WO2019201301A1 - Anti-gitr antibody and use thereof - Google Patents

Abstract

The present invention relates to a novel antibody and antibody fragment that specifically bind to GITR and a composition comprising the antibody or the antibody fragment. Moreover, the present invention relates to nucleic acids encoding the antibody or the antibody fragment thereof, a host cell comprising same, and a related use. Furthermore, the present invention relates to a therapeutic and diagnostic use for the antibody and antibody fragment.

Description

anti-GITR antibody and use thereof

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

Background technique

Glucocorticoid-induced tumor necrosis factor receptor (GITR), also known as CD357 and GITR-D, was first discovered in dopamine-treated mouse T cells (Nocentin, PNAS.1997;94:6216-6221), is the 18th member of the tumor necrosis factor receptor (TNFR) superfamily (TNFRSF18), and other members include CD40, CD27, 4-1BB, OX40 and the like. GITR is activated by its cognate ligand, the GITR ligand (GITRL), which activates the downstream NF-kappa B signaling pathway.

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

GITR ligands (GITRL) are primarily expressed on antigen presenting cells (APCs, including macrophages, B cells, dendritic cells, and endothelial cells). Binding of GITRL on APC to GITR on response T cells triggers GITR signaling, stimulates effector T cell activation and inhibits Treg cell activity. Thus, GITR has several effects on effector T cells and regulatory T cells, including: costimulation and activation of effector T cells, and reduction of Treg cell suppression of effector T cells (Nocentini, Eur. J. Immunol. 2007, 37: 1165-1169).

These effects imply that activation of the GITR signaling pathway can result in an increase in the immune response. Therefore, a substance capable of activating the GITR signaling pathway can activate an immune response, thereby increasing the body's ability to resist tumors and infections, and the GITR molecule is highly expressed in Treg cells in the tumor microenvironment, thereby utilizing the ADCC activity of the GITR antibody to clear the GITR molecule. The expressed Treg cells can further enhance tumor killing activity.

A number of antibodies to GITR have been studied in the prior art (see CN103951753A, CN105829343A, CN106459203A, WO2016196792A1, WO2017068186A9, etc.). Such antibodies are agonists of GITR (i.e., are activating antibodies) that induce or enhance GITR signaling and are effective in treating a variety of GITR-related diseases or conditions that require an enhanced immune response. Since such an anti-GITR antibody is an activated antibody, its activation activity is a more critical indicator of its activity (for example, immunopotentiating activity) relative to its binding affinity to an antigen.

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

Summary of the invention

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

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

(i) combined with GITR with high affinity;

(ii) effective binding to the GITR on the cell surface;

(iii) having agonist activity, for example, capable of efficiently activating the NF-kappaB signaling pathway;

(iv) effective activation of T cells (eg, CD4 T cells);

(v) can effectively mediate the ADCC effect;

(vi) having better anti-tumor activity, for example, capable of reducing tumor volume in a subject without affecting the weight of the subject;

(vii) Combination with an anti-PD-1 antibody can better inhibit tumor activity, for example, to reduce tumor volume in a subject without affecting the subject's body weight.

In some embodiments, the invention provides an antibody or antigen-binding fragment thereof that binds to GITR, comprising three heavy chain CDRs (HCDRs) of the sequence set forth in SEQ ID NO: 17, 18, 19 or 20, and/or 3 light chain CDRs (LCDR) of the sequence set forth in SEQ ID NO: 21, 22, 23 or 24.

In some embodiments, the invention provides a nucleic acid encoding an antibody or fragment thereof of the invention, a vector comprising the nucleic acid, a host cell comprising the vector.

In some embodiments, the invention provides methods of making an antibody or fragment thereof of the invention.

In some embodiments, the invention provides immunoconjugates, pharmaceutical compositions, and combination products comprising an antibody of the invention.

The invention also provides methods of mediating ADCC or activating the NF-kappaB signaling pathway in a subject using an antibody of the invention, and methods of preventing or treating cancer or infection.

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

The invention is further illustrated in the following figures and detailed description. However, the drawings and the specific embodiments are not to be construed as limiting the scope of the present invention, and the modifications which are obvious to those skilled in the art are included in the spirit of the invention and the scope of the appended claims.

DRAWINGS

Figure 1 shows the ability of a chimeric antibody to bind to a CHO-S cell line expressing human GITR (CHO-hGITR) by flow cytometry.

Figure 2 shows the ability of humanized antibodies to bind to a human GITR-expressing CHO-S cell line (CHO-hGITR) by flow cytometry.

Figure 3 shows the ability of humanized antibodies to bind to CHO-S cell line (CHO-cynoGITR) expressing cynomolgus GITR by flow cytometry.

Figure 4 shows the MOA assay for the ability of humanized antibodies to activate Hela-GITR-NF-Kappa B luciferace cell lines.

Figure 5 shows the ability of humanized antibodies to activate IL-4 release from activated CD4 T cells.

Figure 6 shows the ability of humanized antibodies to activate CD4 T cells to release IFN-[gamma].

Figure 7 shows the MOA assay for the determination of humanized antibody ADCC activity.

Figure 8 shows the in vivo pharmacodynamic assay of HZ37G5.

Figure 9 shows the in vivo efficacy test of HZ22F4 molecule.

Figure 10 shows mouse body weight detection.

Detailed description of the invention

I. Definition

Before the present invention is described in detail below, it is to be understood that the invention is not limited to the particular methodology, aspects, and reagents described herein, as these may vary. It is also understood that the terminology used herein is for the purpose of describing the particular embodiment 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, unless otherwise defined.

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

The term "about" when used in connection with a numerical value is meant to encompass a numerical value within the range of the lower limit of 5% less than the specified numerical value and the upper limit of 5% greater than the specified numerical value.

As used herein, the term "and/or" means either of the alternatives or two of the alternatives.

The term "comprising" or "including", as used herein, is meant to include the recited elements, integers or steps, but does not exclude any other elements, integers or steps. As used herein, when the term "comprises" or "comprising" is used, it is intended to encompass a combination of elements, integers or steps. For example, when referring to an antibody variable region that "comprises" a particular sequence, it is also intended to encompass an antibody variable region consisting of that particular sequence.

As used herein, the term "GITR" refers to "glucocorticoid-induced TNF-related genes and/or polypeptides", also referred to in the art as TNF receptor superfamily 18 (TNFRSF18), and refers to any vertebrate source, including Any natural GITR of mammals such as primates (eg, humans, cynomolgus monkeys) and rodents (eg, mice and rats), unless otherwise indicated. The term encompasses "full length", unprocessed GITR, and any form of GITR due to processing in cells. The term also encompasses naturally occurring variants of GITR, such as splice variants or allelic variants. The human GITR amino acid sequence can be found in GenBank Accession No. Q9Y5U5. The amino acid sequence of a particular human GITR polypeptide can be found in SEQ ID NO:41. The amino acid sequence of a specific cynomolgus monkey GITR polypeptide is set forth 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 capable of binding (human or cynomolgus) GITR with sufficient affinity. The protein or fragment thereof such that the antibody can be used as a diagnostic and/or therapeutic agent in a targeted (human or cynomolgus) 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% of the antibody binds to (human or cynomolgus) GITR. About 40%, about 50%, about 60%, about 70%, about 80% or about 90% or more, as measured, for example, by radioimmunoassay (RIA) or biooptical interferometry or MSD assay.

"Antibody fragment" refers to a molecule that is 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; diabodies; linear antibodies; single chain antibodies (eg, scFv); single domain antibodies; Specific antibodies or fragments thereof; camelid antibodies; and bispecific antibodies or multispecific antibodies formed from antibody fragments.

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

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

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 more of the binding of the reference antibody to its antigen in a competition assay. In other words, the reference antibody blocks 50%, 60%, 70%, 80%, 90% or more of the binding of the antibody to its antigen in a competition assay. Numerous types of competitive binding assays can be used to determine whether an antibody competes with another assay such as solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition. Assay (see for example Stahli et al, 1983, Methods in Enzymology 9: 242-253).

An antibody that inhibits (eg, competitively inhibits) binding of a reference antibody to its antigen refers to an antibody that inhibits binding of 50%, 60%, 70%, 80%, 90%, or 95% of the reference antibody to its antigen. . Conversely, the reference antibody inhibits binding of the antibody to its antigen by 50%, 60%, 70%, 80%, 90% or more. The binding of an antibody to its antigen can be measured by affinity (eg, 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 binding at least 50%, 60%, 70%, 80%, 90% or 95% of the reference antibody. Affinity and / or specificity. This can be determined by any method known in the art for determining binding affinity and/or specificity.

A "complementarity determining region" or "CDR region" or "CDR" is a sequence that is hypervariable in an antibody variable domain and that forms a structurally defined loop ("hypervariable loop") and/or contains an antigen contact residue ( The area of the "antigen contact point"). The CDR is primarily responsible for binding to an epitope. 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 LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the exact 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 based on the three-dimensional structure of the antibody and the topology of the CDR loop (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al, Sequences of Proteins of Immunological Interest, 4th edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (imgt.cines.fr/ on the World Wide Web), and based on affinity propagation clustering using a large number of crystal structures North CDR definition.

For example, depending on the CDR determination protocol, the residues of each CDR are as follows.

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

Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses a CDR sequence determined in any of the above manners.

Unless otherwise indicated, in the present invention, when referring to the position of a residue in the variable region of an antibody (including a heavy chain variable region residue and a light chain variable region residue), it is meant according to the Kabat numbering system ( The numbered positions of 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 the antibodies of the invention are bordered by AbM rules, for example 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 vary. That is, the CDR sequences of the same antibody variable region defined under different assignment systems are different. Thus, where an antibody is defined by a particular CDR sequence as defined by the present invention, the scope of the antibody also encompasses an antibody whose variable region sequence comprises the particular CDR sequence, but due to the application of a different protocol (eg Different assignment system rules or combinations result in different claimed CDR boundaries than the specific CDR boundaries defined by the present invention.

Antibodies with different specificities (ie, different binding sites for different antigens) have different CDRs. However, although the CDRs differ between antibodies and antibodies, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, Contact, and North methods, the minimal overlap region can be determined to provide a "minimum binding unit" for antigen binding. The minimum binding unit can be a sub-portion of the CDR. As will be apparent to those skilled in the art, residues of the remainder of the CDR sequences can be determined by the structure of the antibody and protein folding. Accordingly, the invention also contemplates variants of any of the CDRs presented herein. For example, in a variant of a CDR, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues defined by Kabat or Chothia may be replaced by conservative amino acid residues.

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

The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with the binding of PD-1 to one or more of its binding partners (such as PD-L1, PD-L2). Interacting signal transduction. In some embodiments, the PD-1 binding antagonist is a molecule that inhibits PD-1 binding to one or more of its binding partners. In a specific aspect, the PD-1 binding antagonist inhibits PD-1 binding to PD-L1 and/or PD-L2. For example, a PD-1 binding antagonist includes an anti-PD-1 antibody that reduces, blocks, inhibits, abolishes or interferes with signal transduction derived from PD-1 interaction with PD-L1 and/or PD-L2, antigen binding Fragments, immunoadhesins, fusion proteins, oligopeptides and other molecules. In one embodiment, the PD-1 binding antagonist reduces a cell surface protein-mediated negative costimulatory signal (via PD-1 mediated signaling) expressed on or via T lymphocytes, thereby enabling dysfunctional T cells Less dysfunctional (eg, 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 WO2015/095423. 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 the binding of PD-L1 from one or more of its binding partners (such as PD-1, B7-1). Interacting signal transduction. In some embodiments, the PD-L1 binding antagonist is a molecule that inhibits PD-L1 binding to its binding partner. In a specific aspect, the PD-L1 binding antagonist inhibits PD-L1 binding to PD-1 and/or B7-1. In some embodiments, the PD-L1 binding antagonist comprises reducing, blocking, inhibiting, eliminating or interfering with the interaction of PD-L1 from one or more of its binding partners (such as PD-1, B7-1) Signal transduction of anti-PD-L1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules. In one embodiment, the PD-L1 binding antagonist reduces a cell surface protein-mediated negative costimulatory signal (via PD-L1 mediated signaling) expressed on or via T lymphocytes, thereby enabling dysfunctional T cells Less dysfunctional (eg, enhancing effector response to antigen recognition). In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In a specific aspect, the anti-PD-L1 antibody is YW243.55.S70, MDX-1105, MPDL3280A or MEDI4736 as disclosed in WO2015/095423.

The term "PD-L2 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with signals derived from the interaction of PD-L2 with one or more of its binding partners, such as PD-1. divert. In some embodiments, the PD-L2 binding antagonist is a molecule that inhibits PD-L2 binding to one or more of its binding partners. In a specific aspect, the PD-L2 binding antagonist inhibits PD-L2 binding to PD-1. In some embodiments, the PD-L2 antagonist comprises reducing, blocking, inhibiting, eliminating or interfering with signal transduction derived from the interaction of PD-L2 with one or more of its binding partners, such as PD-1. Anti-PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules. In one embodiment, the PD-L2 binding antagonist reduces a cell surface protein-mediated negative costimulatory signal (via PD-L2 mediated signaling) expressed on or via T lymphocytes, thereby enabling dysfunctional T cells Less dysfunctional (eg, 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 an Fc receptor (FcR) that is present on certain cytotoxic cells (eg, NK cells, neutrophils, and macrophages). Secretory immunoglobulins enable these cytotoxic effector cells to specifically bind to target cells carrying the antigen, followed by cytotoxicity to kill the cytotoxic form of the target cells. The primary cell NK cells that mediate ADCC express only FcyRIII, whereas monocytes express FcyRI, FcyRII and FcyRIII. Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991) summarizes FcR expression on hematopoietic cells. In order to assess the ADCC activity of the molecule of interest, an in vitro ADCC assay can be performed, such as that described in U.S. Patent No. 5,500,362 or 5,821,337 or U.S. Patent No. 6,737,056 (Presta). Effector cells that can be used in such assays include PBMC and NK cells. Alternatively, or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example, in animal models such as those disclosed in 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 in the present invention refers to a substance which inhibits or prevents cell function and/or causes cell death or destruction.

"Chemotherapeutic agents" include chemical compounds that are useful in the treatment of cancer. For examples of chemotherapeutic agents, see those disclosed in WO 2015/031667, including but not limited to antineoplastic agents, including alkylating agents; antimetabolites; natural products; antibiotics; enzymes; heterogeneous agents; hormones and antagonists; ; antiandrogens; and non-steroidal antiandrogens.

The term "small molecule drug" refers to a low molecular weight organic compound capable of modulating a biological process.

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

The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that 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 terminus 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 based on the EU numbering system, which is also referred to as the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National. Institutes of Health, Bethesda, MD, 1991.

"Antibody in IgG form" refers to the IgG form to which the heavy chain constant region of an antibody belongs. The heavy chain constant regions of all antibodies of the same type are identical, and the heavy chain constant regions differ between different types of antibodies. For example, an antibody in the IgGl form refers to an Ig domain whose heavy chain constant region Ig domain is IgG1.

The term "therapeutic agent" as used herein encompasses any substance that is effective in preventing or treating a tumor (eg, cancer) and an infection (eg, a chronic infection), including chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies, anti-infective active 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 that, when administered to a patient in single or multiple doses, produces the desired effect in a patient in need of treatment or prevention. An effective amount can be readily determined by the attending physician as a person skilled in the art by considering various factors such as the species of the mammal; its size, age and general health; the particular disease involved; the extent or severity of the disease; Response of an individual patient; specific antibody administered; mode of administration; bioavailability characteristics of the administered formulation; selected dosing regimen; and use of any concomitant therapy.

"Therapeutically effective amount" means an amount effective to achieve the desired therapeutic result at the desired dosage and for the period of time required. The therapeutically effective amount of an antibody or antibody fragment or conjugate or composition thereof can vary depending on a variety of 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 detrimental effects of the antibody or antibody fragment or conjugate or composition thereof are less than a therapeutically beneficial effect. A "therapeutically effective amount" preferably inhibits measurable parameters (eg, tumor growth rate, tumor volume, etc.) of at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, relative to an untreated subject, 60% or 70% and still more preferably at least about 80% or 90%. The ability of a compound to inhibit measurable parameters (eg, cancer) can be evaluated in an animal model system that predicts efficacy in human tumors.

By "prophylactically effective amount" is meant an amount effective to achieve the desired prophylactic result at the desired dosage and for the period of time required. Generally, a prophylactically effective amount will be less than a therapeutically effective amount since the prophylactic dose is administered to a subject prior to the earlier stage of the disease or at an earlier stage of the disease.

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 progeny of such a cell. Host cells include "transformants" and "transformed cells" which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parental cell, but may contain mutations. Mutant progeny of the same function or biological activity selected or selected in the originally transformed cells are included herein.

"Human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source, which utilizes a human antibody library or other human Antibody coding sequence. This definition of a human antibody specifically excludes a humanized antibody comprising a non-human antigen-binding residue.

A "humanized" antibody refers to a chimeric antibody comprising an amino acid residue from a non-human CDR and an amino acid residue from a human FR. In some embodiments, a humanized antibody will comprise substantially all of at least one, typically two variable domains, wherein all or substantially all of the CDRs (eg, CDRs) correspond to those of a non-human antibody, and all Or substantially all of the FRs correspond to those of human antibodies. The humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has been humanized.

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

The term "tumor" refers to all 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, a viral infection, a bacterial infection, a fungal infection, or a protozoan such as a parasitic infection.

The term "chronic infection" refers to an infection in which an infectious agent (eg, a pathogen such as a virus, a bacterium, a protozoan such as a parasite, a fungus, or the like) has induced an immune response in an infected host, but has not yet been as acutely infected. It is also removed or eliminated from the host as in the process. Chronic infections can be persistent, latent or slow. Although acute infections (such as influenza) are usually resolved by the immune system in days or weeks, persistent infections can persist for months, years, decades, or lifetime (eg, hepatitis B) at relatively low levels. In contrast, latent infections are characterized by long-term asymptomatic activity, interrupted by high levels of rapid infection and elevated pathogen levels over time (eg, herpes simplex). Finally, slow infection is characterized by a gradual and continuous increase in disease symptoms, such as a long-term incubation period, followed by an onset of prolonged and progressive clinical processes following the onset of clinical symptoms.

An "immunoconjugate" is an antibody that is 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 directly or indirectly conjugated or fused to an agent, such as a polynucleotide probe or antibody, and that facilitates detection of the agent to which it is conjugated or fused. The label itself may be detectable (eg, a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze a chemical change in a substrate compound or composition that is detectable. The term is intended to encompass the direct labeling of a probe or antibody by coupling (ie, physically linking) a detectable substance to a probe or antibody and indirectly labeling 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 biotinylated DNA probe such that it can be detected with a fluorescently labeled streptavidin protein.

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

An "isolated" antibody is one which has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, such as by, for example, electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (eg, ion exchange or reversed phase) Determined by HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al, J. Chromatogr. B848: 79-87 (2007).

"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 a fragment thereof), including such nucleic acid molecules in a single vector or separate vectors. And such nucleic acid molecules present at one or more positions in the 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 (eg, for optimal alignment, in the first and second amino acid sequences or nucleic acid sequences) Vacancies are introduced in one or both 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% for comparison purposes. , 90%, 100% of the reference sequence length. The amino acid residues or nucleotides at the corresponding amino acid position or nucleotide position are then compared. When the position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecule is identical at this position.

Mathematical algorithms can be used to achieve sequence comparisons and percent identity calculations between two sequences. In a preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm in the GAP program that has been integrated into the GCG software package is used (at http://www.gcg.com) Obtained), using a Blossum 62 matrix or PAM250 matrix and vacancy weights 16, 14, 12, 10, 8, 6 or 4 and length weights 1, 2, 3, 4, 5 or 6, to determine between the two amino acid sequences Percent identity. In yet another preferred embodiment, the GAP program in the GCG software package (available at http://www.gcg.com) is used, using the NWSgapdna.CMP matrix and the vacancy weights of 40, 50, 60, 70 or 80 and The length weights 1, 2, 3, 4, 5 or 6, determine the percent identity between the two nucleotide sequences. A particularly preferred set of parameters (and a set of parameters that should be used unless otherwise stated) is a Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

You can also use the PAM120 weighted remainder table, the gap length penalty of 12, and the gap penalty 4), using the E. Meyers and W. Miller algorithms that have been incorporated into the ALIGN program (version 2.0), ((1989) CABIOS, 4:11- 17) Determine the percent identity between two amino acid sequences or nucleotide sequences.

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

The term "pharmaceutically acceptable adjuvant" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier or stabilizer, and the like, which are administered with the active substance.

The term "pharmaceutical composition" refers to a composition that is present in a form that permits the biological activity of the active ingredient contained therein to be effective, and does not include additional toxicity to the subject to whom the composition is administered. Ingredients.

The term "combination product" refers to a kit of fixed or non-fixed combinations in the form of a dosage unit or a portion for administration in combination, wherein two or more therapeutic agents can be independently at the same time or within a time interval Separate administration, especially when these time intervals allow the combined partner to exhibit collaboration, for example, synergistic effects. The term "fixed combination" refers to the simultaneous administration of an antibody of the invention and a combination partner (eg, other therapeutic agents, eg, an anti-PD1 antibody, an anti-PDL1 antibody, or an anti-PDL2 antibody) to a patient in a single entity or dosage form. The term "non-fixed combination" means that the antibody of the invention and a combination partner (eg, other therapeutic agents, eg, an anti-PD1 antibody, an anti-PDL1 antibody, or an anti-PDL2 antibody) are administered to the patient simultaneously, in parallel, or sequentially as separate entities, without a specific time. Limitations, wherein such administration provides a therapeutically effective level of both compounds in a patient. The latter also applies to cocktail therapy, for example 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 a cancer or infection as described in the present disclosure. Such administration involves co-administering these therapeutic agents in a substantially simultaneous manner, for example, as a single capsule having a fixed ratio of active ingredient. Alternatively, such administration includes co-administration of the individual active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). The powder and/or liquid can be reconstituted or diluted to the desired dose prior to administration. Moreover, such administration also includes the use of each type of therapeutic agent in a sequential manner at substantially the same time or at different times. In either case, the treatment regimen will provide a beneficial effect of the combination of drugs in treating the condition or condition described herein.

As used herein, "treating" refers to slowing, interrupting, arresting, ameliorating, 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 progression of a disease or condition or a symptom of a particular disease or condition. In some embodiments, a subject with a family history of cancer is a candidate 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 a symptom or symptom of a 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 microorganisms at the concentration of administration and the interval of administration, but is not lethal to the host, such as viruses, bacteria, fungi or protozoa, such as parasites. As used herein, the term anti-infective active agent includes antibiotics, antibacterial agents, antiviral agents, antifungal agents, and antiprotozoal agents. In a specific aspect, the anti-infective active agent is non-toxic to the host at the concentration of administration and the interval of administration.

Antibacterial anti-infective active agents or antibacterial agents can be broadly classified as either bactericidal (i.e., directly killed) or bacteriostatic (i.e., preventing division). Antibacterial anti-infective active agents can be further reclassified as narrow-spectrum antibacterial agents (i.e., affecting only subtypes of bacterial subtypes, e.g., Gram-negative, etc.) or broad-spectrum antibacterial agents (i.e., affecting a wide variety of species).

The term "antiviral agent" includes any substance that inhibits or eliminates the growth, pathogenesis, and/or survival of a virus.

The term "antifungal agent" includes any substance that inhibits or eliminates the growth, pathogenesis and/or survival of fungi.

The term "antigenic animal agent" includes any substance that inhibits or eliminates the growth, morbidity and/or survival of a protozoan organism (eg, a parasite).

The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes a vector that is a self-replicating nucleic acid structure and a vector that binds to the genome of a host cell into which it has 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, such as from a fresh, frozen and/or preserved organ or tissue sample or a biopsy sample or a puncture sample; blood or any blood component; body fluids such as cerebrospinal fluid, amniotic fluid (amniotic fluid) ), peritoneal fluid (ascites), or interstitial fluid; cells from the subject's pregnancy or development at any time. Tissue samples may contain compounds that are naturally not intermixed with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. Examples of tumor samples include, but are not limited to, tumor biopsy, fine needle aspirate, bronchial lavage fluid, pleural fluid (thoracic fluid), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulation Plasma protein, 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 tumors sample.

II. Antibody

In some embodiments, the anti-GITR antibody or fragment of the invention binds with high affinity GITR (GITR e.g. human or cynomolgus GITR), for example, in the following equilibrium dissociation constant (K _D) in combination with GITR, the K _{D is} less than about 10 nM, preferably, less than or equal to about 7 nM, more preferably less than or equal to about 6 nM, more preferably less than or equal to about 5 nM, 4.9 nM, 4.8 nM, 4.7 nM, 4.6 nM, 4.5 nM, 4 nM, or of 3 nM, and most preferably, the K _D of less than or equal to about 2.5nM, 2.4nM, 2.3nM, 2.2nM, 2.1nM. In some embodiments, the anti-GITR antibody of the present invention to 1nM-6nM, preferably 1.5nM-5nM, 1.7nM-5nM, 1.8nM-5nM, K D 1.9nM-5nM GITR binding. In some embodiments, the GITR is a human GITR. In some embodiments, the antibody binding affinity is determined using a bio-optical interference assay (eg, Fortebio affinity measurement).

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

In some embodiments, an antibody or fragment thereof of the invention activates the NF-kappaB signaling pathway downstream of the GITR by binding to a GITR molecule on the cell surface. In some embodiments, an antibody of the invention or a fragment thereof has a higher ability to activate than a known anti-GITR antibody, such as the GITR antibody 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 NO: 44 and SEQ ID NO: 54, respectively, in US20130183321A1. In some embodiments, a GITR antibody molecule of the invention is capable of significantly and efficiently activating the NF kappa B signaling pathway compared to a control antibody molecule. In some embodiments, the activation is obtained by luciferase assay.

In some embodiments, an antibody of the invention or a fragment thereof enhances effector T cell function, such as activating effector T cells. In some embodiments, an antibody or fragment thereof of the invention is capable of enhancing proliferation of effector T cells. In some embodiments, an antibody or fragment thereof of the invention increases the secretion/expression of interferon (eg, IFN-[gamma]). In some embodiments, an antibody or fragment thereof of the invention increases secretion/expression of interleukin (eg, IL-2). In some embodiments, the T cell is a CD4 T cell.

In some embodiments, a GITR antibody or fragment thereof of the invention is capable of activating a GITR signaling pathway in a regulatory T cell (eg, a Treg cell), thereby eliminating cells by mediating the ADCC effect. Thus, on the one hand, the inhibitory signal of the effector T cell itself is released, the activity of the CD4 T cell and/or the CD8 T cell is enhanced, and the other aspect is to eliminate the inhibitory effect of the T cell on the effector T cell, thereby maximally activating the effector T cell and enhancing An immune response to tumor cells. In some embodiments, the ADCC activity of the antibody is detected by detecting NF-AT signaling activation. In some embodiments, activation of the NF-AT signal is manifested by expression of a fluorescent reporter gene. In some embodiments, the GITR antibody molecules of the invention are capable of significantly and efficiently activating the NF-AT signaling pathway compared to known anti-GITR antibody molecules. In some embodiments, the GITR antibody molecules of the invention are capable of significantly and efficiently mediating ADCC effects compared to known anti-GITR antibody molecules. In some embodiments, a GITR antibody molecule of the invention is capable of significantly inhibiting or eliminating regulatory T cells compared to known anti-GITR antibody molecules. In some embodiments, the effector T cells of the invention are CD4 T cells. In some embodiments, a known anti-GITR antibody molecule is an anti-GITR antibody as reported in, for example, 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 NO: 44 and SEQ ID NO: 54, respectively, in US20130183321A1.

In some embodiments, an antibody or fragment thereof of the invention (optionally in combination with a therapeutic modality and/or other therapeutic agent, such as a PD-1 axis binding antagonist) is capable of preventing or treating a tumor. In some embodiments, an antibody or fragment thereof of the invention can be used to inhibit or reduce tumor growth (eg, reduce tumor volume). In some embodiments, an antibody or fragment thereof of the invention can also be used to maintain body weight in a tumor patient. In some embodiments, an antibody or fragment thereof of the invention is capable of inhibiting or reducing tumor growth (eg, reducing tumor volume) in combination with a therapeutic modality and/or other therapeutic agent, such as a PD1-axis binding antagonist. In some embodiments, an antibody or fragment thereof of the invention is capable of maintaining the weight of a tumor patient in combination with a therapeutic modality and/or other therapeutic agent, such as a PD1-axis binding antagonist. In some embodiments, an antibody of the invention or a fragment thereof (optionally in combination with a therapeutic modality and/or other therapeutic agent, such as a PD-1 axis binding antagonist) is capable of inhibiting or reducing tumor growth (eg, reducing tumor volume) ), while not affecting the weight of cancer patients. Preferably, the tumor is a gastrointestinal tumor. Preferably, the tumor is a cancer, such as a gastrointestinal cancer, such as gastric cancer, rectal cancer, colon cancer, colorectal cancer, and the like. In some embodiments, an antibody of the invention or a fragment thereof (optionally in combination with a PD-1 axis binding antagonist) is capable of preventing or treating an infection, such as a chronic infection, including a bacterial infection, a fungal infection, a viral infection, a protozoal infection. Wait.

In some embodiments, an anti-GITR antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH), wherein said VH comprises

(i) three complementarity determining regions (CDRs) contained in the VH of any of the antibodies listed in Table B, or

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

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

(i) three complementarity determining regions (CDRs) contained in the VL of any of the antibodies listed in Table B;

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

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

(a) The VH contains

(i) three complementarity determining regions (CDRs) contained in the VH of any of the antibodies listed in Table B, or

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

(b) The VL contains:

(i) three complementarity determining regions (CDRs) contained in the VL of any of the antibodies listed in Table B;

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

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

In a preferred embodiment, VL comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 22, 23 or 24.

In a preferred embodiment, the anti-GITR antibody or antigen-binding fragment thereof of the invention comprises

(i) three complementarity determining region HCDRs of the heavy chain variable region set forth in SEQ ID NO: 17 or 19, and three complementarity determining regions of the light chain variable region set forth in SEQ ID NO: 21 or LCDR, or

(ii) three complementarity determining region HCDRs of the heavy chain variable region set forth in SEQ ID NO: 18 or 20, and three complementarity determining regions of the light chain variable region set forth in SEQ ID NO: 22 or LCDR.

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

(i) said VH comprises a complementarity determining region (CDR) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence selected from SEQ ID NO: 1, 2, 3 or 4, or HCDR1 comprises An amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3 or 4; HCDR2 comprising an SEQ ID NO: 5 Or the amino acid sequence of 6 or consists of the amino acid sequence, or HCDR2 comprises one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence selected from SEQ ID NO: 5 or 6. Amino acid sequence; HCDR3 comprises or consists of the amino acid sequence selected from SEQ ID NO: 7 or 8, or HCDR3 comprises one, two or more than the amino acid sequence selected from SEQ ID NO: 7 or 8. An amino acid sequence of three alterations (preferably amino acid substitutions, preferably conservative substitutions);

and / or

(ii) wherein said VL comprises a complementarity determining region (CDR) LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence selected from SEQ ID NO: 9 or 10, or LCDR1 comprises and is selected from the group consisting of ID NO: amino acid sequence of 9 or 10 having an amino acid sequence of one, two or three changes (preferably amino acid substitution, preferably conservative substitution); LCDR2 comprising an amino acid selected from the group consisting of SEQ ID NO: 11, 12, 13 or 14. The sequence consists either of said amino acid sequence, or LCDR2 comprises one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from SEQ ID NO: 11, 12, 13 or 14. Amino acid sequence; LCDR3 comprises or consists of the amino acid sequence selected from SEQ ID NO: 15 or 16, or LCDR3 comprises one, two or three compared to the amino acid sequence selected from SEQ ID NO: 15 or 16. Amino acid sequence that changes (preferably amino acid substitutions, preferably conservative substitutions).

In a preferred embodiment, the 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) The VH contains

(i) a combination of HCDR1, HCDR2 and HCDR3 as shown in Table A; or

(ii) a variant of the HCDR combination of (i) comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably) on said three CDR regions Conservative substitution);

and / or

(ii) the VL contains

(i) a combination of LCDR1, LCDR2 and LCDR3 as shown in Table A; or

(ii) a variant of the LCDR combination of (i), said variant comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes on said three CDR regions (preferably amino acid substitution, preferably Conservative substitution).

In a preferred embodiment, the 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 said VH comprises a complementarity determining region ( CDR) HCDR1, HCDR2 and HCDR3 comprising (CDR) LCDR1, LCDR2 and LCDR3, wherein the combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprised by the antibody or antigen-binding fragment thereof is as follows (Table A):

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

In some embodiments, an anti-GITR antibody or antigen-binding fragment thereof of the 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 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or an amino acid sequence selected from the group consisting of SEQ ID NO: 17, 18, 19 or 20. 99% identical amino acid sequence or consists of; or

(ii) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 17, 18, 19 or 20;

(iii) comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 17, 18, 19 or 20. Amino acid sequence of an amino acid change (preferably amino acid substitution, more preferably amino acid conservative substitution), preferably, said amino acid change does not occur in the CDR region;

and / or

(b) Light chain variable region VL

(i) comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 22, 23 or 99% identical amino acid sequence or consists of;

(ii) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 22, 23 or 24;

(iii) comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 22, 23 or 24. The amino acid sequence of an amino acid change (preferably an amino acid substitution, more preferably an amino acid conservative substitution), preferably, the amino acid change does not occur in the 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) and a light chain variable region (VL), wherein the antibody or antigen-binding fragment thereof comprises The combination of the heavy chain variable region VH and the light chain variable region VL is shown 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, an anti-GITR antibody or antigen-binding fragment thereof of the invention comprises a heavy chain and/or a light chain, wherein

(a) heavy chain

(i) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and an amino acid sequence selected from the group consisting of SEQ ID NO: 25, 26, 27 or a 98% or 99% identity amino acid sequence or consists of;

(ii) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 25, 26, 27 or 28;

(iii) comprising one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2) compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 25, 26, 27 or 28. Amino acid sequence of amino acid change (preferably amino acid substitution, more preferably amino acid conservative substitution), preferably, the amino acid change does not occur in the CDR region of the heavy chain, and more preferably, the amino acid change does not occur in Heavy chain variable region;

and / or

(b) Light chain

(i) comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and an amino acid sequence selected from the group consisting of SEQ ID NO: 29, 30, 31 or 32, a 98% or 99% identity amino acid sequence or consists of;

(ii) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 29, 30, 31 or 32;

(iii) comprising one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2) compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 29, 30, 31 or 32 Amino acid sequence of amino acid change (preferably amino acid substitution, more preferably amino acid conservative substitution), preferably, the amino acid change does not occur in the CDR region of the light chain, and more preferably, the amino acid change does not occur in Light chain variable region.

In a preferred embodiment, the invention provides an anti-GITR antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the combination of the heavy chain and the light chain comprised by the antibody or antigen-binding fragment thereof is as follows (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, such as METDTLLLWVLLLWVPGSTG (SEQ ID NO: 43).

In one embodiment of the invention, the amino acid changes described herein include substitutions, insertions or deletions of amino acids. 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 FR). More preferably, the amino acid changes described herein 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. A conservative substitution is one in which one amino acid is replaced by another amino acid in the same class, for example, one acidic amino acid is replaced by another acidic amino acid, one basic amino acid is replaced by another basic amino acid, or one neutral amino acid is passed through another neutral amino acid. Replacement. An exemplary substitution is shown in Table D below:

Table D

Primitive residue	Exemplary replacement	Preferred conservative amino acid substitution
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 substitution occurs in the CDR regions of the antibody. Generally, the variant obtained has modifications (e. g., improved) relative to the parent antibody in certain biological properties (e.g., increased affinity) and/or will have certain biological properties that are substantially retained by the parent antibody. An exemplary substitution variant is an affinity matured antibody.

In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent of glycosylation of the antibody. Addition or deletion of a glycosylation site to an antibody can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites. When the antibody comprises an Fc region, the saccharide attached thereto can be altered. In some applications, modifications that remove unwanted glycosylation sites may be useful, such as removal of fucose modules to enhance antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733 ). In other applications, galactosidation modification can be performed to modify complement dependent cytotoxicity (CDC).

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to produce an Fc region variant to enhance the effectiveness of, for example, an antibody in treating cancer or a cell proliferative disorder. An Fc region variant can include a human Fc region sequence (eg, a human IgGl, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (eg, a substitution) at one or more amino acid positions. For examples of Fc variants, see U.S. Patent No. 7,332,581, U.S. Patent No. 6,737,056, U.S. Patent No. 6,737,056, WO2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001), U.S. Patent No. 6,194,551, WO 99/51642 and Idusogie et al. J. Immunol. 164: 4178-4184 (2000), U.S. Patent No. 7,371,826, Duncan & Winter, Nature 322: 738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; And WO 94/29351.

In certain embodiments, it may be desirable to produce a cysteine engineered antibody, such as a "thiocarba", wherein one or more residues of the antibody are replaced with a cysteine residue. Cysteine engineered antibodies can be generated 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 portions known in the art and readily available. Portions suitable for antibody derivatization 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, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly -1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), and dextran or poly(n-ethylene) Pyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyol (such as glycerin), polyvinyl alcohol, and mixtures thereof.

In some embodiments, an anti-GITR antibody or antigen-binding fragment thereof of the invention has one or more of the following characteristics:

(i) showing the same or similar binding affinity and/or specificity to any of the antibodies listed in Table 3 for GITR;

(ii) inhibiting (eg, competitive inhibition) the binding of any of the antibodies listed in Table 3 to GITR;

(iii) an epitope that binds to the same or overlaps with any of the antibodies shown in Table 3;

(iv) competing with any of the antibodies shown in Table 3 to bind GITR;

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

In some embodiments, an anti-GITR antibody of the invention is an antibody in the IgGl form or an antibody in the IgG2 form or an antibody in the IgG4 form.

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 person, as reviewed by Almagro & Fransson, the contents of which are incorporated herein by reference in its 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 prepared using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol 20: 450-459 (2008).

In some embodiments, 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, an anti-GITR antibody of the invention further encompasses an antibody fragment thereof, preferably an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single chain antibody (eg, scFv) or (Fab' ₂ ) Single domain antibody, double antibody (dAb) or 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. The multispecific antibody molecule can have any combination of binding specificities for the aforementioned molecules. The multispecific antibody molecule can be, for example, a trispecific antibody molecule comprising a first binding specificity for GITR and a second and third binding specificity for a molecule of one or more of the following: PD-1, PD- L1 or PD-L2.

II. 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 the nucleic acid or the vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from the group consisting of a yeast cell, a mammalian cell (eg, a CHO cell or a 293 cell), or other cell suitable for use in the preparation 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 comprising an amino acid sequence encoding a light chain and/or a heavy chain of an antibody. An exemplary nucleic acid sequence encoding an antibody heavy chain variable region comprising at least 80%, 85%, 90%, 91%, 92%, 93% of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 33, 34, 35 or 36 a nucleic acid sequence of 94%, 95%, 96%, 97%, 98% or 99% identity, or a nucleic acid sequence selected from the group consisting of SEQ ID NO: 33, 34, 35 or 36. An exemplary nucleic acid sequence encoding an antibody light chain variable region comprises at least 80%, 85%, 90%, 91%, 92%, 93% of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 37, 38, 39 or 40 A nucleic acid sequence of 94%, 95%, 96%, 97%, 98% or 99% identity, or a nucleic acid sequence selected from the group consisting of SEQ ID NO: 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 phage, or yeast artificial chromosomes (YAC). In one embodiment, the vector is a pTT5 vector.

In one embodiment, a host cell comprising the vector is provided. Suitable host cells for cloning or expressing a vector encoding the antibody include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199 and 5,840,523, also to Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ, 2003). , pp. 245-254, which describes the expression of antibody fragments in E. coli). 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 the group consisting of a yeast cell, a mammalian cell, or other cell suitable for use in the preparation of an antibody or antigen-binding fragment thereof. For example, a eukaryotic microorganism such as a filamentous fungus or yeast is a suitable cloning or expression host for a vector encoding an antibody. For example, fungal and yeast strains that have been "humanized" by the glycosylation pathway result in the production of antibodies with partial or complete human glycosylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al, Nat. Biotech. 24: 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 suspension growth can be used. Other examples of useful mammalian host cell lines are the monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney line (293 HEK or 293F or 293 cells such as, for example, Graham et al, J. Gen Virol. 36:59 ( (described in 1977) and so on. 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.; Tumor cell lines such as Y0, NSO and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa). , 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 suitably expressing an antibody or fragment thereof (preferably an antigen-binding fragment) encoding the antibody The host cell is cultured under conditions of a nucleic acid, and the antibody or fragment thereof (preferably an antigen-binding fragment) is optionally isolated. In a certain embodiment, the method further comprises recovering an 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, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, and optionally, under conditions suitable for expression of the antibody The antibody is recovered from the host cell (or host cell culture medium). For recombinant production of an anti-GITR antibody, a nucleic acid encoding an antibody (such as the antibodies described above) is 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 the antibody).

III. Assay

The anti-GITR antibodies provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art. In one aspect, the antibody of the present invention is tested for its 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, and exemplary methods are disclosed herein. In some embodiments, biophotonic interferometry (eg, Fortebio affinity measurement) or MSD assay is used.

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

The invention also provides assays for identifying biologically active anti-GITR antibodies. Biological activity can include, for example, binding to GITR (eg, binding to human and/or cynomolgus GITR), increasing GITR-mediated signal transduction (eg, increasing NFkappa-B signaling pathway), and attenuating cells expressing GITR by ADCC (eg, Treg cells) Enhancing T effector cell function (eg, CD4 effector T cells) (eg, by increasing cytokine production of effector T cells (eg, interferons such as IFN-[gamma] or interleukins such as IL2)). Antibodies having such biological activity in vivo and/or in vitro are also provided.

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

T cell activation can be assayed using methods known in the art. For example, it is determined by the level of a cytokine released after T cell activation, such as interferon (such as IFN-γ) or interleukin (such as IL2). GITR signaling (e.g., NF-kappaB signaling pathway) can also be assayed using methods well known in the art to determine T cell activation. In one embodiment, a transgenic cell expressing a human GITR and a reporter gene comprising a NF-kappa B promoter fused to a reporter gene (e.g., beta luciferase) is generated. Addition of anti-GITR antibodies to cells results in increased NF-kappa B transcription, which is detected using an assay for the reporter gene (eg, a luciferase reporter assay).

The ADCC effect of an antibody can be determined using methods known in the art. For example, activation by NF-AT signal. In one embodiment, a transgenic cell comprising an ADCC effector cell comprising an NF-AT promoter fused to a reporter gene (eg, beta luciferase) is obtained. Co-culture of effector cells with cells that highly express GITR, while the addition of anti-GITR antibodies results in increased NF-AT transcription of effector cells, which is detected using assays for reporter genes (eg, luciferase reporter assay).

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

It will be appreciated that any of the above assays can be performed by replacing or supplementing the anti-GITR antibody with an immunoconjugate of the invention.

It will be appreciated that any of the above assays can be performed using an anti-GITR antibody and another active agent.

IV. Immunoconjugate

In some embodiments, the invention provides an immunoconjugate comprising any of the anti-GITR antibodies and other materials provided herein, such as a cytotoxic agent. In some embodiments, other substances such as therapeutic agents, such as cytotoxic or immunosuppressive agents or chemotherapeutic agents. 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, radioisotopes; growth inhibitors; enzymes and fragments thereof such as nucleases; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including Fragments and/or variants; and various anti-tumor or anti-cancer agents known.

In some embodiments, the immunoconjugate is for use in preventing or treating a tumor, such as a gastrointestinal tumor. In some embodiments, the tumor is a cancer, such as a gastrointestinal cancer, such as gastric cancer, rectal cancer, colon cancer, colorectal cancer, and the like. In some embodiments, the immunoconjugate is used to prevent or treat an infection, such as a chronic infection, such as a bacterial infection, a viral infection, a fungal infection, a protozoal infection, and the like.

V. Pharmaceutical compositions and pharmaceutical preparations

In some embodiments, the invention provides compositions comprising any of the anti-GITR antibodies or fragments thereof, preferably antigen-binding fragments thereof, or immunoconjugates thereof, preferably a composition, which is a pharmaceutical composition. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, a composition, eg, a pharmaceutical composition, comprises an anti-GITR antibody or fragment thereof of the invention, or an immunoconjugate thereof, and one or more additional therapeutic agents (eg, chemotherapeutic agents, cytotoxic agents, A combination of a vaccine, other antibody, anti-infective active agent, small molecule drug or immunomodulatory agent, 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 use in preventing or treating a tumor, such as a gastrointestinal tumor. In some embodiments, the tumor is a cancer, such as a gastrointestinal cancer, such as gastric cancer, rectal cancer, colon cancer, colorectal cancer, and the like. In some embodiments, the composition is for preventing or treating an infection, such as a chronic infection, such as a bacterial infection, a viral infection, a fungal infection, a protozoal infection, and the like.

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

Pharmaceutically acceptable carriers suitable for use in the present invention may 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 used as liquid carriers, especially for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk , glycerin, propylene, glycol, water, ethanol, etc. 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. If desired, the compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. 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.

Preparation can be made by mixing an anti-GITR antibody of the invention having the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th Ed., Osol, A. Ed. (1980)). The pharmaceutical preparations of the anti-GITR antibodies described herein are preferably in the form of a lyophilized formulation or an aqueous solution.

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

The pharmaceutical compositions or formulations of the present invention may further comprise one or more additional active ingredients which are required for the particular indication being treated, preferably those which do not adversely affect each other's complementary activities. . For example, it is 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 (eg, anti- PD-1 antibody or anti-PD-L1 antibody or anti-PD-L2 antibody) and the like. The active ingredient is 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 antibodies in the form of shaped articles such as films or microcapsules.

With regard to other components of the pharmaceutical preparations/pharmaceutical compositions comprising the antibodies of the invention, reference is also made to those disclosed in WO 2015/031667 or WO 2015/187835 and the like.

VI. Combination products

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

In some embodiments, the combination product is for use in preventing or treating a tumor, such as a gastrointestinal tumor. In some embodiments, the tumor is a cancer, such as a gastrointestinal cancer, such as gastric cancer, rectal cancer, colon cancer, colorectal cancer, and the like. In some embodiments, the combination product is for use in preventing or treating an infection, such as a chronic infection, such as a bacterial infection, a viral infection, a fungal infection, a protozoal infection, and the like.

VII. Use

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

A further aspect of the invention provides a method of increasing effector T cell function, such as 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, immunologically conjugated to the subject Compound, pharmaceutical composition or combination product. In some embodiments, the function is an increase in effector T cell proliferation. In some embodiments, the activated T cell is characterized by increased secretion/expression of interferon or interleukin. In some embodiments, the interleukin is IL2. In some embodiments, the interferon is IFN-[gamma].

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

In an aspect, the invention 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 composition or combination product.

In an aspect, the invention 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 composition or combination product.

The invention also provides a method of preventing or treating other conditions or conditions 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 described herein , immunoconjugate, pharmaceutical composition or combination product.

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

In some embodiments, the tumors described herein, eg, cancer, include, but are not limited to, solid tumors, hematological cancers, soft tissue tumors, and metastatic lesions. In some embodiments, the cancers described herein for treatment include but breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, rectal cancer, colorectal cancer, cervical cancer, brain cancer, skin cancer, liver cancer, pancreas Solid tumors such as cancer or stomach 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 PDl) 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 PDl.

In some embodiments, the cancer described herein is colon cancer and metastatic cancer 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 for the manufacture or preparation of a medicament for the treatment of a related disease or condition as referred to herein.

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

In some embodiments, the prophylactic or therapeutic methods described herein further comprise administering to the subject or individual a combination of an antibody molecule or pharmaceutical composition or immunoconjugate disclosed herein, and one or more additional therapies, For example, treatment modalities and/or other therapeutic agents.

In some embodiments, the treatment modality includes surgery (eg, tumor resection); radiation therapy (eg, external particle beam therapy, which involves three-dimensional conformal radiation therapy in which the illumination region is designed), local illumination (eg, pointing to a pre-selected target) Or irradiation of organs) or focused illumination).

In some embodiments, the therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a cytotoxic agent, a vaccine, other antibodies, 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 can be a DNA based vaccine, an RNA based vaccine or a virus transduced vaccine. Cancer vaccines can be prophylactic or therapeutic.

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

Exemplary additional antibodies include, but are not limited to, anti-PD-1 antibodies, anti-PDL1 antibodies, or anti-PDL2 antibodies. A variety of anti-PD-1 antibodies, anti-PDL1 antibodies or anti-PDL2 antibodies are known in the art, see for example WO2007/005874, WO2009/101611, WO2009/114335, WO2010/027827 and WO2011/066342, and the like.

In some embodiments, the antibody combinations described herein can be administered separately, eg, as separate antibodies, or when ligated (eg, as a bispecific or trispecific antibody molecule).

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

Such combination therapies encompasses combined administration (eg, two or more therapeutic agents are included in the same formulation or separate formulations), and administered separately, in which case other therapeutic agents and/or agents may be administered. Administration of the antibodies of the invention occurs previously, simultaneously, and/or after. In one embodiment, administration of the anti-GITR antibody and administration of the additional therapeutic agent are within about one month, or within about one, two or three weeks, or about 1, 2, 3, 4, 5, or 6 days of each other. occur.

The therapeutically effective amount of the pharmaceutical composition comprising the anti-GITR antibody to be employed will depend, for example, on the therapeutic background and objectives. Those skilled in the art will appreciate that appropriate dosage levels for treatment will vary, depending in part on the molecule being delivered, the indication being used, the route of administration, and the weight, body surface area or organ size and/or condition of the patient (age and general health status). In certain embodiments, the clinician can titrate the dose and alter the route of administration to achieve 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 will administer the composition until the dose that achieves the desired effect is achieved. The antibodies of the invention may therefore be administered in a single dose, or in two or more doses (which may comprise the same or different amounts of the desired molecule) over a period of time, or by continuous infusion via an implant device or catheter. The appropriate dose can 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, every month, every two months, every three months, every four months, every five months, or every six months.

The administration route of the pharmaceutical composition is according to known methods, for example, orally, by intravenous injection, intraperitoneal, intracerebral (intrinsic), intraventricular, intramuscular, intraocular, intraarterial, intraportal or intralesional. Route; through a sustained release system or through an implanted device. In certain embodiments, the composition can be administered by bolus injection or by continuous infusion or by an implant device.

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

It will be appreciated that any treatment can be performed by replacing or supplementing the anti-GITR antibody with the immunoconjugate of the invention.

VIII. 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, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (eg, FACS), magnetic binding of antibody molecules, ELISA assays. Method, PCR-technology (for example, RT-PCR). In certain embodiments, the biological sample is a blood, serum or other liquid sample of biological origin. In certain embodiments, the biological sample comprises cells or tissues. In some embodiments, the biological sample is from a hyperproliferative or cancerous lesion.

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

In one embodiment, a cancer or tumor can be diagnosed using an antibody of the invention, for example, to assess (eg, monitor) the treatment or progression, diagnosis, and/or staging of a disease (eg, cancer or tumor) described herein in a subject. In certain embodiments, a labeled anti-GITR antibody is provided. Labels include, but are not limited to, directly detected labels or moieties (such as fluorescent labels, chromophore labels, electron dense labels, chemiluminescent labels, and radioactive labels), as well as indirectly detected portions, such as enzymes or ligands, such as By enzymatic reaction or molecular interaction. Exemplary labels include, but are not limited to, radioisotopes ³² P, ¹⁴ C, ¹²⁵ I, ³ H, and ¹³¹ I, iridium groups such as rare earth chelates or ruthenium and its derivatives, rhodamine and its derivatives, Dan Dansyl, umbelliferone, Luceriferase, for example, firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrogen Pyridazinone, horseradish peroxidase (HR), alkaline phosphatase, beta-galactosidase, glucoamylase, lytic enzyme, carbohydrate oxidase, for example, glucose oxidase, galactose oxidase And glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, and enzymes utilizing hydrogen peroxide dye precursors such as HR, lactoperoxidase, or microperoxide Microperoxidase, biotin/avidin, spin labeling, phage labeling, stable free radicals, and more.

In some embodiments of any of the inventions provided herein, the sample is obtained prior to treatment with an anti-GITR antibody. In some embodiments, the sample is obtained prior to treatment with a 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 (eg, a core biopsy), a surgical specimen (eg, from a surgically resected specimen), or a fine needle aspirate.

In some embodiments, the GITR is detected prior to treatment, for example, prior to initiation of treatment or prior to treatment after the treatment interval.

In some embodiments, a method of treating a tumor or infection is provided, the method comprising: examining a subject (eg, a sample) (eg, a sample of a subject comprising cancer cells) for detecting the presence of a GITR, thereby determining A GITR value, the GITR value is compared to a control value, and if the GITR value is greater than the control value, the subject is administered a therapeutically effective amount of an anti-GITR antibody, optionally in combination with one or more other therapies (eg, as described herein) Anti-GITR antibodies), thus treating tumors or infections.

IX Exemplary Anti-GITR Antibodies of the Invention

These and other aspects and embodiments of the present invention are described in the drawings (hereinafter briefly described below) and the following detailed description of the invention and are illustrated 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 invention is further illustrated by the following examples, which are to be understood by way of illustration and not limitation

Example

Example 1. Preparation of hybridoma cells

Hybridoma technology is achieved by fusing two cells while maintaining the main features of both. These two cells are antigen-immunized mouse spleen cells and mouse myeloma cells, respectively. The main feature of mouse spleen cells (B lymphocytes) immunized by specific antigens is its antibody secretion function, but it cannot be continuously cultured in vitro, and mouse myeloma cells can divide and proliferate indefinitely under culture conditions, that is, The so-called immortality. Under the action of the selective medium, only the hybrid cells in which the B cells are fused with the myeloma cells can have the ability to continue to culture, and form a cell clone having both the secretory function of the antibody and the immortality of the cells. In this experiment, mice were immunized with human GITR protein, and then spleen cells of the mouse were fused with myeloma cells to obtain hybridoma cells capable of expressing positive antibodies.

Hybridoma fusion

Laboratory animals and immune information

Prepare the electrofusion dish: Thoroughly soak the electrofusion dish with 70% ethanol and blow dry in a clean bench.

Isolation of spleen cells: The mice were sacrificed by cervical dislocation, and the body surface was sterilized with 75% alcohol for 5 min, and then placed on the anatomical plate of the mouse in the ultra-clean table, and the left side was placed, and the limbs were fixed with a 7-gauge needle. The spleen was removed by aseptic opening of the abdominal cavity, washed with a basal medium (configuration method as follows), and the connective tissue attached thereto was carefully removed. The spleen was then transferred to another dish containing the basal medium. Press the spleen with a elbow needle, insert a small needle into the spleen, and squeeze with the scorpion to fully release the spleen cells to make a spleen cell suspension. The cell suspension was filtered through a 70 μM cell sieve, washed once with 30 ml of basal medium, and centrifuged at 1200 rpm for 6 min.

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

Electrofusion: Mouse myeloma cells SP2/0 cells (ATCC) were resuspended in 20 ml of basal medium and counted. SP2/0 and spleen cells were mixed at a ratio of 1:2 to 1:1, and centrifuged at 1000 rpm for 6 min. After removing the supernatant, the mixed cells were resuspended in 10 ml of fusion buffer (BTXpress). Further, 15 ml of the fusion buffer was added, and the mixture was centrifuged at 1000 rpm for 5 minutes, and the supernatant was removed. After repeating the above steps, the cells were re-selected with an appropriate amount of fusion buffer, and the mixed cell density was adjusted to 1 × 10 ⁷ cells/ml. The parameters of the electrofusion amplifier are set as follows. 2 ml of the 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

Plate after electrofusion: The cells were allowed to stand in an electric fusion dish for 5 min at room temperature. The cells were transferred to a centrifuge tube, and the cells were diluted to 1 to 2 × 10 ⁴ cells/ml using a screening medium (the configuration method is as follows). 100 μl of the cell suspension was added to each well of a 96-well plate. The screening medium was changed on the 7th day after the fusion. Screening was performed 10 days after culture (or longer, depending on the state of cell growth). Hybridoma cells expressing a specific anti-GITR antibody were screened by FACS (C6 (BD Biosciences)).

Positive hybridoma cell subcloning

Subcloning step: A 96-well plate was prepared, and 200 μl of the basal medium as described above was added to each of the 2nd to 8th columns. The cells of the positive wells selected by the above fusion were made into cell suspensions and added to the first column. 100 μl of the cell suspension of column 1 was added to the second column, and after thorough mixing, 100 μl was added to the next column. The above procedure was repeated until the last column volume became 300 μl; the 96-well plate was allowed to stand for 15 min, and the count was observed under a microscope. The corresponding volume of 100 cells was added to 20 ml of the basal medium as described above, and the plates were mixed and mixed at 200 μl per well. After one week, the microscope was observed under the microscope, and the monoclonal wells were judged and labeled, and the positive wells were picked out.

Cryopreservation of cells: Observe the state of the cells, and wait for the cells to grow well. When the viability is >90%, centrifuge at 1000 rpm for 5 min to remove the supernatant. Resuspend the cells to 1×10 ⁷ cells/ml with cryopreservation solution (45.5% FBS, 44.5% RPMI-1640, 10% DMSO), dispense into a cryotube, place in a programmed cooling box, and freeze at -80 °C. Save.

Example 2. Production and purification of chimeric antibodies

The present invention utilizes molecular biological techniques to obtain antibody sequences in anti-GITR-positive hybridoma cells, and uses the same to construct a human-mouse chimeric antibody.

Hybridoma sequencing

RNA extraction: fresh cells, centrifuged at 300 g for 5 min, the supernatant was removed, and 500 μl of LY buffer (Biomiga) was added to the pellet (20 μl of β-mercaptoethanol was added per 1 ml before use) and mixed until clear. It was added to a DNA removal tube, centrifuged at 13,000 rpm for 2 min, and the flow-through was collected. 100% ethanol was added to the flow through solution at a ratio of 1/2, and mixed 5 times to clarify. Add the clarified solution to the RNA collection tube, centrifuge at 13000 rpm for 1 min to remove the liquid, add 500 μl RB (Recovery Buffer, Recovery Buffer) (Takara), centrifuge at 13000 rpm for 30 s, and add 500 μl RNA Wash Buffer (Biomiga). Ethanol), centrifugation for 30 s, repeat the above process, centrifuge to completely evaporate the ethanol, then add 30 μl of DEPC water to the collection column, centrifuge at 12000 g for 2 min, and collect the eluate. 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 ° C for 5 min, it was quickly cooled on ice. The following reverse transcription system was added to Reaction System I in a total amount of 20 μl:

After slowly mixing, reverse transcription translation was carried out under the following conditions: 42 ° C for 60 min → 95 ° C for 5 min, and then cooled on ice to obtain cDNA.

Link the cDNA to the T vector:

The heavy and light chain variable regions were amplified by PCR, and the PCR reaction system was as follows:

The PCR reaction conditions are as follows:

4.5 μl of the PCR product obtained by the above PCR reaction was added, 0.5 μl of pMD20-T vector (Clontech), 5 μl of Ligation Mighty Mix (Takara), gently mixed, and reacted at 37 ° C for 2 hours to obtain a ligation product.

Table 6. Heavy chain variable region (VH) primers for mouse anti-GITR antibodies (Primer Mix 1)

Table 7. Light chain variable region (VL) primers for mouse anti-GITR antibodies (Primer Mix 2):

Transformed cells:

TOP10 competent cells (Tiangen Biochemical Technology (Beijing) Co., Ltd.) were taken at -80 °C, and thawed on ice. 5 μl of the ligation product obtained above was added to the thawed TOP10 competent cells, mixed, and incubated on ice for 30 min. After heat shock at 42 °C for 90 s, it was rapidly cooled on ice for 2 min, and 900 μl of LB medium (Biotech (Shanghai) Co., Ltd.) was added to the EP tube, and cultured at 37 ° C for 1 h at 220 rpm shaker. After centrifugation at 3000 g for 2 min, 800 μl of the supernatant was aspirated, and the bacteria were suspended and coated on the ampicillin-resistant plate with the remaining medium. Incubate overnight at 37 ° C and pick and clone.

Construction of chimeric antibodies

PCR amplification of the VH and VL regions of the mouse anti-GITR antibody produced by the hybridoma of Example 1 which has been sequenced

The PCR system is as follows:

* For VH amplification, use Primer Mix 1, for VL amplification, apply Primer Mix 2;

^Sequencing the correct pMD20-T plasmid for the above sequencing

The PCR amplification product was recovered by gel cutting.

Homologous recombination reaction:

The homology recombination system is as follows:

The reaction was carried out at 37 ° C for 30 min to obtain a recombinant product. The recombinant product was transformed into TOP10 competent cells, and monoclonal sequencing was picked. The clone containing the plasmid with the correct insertion direction was selected as a positive clone, and the positive clone was preserved.

Expression and purification of chimeric antibodies

A plasmid containing an anti-GITR antibody was extracted from the positive clone obtained above.

293F cells (Invitrogen) were passaged according to the desired transfection volume, and the cell density was adjusted to 1.5 × 10 ⁶ cells/ml one day before transfection. The cell density on the day of transfection was approximately 3 x 10 ⁶ cells/ml. A final volume of 1/10 of F17 medium (Gibco, A13835-01) was used as a transfection buffer, and an appropriate plasmid was added and mixed. Add appropriate polyethyleneimine (PEI) (Polysciences, 23966) to the plasmid (the ratio of plasmid to PEI is 1:3 in 293F cells), mix and incubate for 10 min at room temperature to obtain a DNA/PEI mixture. After resuspending the cells with the DNA/PEI mixture, 36.5 ° C, 8% CO ₂ . After 24 h, a transfection volume of 2% FEED (Sigma) was added and cultured at 36.5 ° C, 120 rpm, 8% CO ₂ . When the culture was continued until day 6 or when the cell viability was ≤ 60%, the cell supernatant was collected for purification.

The gravity column used for purification was treated with 0.5 M NaOH overnight, and the glass bottle and the like were washed with distilled water and then dry-baked at 180 ° C for 4 hours to obtain a purification column. The collected medium was centrifuged at 4500 rpm for 30 min before purification, and the cells were discarded. The supernatant was then filtered using a 0.22 μl filter. Each tube was filled with 1 ml of Protein A and equilibrated with 10 ml of binding buffer (sodium phosphate 20 mM. NaCl 150 mM, pH 7.0). The filtered supernatant was added to the purification column and re-equilibrated with 15 ml of binding buffer. 5 ml of elution buffer (citric acid + sodium citrate 0.1 M, pH 3.5) was added, and the eluate was collected, and 80 μl of Tris-HCl was added per 1 ml of the eluate. The collected antibodies were concentrated by ultrafiltration into PBS (Gibco, 70011-044), and the concentration was measured.

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

The control antibody used in the present invention is a GITR antibody reported in the patent application US20130183321A1 (GITR, INC. (Cambridge, MA, US)), the light and heavy chain sequences thereof are SEQ ID NO: 44 and SEQ ID NO in US20130183321A1, respectively. :54, which will be referred to as TRX518 in the following.

Example 3 Biofilm thin layer interference technique for determining the binding kinetics of the chimeric antibody of the present invention to an antigen

The equilibrium dissociation constant (KD) of the antibody of the present invention in combination with human GITR was determined by biofilm thin layer interferometry (ForteBio). The ForteBio affinity assay was performed according to the existing method (Estep, P et al, High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): p. 270-8).

Half an hour before the start of the experiment, according to the number of samples, take the appropriate amount of AMQ (Pall, 1506091) (for sample detection) or AHQ (Pall, 1502051) (for positive control detection) sensor soaked in SD buffer (PBS 1 ×, BSA 0.1%, Tween-20 0.05%).

100 μl of SD buffer, antibodies (CH22F4, CH37G5 and TRX518), antigens (including human GITR, and cynomolgus GITR, purchased from Acrobiosystems) were added to 96-well black polystyrene half-well microplates (Greiner, 675076). )in. Select the sensor location based on the sample position layout. The instrument setting parameters are as follows: running steps: Baseline, Loading ~ 1 nm, Baseline, Association, and Dissociation; the running time of each step depends on the sample binding and dissociation speed, the rotation speed is 400 rpm, and the temperature is 30 °C. Analysis of K _D values using ForteBio analysis software.

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

Table 8. Affinity constants (equilibrium dissociation constants) for ForteBio detection of antigen-antibody binding

In the above test, a chimeric antibody CH22F4, K _D values were CH37G5 1.96E-09M, 2.04E-09M, TRX518 compared with the control group, the present study the antibody has a K _D value is better.

Example 4 Binding experiment of chimeric antibody and CHO-S cells overexpressing human GITR

In this study, the binding of a gradient-diluted chimeric antibody of the present invention to a CHO-S stable cell strain overexpressing human GITR was detected by flow cytometry.

Encoding human GITR (SEQ ID NO: 41) cDNA was cloned into pCHO1.0 vector (Invitrogen), a plasmid obtained was transfected into CHO-S cells ^{(Invitrogen, ExpiCHO TM Expression System Kit} , NO: A29133), generated CHO-S cells (CHO-hGITR) overexpressing human GITR.

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

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

In the above experiments, the antibodies CH22F4 and CH37G5 all bind to human GITR molecules overexpressed on CHO-S cells with EC50 of 0.4568 nM and 0.9069 nM, respectively, and the binding ability is similar to that of TRX518.

Example 5 Humanization of chimeric antibodies

The chimeric antibody obtained in Example 2 was humanized. And through the following steps for humanization:

1 determining the structure of the CDR ring;

2 Find the closest homologous sequence in each V/J region of the heavy and light chains in the human germline sequence database;

3 screening the human germline that best matches the heavy chain light chain and the lowest amount of back mutation;

4 constructing the CDR regions of the chimeric antibody onto the human framework region;

5 using sequence and structural features to determine the position of the amino acid in the framework region that serves to maintain CDR function;

6 performing a back mutation at the sequence position determined to be important (returning to the input amino acid type);

7 Optimize the amino acids at the risk site.

The CDRs, light chain variable regions and heavy chain variable regions of the two humanized antibodies (HZ22F4 and HZ37G5) obtained in the present invention, and the amino acid sequences of the light and heavy chains are described in Tables 1-3 as described above.

Example 6 Determination of Binding Kinetics of Humanized Antibody to Antigen by ForteBio

The present invention is measured using ForteBio assay humanized antibody binds to human GITR solution equilibrium dissociation constant (K _D). The ForteBio affinity assay was the same as in Example 3 with the exception that the antibodies used were the humanized antibodies HZ22F4 and HZ37G5. In the experiments described in the above assays, the affinities of the antibodies HZ22F4 and HZ37G5 are shown in Table 9:

Table 9. Affinity constants for antigen-antibody binding by ForteBio

In the above test, described herein, humanized antibodies HZ22F4, K _D values were HZ37G5 4.58E-09M, 4.83E-09M, compared to the control antibodies, the present study of humanized antibodies have better K _D value.

Example 7 Binding experiment of humanized antibody and CHO-S cells overexpressing human GITR

This study examined the binding of a gradient-diluted humanized antibody of the present invention to CHO-hGITR cells using flow cytometry. The test method was the same as in Example 4 except that the antibodies used were humanized antibodies HZ22F4 and HZ37G5. The combination is shown in Figure 2.

In the above experiments, the humanized antibodies HZ22F4 and HZ37G5 bind to human GITR overexpressed on CHO-S cells with EC50 of 0.5492 nM and 1.974 nM, respectively, and have similar or better EC50 values than TRX518, ie similar or Better combination ability.

Example 8 Binding experiment of humanized antibody and cells overexpressing cynomolgus monkey GITR

In this study, the binding of a gradient-diluted humanized antibody of the present invention to a CHO-S stable cell line overexpressing cynomolgus monkey GITR was detected by flow cytometry.

Encoding cynomolgus monkey GITR (SEQ ID NO: 42) cDNA was cloned into pCHO1.0 vector (Invitrogen), the plasmid was transfected into CHO-S cells ^{(Invitrogen, ExpiCHO TM Expression System Kit} , NO: A29133), generated CHO-S cells (CHO-cynoGITR) overexpressing cynomolgus GITR. The rest of the test methods were the same as in Example 4 except that the antibodies used were humanized antibodies HZ22F4 and HZ37G5, and the binding conditions are shown in FIG.

In the above experiments, the humanized antibodies HZ22F4 and HZ37G5 bind to the cynomolgus monkey GITR overexpressed on CHO-S cells with EC50 of 0.3533 nM and 0.9931 nM, respectively, and have similar or superior binding ability compared to TRX518.

Example 9 Detection of Biological Activity of Antibodies by MOA Method

The activating antibody against GITR can bind to the cell surface GITR molecule to activate the downstream NF-kappa B signaling pathway. In this study, an internally constructed Hela-GITR-NF kappa B luciferase (hereinafter referred to as Hela-GITR) cell line was detected, and the activation of NF-kappa B signal was detected by detecting the expression of the fluorescent reporter gene, thereby detecting the antibody of the present invention. Activation.

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

The cDNA encoding human GITR (sequence see 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. No.: CCL-2TM), Hela cells (Hela-GITR) that overexpress the human GITR with the NF-kappaB luciferace reporter gene system.

HeLa-GITR cells in logarithmic growth phase were taken, the culture supernatant was discarded, and the cells were washed once with PBS (Gibco). Add appropriate amount of Trypsin (Gibco) and digest at 37 ° C, 5% CO _{2 for 2} min. 4 times Trypsin volume of DMEM medium (ATCC) containing 10% FBS was added, and the cells were transferred to a 50 ml centrifuge tube and counted, 400 g, and centrifuged for 5 min. DMEM medium (ATCC) was added and the cells were resuspended to 1 x 10 ⁵ cells/mL. The cells were added to a 96-well white cell culture plate (Nunclon) at 50 μl/well. At the same time, 50 μl of sample (humanized antibody HZ22F4, HZ37G5 prepared by the present invention) and positive control TRX518) were added to each well. (The antibody dilution method was: the highest antibody concentration was 80 nM, and the dilution was three times in the assay buffer (2% FBS DMEM culture). In the base), a total of 10 concentrations were tested). Incubate for 6 hours in a 37 ° C / 5% CO ₂ incubator.

Detection: Bio-GloTM buffer (promega) was thawed in advance, Bio-GloTM substrate (promega) was added, and mixed to obtain Bio-GloTM reagent. Bio-GloTM reagent, 100 μl/well, was added to the cells cultured for 6 hours as described above. Read immediately.

In the above experiments, the experimental results are shown in Figure 4. The antibodies HZ22F4 and HZ37G5 can effectively activate the NF kappa B signaling pathway with EC50 of 0.3394nM and 0.3208nM, respectively, which is significantly better than TRX518 (1.139nM). Activation ability.

Example 10 CD4T Activated Cell Experiment

In this study, antibodies and activated CD4T cells were incubated together to detect the relative expression of IL-2 and IFN-γ in the system, thereby reflecting the activation of CD4 T cells by different antibodies.

PBMC separation: Take 50 ml of fresh blood from the donor, add 2.5 times PBS, gently add to FiColl (Thermo), divide into 4 tubes, centrifuge at 400 g for 30 min, increase the speed to 7, and decelerate to 0. At the end of centrifugation, the tube was gently removed, and the middle white cloud-like cell population was aspirated into PBS and washed twice with PBS.

CD4 ⁺ T cell isolation: PBMC cells isolated as described above were isolated and enriched for CD4 ⁺ T cells according to the EasySep Human CD4 ⁺ T Cell Enrichment Kit (stem cell) instructions and used in T cell culture medium (recipe is shown in the table below). Hanging.

CD4 ⁺ T cell activation: taking the above-described CD4 ⁺ T cells isolated and cell density was adjusted by a T cell culture medium of 1 * 10 ⁶ / ml, according cells: beads (1: 1) added Dynabeads Human T-Activator CD3 / CD28 (Invitrogen), cultured in a 5% CO ₂ incubator at 37 ° C for one week.

T cell activation assay: Nunc 96 well flat bottom plate (Nunclon) was taken, 100 μl/well was added to PBS diluted 0.25 μg/ml of Purified NA/LE Mouse anti-human CD3Clone UCHT1 (BD Biosciences), and coated at 37 ° C for 2 hours to remove the package. Being liquid. The above-mentioned activated CD4 ⁺ T cells were removed for one week, washed twice with T cell culture medium, and the cell density was adjusted to 1*10 ⁶ /ml, and 100 μl of the cell suspension per well was added to the 96-well flat bottom plate. The antibody of the present invention (HZ22F4, HZ37G5) and TRX518 100 μl were simultaneously added (the antibody dilution method was: the highest antibody concentration was 400 nM, three-fold dilution in T cell medium, a total of 10 concentrations were tested) and Purified NA/LE Mouse antibody Human CD28Clone CD28 (BD Biosciences) (final concentration 2 μg/ml) was cultured for 3 and 5 days, and IL-2 and IFN-γ were detected with Human IL-2 Kit 1000 Test and Human IFN gamma 1000 test kit (both purchased from cisbio). Relative expression level (in DeltaF%).

The experimental results are shown in Tables 10 and 11 and Figures 5 and 6. The antibodies of the invention all efficiently activate CD4 ⁺ T cells in vitro. The data unit in the table is the DeltaF% average.

Table 10. Relative expression levels of IL2 (DeltaF% average)

Table 11. Relative expression levels of IFN-γ (DeltaF% average)

Example 11 ADCC effect experiment

The IgG1 subtype antibody against GITR can bind to the GITR molecule with high expression on the surface of Treg, which in turn mediates the ADCC effect to clear Treg. In this study, Promega's Jurkat-ADCCNF-AT luciferase effector cell line (hereinafter referred to as ADCC effector cell) was used as a test cell strain and CHO-hGITR cells (as described above) as target cells, and the antibody of the present invention was incubated, and the test was carried out. The expression of the light reporter gene reflects the activation of the NF-AT signal, thereby detecting the ADCC activity of the antibody.

ADCC effector cells in logarithmic growth phase (cultured according to Promega instructions), centrifuged to remove supernatant, washed twice with PBS, and resuspended in assay medium (5% low IgG serum in 1640 medium (Gibco)) to adjust cell density For 2*10 ⁷ /ml, the CHO-hGITR target cells prepared and incubated as described above were centrifuged, and the supernatant was centrifuged, and the cell density was adjusted to 2*10 ⁶ /ml by resuspending in the assay medium, and the cells were subjected to 1: 1 ratio was mixed and added to a 96-well white cell culture plate (Nunclon) at 50 μl/well. At the same time, 50 μl of sample (humanized antibody HZ22F4, HZ37G5 prepared by the present invention) and positive control antibody (TRX518) were added to each well. (The antibody dilution method was: the maximum antibody concentration was 66.66 nM, and the dilution was three times in the detection medium. A total of 12 concentrations were tested). Incubate for 12 hours in a 37 ° C / 5% CO ₂ incubator.

Detection: Bio-GloTM buffer (promega) was thawed in advance, Bio-GloTM substrate (promega) was added, and mixed to obtain Bio-GloTM reagent. Bio-GloTM reagent, 100 μl/well, was added to the cells cultured for 12 hours as described above. Read immediately.

In the above experiments, the experimental results are shown in Figure 7. The antibodies HZ22F4 and HZ37G5 can effectively activate the NF-AT signaling pathway downstream of ADCC. The EC50 is 0.02958nM and 0.04056nM, respectively. Compared with TRX518, it has a significantly better EC50. Value.

Example 12 Antitumor Efficacy Test

In this experiment, MC38 cells were used to inoculate hGITR transgenic mice to determine the anti-tumor effect of the GITR antibody of the present invention.

Human GITR transgenic mice:

Female C57B1/6 background human GITR transgenic mice (approximately 5 weeks old) were purchased from Biotech Test Animal Technology Co., Ltd. The mice were domesticated for 7 days after arrival and the study was started.

cell:

Mouse MC38 cells were purchased from Nanjing Yinhe Biomedical Co., Ltd. and routinely subcultured for subsequent in vivo experiments in strict accordance with the instructions. The cells were collected by centrifugation, resuspended in sterile PBS and adjusted to a cell density of 5 x 10 ⁶ /ml. A MC38-hGITR tumor-bearing mouse model was established by subcutaneous inoculation of 0.2 ml of cell suspension on day 0 into the right abdomen region of human GITR transgenic mice.

Dosing:

Six days after tumor cell inoculation, the tumor volume of each mouse was measured, and mice with tumor volume ranging from 87.4 mm ³ to 228.4 mm ³ were selected and grouped according to the average tumor volume group (5 mice per group). As shown in Table 12, Antibody C is a monoclonal antibody "Antibody C" against anti-PD-1 (WO2017/133540). The mice were administered on the 6th, 10th, 13th and 17th day after the inoculation, and the tumor volume and body weight of each group were monitored during the administration period. The monitoring frequency was 2 times/week and the monitoring was continued for 5 weeks. Body weight and tumor volume were measured before each administration, and the relative tumor inhibition rate (TGI%) was calculated on the 24th day after inoculation, and the formula was calculated as follows: TGI%=100%* (h-IgG control tumor volume – treatment group tumor volume) / (h-IgG control tumor volume - tumor volume before administration of h-IgG control group), wherein the average tumor volume before administration of the h-IgG control group was 106 mm ³ . Tumor volume determination: The maximum long axis (L) and the largest broad axis (W) of the tumor were measured using a vernier caliper, and the tumor volume was calculated as follows: V = L x W ² /2. The weight was measured using an electronic balance.

Table 12. Experimental Design Table

* Dosing every 3 or 4 days for a total of 4 times.

^h-IgG was purchased from Equitech-Bio, article number: SLH56-0001.

The results of tumor inhibition rate are shown in Figures 8, 9 and Table 13. On the 24th day after inoculation, HZ37G5 and HZ22F4 showed good tumor inhibition rates, 53% and 50%, respectively, which was better than TRX518 group. 36%. HZ37G5 and HZ22F4 combined with anti-PD-1 antibody Antibody C, the tumor inhibition effect was significantly better than Antibody C, HZ37G5, HZ22F4, and TRX518 single drug group. Among them, 4 mice in the combination of HZ37G5 and Antibody C, and 5 mice in the combination of HZ22F4 and Antibody C completely disappeared. At the same time, we tested the body weight of the mice, and the results showed no significant difference in the body weight of the mice as shown in FIG. Therefore, the antibody against the GITR molecule of the present invention has a significant inhibitory effect on tumors, and when the antibody is combined with the PD-1 antibody, the inhibitory effect on the tumor is more remarkable.

Table 13. Tumor inhibition rate on day 24

Claims (23)

1. An antibody or antigen-binding fragment thereof that binds to GITR, which comprises

   (i) three complementarity determining region HCDRs of the heavy chain variable region set forth in SEQ ID NO: 17 or 19, and three complementarity determining regions of the light chain variable region set forth in SEQ ID NO: 21 or LCDR, or

   (ii) three complementarity determining region HCDRs of the heavy chain variable region set forth in SEQ ID NO: 18 or 20, and three complementarity determining regions of the light chain variable region set forth in SEQ ID NO: 22 or LCDR.
2. An antibody or antigen-binding fragment thereof that binds to GITR, comprising three complementarity determining region HCDRs of the heavy chain variable region, and three complementarity determining regions LCDR of the light chain variable region, wherein

   (i) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1 or 3 or 4, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 5, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7, and LCDR1 comprises SEQ ID NO: the amino acid sequence shown in 9, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 11, 13 or 14, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 15;

   (ii) HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 2, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 6, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 8, and LCDR1 comprises SEQ ID NO: The amino acid sequence shown, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16.
3. An antibody or antigen-binding fragment thereof that binds to GITR, comprising a heavy chain variable region and/or a light chain variable region, wherein

   The heavy chain variable region comprises:

   (i) three complementarity determining regions (HCDRs) contained in the VH of any of the antibodies listed in Table B;

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

   (iii) a variant of the HCDR combination of (i) or (ii) comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes in said three CDR regions (preferably Amino acid substitution, preferably conservative substitution); or

   (iv) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence selected from SEQ ID NO: 1, 2, 3 or 4, or HCDR1 comprises and is selected from the group consisting of SEQ ID NO: 1, 2 An amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of 3 or 4; HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5 or 6, or Amino acid sequence composition, or HCDR2 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from SEQ ID NO: 5 or 6; HCDR3 comprises a mutation selected from the group consisting of SEQ ID NO: The amino acid sequence of 7 or 8 consists of or consists of the amino acid sequence, or HCDR3 comprises one, two or three changes (preferably amino acid substitution, preferably conservative) compared to the amino acid sequence selected from SEQ ID NO: 7 or 8. Alternate amino acid sequence;

   and / or

   The light chain variable region comprises:

   (i) three complementarity determining regions (LCDR) contained in the VL of any of the antibodies listed in Table B;

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

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

   (iv) LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence selected from SEQ ID NO: 9 or 10, or LCDR1 comprises an amino acid sequence selected from SEQ ID NO: 9 or 10. An amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions); LCDR2 comprises or consists of an amino acid sequence selected from SEQ ID NO: 11, 12, 13 or 14 or LCDR2 An amino acid sequence comprising one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 11, 12, 13 or 14; LCDR3 comprising a selected from the group consisting of SEQ ID NO: The amino acid sequence of 15 or 16 consists of or consists of the amino acid sequence, or LCDR3 comprises one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence selected from SEQ ID NO: 15 or 16. Amino acid sequence.
4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising

   (i) a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17 or 19, and/or comprising an amino acid represented by SEQ ID NO: 21 or a light chain variable region of an amino acid sequence having at least 90% sequence identity, or

   (ii) a heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18 or 20, and/or comprising the amino acid set forth in SEQ ID NO: 22 or 24. a light chain variable region of an amino acid sequence having at least 90% sequence identity, or

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

   (iv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18 or 20, and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22 or 24.
5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, comprising

   (i) a heavy chain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 25 or 27, and/or comprising at least the amino acid sequence set forth in SEQ ID NO: 29 or 31 a light chain of 90% sequence identity amino acid sequence, or

   (ii) a heavy chain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 26 or 28, and/or comprising at least the amino acid sequence set forth in SEQ ID NO: 30 or 32 a light chain of 90% sequence identity amino acid sequence, or

   (iii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 25 or 27, and/or a light chain comprising the amino acid sequence of SEQ ID NO: 29 or 31, or

   (iv) a heavy chain comprising the amino acid sequence of SEQ ID NO: 26 or 28, and/or a light chain comprising the amino acid sequence of SEQ ID NO: 30 or 32.
6. The GITR-binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, which has one or more of the following characteristics:

   (i) showing the same or similar binding affinity and/or specificity to any of the antibodies listed in Table 3 for GITR;

   (ii) inhibiting (eg, competitive inhibition) the binding of any of the antibodies listed in Table 3 to GITR;

   (iii) an epitope that binds to the same or overlaps with any of the antibodies shown in Table 3;

   (iv) competing with any of the antibodies shown in Table 3 to bind GITR;

   (v) having one or more of the biological properties of any of the antibody molecules listed in Table 3.
7. The GITR-binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which has one or more of the following characteristics:

   (i) combining GITR with high affinity, such as human GITR;

   (ii) having agonist activity, for example, capable of efficiently activating the NF-kappaB signaling pathway;

   (iii) capable of effectively mediating the ADCC effect;

   (iv) being capable of activating T cells, such as CD4 T cells;

   (v) having better anti-tumor activity, for example, capable of reducing tumor volume in a subject without affecting the weight of the subject;

   (vi) Combination with an anti-PD-1 antibody can better inhibit tumor activity, for example, to reduce tumor volume in a subject without affecting the subject's body weight.
8. The GITR-binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antibody is an antibody or antigen-binding fragment in the form of IgG1 or IgG2 or IgG4.
9. The GITR-binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antibody is a monoclonal antibody.
10. The GITR-binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antibody is a humanized antibody or a human antibody or a chimeric antibody.
11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv) Or (Fab') ₂ , a single domain antibody, a diabody (dAb) or a linear antibody.
12. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein the antibody is a bispecific or multispecific antibody molecule, preferably a bispecific antibody molecule with GITR and PD-1, PD-L1 Or PD-L2 binding.
13. An isolated nucleic acid encoding the GITR-binding antibody of any one of claims 1 to 12, or any one or more of its strands or fragments thereof.
14. A vector comprising the nucleic acid of claim 13, preferably the vector is an expression vector.
15. A host cell comprising the nucleic acid of claim 13 or the vector of claim 14, preferably the host cell is prokaryotic or eukaryotic, more preferably selected from the group consisting of a yeast cell, a mammalian cell (e.g., a 293 cell or a CHO cell). Or suitable for the preparation of antibodies or other antigen-binding fragments thereof.
16. A method of producing an antibody or antigen-binding fragment thereof that binds to GITR, the method comprising a nucleic acid suitable for expression of the antibody or antigen-binding fragment thereof encoding the GITR according to any one of claims 1 to 12, or the method of claim 13. Culturing the host cell of claim 15 under conditions of a nucleic acid, 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 .
17. An immunoconjugate comprising the GITR-binding antibody or antigen-binding fragment thereof of any one of claims 1 to 12 and other substances, such as a cytotoxic agent or a label.
18. A pharmaceutical composition comprising the GITR-binding antibody or antigen-binding fragment thereof of any one of claims 1 to 12 or the immunoconjugate of claim 17, and optionally one or more additional therapeutic agents, such as an anti- PD-1 antibody, anti-PD-L1 antibody or anti-PD-L2 antibody, and optionally a pharmaceutical excipient.
19. A combination product comprising the GITR-binding antibody or antigen-binding fragment thereof of any one of claims 1 to 12 or the immunoconjugate of claim 17, and one or more other therapeutic agents, such as a chemotherapeutic agent, cytotoxicity Agents, vaccines, other antibodies, anti-infective agents, small molecule drugs or immunomodulators, preferably PD-1 axis binding antagonists, such as anti-PD-1 antibodies, anti-PD-L1 antibodies or anti-PD-L2 antibodies.
20. A method of mediating ADCC or activating an effector T cell or activating a NF-kappaB signaling pathway or eliminating a regulatory T cell (eg, a Treg cell) in a subject, the method comprising administering to the subject an effective amount of claim 1 The GITR-binding antibody or antigen-binding fragment thereof, or the immunoconjugate of claim 17, or the pharmaceutical composition of claim 18, or the combination of claim 19.
21. A method of preventing or treating a tumor or infection in a subject, the method comprising administering to the subject an effective amount of the GITR-binding antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, or claim 17 The immunoconjugate, or the pharmaceutical composition of claim 18, or the combination of claim 19, preferably, the tumor is a cancer, such as a gastrointestinal cancer, such as gastric cancer, rectal cancer, colon cancer, colorectal cancer Preferably, the infection is, for example, a bacterial infection, a viral infection, a fungal infection or a protozoal infection, preferably the infection is a chronic infection.
22. The method of claim 21, further comprising administering to the subject one or more therapies, such as a treatment modality and/or other therapeutic agent, preferably, the remedy comprising surgery and/or radiation therapy, And/or other therapeutic agents are selected from the group consisting of chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies, anti-infective active agents, small molecule drugs or immunomodulators, preferably PD-1 axis binding antagonists, such as anti-PD-1 antibodies or Anti-PD-L1 antibody or anti-PD-L2 antibody.
23. A method of detecting GITR in a sample, the method comprising

    (a) contacting the sample with any of the GITR-binding antibodies or antigen-binding fragments thereof according to any one of claims 1 to 12;

    (b) detecting the formation of a complex between the antibody or antigen-binding fragment thereof that binds to GITR and GITR; optionally, the antibody that binds to the GITR is detectably labeled.

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