancer, pancreatic cancer, or gastric cancer, as well as blood cancers such as leukemia and lymphoma. In some embodiments, the cancer is a gastrointestinal cancer, such as gastric cancer, rectal cancer, colon cancer, colorectal cancer, and the like.

Treatment of metastatic cancer (e.g., metastatic cancer expressing GITR or PD 1) can be achieved using the antibody molecules described herein.

In one embodiment, the tumor is a cancer that expresses elevated levels of GITR and/or PD 1.

In some embodiments, the cancer described herein is colon cancer and metastatic cancers thereof.

In some embodiments, the infection is acute or chronic. In some embodiments, the chronic infection is a persistent infection, a latent infection, or a slow infection. In some embodiments, the chronic infection is caused by a pathogen selected from the group consisting of bacteria, viruses, fungi, and protozoa.

In other aspects, the invention provides the use of an anti-GITR antibody, or fragment thereof, in the manufacture or preparation of a medicament for the treatment of a related disease or disorder mentioned herein.

In some embodiments, an antibody or antibody fragment or immunoconjugate or composition or product of the invention delays onset of a disorder and/or symptoms associated with a disorder.

In some embodiments, the methods of prevention or treatment described herein further comprise administering to the subject or individual the antibody molecule or pharmaceutical composition or immunoconjugate disclosed herein in combination with one or more other therapies, e.g., a therapeutic modality and/or other therapeutic agents.

In some embodiments, the treatment modality includes surgery (e.g., tumor resection); radiation therapy (e.g., external particle beam therapy, which involves three-dimensional conformal radiation therapy in which an irradiation region is designed), localized irradiation (e.g., irradiation directed at a preselected target or organ) or focused irradiation), and the like.

In some embodiments, the therapeutic agent is selected from a chemotherapeutic agent, a cytotoxic agent, a vaccine, another antibody, an anti-infective active agent, a small molecule drug, or an immunomodulatory agent.

Exemplary vaccines include, but are not limited to, cancer vaccines. The vaccine may be a DNA-based vaccine, an RNA-based vaccine or a virus transduction-based vaccine. Cancer vaccines can be prophylactic or therapeutic.

Exemplary anti-infective actives include, but are not limited to, antiviral agents, antifungal agents, antiprotozoal agents, antibacterial agents, such as the nucleoside analogs zidovudine (AST), ganciclovir, foscarnet, or cidovir, as described above.

Exemplary other antibodies include, but are not limited to, anti-PD-1 antibodies, anti-PDL 1 antibodies, or anti-PDL 2 antibodies. Various anti-PD-1, anti-PDL 1, or anti-PDL 2 antibodies are known in the art, see, e.g., WO2007/005874, WO2009/101611, WO2009/114335, WO2010/027827, and WO2011/066342, among others.

In some embodiments, the antibody combinations described herein can be administered separately, e.g., as individual antibodies separately, or when linked (e.g., as a bispecific or trispecific antibody molecule).

Further therapies or therapeutic agents that can be combined with anti-GITR antibodies or fragments thereof can be found in WO 2015/031667.

Such combination therapies encompass combined administration (e.g., two or more therapeutic agents contained in the same formulation or separate formulations), and separate administration, in which case administration of the antibody of the invention can occur prior to, concurrently with, and/or after administration of the other therapeutic agent and/or agents. In one embodiment, the administration of the anti-GITR antibody and the administration of the additional therapeutic agent occur within about one month, or within about one, two, or three weeks, or within about 1, 2, 3, 4,5, or 6 days of each other.

The therapeutically effective amount of the anti-GITR antibody-containing pharmaceutical composition to be employed will depend, for example, on the therapeutic context and objectives. Those skilled in the art will appreciate that the appropriate dosage level for treatment will vary depending in part on the following factors: the molecule delivered, the indication for which it is to be used, the route of administration, and the weight, body surface area or organ size and/or condition (age and general health) of the patient. In certain embodiments, a clinician may titrate the dosage and modify the route of administration to obtain the optimal therapeutic effect.

The frequency of administration will depend on the pharmacokinetic parameters of the particular anti-GITR antibody in the formulation used. Typically, the clinician administers the composition until a dosage is reached that achieves the desired effect. The antibodies of the invention may thus be administered in a single dose, or in two or more doses (which may contain the same or different amounts of the desired molecule) over a period of time, or by continuous infusion through an implanted device or catheter. Appropriate dosages may be determined by using appropriate dose response data. In certain embodiments, the antibody can be administered to the patient over an extended period of time. In certain embodiments, the antibody is administered every two weeks, monthly, every two months, every three months, every four months, every five months, or every six months.

The route of administration of the pharmaceutical composition is according to known methods, e.g., oral, by intravenous injection, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; by a sustained release system or by an implanted device. In certain embodiments, the composition may be administered by bolus injection or by continuous infusion or by an implanted device.

The composition may also be administered topically via an implant membrane, sponge, or another suitable material onto which the desired molecule is absorbed or encapsulated. In certain embodiments, when an implant device is used, the device may be implanted into any suitable tissue or organ and the desired molecule may be delivered via diffusion, timed-release bolus, or continuous administration.

It will be appreciated that any treatment can be carried out using the immunoconjugates of the invention in place of or in addition to an anti-GITR antibody.

Methods and compositions for diagnosis and detection

In certain embodiments, any of the anti-GITR antibodies or antigen-binding fragments thereof provided herein can be used to detect the presence of GITR in a biological sample. The term "detection" as used herein includes quantitative or qualitative detection, exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays, PCR-techniques (e.g., RT-PCR). In certain embodiments, the biological sample is blood, serum, or other liquid sample of biological origin. In certain embodiments, the biological sample comprises a cell or tissue. In some embodiments, the biological sample is from a hyperproliferative or cancerous lesion.

In one embodiment, anti-GITR antibodies are provided for use in diagnostic or detection methods. In another aspect, methods of detecting the presence of GITR in a biological sample are provided. In certain embodiments, the method comprises detecting the presence of a GITR protein in the biological sample. In certain embodiments, the GITR is human GITR. In certain embodiments, the method comprises contacting the biological sample with an anti-GITR antibody as described herein under conditions that allow the anti-GITR antibody to bind to GITR, and detecting whether a complex is formed between the anti-GITR antibody and GITR. The formation of a complex indicates the presence of GITR. The method may be an in vitro or in vivo method. In one embodiment, the anti-GITR antibody is used to select a subject suitable for treatment with the anti-GITR antibody, e.g., where GITR is a biomarker for selecting the subject.

In one embodiment, an antibody of the invention can be used to diagnose cancer or tumor, e.g., to assess (e.g., monitor) the treatment or progression of, diagnosis and/or stage of a disease (e.g., cancer or tumor) described herein in a subject. In certain embodiments, labeled anti-GITR antibodies are provided. Labels include, but are not limited to, labels or moieties that are detected directly (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as moieties that are detected indirectly, such as enzymes or ligands, for example, by enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes ³² P、 ¹⁴ C、 ¹²⁵ I、 ³ H and ¹³¹ fluorophores such as rare earth chelates or luciferin and derivatives thereof, rhodamine and derivatives thereof, dansyl (dansyl), umbelliferone (umbelliferone), luciferase (luceriferase), e.g., firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2, 3-dihydrophthalazinedione, horseradish peroxidase (HR), alkaline phosphatase, beta-galactosidaseGlucoamylases, lytic enzymes, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, and enzymes that oxidize dye precursors with hydrogen peroxide such as HR, lactoperoxidase, or microperoxidase (microperoxidase), biotin/avidin, spin labels, phage labels, stable free radicals, and the like.

In some embodiments of any of the inventions provided herein, the sample is obtained prior to treatment with the anti-GITR antibody. In some embodiments, the sample is obtained prior to treatment with the cancer drug. In some embodiments, the sample is obtained after the cancer has metastasized. In some embodiments, the sample is formalin fixed, paraffin coated (FFPE). In some embodiments, the sample is a biopsy (e.g., core biopsy), a surgical specimen (e.g., a specimen from a surgical resection), or a fine needle aspirate.

In some embodiments, GITR is detected prior to treatment, e.g., prior to initiation of treatment or prior to some treatment following a treatment interval.

In some embodiments, there is provided a method of treating a tumor or infection, the method comprising: the method comprises testing a subject (e.g., a sample) (e.g., a sample of a subject comprising cancer cells) for the presence of GITR, thereby determining a GITR value, comparing the GITR value to a control value, and if the GITR value is greater than the control value, administering to the subject a therapeutically effective amount of an anti-GITR antibody (e.g., an anti-GITR antibody described herein), optionally in combination with one or more other therapies, thereby treating the tumor or infection.

IX exemplary anti-GITR antibodies of the invention

These and other aspects and embodiments of the invention are described in the accompanying drawings (brief description of the drawings follows) and in the following detailed description of the invention and are exemplified in the following examples. Any or all of the features discussed above and throughout this application may be combined in various embodiments of the invention. The following examples further illustrate the invention, however, it is to be understood that the examples are described by way of illustration and not limitation, and that various modifications may be made by those skilled in the art.

Examples

Example 1 preparation of hybridoma cells

Hybridoma technology is a technique that maintains the main characteristics of both cells simultaneously by fusing them. These two cells are antigen immunized mouse spleen cells and mouse myeloma cells, respectively. The main characteristic of mouse splenocytes (B lymphocytes) immunized with a specific antigen is their antibody-secreting function, but they cannot be cultured continuously in vitro, and mouse myeloma cells can divide and proliferate indefinitely under culture conditions, i.e., they are so-called immortal. Under the action of the selection medium, only the hybrid cell formed by fusing the B cell and the myeloma cell can have the capacity of continuous culture, and cell clone which has both the antibody secretion function and the cell immortal maintaining property is formed. In the experiment, a mouse is immunized by the human GITR protein, and then spleen cells of the mouse are obtained to be fused with myeloma cells, so that hybridoma cells capable of expressing positive antibodies are obtained.

Hybridoma fusion

Laboratory animal and immune information

Preparing an electrofusion dish: the electrofusion dish was thoroughly soaked in 70% ethanol and blown dry in a clean bench for use.

Isolation of splenocytes: the mice were sacrificed by cervical dislocation, the body surface was sterilized with 75% alcohol for 5min, immediately placed on a mouse dissecting plate in a clean bench, left recumbent, and four limbs were fixed with a 7-gauge needle. The spleen was removed by aseptically opening the abdominal cavity, washed with basal medium (prepared as in the table below), and carefully removed of surrounding connective tissue. The spleen was then transferred to another dish containing basal medium. Pressing spleen with elbow needle, inserting hole on spleen with small needle, and squeezing with forceps to release splenocytes completely to obtain splenocyte suspension. The cell suspension was filtered through a 70 μ M cell screen and washed once with 30ml of basal medium and centrifuged at 1200rpm for 6 min.

And (3) cracking red blood cells: the supernatant was removed and the cells were resuspended in 10ml of RBC lysis buffer (GIBCO). Then 20ml of RBC lysis buffer was added. The suspension was left to stand for 5min and centrifuged at 1100rpm for 6 min. After removing the supernatant, the cells were resuspended in 10ml of basal medium, then 30ml of basal medium was added and centrifuged at 1100rpm for 6 min. After removal of the supernatant, the cells were resuspended in 20ml of basal medium and counted.

Electrofusion: mouse myeloma cells SP2/0 cells (ATCC) were resuspended in 20ml of basal medium and counted. SP2/0 and splenocytes were mixed at a ratio of 1: 2 to 1: 1, and centrifuged at 1000rpm for 6 min. After removal of the supernatant, the pooled cells were resuspended in 10ml of fusion buffer (BTXpress). Then, 15ml of the fusion buffer was added thereto, and the mixture was centrifuged at 1000rpm for 5min to remove the supernatant. After repeating the above steps once, reselecting the cells with appropriate amount of fusion buffer, and adjusting the density of the mixed cells to 1 × 10 ⁷ Individual cells/ml. The parameters of the electrofusion apparatus are set as follows. 2ml of cell suspension was added to each electrofusion dish for electrofusion.

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

Plating after electrofusion: the cells were allowed to stand in the electrofusion dish at room temperature for 5 min. Transferring the cells into a centrifuge tube, and diluting the cells to 1-2 × 10 with a screening medium (the configuration method is shown in the following table) ⁴ Individual cells/ml. 100. mu.l of cell suspension was added to each well of a 96-well plate. Selection medium was changed 7 days after the fusion. Screening was performed after 10 days of culture (or longer, depending on the cell growth state). Hybridoma cells expressing specific anti-GITR antibodies were selected by FACS (C6(BD Biosciences)).

Positive hybridoma cell subcloning

A subcloning step: a96-well plate was prepared and 200. mu.l of the basal medium described above was added to each well of columns 2 to 8. The cells of the positive well selected by the fusion are made into a cell suspension and added to column 1. Add 100. mu.l of the cell suspension from column 1 to column 2, mix well and add 100. mu.l to the next column. Repeating the above steps until the volume of the last column becomes 300 μ l; standing the 96-well plate for 15min, and observing and counting under a microscope. A volume corresponding to 100 cells was added to 20ml of basal medium as described above and plated well with 200. mu.l per well. After one week, observing under a microscope, judging and marking the monoclonal hole, and selecting a positive hole to be detected.

Freezing and storing cells: observing cell state, when cell growth is good and activity is more than 90%, centrifuging at 1000rpm for 5min, and removing supernatant. With frozen stock solution (45.5)% FBS, 44.5% RPMI-1640, 10% DMSO) to 1X 10 ⁷ And (4) packaging each cell/ml into a freezing tube, and putting the freezing tube into a programmed cooling box for freezing at-80 ℃.

EXAMPLE 2 production and purification of chimeric antibodies

The invention utilizes molecular biology technology to obtain an antibody sequence in anti-GITR positive hybridoma cells, and constructs a human-mouse chimeric antibody by utilizing the antibody sequence.

Sequencing of hybridomas

RNA extraction: fresh cells, 300g centrifuged for 5min, the supernatant removed, and the pellet added 500. mu.l LY buffer (Biomiga) (20. mu.l. beta. mercaptoethanol per 1ml prior to use) and mixed until clear. Added to the DNA removal tube, centrifuged at 13000rpm for 2min, and the flow-through was collected. Adding 100% ethanol into the fluid according to the proportion of 1/2, and mixing for 5 times until the fluid is clear. The clarified solution was added to an RNA collection tube, centrifuged at 13000rpm for 1min to remove the liquid, 500. mu.l RB (Recovery Buffer) (Takara), centrifuged at 13000rpm for 30s, then 500. mu.l RNA washing Buffer (Biomiga) (ethanol was added before use), centrifuged for 30s, after repeating the above process, centrifuged to completely evaporate the ethanol, 30. mu.l DEPC water was added to the collection column, centrifuged at 12000g for 2min, and the eluate was collected. The RNA concentration was determined.

cDNA was obtained by reverse transcription using PrimeScript II 1st Strand cDNA Synthesis Kit (Takara):

the reaction system I is configured as follows:

after incubation at 65 ℃ for 5min, the cells were rapidly cooled on ice. To reaction I was added the following reverse transcription system in a total amount of 20. mu.l:

after slowly mixing, carrying out reverse transcription and translation according to the following conditions: 60min at 42 → 5min at 95 ℃ and cooling on ice to obtain cDNA.

Ligation of cDNA to T vector:

respectively amplifying heavy chain and light chain variable regions by PCR, wherein the PCR reaction system comprises the following steps:

the PCR reaction conditions were as follows:

mu.l of the PCR product obtained by the above PCR reaction was taken, and 0.5. mu.l of pMD20-T vector (Clontech) and 5. mu.l of light night Mix (Takara) were added thereto, gently mixed, and reacted at 37 ℃ for 2 hours to obtain a Ligation product.

TABLE 6 heavy chain variable region (VH) primers (Primer Mix 1) for mouse anti-GITR antibody

TABLE 7 light chain variable region (VL) primers for mouse anti-GITR antibodies (Primer Mix 2):

transforming cells:

TOP10 competent cells (Tiangen Biochemical technology (Beijing) Co., Ltd.) were removed at-80 ℃ and thawed on ice, 5. mu.l of the ligation product obtained above was added to thawed TOP10 competent cells, mixed well and incubated on ice for 30 min. After heat shock at 42 ℃ for 90s, the mixture was rapidly cooled on ice for 2min, and 900. mu.l of LB medium (Biotechnology engineering, Shanghai, Ltd.) was added to the EP tube, and shaking culture was carried out at 37 ℃ and 220rpm for 1 h. Centrifugation at 3000g for 2min, aspiration of 800. mu.l of supernatant, resuspension of the cells with the remaining medium and plating on ampicillin resistant plates. Cultured overnight at 37 ℃ and then cloned for sequencing.

Construction of chimeric antibodies

PCR amplification of sequenced murine anti-GITR antibody VH and VL regions from the hybridoma of example 1

The PCR system was as follows:

for VH amplification, Primer Mix 1 was applied, for VL amplification, Primer Mix 2 was applied;

the pMD20-T plasmid with correct sequencing

Cutting the gel and recovering PCR amplification products.

Homologous recombination reaction:

the homologous recombination system is as follows:

reacting at 37 ℃ for 30min to obtain a recombinant product. Transforming TOP10 competent cells with the recombinant product, picking single clone for sequencing, selecting the clone containing the plasmid with the correct insertion direction as a positive clone, and storing the positive clone.

Expression and purification of chimeric antibodies

Plasmids containing anti-GITR antibodies were extracted from the positive clones obtained above.

293F cells (Invitrogen) were passaged according to the desired transfection volume, and the cell density was adjusted to 1.5X 10 the day before transfection ⁶ Individual cells/ml. Cell density at day of transfection was approximately 3X 10 ⁶ Individual cells/ml. Taking the final volume 1/10F 17 medium (Gibco, A13835-01) was used as a transfection buffer, and the appropriate plasmid was added and mixed well. Appropriate Polyethylenimine (PEI) (Polysciences, 23966) was added to the plasmid (plasmid to PEI ratio 1: 3 in 293F cells), mixed well and incubated at room temperature for 10min to obtain a DNA/PEI mixture. After resuspending the cells with the DNA/PEI mixture, 8% CO at 36.5 ℃ ₂ .24 h later, the cells were supplemented with 2% FEED (Sigma) in transfection volume, 120rpm, 8% CO at 36.5 ℃ ₂ Culturing under the condition. Continuously culturing for 6 days or collecting cell supernatant for purification when cell activity is less than or equal to 60%.

The gravity column used for purification was treated with 0.5M NaOH overnight, and the column was dried at 180 ℃ for 4 hours after washing with distilled water in a glass bottle or the like to obtain a purification column. The collected medium was centrifuged at 4500rpm for 30min before purification, and the cells were discarded. The supernatant was filtered using a 0.22. mu.l filter. Each tube was filled with 1ml of Protein A and equilibrated with 10ml of binding buffer (sodium phosphate 20mM. NaCl 150mM, pH 7.0). The filtered supernatant was applied to a purification column and re-equilibrated with 15ml of binding buffer. 5ml of elution buffer (citric acid + sodium citrate 0.1M, pH3.5) was added, and the eluate was collected, and 80. mu.l of Tris-HCl was added to 1ml of the eluate. The collected antibodies were concentrated by ultrafiltration and exchanged into PBS (Gibco, 70011-.

The amino acid sequences of the CDRs, light chain variable region and heavy chain variable region, light chain and heavy chain of 2 chimeric antibodies (CH22F4 and CH37G5) obtained in the present invention, and the sequence numbers are shown in tables 1 to 3 above.

The control antibody used in the present invention is a GITR antibody reported in patent application US20130183321a1(GITR, INC. (Cambridge, MA, US)), whose light and heavy chain sequences are respectively SEQ ID NOs: 44 and SEQ ID NO: 54, hereinafter referred to simply as TRX 518.

Example 3 determination of binding kinetics of the chimeric antibodies of the invention to antigens by biofilm thin layer interferometry

The equilibrium dissociation constant (KD) for binding of the antibodies of the invention to human GITR was determined using a biofilm thin layer interferometry technique (ForteBio). ForteBio affinity assays were performed according to the current protocol (Estep, P et al, High throughput solution Based measurement of antibody-antibody affinity and affinity binding. MAbs, 2013.5 (2): pages 270-8).

Half an hour before the start of the experiment, depending on the sample size, appropriate AMQ (Pall, 1506091) (for sample detection) or AHQ (Pall, 1502051) (for positive control detection) sensors were soaked in SD buffer (PBS 1 ×, BSA 0.1%, Tween-200.05%).

100 μ l of SD buffer, antibodies (CH22F4, CH37G5, and TRX518), antigens (including human GITR, and cynomolgus GITR, all purchased from Acrobiosystems) were added to 96-well black polystyrene half-well microplates (Greiner, 675076), respectively. Sensor locations were selected based on sample location plating. The instrument setup parameters were as follows: the operation steps are as follows: baseline, Loading-1 nm, Baseline, Association and Dissociation; the run time for each step depends on the sample binding and dissociation speed, 400rpm, and 30 ℃. Analysis of K Using ForteBio analysis software _D The value is obtained.

In the experiments described in the above assays, the affinities of the antibodies CH22F4, CH37G5 are shown in table 8:

TABLE 8 ForteBio assays affinity constants for antigen antibody binding (equilibrium dissociation constants)

In the above experiments, K of chimeric antibodies CH22F4, CH37G5 _D The values were 1.96E-09M and 2.04E-09M, respectively, and the antibodies in this study had superior K compared to the TRX518 in the control group _D The value is obtained.

Example 4 binding experiments of chimeric antibodies and CHO-S cells overexpressing human GITR

This study examined the binding of a chimeric antibody of the invention diluted in gradient to a CHO-S stable cell line overexpressing human GITR on its surface using a flow cytometer.

The cDNA encoding human GITR (SEQ ID NO: 41) was cloned into pCHO1.0 vector (Invitrogen), and the obtained plasmid was transfected into CHO-S cells (Invitrogen, ExpicCHO) ^TM Expression System Kit，The goods number is: a29133) CHO-S cells overexpressing human GITR (CHO-hGITR) were generated.

CHO-hGITR cells were counted and diluted to 2X 10 ⁶ Cells/ml, 100. mu.l/well were added to a U-bottom 96-well plate. Centrifuge at 400g for 5min and remove the cell culture medium. Samples (chimeric antibodies CH22F4, CH37G5, and TRX518, respectively) (antibody dilution method: maximum antibody concentration 400nM, three times diluted in PBS, total 12 concentrations tested) were added to the U plate and the cells resuspended, 100. mu.l/well, and allowed to stand on ice for 30 min. The supernatant was removed by centrifugation at 400g for 5min and the cells were washed 1 time with PBS. PBS was removed by centrifugation at 400g for 5min, 100. mu.l of anti-human Fc PE-labeled secondary antibody (SoutherBiotech; 2040-09) (1: 200 diluted in PBS) was added to each well, and incubated on ice for 30min in the absence of light. The supernatant was removed by centrifugation at 400g for 5min and the cells were washed 1 time with PBS. Cells were resuspended in 80. mu.l of 1 XPBS and examined by FACS.

In the experiments described in the above assays, the binding of the antibodies CH22F4, CH37G5 and CHO-hGITR cells is shown in FIG. 1.

In the above assay, antibodies CH22F4, CH37G5 both bound human GITR molecules overexpressed on CHO-S cells, with EC50 at 0.4568nM and 0.9069nM, respectively, and similar binding capacity compared to TRX 518.

Example 5 humanization of chimeric antibodies

The chimeric antibody obtained in example 2 was humanized. And carrying out humanization through the following steps:

determining a CDR ring structure;

finding the closest homologous sequence for each V/J region of the heavy chain and the light chain in a human germline sequence database;

screening the human species line which is most matched with the heavy chain and the light chain and the lowest amount of back mutation;

constructing the CDR region of the chimeric antibody to the human framework region;

determining the amino acid position in the skeleton region to maintain the CDR function;

sixthly, carrying out reversion mutation (returning to the input amino acid type) at the sequence position determined to be important;

seventhly, optimizing amino acid of the risk position.

The amino acid sequences of the CDRs, light chain variable region and heavy chain variable region, light chain and heavy chain of the 2 humanized antibodies (HZ22F4 and HZ37G5) obtained in the present invention are shown in tables 1-3 as described above.

Example 6 ForteBio assays binding kinetics of humanized antibodies to antigens

Equilibrium dissociation constant (K) for binding of humanized antibodies of the invention to human GITR was determined using the ForteBio assay _D ). ForteBio affinity assay method the same as in example 3 except that the antibodies used were humanized antibodies HZ22F4 and HZ37G 5. In the experiments described in the above assays, the affinities of the antibodies HZ22F4 and HZ37G5 are shown in table 9:

TABLE 9 ForteBio assays affinity constants for antigen antibody binding

In the above assay, K of the humanized antibodies HZ22F4, HZ37G5 described herein _D The values were 4.58E-09M and 4.83E-09M, respectively, and the humanized antibodies in this study had superior K compared to the control antibody _D The value is obtained.

Example 7 binding experiments of humanized antibodies and CHO-S cells overexpressing human GITR

This study examined the binding of the humanized antibody of the present invention to CHO-hGITR cells using a flow cytometer in a gradient dilution. The assay was as in example 4, except that the antibodies used were humanized antibodies HZ22F4 and HZ37G 5. The bonding situation is shown in fig. 2.

In the above assay, the humanized antibodies HZ22F4, HZ37G5 bind to human GITR overexpressed on CHO-S cells, EC50 being 0.5492nM, 1.974nM, respectively, with similar or superior EC50 values, i.e. similar or superior binding capacity, compared to TRX 518.

Example 8 binding experiments of humanized antibodies and cells overexpressing cynomolgus GITR

In this study, the binding of the humanized antibody of the present invention diluted in gradient to a CHO-S stable cell line overexpressing cynomolgus GITR on the surface was examined using a flow cytometer.

The cDNA encoding cynomolgus monkey GITR (SEQ ID NO: 42) was cloned into pCHO1.0 vector (Invitrogen), and the plasmid was transfected into CHO-S cells (Invitrogen, ExpicCHO) ^TM Expression System Kit, Commodity number: a29133) CHO-S cells overexpressing cynomolgus GITR (CHO-cynoGITR) were generated. The remainder of the assay was the same as in example 4, except that the antibodies used were humanized antibodies HZ22F4 and HZ37G5, the binding of which is shown in FIG. 3.

In the above experiments, humanized antibodies HZ22F4, HZ37G5 bound cynomolgus GITR overexpressed on CHO-S cells, EC50 was 0.3533nM, 0.9931nM, respectively, with similar or superior binding capacity compared to TRX 518.

Example 9MOA assay for the detection of biological Activity of antibodies

An activating antibody directed against GITR can bind to cell surface GITR molecules to activate the downstream NF-kappa B signaling pathway. The research uses a detection cell strain of internal constructed Hela-GITR-NF kappa B luciferase (hereinafter referred to as Hela-GITR) to detect the activation condition of NF-kappa B signals through detecting the expression of a fluorescent reporter gene, thereby detecting the activation effect of the antibody.

Construction of Hela-GITR-NF kappa B luciferase cell line

A cDNA encoding human GITR (sequence shown in SEQ ID NO: 41) was cloned into pCHO1.0 vector (Invitrogen), and the obtained plasmid and NF-kappaB luciferace reporter plasmid (Promega) were co-transfected into Hela cells (ATCC, cat # CCL-2TM), resulting in Hela cells (Hela-GITR) overexpressing human GITR with the NF-kappaB lucifere reporter gene system.

Hela-GITR cells were taken in the logarithmic growth phase, the culture supernatant was discarded, and the cells were washed once with PBS (Gibco). Adding appropriate amount of Trypsin (Gibco) at 37 deg.C and 5% CO ₂ Digesting for 2 min. Add 4 Trypsin volumes of 10% FBS in DMEM medium (ATCC), transfer cells to 50ml centrifuge tubes and count, 400g, centrifuge for 5 min. DMEM medium (ATCC) was added and the cells were resuspended to 1X 10 ⁵ Individual cells/mL. Cells were added to 96-well white cell culture plates (Nunclon), 50. mu.l/well. Simultaneously added per holeMu.l of sample (humanized antibodies HZ22F4, HZ37G5 prepared by the present invention) and positive control TRX518) (antibody dilution method: the highest antibody concentration was 80nM, three-fold diluted in assay buffer (2% FBS DMEM medium), for a total of 10 concentrations tested). At 37 deg.C/5% CO ₂ The culture was carried out in an incubator for 6 hours.

And (3) detection: Bio-GloTM buffer (promega) was thawed in advance, and the Bio-GloTM substrate (promega) was added thereto and mixed to obtain a Bio-GloTM reagent. To the cells cultured for 6 hours as described above, Bio-GloTM reagent was added at 100. mu.l/well. The reading is immediate.

In the above experiments, as shown in fig. 4, the antibodies HZ22F4 and HZ37G5 both can effectively activate the NF kappa B signaling pathway, EC50 is 0.3394nM and 0.3208nM, respectively, and has significantly better activation ability than TRX518(1.139 nM).

Example 10 CD4T activated cell assay

In the study, the antibody and activated CD4T cells are incubated together, and the activation effect of different antibodies on CD4T cells is reflected by detecting the relative expression amount of IL-2 and IFN-gamma in a system.

PBMC separation: 50ml of fresh blood of a donor is taken, 2.5 times of PBS is added, the blood is gently added into FiColl (thermo), the mixture is divided into 4 tubes, 400g of the mixture is centrifuged for 30min, the ascending speed is 7, and the deceleration is 0. And after the centrifugation is finished, gently taking out the centrifuge tube, sucking the middle white cloudy cell population into PBS, and washing the cells for 2 times by using the PBS.

CD4 ⁺ T cell isolation: PBMC cells isolated as described above were taken and processed according to EasySep Human CD4 ⁺ T Cell Enrichment Kit (stem Cell) Specification isolation enriched CD4 ⁺ T cells and resuspended using T cell culture medium (recipe see table below).

CD4 ⁺ Activation of T cells: collecting CD4 obtained by the above separation ⁺ T cells, and adjusting cell density to 1 x 10 with T cell culture medium ⁶ Per ml, in terms of cells: beads (1: 1) were added to Dynabeads Human T-Activator CD3/CD28(Invitrogen), 5% CO at 37 deg.C ₂ Culturing in an incubator for one week.

T cell activation experiments: a Nunc 96 well flat bottom plate (Nunclon) was prepared, and 0.25. mu.g/ml of Purified NA/LE Mouse anti-human CD3 Clone UCHT1(BD Biosciences) diluted with PBS was added to 100. mu.l/well, and the mixture was coated at 37 ℃ for 2 hours, and then the coating solution was removed. Activating the above CD4 for one week ⁺ T cells were stripped of beads, washed twice with T cell media, and cell density adjusted to 1 x 10 ⁶ Mu.l of cell suspension per well volume was added to the above 96-well flat-bottom plate, while adding the antibody of the present invention (HZ22F4, HZ37G5) and TRX 518100. mu.l (antibody dilution method: maximum antibody concentration 400nM, three-fold dilution in T cell medium, total 10 concentrations tested) and Purified NA/LE Mouse anti-Human CD28 Clone CD28(BD Biosciences) (final concentration 2. mu.g/ml), cultured for 3 and 5 days, and the relative expression amounts of IL-2 and IFN-. gamma. (in terms of DeltaF%) were measured using Human IL-2 Kit 1000 Test and Human IFN gamma 1000 Test kits (both purchased from cisbio), respectively.

The results of the experiment are shown in tables 10 and 11 and FIGS. 5 and 6. The antibody of the invention can effectively activate CD4 in vitro ⁺ T cells. Where the data units in the table are DeltaF% averages.

TABLE 10 relative expression of IL2 (DeltaF% average)

TABLE 11 relative expression of IFN-. gamma.s (DeltaF% average)

Example 11 ADCC Effect experiment

IgG1 subtype antibodies aiming at the GITR can be combined with the high-expression GITR molecules on the surface of the Tregs so as to mediate ADCC effect to clear the Tregs. In the research, Jurkat-ADCCNF-AT luciferase effector cell strain (ADCC effector cell) of Promega is used as a detection cell strain to be incubated with CHO-hGITR cell (as described above) as a target cell and the antibody of the invention, and the activation condition of NF-AT signals is reacted by detecting the expression of a fluorescent reporter gene, so that the ADCC activity of the antibody is detected.

ADCC effector cells (cultured according to the instructions of Promega), centrifuged to remove supernatant, washed 2 times with PBS, resuspended in assay medium (1640 medium with 5% low IgG serum (Gibco)) to a cell density of 2 x 10 ⁷ Perml, CHO-hGITR target cells prepared and incubated as described above were removed, centrifuged to remove supernatant, and resuspended in assay medium to an adjusted cell density of 2 x 10 ⁶ Perml, the cells were mixed at a ratio of 1: 1 and added to a 96-well white cell culture plate (Nunclon), 50. mu.l/well. At the same time, 50. mu.l of the sample (humanized antibodies HZ22F4, HZ37G5 prepared according to the invention) and a positive control antibody (TRX518) were added to each well (antibody dilution method: the highest final antibody concentration was 66.66nM, three-fold dilution in the detection medium, 12 concentrations in total were tested). At 37 deg.C/5% CO ₂ The culture was carried out in an incubator for 12 hours.

And (3) detection: Bio-GloTM buffer (promega) was thawed in advance, and Bio-GloTM substrate (promega) was added thereto and mixed well to obtain a Bio-GloTM reagent. To the cells cultured for 12 hours as described above, Bio-GloTM reagent was added at 100. mu.l/well. The reading is immediate.

In the above experiments, as shown in fig. 7, the antibodies HZ22F4 and HZ37G5 both can effectively activate the NF-AT signal pathway downstream of ADCC, and EC50 is 0.02958nM and 0.04056nM, respectively, and has significantly better EC50 value than TRX 518.

EXAMPLE 12 antitumor drug efficacy test

This experiment used MC38 cells to inoculate hGITR transgenic mice to determine the anti-tumor effect of the GITR antibodies of the invention.

Human GITR transgenic mice:

female C57Bl/6 background human GITR transgenic mice (approximately 5 weeks old) were purchased from Poiosael chart laboratory animal technology, Inc. Mice were acclimated for 7 days after arrival and then the study was started.

Cell:

mouse MC38 cells were purchased from southKyanghe bio-medicine limited, and was subjected to conventional subculture for subsequent in vivo experiments strictly according to the instructions. Cells were harvested by centrifugation, resuspended in sterile PBS and adjusted to a cell density of 5X 10 ⁶ One per ml. A MC38-hGITR tumor-bearing mouse model was established by inoculating 0.2ml of cell suspension subcutaneously into the right abdominal region of human GITR transgenic mice on day 0.

Administration:

tumor volume of each mouse is detected 6 days after tumor cell inoculation, and the tumor volume is selected to be 87.4mm ³ ～228.4mm ³ Mice within the range were grouped by mean tumor volume (5 mice per group) and the dosage and mode of administration were as shown in table 12, where the Antibody C is the monoclonal Antibody "Antibody C" against PD-1 (WO 2017/133540). The administration was carried out on

days

6, 10, 13 and 17 after the inoculation, and the tumor volume and body weight change of each group of mice were monitored during the administration period, with a frequency of 2 times per week for 5 weeks. Body weight and tumor volume were measured before each dose, and the relative tumor inhibition rate (TGI%) was calculated at day 24 after inoculation as follows: TGI% (% 100% ³ . Tumor volume determination: the maximum long axis (L) and maximum wide axis (W) of the tumor were measured with a vernier caliper, and the tumor volume was calculated according to the following formula: v ═ lxw ² /2. Body weight was measured using an electronic balance.

TABLE 12 Experimental design Table

Administration was performed every 3 or 4 days for 4 times.

Lambda h-IgG purchased from Equitech-Bio, cat #: SLH 56-0001.

The tumor inhibition rate results are shown in fig. 8, 9 and table 13: on day 24 post-inoculation, HZ37G5 and HZ22F4 showed good tumor suppression rates with single drug, respectively: 53% and 50%, better than 36% of the TRX518 group. The combined administration of HZ37G5 and HZ22F4 and anti-PD-1 Antibody Antibody C has obviously better tumor inhibition effect than the single-drug groups of Antibody C, HZ37G5, HZ22F4 and TRX 518. Wherein 4 tumors of 5 mice of the HZ37G5 and Antibody C combination and the HZ22F4 and Antibody C combination completely disappeared. Meanwhile, the body weight of the mice is detected, and the result shows that the body weight of the mice has no significant difference as shown in figure 10. Therefore, the antibody against GITR molecules of the present invention has a significant tumor-inhibiting effect, and the tumor-inhibiting effect is more significant when the antibody is administered in combination with the PD-1 antibody.

TABLE 13 tumor inhibition by day 24

Claims

1. A GITR-binding antibody or antigen-binding fragment thereof, comprising 3 complementarity determining regions (HCDRs) of a heavy chain variable region, and 3 complementarity determining regions (LCDRs) of a light chain variable region, wherein

(i) The HCDR1 consists of an amino acid sequence shown by SEQ ID NO. 1, the HCDR2 consists of an amino acid sequence shown by SEQ ID NO.5, the HCDR3 consists of an amino acid sequence shown by SEQ ID NO. 7, the LCDR1 consists of an amino acid sequence shown by SEQ ID NO. 9, the LCDR2 consists of an amino acid sequence shown by SEQ ID NO. 11, and the LCDR3 consists of an amino acid sequence shown by SEQ ID NO. 15; or

(ii) The HCDR1 consists of an amino acid sequence shown by SEQ ID NO. 2, the HCDR2 consists of an amino acid sequence shown by SEQ ID NO.6, the HCDR3 consists of an amino acid sequence shown by SEQ ID NO. 8, the LCDR1 consists of an amino acid sequence shown by SEQ ID NO. 10, the LCDR2 consists of an amino acid sequence shown by SEQ ID NO. 12, and the LCDR3 consists of an amino acid sequence shown by SEQ ID NO. 16; or

(iii) The HCDR1 consists of an amino acid sequence shown by SEQ ID NO. 3, the HCDR2 consists of an amino acid sequence shown by SEQ ID NO.5, the HCDR3 consists of an amino acid sequence shown by SEQ ID NO. 7, the LCDR1 consists of an amino acid sequence shown by SEQ ID NO. 9, the LCDR2 consists of an amino acid sequence shown by SEQ ID NO. 13, and the LCDR3 consists of an amino acid sequence shown by SEQ ID NO. 15; or

(iv) The HCDR1 consists of the amino acid sequence shown by SEQ ID NO. 2, the HCDR2 consists of the amino acid sequence shown by SEQ ID NO.6, the HCDR3 consists of the amino acid sequence shown by SEQ ID NO. 8, the LCDR1 consists of the amino acid sequence shown by SEQ ID NO. 10, the LCDR2 consists of the amino acid sequence shown by SEQ ID NO. 12, and the LCDR3 consists of the amino acid sequence shown by SEQ ID NO. 16.

2. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region, wherein the heavy chain variable region comprises or consists of an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NO 17, 18, 19 or 20.

3. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region, wherein the heavy chain variable region comprises or consists of the amino acid sequence of SEQ ID NO 17, 18, 19 or 20.

4. The antibody or antigen-binding fragment thereof of any one of claims 1 to 3, comprising a light chain variable region, wherein the light chain variable region comprises or consists of an amino acid sequence having at least 90% identity to an amino acid sequence selected from SEQ ID NO 21, 22, 23 or 24.

5. The antibody or antigen-binding fragment thereof of any one of claims 1 to 3, comprising a light chain variable region, wherein the light chain variable region comprises or consists of the amino acid sequence of SEQ ID NO 21, 22, 23 or 24.

6. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region and a light chain variable region, wherein

The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 17, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 21;

the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 18, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 22;

the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 19, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 23; or

The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 20 and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 24.

7. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain comprising or consisting of an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NO 25, 26, 27 or 28.

8. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO 25, 26, 27 or 28.

9. The antibody or antigen-binding fragment thereof of claim 1 or 7 or 8, comprising a light chain comprising or consisting of an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NO 29, 30, 31 or 32.

10. The antibody or antigen-binding fragment thereof of claim 1 or 7 or 8, comprising a light chain comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO 29, 30, 31 or 32.

11. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain and a light chain, wherein

The heavy chain comprises or consists of the amino acid sequence shown as SEQ ID NO. 25 and the light chain comprises or consists of the amino acid sequence shown as SEQ ID NO. 29; or

The heavy chain comprises or consists of the amino acid sequence shown as SEQ ID NO. 26 and the light chain comprises or consists of the amino acid sequence shown as SEQ ID NO. 30; or

The heavy chain comprises or consists of the amino acid sequence shown as SEQ ID NO. 27 and the light chain comprises or consists of the amino acid sequence shown as SEQ ID NO. 31; or

The heavy chain comprises or consists of the amino acid sequence shown as SEQ ID NO. 28 and the light chain comprises or consists of the amino acid sequence shown as SEQ ID NO. 32.

12. The antibody or antigen-binding fragment thereof of claim 1 or 6, having one or more of the following properties:

(i) exhibits the same binding affinity and/or specificity for GITR as any one of the antibodies of claim 11;

(ii) competitively inhibiting the binding of any of the antibodies of claim 11 to GITR;

(iii) an epitope that binds to the same or an overlapping epitope as any one of the antibodies of claim 11.

13. The antibody or antigen-binding fragment thereof of any one of claims 1, 6, or 11, having one or more of the following properties:

(i) binds to human GITR;

(ii) having agonist activity;

(iii) can effectively mediate ADCC effect;

(iv) is capable of activating T cells;

(v) has anti-tumor activity without affecting the body weight of the subject;

(vi) combined with anti-PD-1 antibodies can inhibit tumor activity without affecting the subject's body weight.

14. The antibody or antigen-binding fragment thereof of claim 13, wherein the agonist activity is capable of effectively activating the NF-kappaB signaling pathway.

15. The antibody or antigen-binding fragment thereof of claim 13, wherein the T cell is a CD4T cell.

16. The antibody or antigen-binding fragment thereof of claim 13, wherein the anti-tumor activity is capable of reducing tumor volume in a subject.

17. The antibody or antigen-binding fragment thereof of claim 13, wherein said inhibition of tumor activity is an ability to reduce tumor volume in a subject.

18. The antibody or antigen-binding fragment thereof of any one of claims 1, 6, or 11, wherein the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment of the IgG1 format or the IgG2 format or the IgG4 format.

19. The antibody or antigen-binding fragment thereof of any one of claims 1, 6, or 11, wherein the antibody is a monoclonal antibody.

20. The antibody or antigen binding fragment thereof of any one of claims 1, 6, or 11, wherein the antibody is a humanized or human antibody or a chimeric antibody.

21. The antibody or antigen-binding fragment thereof of any one of claims 1, 6, or 11, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of: fab, Fab '-SH, Fv, single-chain antibody, (Fab') ₂ Or a diabody dAb.

22. The antibody or antigen-binding fragment thereof of claim 21, wherein the single chain antibody is an scFv.

23. The antibody or antigen-binding fragment thereof of claim 1, 6, or 11, wherein the antibody is a bispecific or multispecific antibody molecule.

24. The antibody or antigen-binding region fragment thereof of claim 23, wherein the bispecific antibody molecule binds to GITR and PD-1, PD-L1, or PD-L2.

25. An isolated nucleic acid encoding the GITR-binding antibody of any one of claims 1 to 24.

26. A vector comprising the nucleic acid of claim 25.

27. The vector of claim 26, wherein the vector is an expression vector.

28. A host cell comprising the nucleic acid of claim 25 or the vector of claim 26 or 27.

29. The host cell of claim 28, wherein the host cell is prokaryotic or eukaryotic.

30. The host cell of claim 28, wherein the host cell is selected from the group consisting of a yeast cell, a mammalian cell, or other cell suitable for use in the production of an antibody or antigen-binding fragment thereof.

31. The host cell of claim 30, wherein the mammalian cell is a 293 cell or a CHO cell.

32. A method of making a GITR-binding antibody or antigen-binding fragment thereof, the method comprising culturing the host cell of any one of claims 28-31 under conditions suitable for expression of a nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1-24 or the nucleic acid of claim 25, optionally isolating the antibody or antigen-binding fragment thereof, optionally the method further comprising recovering the GITR-binding antibody or antigen-binding fragment thereof from the host cell.

33. An immunoconjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 24 and an additional agent.

34. The immunoconjugate of claim 33, wherein the other agent is a cytotoxic agent or a label.

35. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 24 or the immunoconjugate of claim 31 or 32.

36. The pharmaceutical composition of claim 35, further comprising one or more additional therapeutic agents.

37. The pharmaceutical composition of claim 36, wherein the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody.

38. The pharmaceutical composition of any one of claims 35 to 37, further comprising a pharmaceutical excipient.

39. A combination comprising the GITR-binding antibody or antigen-binding fragment thereof of any one of claims 1 to 24 or the immunoconjugate of claim 33 or 34 or the pharmaceutical composition of any one of claims 35 to 38, and one or more other therapeutic agents.

40. The combination product of claim 39 wherein the additional therapeutic agent is selected from the group consisting of chemotherapeutic agents, cytotoxic agents, vaccines, additional antibodies, anti-infective agents, small molecule drugs, or immunomodulators.

41. The combination of claim 40, wherein the immunomodulatory agent is a PD-1 axis binding antagonist.

42. The combination of claim 41, wherein the PD-1 axis binding antagonist is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody.

43. Use of the GITR-binding antibody or antigen-binding fragment thereof of any one of claims 1 to 24, or the immunoconjugate of claim 33 or 34, or the pharmaceutical composition of any one of claims 35 to 38, or the combination product of any one of claims 39 to 42, for the manufacture of a medicament for the prevention or treatment of a tumor or infection in a subject.

44. The use of claim 43, wherein the tumor is a cancer.

45. The use of claim 44, wherein the cancer is a gastrointestinal cancer.

46. The use of claim 44, wherein the cancer is gastric cancer, rectal cancer, colon cancer, or colorectal cancer.

47. The use of claim 43, wherein the infection is a bacterial infection, a viral infection, a fungal infection, or a protozoal infection.

48. The use of claim 43, wherein the infection is a chronic infection.

49. The use of claim 47, wherein the infection is a chronic infection.

50. The use of any one of claims 41 to 49, wherein the medicament is administered in combination with one or more therapies.

51. The use of claim 50, wherein the therapy is a therapeutic modality and/or other therapeutic agent.

52. The use of claim 51, wherein the treatment modality comprises surgical therapy and/or radiotherapy, and/or the other therapeutic agent is selected from the group consisting of chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies, anti-infective active agents, small molecule drugs, or immunomodulators.

53. The use of claim 52, wherein the immunomodulatory agent is a PD-1 axis binding antagonist.

54. The use of claim 53, wherein the PD-1 axis binding antagonist is an anti-PD-1 antibody or an anti-PD-L1 antibody or an anti-PD-L2 antibody.

55. Use of any GITR-binding antibody or antigen-binding fragment thereof of any one of claims 1 to 22 in the preparation of a detection reagent for detecting GITR in a sample, wherein the detecting comprises

(a) Contacting the sample with any GITR-binding antibody or antigen-binding fragment thereof of any one of claims 1 to 22; and

(b) detecting the formation of a complex between the antibody or antigen-binding fragment thereof that binds GITR and GITR.

56. The use of claim 55, wherein the antibody is detectably labeled.