TWI743469B - Antibodies against gitr and use thereof - Google Patents

Abstract

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.

Description

Anti-GITR antibody and its use

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

Glucocorticoid induced tumor necrosis factor receptor (GITR), also known as CD357 and GITR-D, was first discovered in doxorubicin-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 so on. GITR is activated by its homologous ligand, GITR ligand (GITRL), which can activate the downstream NF-kappa B signaling pathway.

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

GITR ligand (GITRL) is mainly expressed on antigen presenting cells (APC, including macrophages, B cells, dendritic cells and endothelial cells). The combination of GITRL on APC and GITR on responder T cells triggers GITR signal transduction, stimulates the activation of effector T cells and inhibits the activity of Treg cells. Therefore, GITR has several effects on effector T cells and regulatory T cells, including costimulation and activation of effector T cells, reducing the inhibition of effector T cells by Treg cells, etc. (Nocentini, Eur. J. Immunol. 2007, 37: 1165-1169).

These effects mean that the activation of the GITR signaling pathway can lead to an enhanced immune response. Therefore, substances that can activate the GITR signaling pathway can activate the body's immune response, thereby increasing the body's ability to resist tumors and infections. At the same time, GITR molecules are highly expressed in Treg cells in the tumor microenvironment. Therefore, the ADCC activity of GITR antibodies can be used to eliminate GITR molecules. The expressed Treg cells can further enhance tumor killing activity.

Various GITR antibodies have been studied in the prior art (see CN103951753A, CN105829343A, CN106459203A, WO2016196792A1, WO2017068186A9, etc.). Such antibodies are GITR agonists (ie, activating antibodies), can induce or enhance GITR signal transduction, and are effective in the treatment of various GITR-related diseases or disorders that require an enhanced immune response. Since such an anti-GITR antibody is an activated antibody, relative to its binding affinity to the antigen, the level of its activation activity is a more critical indicator of its activity (for example, immune enhancement activity).

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

The present invention therefore provides a new GITR-binding antibody and its antigen-binding fragment.

In some embodiments, the anti-GITR antibodies of the present invention have one or more of the following characteristics: (i) bind to GITR with high affinity; (ii) effectively bind to GITR on the cell surface; (iii) have agonist activity, such as It can effectively activate the NF-kappaB signaling pathway; (iv) can effectively activate T cells (such as CD4 T cells); (v) can effectively mediate the ADCC effect; (vi) have better anti-tumor activity, for example, can reduce The tumor volume does not affect the weight of the subject at the same time; (vii) the combination with the anti-PD-1 antibody can better inhibit tumor activity, for example, it can reduce the tumor volume in the subject without affecting the weight of the subject.

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

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

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

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

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

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

The present invention is further illustrated in the following drawings and specific embodiments. However, these drawings and specific embodiments should not be considered as limiting the scope of the present invention, and changes easily conceived by those skilled in the art will be included in the spirit of the present invention and the scope of protection of the appended patents.

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

Figure 2 shows the flow cytometry determination of the binding ability of the humanized antibody to the CHO-S cell line expressing human GITR (CHO-hGITR).

Figure 3 shows the binding ability of the humanized antibody to the CHO-S cell line (CHO-cynoGITR) expressing cynomolgus monkey GITR (CHO-cynoGITR) measured by flow cytometry.

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

Figure 5 shows the detection of the ability of humanized antibodies to activate CD4 T cells to release IL-2.

Figure 6 shows the detection of the ability of humanized antibodies to activate CD4 T cells to release IFN-γ.

Figure 7 shows the MOA method to measure ADCC activity of humanized antibodies.

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

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

Figure 10 shows the weight detection of mice.

Detailed description of the invention

I. Definition

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

To interpret this specification, the following definitions will be used, and as long as appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terms used herein are only for describing specific embodiments and are not intended to be limiting.

The term "about" when used in conjunction with a numerical value means to cover a numerical value within a range that has a lower limit that is 5% smaller than the specified numerical value and an upper limit that is 5% larger than the specified numerical value.

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

As used herein, the term "comprising" or "including" means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps. In this document, when the term "comprising" or "including" is used, unless otherwise specified, it also covers the combination of the stated elements, integers or steps. For example, when referring to an antibody variable region that "comprises" a specific sequence, it is also intended to encompass the antibody variable region composed of the specific sequence.

As used herein, the term "GITR" refers to "glucocorticoid-induced TNF-related genes and/or polypeptides", also known in the art as TNF receptor superfamily 18 (TNFRSF18), and refers to any vertebrate source, including Any natural GITR of mammals such as primates (e.g., humans, cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise stated. The term encompasses "full length", unprocessed GITR and any form of GITR due to processing in the cell. The term also encompasses naturally occurring variants of GITR, such as splice variants or allelic variants. The amino acid sequence of human GITR can be found in GenBank accession number Q9Y5U5. The amino acid sequence of a specific human GITR polypeptide can be found in SEQ ID NO:41. See SEQ ID NO:42 for the amino acid sequence of a specific cynomolgus monkey GITR polypeptide.

The terms "anti-GITR antibody", "anti-GITR", "GITR antibody" or "GITR-binding antibody" as used herein refer to antibodies that can bind (human or cynomolgus) GITR with sufficient affinity The protein or fragments thereof such that the antibody can be used as a diagnostic and/or therapeutic agent in targeting (human or cynomolgus) GITR. In one embodiment, the degree of binding of an anti-GITR antibody to a non-(human or cynomolgus) GITR protein is less than about 10%, about 20%, or about 30% of the binding of the antibody 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 different from an intact antibody, which contains a part of an intact antibody and binds to the 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 (such as 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 the part 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 blocks More than 50% of the binding of the antibody to its antigen is broken.

An antibody that competes with a reference antibody for binding to its antigen refers to an antibody that blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody to its antigen in a competition assay. Conversely, the reference antibody blocks 50%, 60%, 70%, 80%, 90%, or 95% of the binding of the antibody to its antigen in the competition assay. Numerous types of competitive binding assays can be used to determine whether one antibody competes with another, such as: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay ( See, for example, Stahli et al., 1983, Methods in Enzymology 9: 242-253).

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

An antibody that shows the same or similar binding affinity and/or specificity as the reference antibody refers to an antibody that can have at least 50%, 60%, 70%, 80%, 90%, or more than 95% of the binding 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 an antibody variable domain that is hypervariable in sequence and forms a structurally defined loop ("hypervariable loop") and/or contains antigen contact residues ( "Antigen contact point") area. CDR is mainly responsible for binding to antigen epitopes. The CDRs of the heavy chain and light chain are usually referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. The CDRs located in the variable domain of the antibody heavy chain are called HCDR1, HCDR2, and HCDR3, and the CDRs located in the variable domain of the antibody light chain are called LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundary of each CDR can be determined using any one or a combination of many well-known antibody CDR assignment systems. Assignment systems include, for example, Chothia (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", based on the three-dimensional structure of antibodies and the topology of CDR loops. 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, USDepartment of Health and Human Services, National Institutes of Health (1987) )), AbM (University of Bath), Contact (University College London), International ImmunoGeneTics database (IMGT) (on the World Wide Web imgt.cines.fr/on), and affinity propagation clustering based on the use of a large number of crystal structures Clustering) North CDR definition.

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

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

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

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

In one embodiment, the CDR of the antibody of the present invention is bounded by AbM rules, as shown in Table 1, for example.

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 be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different. Therefore, when it comes to defining antibodies with specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences include the specific CDR sequences, but due to the application of different schemes (for example, Different assignment system rules or combinations) cause the claimed CDR boundary to be different from the specific CDR boundary defined in the present invention.

Antibodies with different specificities (ie, different binding sites for different antigens) have different CDRs. However, although CDRs are different from antibody to antibody, there are only a limited number of amino acid positions within the CDR that directly participate in antigen binding. Using at least two of the Kabat, Chothia, AbM, Contact, and North methods, the minimum overlap area can be determined, thereby providing the "minimum binding unit" for antigen binding. The minimum binding unit can be a sub-portion of the CDR. As those skilled in the art know, the structure of the antibody and protein folding can determine the residues of the rest of the CDR sequence. Therefore, the present invention also considers any CDR variants given herein. For example, in a CDR variant, the amino acid residue of the smallest binding unit can remain unchanged, while the remaining CDR residues defined by Kabat or Chothia can be replaced by conserved amino acid residues.

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

The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with the interaction of PD-1 with one or more of its binding partners (such as PD-L1, PD-L2) Signal transduction. In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners. In a specific aspect, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies that reduce, block, inhibit, eliminate, or interfere with signal transduction derived from the interaction of PD-1 with PD-L1 and/or PD-L2, and their antigen binding Fragments, immunoadhesins, fusion proteins, oligopeptides and other molecules. In one embodiment, the PD-1 binding antagonist reduces negative costimulatory signals (via PD-1 mediated signaling) mediated by or via cell surface proteins expressed on T lymphocytes, thereby rendering dysfunctional T cells Less dysfunctional (e.g. enhanced 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 disclosed in WO2015/095423. In one embodiment, the anti-PD-1 antibody is "Antibody C" (WO2017/133540).

The term "PD-L1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with the interaction of PD-L1 with one or more of its binding partners (such as PD-1, B7-1) Signal transduction. In some embodiments, the PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In a specific aspect, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and/or B7-1. In some embodiments, PD-L1 binding antagonists include reducing, blocking, inhibiting, eliminating or interfering with signals derived from the interaction of PD-L1 with one or more of its binding partners (such as PD-1, B7-1) Transduced anti-PD-L1 antibodies, antigen-binding fragments, immunoadhesins, fusion proteins, oligopeptides and other molecules. In one embodiment, the PD-L1 binding antagonist reduces negative costimulatory signals (via PD-L1 mediated signaling) mediated by or via cell surface proteins expressed on T lymphocytes, thereby rendering dysfunctional T cells Less dysfunctional (e.g. enhanced 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 disclosed in WO2015/095423.

The term "PD-L2 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with signal transduction derived from the interaction of PD-L2 with one or more of its binding partners (such as PD-1) . In some embodiments, the PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to one or more of its binding partners. In a specific aspect, the PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1. In some embodiments, PD-L2 antagonists include anti-PD that reduces, blocks, inhibits, eliminates, or interferes with signal transduction derived from the interaction of PD-L2 with one or more of its binding partners (such as PD-1) -L2 antibody, its antigen-binding fragment, immunoadhesin, fusion protein, oligopeptide and other molecules. In one embodiment, the PD-L2 binding antagonist reduces negative costimulatory signals (via PD-L2 mediated signaling) mediated by or via cell surface proteins expressed on T lymphocytes, thereby rendering dysfunctional T cells Less dysfunctional (e.g. enhanced 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 those that bind to Fc receptors (FcR) present on certain cytotoxic cells (such as NK cells, neutrophils, and macrophages) Secreted immunoglobulins enable these cytotoxic effector cells to specifically bind to target cells carrying antigens, and then use cytotoxins to kill the cytotoxic form of the target cells. The main cells that mediate ADCC, NK cells, only express Fc γ RIII, while monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991) page 464 Table 3 summarizes FcR expression on hematopoietic cells. In order to evaluate the ADCC activity of the molecule of interest, an in vitro ADCC assay can be performed, such as described in US Patent No. 5,500,362 or 5,821,337 or US Patent No. 6,737,056 (Presta). Useful effector cells for 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 an animal model such as that 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" used in the present invention refers to a substance that inhibits or prevents cell function and/or causes cell death or destruction.

"Chemotherapeutic agents" include chemical compounds useful in the treatment of cancer. For examples of chemotherapeutic agents, see those disclosed in WO2015/031667, including but not limited to anti-tumor agents, including alkylating agents; antimetabolites; natural products; antibiotics; enzymes; miscellaneous agents; hormones and antagonists; anti-estrogens ; Anti-androgen; and non-steroidal anti-androgen.

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

The "functional Fc region" possesses the "effector function" of the native sequence Fc region. Exemplary "effector functions" include Clq binding; CDC; Fc receptor binding; ADCC; phagocytosis; down-regulation of cell surface receptors (eg, B cell receptor; BCR), and the like. Such effector functions generally require that the Fc region be combined with a binding domain (e.g., 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 the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In certain embodiments, the Fc region of a human IgG heavy chain extends from Cys226 or Pro230 to the carbonyl end of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified, the numbering of amino acid residues in the Fc region or the constant region is based on the EU numbering system, which is also called 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 the form of IgG" refers to the form of IgG to which the constant region of the heavy chain of the antibody belongs. The heavy chain constant regions of all antibodies of the same type are the same, and the heavy chain constant regions of antibodies of different types are different. For example, an antibody in the form of IgG1 means that the Ig domain of its heavy chain constant region is the Ig domain of IgG1.

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

The term "effective amount" refers to the amount or dose of the antibody or fragment or conjugate or composition of the present invention that, after administration to the patient in single or multiple doses, produces the desired effect in the patient in need of treatment or prevention. The effective amount can be easily determined by the attending physician as a person skilled in the art by considering the following factors: such as mammalian species; its size, age, and general health; the specific disease involved; the degree or severity of the disease; The response of the individual patient; the specific antibody administered; the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; and the use of any concomitant therapy.

"Therapeutically effective amount" refers to the amount that is effective to achieve the desired therapeutic result at the required dose and for the required period of time. The therapeutically effective amount of the antibody or antibody fragment or its conjugate or composition can vary according to various factors such as 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 an amount in which any toxic or deleterious effects of the antibody or antibody fragment or its conjugate or composition are not as good as the therapeutically beneficial effects. Relative to an untreated subject, a "therapeutically effective amount" preferably inhibits measurable parameters (such as tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50% , 60% or 70% and still more preferably at least about 80% or 90%. The ability of a compound to inhibit a measurable parameter (e.g., cancer) can be evaluated in an animal model system that predicts efficacy in human tumors.

"Prophylactically effective amount" refers to an amount that effectively achieves the desired preventive result at the required dose and for the required period of time. Generally, since the prophylactic dose is used in the subject before or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

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

"Human antibody" refers to an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by human or human cells or derived from a non-human source, which uses Human antibody library or other human antibody coding sequences. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

A "humanized" antibody refers to a chimeric antibody comprising amino acid residues derived from non-human CDR and amino acid residues derived from human FR. In some embodiments, a humanized antibody will comprise substantially all of at least one, and usually two, variable domains, wherein all or substantially all of the CDRs (e.g., CDRs) correspond to those of the non-human antibody, and all Or substantially all FRs correspond to those of human antibodies. The humanized antibody may optionally contain at least a part of the constant region of an antibody derived from a human antibody. A "humanized form" of an antibody (e.g., 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 mammals that is usually characterized by unregulated cell growth.

The term "tumor" refers to the growth and proliferation of all neoplastic cells, 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 diseases" refers to diseases caused by pathogens, including, for example, viral infections, bacterial infections, fungal infections, or protozoan infections such as parasites.

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

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

The term "label" as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and facilitates the detection of the reagent to which it is conjugated or fused. The label itself can be detectable (for example, a radioisotope label or a fluorescent label) or, in the case of enzymatic labeling, can catalyze a chemical change of a detectable substrate compound or composition. The term is intended to cover the direct labeling of the probe or antibody by coupling (ie, physically linking) a detectable substance to the probe or antibody and the indirect labeling of the probe or antibody by reaction with another reagent that is directly labeled. Examples of indirect labels include detection of primary antibodies using fluorescently labeled secondary antibodies and end labeling of DNA probes with biotin so that they can be detected with fluorescently labeled streptavidin.

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

An "isolated" antibody is an antibody that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to more than 95% or 99% purity, such as by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse Phase HPLC) confirmed. For a review of methods used to assess 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 that encode antibody heavy or light chains (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors , And such nucleic acid molecules present at one or more locations 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 (for example, the first and second amino acid sequences can be used for optimal alignment. Or introduce gaps in one or both of the nucleic acid sequences or discard non-homologous sequences for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the compared reference sequence is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the reference sequence length. The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at this position.

Mathematical algorithms can be used to achieve sequence comparison between two sequences and calculation of percent identity. In a preferred embodiment, the Needlema and Wunsch ((1970) J.Mol.Biol.48:444-453) algorithm (at http://www.gcg. com), using Blossum 62 matrix or PAM250 matrix and gap weight 16, 14, 12, 10, 8, 6 or 4 and length weight 1, 2, 3, 4, 5 or 6, to determine two amino acid sequences The percentage of identity between. In another preferred embodiment, the GAP program in the GCG software package (available at http://www.gcg.com) is used, the NWSgapdna.CMP matrix and the gap weight 40, 50, 60, 70, or 80 are used. And length weights 1, 2, 3, 4, 5, or 6, determine the percent identity between two nucleotide sequences. A particularly good set of parameters (and a set of parameters that should be used unless otherwise specified) is the 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, gap length penalty 12, gap penalty 4), using 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, for example to identify other family member sequences or related sequences.

The term "pharmaceutical excipients" refers to diluents, adjuvants (for example Freund's adjuvant (complete and incomplete)), excipients, carriers or stabilizers, etc. administered together with the active substance.

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

The term "combination product" refers to a fixed or non-fixed combination in the form of a dosage unit or a kit of parts for combined administration, in which two or more therapeutic agents can be separated independently at the same time or within a time interval Administration, especially when these time intervals allow the combination partner to exhibit cooperation, for example, a synergistic effect. The term "fixed combination" means that the antibody of the present invention and the combination partner (for example, other therapeutic agents, such as anti-PD1 antibody, anti-PDL1 antibody or anti-PDL2 antibody) are simultaneously administered to a patient in the form of a single entity or dosage. The term "non-fixed combination" means that the antibody of the present invention and the combination partner (such as other therapeutic agents, such as anti-PD1 antibody, anti-PDL1 antibody or anti-PDL2 antibody) are administered to the patient simultaneously, concurrently or sequentially as separate entities, without a specific time. Limitations, where such administration provides therapeutically effective levels of the two compounds in the patient. The latter also applies to cocktail therapy, such as the administration of three or more therapeutic agents. In a preferred embodiment, the drug combination is a non-fixed combination.

The term "combination therapy" refers to the administration of two or more therapeutic agents to treat cancer or infection as described in this disclosure. Such administration includes co-administration of these therapeutic agents in a substantially simultaneous manner, for example, in a single capsule having a fixed ratio of active ingredients. Alternatively, such administration includes co-administration of the respective 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 before administration. In addition, such administration also includes the use of each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the treatment regimen will provide the beneficial effects of the drug combination in the treatment of the conditions or conditions described herein.

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

As used herein, "prevention" includes the inhibition of the occurrence or development of a disease or condition or the symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for prophylactic regimens. Generally, in the context of cancer, the term "prevention" refers to the administration of drugs before the onset of signs or symptoms of cancer, especially in subjects at risk of cancer.

The term "anti-infective active agent" includes any molecule that specifically inhibits or eliminates the growth of microorganisms, such as viruses, bacteria, fungi, or protozoa, such as parasites, at the applied concentration and dosing interval, but is not lethal to the host. As used herein, the term anti-infective 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 applied concentration and dosing interval.

Antibacterial anti-infective actives or antibacterial agents can be broadly classified as bactericidal (ie, direct killing) or bacteriostatic (ie, preventing division). Antibacterial anti-infective active agents can be further classified as narrow-spectrum antibacterial agents (i.e., affect only small bacterial subtypes, for example, gram-negative, etc.) or broad-spectrum antibacterial agents (i.e., affect a wide range of species).

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

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

The term "antiprotozoal agent" includes any substance that inhibits or eliminates the growth, morbidity, and/or survival of protozoan organisms (e.g., parasites).

The term "vector" when used herein refers to a nucleic acid molecule capable of multiplying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which it has been introduced. Some vectors can direct the expression of nucleic acids 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 can be solid tissue, such as fresh, frozen, and/or preserved organ or tissue samples or biopsy samples or puncture samples; 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 not naturally mixed with tissues in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and so on. Examples of tumor samples include, but are not limited to, tumor biopsy, fine needle aspirates, bronchial lavage fluid, pleural fluid (pleural fluid), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulation Plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples, such as formalin-fixed, paraffin-embedded tumor samples or frozen tumors sample.

II. Antibodies

In some embodiments, the anti-GITR antibody or fragment thereof of the present invention binds to GITR (such as human GITR or cynomolgus GITR) with high affinity, for example, binds to GITR with the following equilibrium dissociation constant (K _D ), the K _D Less than about 10nM, preferably less than or equal to about 7nM, more preferably less than or equal to about 6nM, more preferably less than or equal to about 5nM, 4.9nM, 4.8nM, 4.7nM, 4.6nM, 4.5nM, 4nM, or 3nM, optimally, the K _{D is} 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 binds to GITR with _{a K D of} 1 nM-6nM, preferably 1.5nM-5nM, 1.7nM-5nM, 1.8nM-5nM, 1.9nM-5nM. In some embodiments, GITR is human GITR. In some embodiments, antibody binding affinity is determined using a bio-optical interferometry (eg, Fortebio affinity measurement).

In some embodiments, the antibodies of the present invention or fragments thereof bind to cells expressing GITR, for example, at less than or equal to about 2nM, 1.9nM, 1.5nM, 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM , 0.4nM EC50. In some embodiments, the binding is determined by flow cytometry (e.g., FACS). In some embodiments, the GITR-expressing cell is a GITR-expressing CHO cell (e.g., CHO-S cell). In some embodiments, the GITR is human GITR or cyno GITR.

In some embodiments, the antibody or fragment thereof of the present invention activates the NF-kappaB signaling pathway downstream of GITR by binding to GITR molecules on the cell surface. In some embodiments, the activation ability of the antibody or fragment thereof of the present invention is superior to known anti-GITR antibodies, such as the GITR antibody reported in patent application US20130183321A1 (GITR, INC. (Cambridge, MA, US)). In one embodiment, the light chain and heavy chain sequences of the known anti-GITR antibody molecule are SEQ ID NO: 44 and SEQ ID NO: 54 in US20130183321A1, respectively. In some embodiments, the GITR antibody molecule of the present invention can significantly and effectively activate the NF kappa B signaling pathway compared to the control antibody molecule. In some embodiments, the activation is obtained by luciferase detection.

In some embodiments, the antibodies of the invention or fragments thereof enhance effector T cell functions, such as activation of effector T cells. In some embodiments, the antibodies or fragments thereof of the present invention can enhance the proliferation of effector T cells. In some embodiments, the antibodies of the invention or fragments thereof increase the secretion/expression of interferons (eg, IFN-γ). In some embodiments, the antibodies of the invention or fragments thereof increase the secretion/expression of interleukins (e.g., IL-2). In some embodiments, the T cells are CD4 T cells.

In some embodiments, the GITR antibody or fragment thereof of the present invention can activate the GITR signaling pathway in regulatory T cells (such as Treg cells), thereby eliminating the cells by mediating the ADCC effect. Therefore, on the one hand, the inhibitory signal of effector T cells is relieved, and the activity of CD4 T cells and/or CD8 T cells is enhanced, and on the other hand, the inhibitory effect of regulatory T cells on effector T cells is eliminated, thereby maximizing the activation of effector T cells and enhancing Immune response to tumor cells. In some embodiments, the ADCC activity of the antibody is detected by detecting NF-AT signal activation. In some embodiments, the activation of the NF-AT signal is reflected by the expression of a fluorescent reporter gene. In some embodiments, the GITR antibody molecule of the present invention can significantly and effectively activate the NF-AT signaling pathway compared with known anti-GITR antibody molecules. In some embodiments, the GITR antibody molecule of the present invention can significantly and effectively mediate the ADCC effect compared to known anti-GITR antibody molecules. In some embodiments, the GITR antibody molecule of the present invention can significantly and effectively inhibit or eliminate 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, the known anti-GITR antibody molecule is, for example, the anti-GITR antibody reported in patent application US20130183321A1 (GITR, INC. (Cambridge, MA, US)). In one embodiment, the light chain and heavy chain sequences of the known anti-GITR antibody molecule are SEQ ID NO: 44 and SEQ ID NO: 54 in US20130183321A1, respectively.

In some embodiments, the antibodies or fragments thereof of the present invention (optionally combined with treatment modalities and/or other therapeutic agents, such as PD-1 axis binding antagonists) can prevent or treat tumors. In some embodiments, the antibodies or fragments thereof of the present invention can be used to inhibit or reduce tumor growth (e.g., reduce tumor volume). In some embodiments, the antibodies or fragments thereof of the present invention can also be used to maintain the body weight of tumor patients. In some embodiments, the antibody or fragment thereof of the present invention can be combined with a treatment modality and/or other therapeutic agents, such as a PD1-axis binding antagonist, to inhibit or reduce tumor growth (e.g., reduce tumor volume). In some embodiments, the antibodies or fragments of the present invention can be combined with treatment modalities and/or other therapeutic agents, such as PD1-axis binding antagonists, to maintain the weight of tumor patients. In some embodiments, the antibodies or fragments of the present invention (optionally combined with treatment modalities and/or other therapeutic agents, such as PD-1 axis binding antagonists) can inhibit or reduce tumor growth (e.g., reduce tumor volume ), and does not affect the weight of tumor patients. Preferably, the tumor is a gastrointestinal tumor. Preferably, the tumor is cancer, such as gastrointestinal cancer, such as gastric cancer, rectal cancer, colon cancer, colorectal cancer and the like. In some embodiments, the antibodies of the present invention or fragments thereof (optionally combined with PD-1 axis binding antagonists) can prevent or treat infections, such as chronic infections, including bacterial infections, fungal infections, viral infections, and protozoan infections Wait.

In some embodiments, the anti-GITR antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH), wherein the VH comprises (i) three of the VH of any antibody listed in Table B Complementarity determining region (CDR), or (ii) relative to the sequence of (i), contains at least one and no more than 5, 4, 3, 2 or 1 amino acid changes in the three CDR regions (compared to Preferred amino acid substitutions, preferred conservative substitutions).

In some embodiments, the anti-GITR antibody or antigen-binding fragment thereof of the present invention comprises a light chain variable region (VL), wherein the VL comprises: (i) the three contained in the VL of any antibody listed in Table B A complementarity determining region (CDR); or (ii) relative to the sequence of (i), at least one and no more than 5, 4, 3, 2 or 1 amino acid changes are included in the three CDR regions ( Preferred are amino acid substitutions, preferred conservative substitutions).

In some embodiments, the anti-GITR antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region VH and a light chain variable region VL, wherein (a) the VH comprises (i) any antibody listed in Table B The three complementarity determining regions (CDR) contained in the VH, or (ii) relative to the sequence of (i), the three CDR regions include at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably amino acid substitution, preferably conservative substitution) sequence; and/or (b) the VL includes: (i) three contained in the VL of any antibody listed in Table B A complementarity determining region (CDR); or (ii) relative to the sequence of (i), at least one and no more than 5, 4, 3, 2 or 1 amino acid changes are included in the three CDR regions ( Preferred are amino acid substitutions, preferred conservative substitutions).

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

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

In a preferred embodiment, the anti-GITR antibody or antigen-binding fragment thereof of the present invention comprises (i) the three complementarity determining regions HCDR of the heavy chain variable region as shown in SEQ ID NO: 17 or 19, and the HCDR as shown in SEQ ID NO: 17 or 19. The 3 complementarity determining regions LCDR of the light chain variable region shown in NO: 21 or 23, or (ii) the 3 complementarity determining regions HCDR of the heavy chain variable region shown in SEQ ID NO: 18 or 20, and The three complementarity determining regions LCDR of the light chain variable region shown in SEQ ID NO: 22 or 24.

In some embodiments, the anti-GITR antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein (i) the VH comprises a complementarity determining region ( CDR) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises an amino acid sequence selected from SEQ ID NO: 1, 2, 3 or 4, or consists of said amino acid sequence, or HCDR1 comprises and is selected from SEQ ID NO: Compared with the amino acid sequence of 1, 2, 3 or 4, the amino acid sequence has one, two or three changes (preferably amino acid substitution, preferably conservative substitution); HCDR2 includes a sequence selected from SEQ ID NO: The amino acid sequence of 5 or 6 or consists of the amino acid sequence, or HCDR2 contains one, two or three changes (compared to the amino acid sequence selected from SEQ ID NO: 5 or 6). HCDR3 comprises or consists of an amino acid sequence selected from SEQ ID NO: 7 or 8, or HCDR3 includes and is selected from SEQ ID NO: 7 or 8. ID NO: 7 or 8 amino acid sequence with one, two or three changes (preferably amino acid substitution, preferably conservative substitution) compared to the amino acid sequence; and/or (ii) said VL comprises complementarity determining regions (CDR) LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises or consists of an amino acid sequence selected from SEQ ID NO: 9 or 10, or LCDR1 comprises and is selected from SEQ ID NO ：Amino acid sequence with one, two or three changes (preferably amino acid substitution, preferably conservative substitution) compared to the amino acid sequence of 9 or 10; LCDR2 includes SEQ ID NO: 11, 12 The amino acid sequence of, 13 or 14 or consists of the amino acid sequence, or LCDR2 contains one, two or three amino acid sequences selected from SEQ ID NO: 11, 12, 13 or 14 A modified amino acid sequence (preferably amino acid substitution, preferably conservative substitution); LCDR3 includes an amino acid sequence selected from SEQ ID NO: 15 or 16 or consists of the amino acid sequence, or LCDR3 includes Compared with the amino acid sequence selected from SEQ ID NO: 15 or 16, an amino acid sequence having one, two or three changes (preferably amino acid substitution, preferably conservative substitution).

In a preferred embodiment, the present invention provides an anti-GITR antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein (a) the VH comprises (i) The combination of HCDR1, HCDR2 and HCDR3 shown in Table A; or (ii) a variant of the HCDR combination of (i), the variant comprising at least one and no more than 5 in the three CDR regions , 4, 3, 2 or 1 amino acid change (preferably amino acid substitution, preferably conservative substitution); and/or (ii) the VL includes (i) LCDR1, LCDR2 and LCDR3 shown in Table A Or (ii) a variant of the LCDR combination of (i), said variant comprising a total of at least one and no more than 5, 4, 3, 2 or 1 amino acid changes in the three CDR regions (Amino acid substitution is preferred, conservative substitution is preferred).

In a preferred embodiment, the present invention provides an anti-GITR antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein the VH comprises a complementarity determining region (CDR) HCDR1, HCDR2, and HCDR3, the VL includes (CDR) LCDR1, LCDR2, and LCDR3, and the combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 contained in the antibody or antigen-binding fragment thereof is as follows ( Table A) shows:

In some embodiments, the anti-GITR antibody or antigen-binding fragment thereof of the present invention comprises heavy chain variable region VH and/or light chain variable region VL, wherein (a) heavy chain variable region VH(i) comprises The amino acid sequence selected from SEQ ID NO: 17, 18, 19 or 20 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity Amino acid sequence or consisting of; or (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 17, 18, 19 or 20; or (iii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: : The amino acid sequence of 17, 18, 19 or 20 has one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes ( Preferably, amino acid substitution, more preferably amino acid conservative substitution) of the amino acid sequence, preferably, the amino acid change does not occur in the CDR region; and/or (b) the light chain variable region VL (i) comprising an amino acid sequence selected from SEQ ID NO: 21, 22, 23 or 24 that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% % Or 99% identical amino acid sequence; (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 21, 22, 23 or 24; or (iii) comprising and selecting Compared with the amino acid sequence of SEQ ID NO: 21, 22, 23 or 24, amines having one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2, 1) For amino acid sequences with base acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions), preferably, the amino acid changes do not occur in the CDR region.

In a preferred embodiment, the present invention provides an anti-GITR antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the antibody or antigen-binding fragment thereof The combination of the heavy chain variable region VH and the light chain variable region VL included is shown in the following table (Table B):

In some embodiments, the anti-GITR antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and/or a light chain, wherein (a) the heavy chain (i) comprises and is selected from SEQ ID NO: 25, 26, 27 or 28 The amino acid sequence has or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical ; (Ii) comprising or consisting of an amino acid sequence selected from SEO ID NO: 25, 26, 27 or 28; or (iii) comprising an amino acid sequence selected from SEQ ID NO: 25, 26, 27 or 28 The acid sequence has one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid replacement, more 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 the heavy chain variable. And/or (b) the light chain (i) contains at least 85%, 90%, 91%, 92%, 93% with an amino acid sequence selected from SEQ ID NO: 29, 30, 31 or 32 , 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence or consisting of it; (ii) comprising an amino group selected from SEQ ID NO: 29, 30, 31 or 32 The acid sequence or consists of it; or (iii) contains one or more (preferably not more than 20 or 10) amino acid sequence selected from SEQ ID NO: 29, 30, 31 or 32 compared to the amino acid sequence. More preferably, no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid replacement, more preferably amino acid conservative replacement) amino acid sequence, preferably, the amino acid sequence The acid change does not occur in the CDR region of the light chain, and more preferably, the amino acid change does not occur in the light chain variable region.

In a preferred embodiment, the present invention provides an anti-GITR antibody or antigen-binding fragment thereof, which comprises a heavy chain and a light chain, wherein the combination of the heavy chain and light chain contained in the antibody or the antigen-binding fragment thereof is as follows ( Table C) shows:

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

In one embodiment of the present invention, the amino acid changes described herein include amino acid substitutions, insertions or deletions. Preferably, the amino acid described herein is changed to amino acid substitution, preferably conservative substitution.

In a preferred embodiment, the amino acid changes described in the present invention occur in regions outside the CDR (for example, in the FR). More preferably, the amino acid changes described in the present invention occur in regions outside the variable region of the heavy chain and/or outside the variable region of the light chain.

In some embodiments, the substitutions are conservative substitutions. Conservative substitution refers to the replacement of an amino acid by another amino acid in the same category, for example, an acidic amino acid is replaced by another acidic amino acid, and a basic amino acid is replaced by another basic amino acid. , Or one neutral amino acid is replaced by another neutral amino acid. Exemplary permutations are shown in Table D below:

In certain embodiments, the substitution occurs in the CDR region of the antibody. Generally, the obtained variant has a modification (e.g., improvement) in certain biological properties (e.g., increased affinity) relative to the parent antibody and/or will have certain biological properties substantially retained of the parent antibody . An exemplary substitution variant is an affinity matured antibody.

In certain embodiments, the antibodies provided herein are modified to increase or decrease the degree to which the antibody is glycosylated. The addition or deletion of glycosylation sites of the antibody can be conveniently achieved by changing the amino acid sequence to create or remove one or more glycosylation sites. When the antibody contains an Fc region, the carbohydrate attached to it can be changed. In some applications, modifications to remove unwanted glycosylation sites may be useful, such as removing fucose moieties to improve antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC277:26733 ). In other applications, galactosylation can be modified to modify complement dependent cytotoxicity (CDC).

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein to generate Fc region variants, so as to enhance the effectiveness of the antibody in the treatment of cancer or cell proliferative diseases, for example. . Fc region variants may include human Fc region sequences (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) that contain amino acid modifications (e.g., substitutions) 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; WO 2004/056312 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 necessary to produce cysteine engineered antibodies, such as "thioMAb", in which one or more residues of the antibody are replaced with cysteine residues. Cysteine engineered antibodies can be generated, for example, as described in U.S. Patent No. 7,521,541.

In certain embodiments, the antibodies provided herein can be further modified to contain other non-protein moieties known and readily available in the art. The part suitable for antibody derivatization includes, but is 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 copolymer, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly -1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), and dextran or poly(n -Vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyol (e.g. glycerin), polyvinyl alcohol, and mixtures thereof.

In some embodiments, the anti-GITR antibody or antigen-binding fragment thereof of the present invention has one or more of the following characteristics: (i) shows the same or similar binding affinity and/or specificity for GITR as any antibody listed in Table 3 (Ii) inhibit (for example, competitively inhibit) the binding of any antibody listed in Table 3 to GITR; (iii) bind the same or overlapping epitope with any antibody listed in Table 3; (iv) and Any antibody shown in Table 3 competes for binding to GITR; (v) has one or more biological properties of any antibody molecule listed in Table 3.

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

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 fully incorporated herein by reference (Almagro JC and Fransson J (2008) Frontiers in Bioscience 13: 1619-1633 ).

In some embodiments, the anti-GITR antibody is a human antibody. Various techniques known in the art can be used to prepare human antibodies. 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 part of the framework sequence of the anti-GITR antibody is a human consensus framework sequence. In one embodiment, the anti-GITR antibody of the present invention also covers its antibody fragments, preferably selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single-chain antibodies (such as 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. Multispecific antibody molecules can have any combination of binding specificities for the aforementioned molecules. The multispecific antibody molecule may be, for example, a trispecific antibody molecule, which includes a first binding specificity for GITR and a second and third binding specificity for one or more of the following molecules: PD-1, PD-L1 or PD-L2.

II. The nucleic acid of the present invention and the host cell containing it

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 yeast cells, mammalian cells (such as CHO cells or 293 cells), or other cells suitable for preparing antibodies or antigen-binding fragments 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 include a nucleic acid encoding the amino acid sequence of the light chain variable region and/or the heavy chain variable region of an antibody, or a nucleic acid encoding the amino acid sequence of the light chain and/or heavy chain of the antibody. An exemplary nucleic acid sequence encoding the variable region of an antibody heavy chain comprises at least 80%, 85%, 90%, 91%, 92%, 93% of a nucleic acid sequence selected from SEQ ID NO: 33, 34, 35, or 36 , 94%, 95%, 96%, 97%, 98%, or 99% identical nucleic acid sequence, or comprising a nucleic acid sequence selected from SEQ ID NO: 33, 34, 35, or 36. Exemplary nucleic acid sequence encoding the variable region of the antibody light chain comprises at least 80%, 85%, 90%, 91%, 92%, 93% with a nucleic acid sequence selected from SEQ ID NO: 37, 38, 39 or 40 , 94%, 95%, 96%, 97%, 98%, or 99% identical nucleic acid sequence, or comprising a nucleic acid sequence selected from 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 the selection or expression of antibody-encoding vectors include the prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. For the 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, and see also Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo, editor, Humana Press, Totowa, NJ, 2003) , Pages 245-254, which describe the expression of antibody fragments in E. coli). After expression, the antibody can be separated 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 yeast cells, mammalian cells, or other cells suitable for preparing antibodies or antigen-binding fragments thereof. For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable pure strains or expression hosts for antibody-encoding vectors. For example, fungal and yeast strains where the glycosylation pathway has been "humanized" result in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006). Suitable host cells for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, a mammalian cell line modified to be suitable for growth in suspension can be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney line (293HEK or 293F or 293 cells, such as, for example, Graham et al., J. Gen Virol. 36:59 ( 1977) described in) 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.; and bone marrow) Tumor cell lines such as Y0, NS0 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, Volume 248 (BKCLo, ed., Humana Press, Totowa , NJ), pages 255-268 (2003).

In one embodiment, the present invention provides a method for preparing an anti-GITR antibody or a fragment thereof (preferred antigen-binding fragment), wherein the method includes a method suitable for expressing the antibody or fragment thereof (preferred antigen-binding fragment). ) The host cell is cultured under the condition of nucleic acid, and the antibody or fragment thereof (preferably antigen-binding fragment) is isolated as necessary. In a certain embodiment, the method further comprises recovering the anti-GITR antibody or fragment thereof (preferably an antigen-binding fragment) from the host cell.

In one embodiment, there is provided a method of preparing an anti-GITR antibody, wherein the method comprises, under conditions suitable for expression of the antibody, culturing a host cell containing a nucleic acid encoding the antibody, as provided above, and optionally The antibody is recovered from the host cell (or host cell culture medium). In order to recombinantly produce anti-GITR antibodies, nucleic acids encoding antibodies (such as the antibodies described above) are isolated and inserted into one or more vectors for further selection and/or expression in host cells. Such nucleic acids are easy to isolate and sequence using conventional procedures (for example, by using oligonucleotide probes that can specifically bind to genes encoding antibody heavy and light chains).

III. Assay

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

On the other hand, competition assays 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 competitive antibodies bind to the same or overlapping epitopes (e.g., linear or conformational epitopes) as bound by any of the anti-GITR antibodies disclosed herein. See Morris (1996) "Epitope Mapping Protocols", Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ) for detailed exemplary methods for locating epitopes bound by antibodies.

The present invention also provides an assay method for identifying anti-GITR antibodies with biological activity. Biological activities may include, for example, binding to GITR (such as binding to human and/or cyno GITR), increasing GITR-mediated signal transduction (such as increasing NFkappa-B signaling pathway), and reducing GITR-expressing cells (such as Treg) by ADCC. Cells), enhancing the function of T effector cells (e.g. CD4 effector T cells) (e.g. by increasing the cytokine production of effector T cells (e.g. interferons such as IFN-γ or interleukins such as IL2)). An antibody having such biological activity in vivo and/or in vitro is also provided.

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

Methods known in the art can be used to determine T cell activation. For example, it can be determined by the level of cytokines released after activation of T cells, such as interferons (such as IFN-γ) or interleukins (such as IL2). Methods known in the art can also be used to measure GITR signaling (for example, the NF-kappaB signaling pathway) to measure T cell activation. In one embodiment, a transgenic cell expressing human GITR and a reporter gene (comprising the NF-kappa B promoter fused to a reporter gene (e.g., β-luciferase)) is generated. The addition of an anti-GITR antibody to the cells leads to an increase in NF-kappa B transcription, which is detected using an assay for a reporter gene (for example, a luciferase reporter gene assay).

Methods known in the art can be used to determine the ADCC effect of antibodies. For example, it is activated by NF-AT signal. In one embodiment, a transgenic cell of ADCC effector cell (comprising an NF-AT promoter fused to a reporter gene (e.g. β-luciferase)) is obtained. Co-cultivation of effector cells and cells expressing GITR, and the addition of anti-GITR antibody at the same time leads to an increase in effector cell NF-AT transcription, which is detected by an assay for reporter genes (for example, a luciferase reporter gene assay).

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

It is understood that the immunoconjugates of the invention can be used to replace or supplement anti-GITR antibodies to perform any of the above-mentioned assays.

It is understood that any of the aforementioned assays can be performed using anti-GITR antibodies and other active agents.

IV. Immunoconjugates

In some embodiments, the invention provides immunoconjugates comprising any of the anti-GITR antibodies provided herein and other substances, such as cytotoxic agents. 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 such as nucleic acid hydrolases; 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 known anti-tumor or anti-cancer agents.

In some embodiments, the immunoconjugate is used to prevent or treat tumors, such as gastrointestinal tumors. In some embodiments, the tumor is cancer, such as 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 infections, such as chronic infections, such as bacterial infections, viral infections, fungal infections, protozoan infections, and the like.

V. Pharmaceutical compositions and pharmaceutical preparations

In some embodiments, the present invention provides a composition comprising any of the anti-GITR antibodies or fragments (preferably antigen-binding fragments thereof) or immunoconjugates thereof described herein, preferably the composition is a pharmaceutical composition. In one embodiment, the composition further comprises pharmaceutical excipients. In one embodiment, the composition, for example, a pharmaceutical composition, comprises the anti-GITR antibody of the present invention or a fragment or immunoconjugate thereof, and one or more other therapeutic agents (e.g., chemotherapeutics, cytotoxic 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) in combination.

In some embodiments, the composition is used to prevent or treat tumors, such as gastrointestinal tumors. In some embodiments, the tumor is cancer, such as gastrointestinal cancer, such as gastric cancer, rectal cancer, colon cancer, colorectal cancer, and the like. In some embodiments, the composition is used to prevent or treat infections, such as chronic infections, such as bacterial infections, viral infections, fungal infections, protozoan infections, and the like.

The present invention also includes compositions (including pharmaceutical compositions or pharmaceutical preparations) comprising anti-GITR antibodies or immunoconjugates thereof and compositions (including pharmaceutical compositions or pharmaceutical preparations) comprising polynucleotides encoding anti-GITR antibodies. In certain embodiments, the composition comprises one or more GITR-binding antibodies or fragments thereof or one or more polynucleotides encoding one or more GITR-binding antibodies or fragments thereof. These compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.

Pharmaceutical carriers suitable for the present invention can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the pharmaceutical composition is administered intravenously, water is the preferred carrier. It is also possible to use saline solutions and aqueous dextrose and glycerol solutions as liquid carriers, especially for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicone, sodium stearate, glyceryl monostearate, talc, sodium chloride, dry skim Milk, glycerin, propylene, glycol, water, ethanol, etc. For the use of excipients and their uses, 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 composition may also contain small amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can 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.

It can be prepared by mixing the anti-GITR antibody of the present invention with the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980)). The anti-GITR antibody pharmaceutical preparation is preferably in the form of a lyophilized preparation or an aqueous solution.

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

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

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

Regarding other components of the pharmaceutical preparation/pharmaceutical composition containing the antibody of the present invention, one can also refer to those disclosed in WO2015/031667 or WO2015/187835 and the like.

VI. Combination products

In some embodiments, the present invention also provides a combination product, which comprises the antibody of the present invention or an antigen-binding fragment thereof, or an immunoconjugate thereof, and one or more other therapeutic agents (e.g., chemotherapeutics, other antibodies, cytotoxicity Agents, vaccines, anti-infective agents, small molecule drugs or immunomodulators, etc.). In some embodiments, other antibodies such as anti-PD-1 antibodies or anti-PD-L1 antibodies or anti-PD-L2 antibodies.

In some embodiments, the combination product is used to prevent or treat tumors, such as gastrointestinal tumors. In some embodiments, the tumor is cancer, such as gastrointestinal cancer, such as gastric cancer, rectal cancer, colon cancer, colorectal cancer, and the like. In some embodiments, the combination product is used to prevent or treat infections, such as chronic infections, such as bacterial infections, viral infections, fungal infections, protozoan infections, and the like.

VII. Purpose

One aspect of the present invention provides a method for activating the NF-KappaB signaling pathway in a subject, which comprises administering to the subject an effective amount of the anti-GITR antibody or antigen-binding fragment, immunoconjugate, pharmaceutical composition or combination product of the present invention.

Another aspect of the present invention provides a method for improving the function of effector T cells in a subject, such as activating effector T cells, which comprises administering to the subject an effective amount of the anti-GITR antibody or antigen-binding fragment thereof, immunoconjugates, and drugs of the present invention. Composition or combination product. In some embodiments, the function is enhanced effector T cell proliferation. In some embodiments, the activated T cells are manifested by increased secretion/expression of interferon or interleukin. In some embodiments, the interleukin is IL2. In some embodiments, the interferon is IFN-γ.

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

One aspect of the present invention also provides a method of preventing or treating tumors in a subject, the method comprising administering to the subject an effective amount of any anti-GITR antibody or fragment thereof, immunoconjugate, pharmaceutical composition, or Combination products.

One aspect of the present invention also provides a method of preventing or treating infection in a subject, the method comprising administering to the subject an effective amount of any anti-GITR antibody or fragment thereof, immunoconjugate, pharmaceutical composition, or Combination products.

The present invention also provides a method for preventing or treating other pathologies or conditions related to Treg proliferation in a subject, the method comprising administering to the subject an effective amount of any anti-GITR antibody or fragments thereof, immunoconjugates described herein Compound, pharmaceutical composition or combination product.

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

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

The antibody molecules described herein can be used to achieve the treatment of metastatic cancer (e.g., metastatic cancer expressing GITR or PD1).

In one embodiment, the tumor is a cancer that expresses elevated levels of GITR and/or PD1.

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

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 bacteria, viruses, fungi, and protozoa.

In other aspects, the present invention provides the use of anti-GITR antibodies or fragments thereof in the production or preparation of medicaments for the treatment of related diseases or disorders mentioned herein.

In some embodiments, the antibodies or antibody fragments or immunoconjugates or compositions or products of the invention delay the onset of the disorder and/or symptoms associated with the disorder.

In some embodiments, the prevention or treatment methods described herein further include administering to the subject or subject combination the antibody molecules or pharmaceutical compositions or immunoconjugates disclosed herein, as well as one or more other therapies, such as treatment modalities and / Or other therapeutic agents.

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

In some embodiments, the therapeutic agent is selected from chemotherapeutics, cytotoxic agents, vaccines, other antibodies, anti-infective agents, small molecule drugs, or immunomodulators.

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-transduction-based vaccine. Cancer vaccines can be prophylactic or therapeutic.

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

Exemplary other 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.

In some embodiments, the antibody combinations described herein may be administered separately, for example, as separate antibodies, or when linked (for example, as a bispecific or trispecific antibody molecule).

More therapies or therapeutic agents that can be combined with anti-GITR antibodies or fragments thereof can be found in WO2015/031667.

Such combination therapies encompass combined administration (for example, two or more therapeutic agents are contained in the same formulation or separate formulations), and separate administration, in which case, additional therapeutic agents and/or agents may be administered. The administration of the antibody of the invention occurs before, at the same time, and/or afterwards. In one embodiment, the administration of the anti-GITR antibody and the administration of the other therapeutic agent are within about one month of each other, or within about one, two or three weeks, or about 1, 2, 3, 4, 5, or 6 days of each other Happen within.

The therapeutically effective amount of the anti-GITR antibody-containing pharmaceutical composition to be used will, for example, depend on the therapeutic background and goals. Those skilled in the art will understand that the appropriate dosage level for treatment will vary in part based on the following factors: the molecule delivered, the indication for use, the route of administration, and the patient's body weight, body surface area or organ size and/or condition (Age and general health status). In certain embodiments, the clinician can titrate the dose and change the route of administration to obtain the best therapeutic effect.

The frequency of dosing will depend on the pharmacokinetic parameters of the specific anti-GITR antibody in the formulation used. Generally, the clinician administers the composition until the dose that achieves the desired effect is reached. The antibody of the present invention can therefore be administered in a single dose, or in two or more doses (which may contain the same or different amounts of the desired molecule) within a certain period of time, or by continuous infusion via an implanted 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 for 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, oral, by intravenous injection, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal or lesion Internal approach; by a sustained release system or by an implanted device. In certain embodiments, the composition can be administered by bolus injection or by continuous infusion or by implantation devices.

The composition may also be applied topically via an implanted membrane, sponge, or another suitable material on which the desired molecule is absorbed or encapsulated. In certain embodiments, when an implanted 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 is understood that the immunoconjugate of the present invention can be used to replace or supplement the anti-GITR antibody for any treatment.

VIII. Methods and compositions for diagnosis and detection

In certain embodiments, any anti-GITR antibody or antigen-binding fragment thereof provided herein can be used to detect the presence of GITR in a biological sample. When the term "detection" is used herein, it includes quantitative or qualitative detection. Exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (for example, FACS), antibody molecule complexed magnetic beads, ELISA assays Method, PCR-technology (for example, RT-PCR). In certain embodiments, the biological sample is blood, serum, or other liquid samples 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 method of diagnosis or detection is provided. In another aspect, a method of detecting the presence of GITR in a biological sample is provided. In certain embodiments, the method comprises detecting the presence of GITR protein in a biological sample. In certain embodiments, GITR is human GITR. In certain embodiments, the method includes contacting a biological sample with an anti-GITR antibody as described herein under conditions that allow the anti-GITR antibody to bind to GITR, and detecting whether a complex is formed between the anti-GITR antibody and GITR . The formation of 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 the anti-GITR antibody, for example where GITR is a biomarker used to select the subject.

In one embodiment, the antibodies of the invention can be used to diagnose cancer or tumors, for example, to evaluate (e.g., monitor) the treatment or progression, diagnosis, and/or staging of the diseases described herein (e.g., cancer or tumor) in a subject. In certain embodiments, labeled anti-GITR antibodies are provided. Labels include, but are not limited to, labels or parts that are directly detected (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and parts that are indirectly detected, such as enzymes or ligands, such as , Through enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes ³² P, ¹⁴ C, ¹²⁵ I, ³ H and ¹³¹ I, fluorophores such as rare earth chelates or luciferin and its derivatives, rhodamine and its derivatives, dan Dansyl, umbelliferone, luceriferase, for example, firefly luciferase and bacterial luciferase (US Patent No. 4,737,456), luciferin, 2,3-dihydro Phthalazinedione, horseradish peroxidase (HR), alkaline phosphatase, β-galactosidase, glucoamylase, lyase, carbohydrate oxidase, for example, glucose oxidase, galactose oxidase, And glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, and enzymes that use hydrogen peroxide to oxidize dye precursors such as HR, lactoperoxidase, or microperoxidase ( microperoxidase), biotin/avidin, spin labeling, phage labeling, stable free radicals, etc.

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

In some embodiments, GITR is detected before treatment, for example, before initiating treatment or before some treatment after the treatment interval.

In some embodiments, there is provided a method of treating tumors or infections, the method comprising: testing a subject (e.g., a sample) (e.g., a sample of a subject containing cancer cells) for the presence of GITR, thereby determining the GITR value, and determining the GITR value. The value is compared with the control value, and if the GITR value is greater than the control value, then a therapeutically effective amount of an anti-GITR antibody (eg, an anti-GITR antibody described herein) in combination with one or more other therapies as needed is administered to the subject, thereby treating the tumor Or infection.

IX Exemplary anti-GITR antibodies of the invention

These and other aspects and embodiments of the invention are described in the accompanying drawings (the brief description of the drawings immediately follows) and the following detailed description of the invention and are exemplified in the following examples. Any or all of the features discussed above and throughout this application can be combined in various embodiments of the invention. The following examples further illustrate the present invention, however, it should be understood that the examples are described in an illustrative rather than a limiting manner, and various modifications can be made by those skilled in the art.

Example

Example 1. Preparation of fusion tumor cells

Fusion tumor technology is to fuse two kinds of cells while maintaining the main characteristics of both. These two kinds of cells are mouse spleen cells immunized with antigen and mouse myeloma cells. The main feature of mouse spleen cells (B lymphocytes) immunized with specific antigens is its antibody secretion function, but they cannot be continuously cultured in vitro. Mouse myeloma cells can divide and proliferate indefinitely under culture conditions, that is, they have The so-called immortality. Under the action of the selective medium, only the hybrid cells fused with B cells and myeloma cells can have the ability to continue culture, forming a pure cell line with both the function of antibody secretion and the maintenance of cell immortality. In this experiment, mice were immunized with human GITR protein, and then spleen cells and myeloma cells from the mice were fused to obtain fusion tumor cells that can express positive antibodies.

Fusion tumor fusion

Laboratory animal and immune information

Preparation of the electrofusion dish: Thoroughly soak the electrofusion dish with 70% ethanol, and dry it in an ultra-clean table for later use.

Separate spleen cells: sacrifice the mice by cervical dislocation, disinfect the body surface with 75% alcohol for 5 minutes, then put them on the mouse dissection board in the ultra-clean bench, lying on the left side, and fix the limbs with a 7-gauge needle. Open the abdominal cavity aseptically and take out the spleen, wash it with basal medium (the configuration method is as follows), and carefully remove the surrounding connective tissue. The spleen was then transferred to another petri dish containing basal medium. Press the spleen with an elbow needle, insert a hole on the spleen with a small needle, and squeeze with tweezers to fully release the spleen cells to make a spleen cell suspension. The cell suspension was filtered through a 70μM cell sieve and washed with 30ml basal medium, and centrifuged at 1200rpm for 6min.

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

Electrofusion: Resuspend mouse myeloma SP2/0 cells (ATCC) with 20ml of basal medium and count them. Mix SP2/0 and spleen cells at a ratio of 1:2 to 1:1, and centrifuge at 1000 rpm for 6 minutes. After removing the supernatant, the mixed cells were resuspended in 10 ml fusion buffer (BTXpress). Then add 15 ml of fusion buffer, centrifuge at 1000 rpm for 5 min, and remove the supernatant. After repeating the above steps, resuspend the cells in an appropriate amount of fusion buffer and adjust the mixed cell density to 1×10 ⁷ cells/ml. The parameters of the electrofusion instrument are set as follows. Add 2ml of cell suspension to each electrofusion dish for electrofusion.

Plating after electrofusion: cells are allowed to stand for 5 min at room temperature in an electrofusion dish. Transfer the cells to a centrifuge tube, and dilute the cells to 1~2×10 ⁴ cells/ml with the selection medium (the configuration method is as follows). Add 100μl of cell suspension to each well of a 96-well plate. The selection medium was replaced on the 7th day after fusion. Screening is performed after the 10th day of culture (or longer, depending on the cell growth status). FACS (C6 (BD Biosciences)) was used to screen out fusion tumor cells expressing specific anti-GITR antibodies.

Subselection of positive fusion tumor cells

Sub-selection step: prepare a 96-well plate, and add 200 μl of the basal medium as described above to each well in the second to eighth columns. The cells in the positive wells screened by the above fusion were made into cell suspension and added to the first column. Add 100 μl of the cell suspension from column 1 to column 2, mix well and add 100 μl to the next column. Repeat the above steps until the volume of the last column becomes 300μl; let the 96-well plate stand for 15min, observe and count under the microscope. Take the volume corresponding to 100 cells and add 20ml of the basal medium as described above, and mix and spread the plate, 200μl per well. Observe under the microscope one week later, judge and mark the simple plant hole, and pick out the positive hole to be tested.

Cryopreservation of cells: Observe the cell status, and when the cells grow well and their viability is >90%, centrifuge at 1000 rpm for 5 minutes to remove the supernatant. Resuspend the cells with freezing solution (45.5% FBS, 44.5% RPMI-1640, 10% DMSO) to 1×10 ⁷ cells/ml, divide them into cryovials, put them in the program cooling box, and freeze at -80℃ live.

Example 2. Production and purification of chimeric antibodies

The present invention uses molecular biology technology to obtain the antibody sequence in the anti-GITR positive fusion tumor cell, and uses it to construct a human-mouse chimeric antibody.

Fusion tumor sequencing

RNA extraction: fresh cells, centrifuge at 300g for 5min, remove the supernatant, add 500 μl LY buffer (Biomiga) to the pellet (add 20 μl β-mercaptoethanol per 1 ml before use), and mix until clear. Add to the DNA removal tube, centrifuge at 13000 rpm for 2 min, and collect the flow-through. Add 100% ethanol to the flow-through liquid at a ratio of 1/2, and mix 5 times until it is clear. 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) (Takara), centrifuge at 13000 rpm for 30 seconds, and then add 500 μl RNA wash buffer (Biomiga) (Add ethanol before use), centrifuge for 30 seconds, repeat the above process, after centrifugation to completely volatilize to remove ethanol, add 30 μl DEPC water to the collection column, centrifuge at 12000g for 2 minutes, and collect the eluate. Determine the RNA concentration.

Use PrimeScript II 1st Strand cDNA Synthesis Kit (Takara) to obtain cDNA by reverse transcription: Configure reaction system I as follows:

After incubating at 65°C for 5 min, quickly place on ice to cool. Add the following reverse transcription system to reaction system I, the total amount is 20 μl :

After mixing slowly, perform reverse transcription and translation according to the following conditions: 42°C 60min→95°C 5min, then cool on ice to obtain cDNA.

Connect the cDNA to the T vector: PCR to amplify the variable regions of the heavy chain and light chain respectively, and the PCR reaction system is as follows:

The PCR reaction conditions are as follows:

Take 4.5 μl of the PCR product obtained in the above PCR reaction, add 0.5 μl pMD20-T vector (Clontech), 5 μl Ligation Mighty Mix (Takara), gently mix, and react at 37° C. for 2 hours to obtain the ligation product.

Transformed cells:

Take out TOP10 competent cells (Tiangen Biochemical Technology (Beijing) Co., Ltd.) at -80℃, melt on ice, take 5 μl of the ligation product obtained above and add to the melted TOP10 competent cells, mix well and incubate on ice for 30 minutes . After a heat shock at 42°C for 90 seconds, it was quickly cooled on ice for 2 minutes, and 900 μl of LB medium (Sanggong Bioengineering (Shanghai) Co., Ltd.) was added to the EP tube, and cultured on a shaker at 37°C at 220 rpm for 1 hour. Centrifugation at 3000g for 2min, 800 μ l supernatant was removed by suction, with the remainder of the culture medium cells were resuspended and plated on ampicillin-resistant plates amine. Incubate overnight at 37°C, and select pure strains for sequencing.

Construction of chimeric antibodies

PCR amplifies the VH and VL regions of the murine anti-GITR antibody produced by the fusion tumor in Example 1 that have been sequenced

The PCR system is as follows:

The gel is cut to recover the PCR amplified product.

Homologous recombination reaction: The homologous recombination system is as follows:

React at 37°C for 30 min to obtain a recombinant product. The recombinant product was transformed into TOP10 competent cells, and simple strains were selected for sequencing, the pure strain containing the plasmid with the correct insertion direction was selected as the positive pure strain, and the positive pure strain was saved.

Expression and purification of chimeric antibodies

The plasmid containing the anti-GITR antibody was extracted from the positive pure strain obtained above.

60%時，收集細胞上清進行純化。 Passage 293F cells (Invitrogen) according to the required transfection volume, and adjust the cell density to 1.5×10 ⁶ cells/ml the day before transfection. The cell density on the day of transfection was approximately 3×10 ⁶ cells/ml. Take 1/10 of the final volume of F17 medium (Gibco, A13835-01) as the transfection buffer, add appropriate plasmids, and mix. Add appropriate polyethyleneimine (PEI) (Polysciences, 23966) to the plasmid (the ratio of plasmid to PEI is 1:3 in 293F cells), mix and incubate at room temperature for 10 minutes to obtain a DNA/PEI mixture. After resuspending the cells with DNA/PEI mixture, 36.5°C, 8% CO ₂ . After 24 hours, 2% transfection volume of FEED (Sigma) was added, and cultured at 36.5℃, 120rpm, 8% CO ₂ conditions. Continuous culture to the 6th day or cell viability

At 60%, the cell supernatant was collected for purification.

The gravity column used for purification was treated with 0.5M NaOH overnight, and the glass bottles were washed with distilled water and then dry-baked at 180°C for 4 hours to obtain a purification column. Before purification, the collected medium was centrifuged at 4500 rpm for 30 min, and the cells were discarded. Filter the supernatant with a 0.22 μl filter. Each tube is filled with 1ml of Protein A and equilibrated with 10ml of binding buffer (sodium phosphate 20mM, NaCl 150mM, pH7.0). Add the filtered supernatant to the purification column and re-equilibrate with 15ml of binding buffer. Add 5ml of elution buffer (citric acid+sodium citrate 0.1M, pH 3.5), collect the eluate, add 80 μl Tris-HCl per 1ml of the eluate. The collected antibodies were concentrated by ultrafiltration and exchanged into PBS (Gibco, 70011-044), and the concentration was detected.

For the CDR, light chain variable region and heavy chain variable region of the two chimeric antibodies (CH22F4 and CH37G5) obtained in the present invention, the amino acid sequences of the light chain and the heavy chain, and the sequence numbers, please refer to Table 1-3 above .

The control antibody used in the present invention is the GITR antibody reported in the patent application US20130183321A1 (GITR, INC. (Cambridge, MA, US)), and its light chain and heavy chain sequences are respectively SEQ ID NO: 44 and SEQ ID NO in US20130183321A1 ：54, hereinafter referred to as TRX518 for short.

Example 3 Measurement of the binding kinetics of the chimeric antibody of the present invention with the antigen by the biofilm thin-layer interference technique

The equilibrium dissociation constant (KD) of the antibody of the present invention bound to human GITR is determined using the biofilm thin-layer interferometry technique (ForteBio). ForteBio affinity determination is performed according to existing methods (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 an appropriate amount of AMQ (Pall, 1506091) (for sample detection) or AHQ (Pall, 1502051) (for positive control detection) and soak the sensor in SD buffer (PBS 1×, BSA 0.1%, Tween-20 0.05%).

Take 100μl of SD buffer, antibodies (CH22F4, CH37G5 and TRX518), antigens (including human GITR, and cyno GITR, all purchased from Acrobiosystems) were added to 96-well black polystyrene half-well microplate (Greiner, 675076 )middle. According to the sample position layout, select the sensor position. The instrument setting parameters are as follows: Operating steps: Baseline, Loading~1nm, Baseline, Association, and Dissociation; the running time of each step depends on the sample binding and dissociation speed, the speed is 400rpm, and the temperature is 30℃. _{The K D} value was analyzed using ForteBio analysis software.

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

In the above experiments, the K _D values of the chimeric antibodies CH22F4 and CH37G5 were 1.96E-09M and 2.04E-09M, respectively. Compared with the TRX518 of the control group, the antibody in this study had a better K _D value.

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

In this study, flow cytometry was used to detect the binding of the serially diluted chimeric antibody of the present invention to the stable CHO-S cell line overexpressing human GITR on the surface.

The cDNA encoding human GITR (SEQ ID NO: 41) was cloned into pCHO1.0 vector (Invitrogen), and the obtained plasmid was transfected into CHO-S cells (Invitrogen, ExpiCHO ^TM Expression System Kit, catalog number: A29133), Generate CHO-S cells (CHO-hGITR) overexpressing human GITR.

The CHO-hGITR cells were counted and diluted to 2×10 ⁶ cells/ml, and 100 μl/well was added to the U-shaped bottom 96-well plate. Centrifuge at 400g for 5 minutes to remove the cell culture medium. Add the samples (respectively the chimeric antibodies CH22F4, CH37G5, and TRX518) (antibody dilution method: the highest antibody concentration is 400nM, three-fold dilution in PBS, a total of 12 concentrations tested) into the U-shaped plate and resuspend the cells, 100μl/well, let stand on ice for 30min. Centrifuge at 400g for 5min to remove the supernatant, and wash the cells with PBS once. Centrifuge at 400 g for 5 min to remove PBS, add 100 μl anti-human Fc PE-labeled secondary antibody (SoutherBiotech; 2040-09) (diluted in PBS at 1:200) to each well, and incubate for 30 min on ice in the dark. Centrifuge at 400g for 5min to remove the supernatant, and wash the cells with PBS once. Resuspend the cells with 80μl 1×PBS and FACS detection.

In the experiment 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 both bind to human GITR molecules overexpressed on CHO-S cells, with EC50 of 0.4568nM and 0.9069nM, respectively. Compared with TRX518, the binding ability is similar.

Example 5 Humanization of Chimeric Antibodies

The chimeric antibody obtained in Example 2 was humanized. And go through the following steps for humanization: ①Determine the structure of the CDR loop; ②Find the closest homologous sequence for each V/J region of the heavy chain and light chain in the human germline sequence database; Matching human germline and the lowest amount of back mutation; ④Construct the CDR region of the chimeric antibody onto the human framework region; ⑤Use sequence and structural features to determine the amino acid positions in the framework region that maintain the CDR function; ⑥ Make back mutations at the important sequence positions (return to the input amino acid type); ⑦ Optimize the amino acids at risk sites.

The CDR, light chain variable region and heavy chain variable region of the two humanized antibodies (HZ22F4 and HZ37G5) obtained in the present invention. For the amino acid sequences of the light chain and the heavy chain, please refer to Table 1-3 as described above .

Example 6 ForteBio measures the binding kinetics of humanized antibodies to antigens

The ForteBio assay was used to determine the equilibrium dissociation constant (K _D ) of the humanized antibody of the present invention binding to human GITR. The ForteBio affinity determination method is the same as in Example 3, except that the antibodies used are humanized antibodies HZ22F4 and HZ37G5. In the experiment described in the above assay, the affinities of antibodies HZ22F4 and HZ37G5 are shown in Table 9: Table 9. ForteBio's affinity constants for detecting antigen-antibody binding

In the above experiments, the K _D values of the humanized antibodies HZ22F4 and HZ37G5 described herein are 4.58E-09M and 4.83E-09M, respectively. Compared with the control antibody, the humanized antibody in this study has better K _D value.

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

In this study, a flow cytometer was used to detect the binding of the humanized antibody of the present invention to CHO-hGITR cells in serial dilutions. The test method is the same as in Example 4, except that the antibodies used are humanized antibodies HZ22F4 and HZ37G5. The combination is shown in Figure 2.

In the above experiment, the humanized antibodies HZ22F4 and HZ37G5 bind to human GITR overexpressed on CHO-S cells, with EC50 of 0.5492nM and 1.974nM, respectively. Compared with TRX518, they have similar or better EC50 values, that is, similar or Better combining ability.

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

In this study, a flow cytometer was used to detect the binding of the humanized antibody of the present invention in serial dilutions to the stable CHO-S cell line overexpressing cynomolgus monkey GITR on the surface.

The cDNA encoding cynomolgus monkey GITR (SEQ ID NO: 42) was cloned into pCHO1.0 vector (Invitrogen), and the plasmid was transfected into CHO-S cells (Invitrogen, ExpiCHO ^TM Expression System Kit, catalog number: A29133), Generate CHO-S cells (CHO-cynoGITR) overexpressing cyno GITR. The rest of the test methods are the same as in Example 4, except that the antibodies used are humanized antibodies HZ22F4 and HZ37G5, and the binding conditions are shown in Figure 3.

In the above experiment, 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. Compared with TRX518, they have similar or better binding capacity.

Example 9 MOA method to detect the biological activity of antibodies

The activating antibody against GITR can bind to GITR molecules on the cell surface 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 used to detect the activation of the NF-kappa B signal by detecting the expression of the fluorescent reporter gene, thereby testing the present invention Activation of antibodies.

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

The cDNA encoding human GITR (see SEQ ID NO: 41 for the sequence) was cloned into pCHO1.0 vector (Invitrogen), and the obtained plasmid and NF-kappaB luciferace reporter gene plasmid (Promega) were co-transfected into Hela cells (ATCC). , Item No.: CCL-2TM), to produce Hela cells (Hela-GITR) that overexpress human GITR and carry the NF-kappaB luciferace reporter gene system.

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

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

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

Example 10 CD4 T activated cell experiment

In this study, antibodies and activated CD4 T cells were incubated together, and the relative expression levels of IL-2 and IFN-γ in the system were detected to reflect the activation effects of different antibodies on CD4 T cells.

PBMC separation: Take 50ml of fresh blood from the donor, add 2.5 times PBS, gently add to FiColl (Thermo), divide into 4 tubes, centrifuge at 400g for 30min, rise at 7, and deceleration at 0. At the end of centrifugation, gently take out the centrifuge tube, suck the middle white cloud-like cell group into PBS, and wash with PBS twice.

CD4 ⁺ T cell separation: Take the PBMC cells separated as described above, ^{separate and enrich CD4 + T cells according to the instructions of the EasySep Human CD4 +} T Cell Enrichment Kit (stem cell), and resuspend them in T cell culture medium (see the following table for the recipe) .

CD4 ⁺ T cell activation: Take the isolated CD4 ⁺ T cells and adjust the cell density to 1*10 ⁶ /ml with T cell culture medium. Add Dynabeads Human T-Activator CD3/CD28 ( Invitrogen), cultured in a 37°C, 5% CO ₂ incubator for one week.

T activation assay Cells: Take Nunc 96 well flat bottom plates (Nunclon), 100 μ l / well of diluted in PBS 0.25 μ g / ml of the Purified NA / LE Mouse anti-human CD3 Clone UCHT1 (BD Biosciences), 37 ℃ coated 2 After hours, remove the coating liquid. ^{Remove the beads of the above-mentioned CD4 +} T cells activated for one week, wash twice with T cell culture medium, and adjust the cell density to 1*10 ⁶ /ml, and add 100μl of cell suspension per well to the above 96-well flat bottom plate. Add 100μl of the antibody of the present invention (HZ22F4, HZ37G5) and TRX518 (antibody dilution method: the highest antibody concentration is 400nM, three-fold dilution in T cell culture medium, a total of 10 concentrations tested) and Purified NA/LE Mouse anti-human CD28 clone CD28 (BD Biosciences) (final concentration of 2 μ g / ml), and cultured for 3 and 5 days, were detected by Human IL-2 kit 1000 Test and Human IFN gamma 1000 test kit (all from cisbio) IL-2 And IFN-γ relative expression (in DeltaF%).

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

Example 11 ADCC effect experiment

The IgG1 subtype antibody against GITR can bind to the highly expressed GITR molecules on the surface of Treg, and then mediate 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 the test cell line and the target cell CHO-hGITR cell (as described above) was incubated with the antibody of the present invention. The expression of the fluorescent reporter gene reflects the activation of the NF-AT signal, thereby detecting the ADCC activity of the antibody.

Take the ADCC effector cells in the logarithmic growth phase (cultured according to Promega's instructions), centrifuge to remove the supernatant, wash twice with PBS, and resuspend in the detection medium (5% low IgG serum 1640 medium (Gibco)) to adjust the cell density to 2* 10 ⁷ /ml, take the CHO-hGITR target cells prepared and incubated as described above, centrifuge to remove the supernatant, resuspend in the detection medium to adjust the cell density to 2*10 ⁶ /ml, mix the above cells at a ratio of 1:1, And added to 96-well white cell culture plate (Nunclon), 50μl/well. At the same time, add 50μl of sample (humanized antibodies HZ22F4, HZ37G5 prepared by the present invention) and positive control antibody (TRX518) to each well, (antibody dilution method: the highest antibody final concentration is 66.66nM, three-fold dilution in the detection medium, total 12 concentrations were tested). Incubate for 12 hours in a 37°C/5% CO _{2 incubator.}

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

In the above experiments, the experimental results are shown in Figure 7. Antibodies HZ22F4 and HZ37G5 can effectively activate the NF-AT signaling pathway downstream of ADCC, with EC50 of 0.02958nM and 0.04056nM, respectively. Compared with TRX518, they have significantly better EC50 value.

Example 12 Anti-tumor 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 C57Bl/6 background human GITR transgenic mice (approximately 5 weeks old) were purchased from Biocytometer Laboratory Animal Technology Co., Ltd. The mice were acclimated for 7 days after arrival, and then the study began.

Cell:

Mouse MC38 cells were purchased from Nanjing Yinhe Biomedicine Co., Ltd., and routinely subcultured in strict accordance with the instructions for subsequent in vivo experiments. Collect the cells by centrifugation, resuspend the cells in sterile PBS and adjust the cell density to 5×10 ⁶ cells/ml. On day 0, 0.2ml of cell suspension was subcutaneously inoculated into the right abdominal region of human GITR transgenic mice to establish MC38-hGITR tumor-bearing mouse model.

Administration:

The tumor volume of each mouse was measured 6 days after tumor cell inoculation, and the mice with tumor volume in the ^{range of 87.4mm 3} ~ 228.4mm ^{3 were} divided into groups according to the tumor volume (5 mice per group), and the dosage and The method is shown in Table 12, where Antibody C is the anti-PD-1 monoclonal antibody "Antibody C" (WO2017/133540). They were administered on the 6, 10, 13, and 17 days after the inoculation, and the tumor volume and body weight changes of the mice in each group were monitored during the administration. The monitoring frequency was 2 times per week, and the monitoring was continuous for a week. The 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. The calculation formula is as follows: TGI%=100% * (h-IgG control group tumor volume-treatment group tumor volume )/(h-IgG control group tumor volume-h-IgG control group pre-dose tumor volume), in which the average value of h-IgG control group pre-dose tumor volume is 106 mm ³ . Tumor volume measurement: use a vernier caliper to determine the largest long axis (L) and largest wide axis (W) of the tumor. The tumor volume is calculated according to the following formula: V=L×W ² /2. An electronic balance is used to determine body weight.

The results of tumor suppression rate are shown in Figure 8, Figure 9 and Table 13. On the 24th day after vaccination, HZ37G5 and HZ22F4 single drugs both showed good tumor suppression rates, which were 53% and 50%, respectively, better than 36% of the TRX518 group. HZ37G5 and HZ22F4 combined with anti-PD-1 antibody Antibody C, the tumor suppression effect was significantly better than Antibody C, HZ37G5, HZ22F4, and TRX518 single agent group. Among the 5 mice in the HZ37G5 and Antibody C combined medication group, and in the HZ22F4 and Antibody C combined medication group, 4 tumors disappeared completely. At the same time, we tested the body weight of the mice, and the results showed that there was no significant difference in the body weight of the mice as shown in Figure 10. Therefore, the antibody against the GITR molecule of the present invention has a significant inhibitory effect on tumors, and when the antibody and PD-1 antibody are used in combination, the inhibitory effect on tumors is more significant.

<110> Cinda Biopharmaceutical (Suzhou) Co., Ltd.

<120> Anti-GITR antibody and its use

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Claims (34)

An antibody or antigen-binding fragment thereof that binds to GITR, comprising three complementarity determining regions HCDR1, HCDR2, HCDR3 of the heavy chain variable region, and 3 complementarity determining regions LCDR1, LCDR2, LCDR3 of the light chain variable region, wherein, HCDR1 includes the amino acid sequence shown in SEQ ID NO: 2, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 6, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 8, and LCDR1 includes SEQ ID NO : The amino acid sequence shown in 10, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 12, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 16. The GITR-binding antibody or antigen-binding fragment thereof as described in the first item of the scope of patent application, which comprises the heavy chain variable region of the amino acid sequence shown in SEQ ID NO: 20, and comprises the heavy chain variable region shown in SEQ ID NO: 24 The light chain variable region of the amino acid sequence. The GITR-binding antibody or antigen-binding fragment thereof as described in the first item of the scope of patent application, which comprises the heavy chain of the amino acid sequence shown in SEQ ID NO: 28 and the amino group shown in SEQ ID NO: 32 The acid sequence of the light chain. The antibody or antigen-binding fragment thereof that binds to GITR as described in item 1 of the scope of patent application, wherein the antibody is an antibody or antigen-binding fragment in the form of IgG1, IgG2, or IgG4. The GITR-binding antibody or antigen-binding fragment thereof as described in item 1 of the scope of patent application, wherein the antibody is a monoclonal antibody. The antibody or antigen-binding fragment thereof that binds to GITR as described in any one of items 1 to 5 in the scope of the patent application, wherein the antibody is a humanized antibody or a human antibody or a chimeric antibody. The antibody or antigen-binding fragment thereof that binds to GITR as described in item 1 of the scope of patent application, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, scFv, (Fab' ) _2. Single domain antibody, double antibody (dAb) or linear antibody. The GITR-binding antibody or antigen-binding fragment thereof as described in item 1 of the scope of the patent application, wherein the antibody is a bispecific antibody. The antibody or antigen-binding fragment thereof that binds to GITR as described in item 8 of the scope of patent application, wherein the bispecific antibody binds to GITR and PD-1, PD-L1 or PD-L2. An isolated nucleic acid encoding the GITR-binding antibody or antigen-binding fragment thereof as described in any one of items 1 to 9 in the scope of the patent application. A vector containing the nucleic acid described in item 10 of the scope of the patent application. A host cell containing the nucleic acid described in item 10 of the scope of patent application or the vector described in item 11 of the scope of patent application. A method for preparing an antibody or antigen-binding fragment thereof that binds to GITR, the method comprising: Culture the host cell as described in item 12 of the scope of patent application under the conditions of the nucleic acid described in item 10 of the scope of patent application. According to the method described in item 13 of the scope of the patent application, the method further comprises isolating the antibody or antigen-binding fragment thereof. According to the method described in item 13 or 14 of the scope of the patent application, the method further comprises recovering the GITR-binding antibody or antigen-binding fragment thereof from the host cell. An immunoconjugate comprising the GITR-binding antibody or antigen-binding fragment thereof as described in any one of items 1 to 9 of the scope of patent application and other substances, and the other substances are cytotoxic agents or markers. A pharmaceutical composition comprising the GITR-binding antibody or antigen-binding fragment thereof as described in any one of the scope of the patent application or the immunoconjugate as described in the 16th item of the scope of the patent application. The pharmaceutical composition as described in item 17 of the scope of patent application, which further comprises pharmaceutical excipients. A combination product comprising the GITR-binding antibody or antigen-binding fragment thereof as described in any one of the scope of the patent application or the immunoconjugate as described in the 16th patent application, and one or Various other therapeutic agents. The combination product according to item 19 of the scope of patent application, wherein the other therapeutic agent is selected from chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies, anti-infective agents, small molecule drugs or immunomodulators. The combination product according to item 19 of the scope of patent application, wherein the other therapeutic agent is selected from PD-1 axis binding antagonists. The combination product according to item 19 of the scope of patent application, wherein the other therapeutic agent is selected from an anti-PD-1 antibody, an anti-PD-L1 antibody or an anti-PD-L2 antibody. An effective amount of the antibody or antigen-binding fragment thereof that binds to GITR as described in any one of the scope of patent application 1 to 9, the immunoconjugate as described in item 16 of the scope of patent application, as described in the scope of patent application The pharmaceutical composition according to any one of items 17 or 18, or the combination product according to any one of items 19 to 22 in the scope of the patent application is used in the preparation of medicines Purpose: The drug is used to mediate ADCC or activate effector T cells or activate NF-kappaB signaling pathway or eliminate regulatory T cells in a subject. The use according to item 23 of the scope of patent application, wherein the regulatory T cell is a Treg cell. An effective amount of the antibody or antigen-binding fragment thereof that binds to GITR as described in any one of the scope of patent application 1 to 9, the immunoconjugate as described in item 16 of the scope of patent application, as described in the scope of patent application Use of the pharmaceutical composition according to any one of items 17 or 18, or the combination product according to any one of items 19 to 22 in the scope of the patent application, in the preparation of a medicine for the treatment of tumors or infections in a subject . The use according to item 25 of the scope of patent application, wherein the tumor is cancer, and/or the infection is a bacterial infection, a viral infection, a fungal infection, or a protozoan infection. The use according to item 26 of the scope of patent application, wherein the cancer is gastrointestinal cancer. The use according to item 27 of the scope of patent application, wherein the gastrointestinal cancer is selected from the group consisting of gastric cancer, rectal cancer, colon cancer, and colorectal cancer. The use described in item 25 of the scope of patent application, wherein the infection is a chronic infection. The use according to any one of the 25th to 29th scope of the patent application, which also includes the combined administration of one or more therapies to the subject. The use as described in item 30 of the scope of the patent application, wherein the therapy is a treatment modality and/or other therapeutic agents. The use according to item 30 of the scope of patent application, wherein the treatment method includes surgical treatment and/or radiotherapy, and/or other therapeutic agents are selected from chemotherapeutics, cytotoxic agents, vaccines, other antibodies, anti-infective agents, Small molecule drugs or immunomodulators. The use according to item 31 of the scope of patent application, wherein the therapeutic agent is a PD-1 axis binding antagonist. The use according to item 31 of the scope of the patent application, wherein the therapeutic agent is an anti-PD-1 antibody or an anti-PD-L1 antibody or an anti-PD-L2 antibody.

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