TW201625697A - Compositions and methods of use for augmented immune response, and cancer therapy - Google Patents

Abstract

The present invention provides antibody compositions, including, e.g., antibodies, engineered antibodies and antibody fragments that bind to a tumor necrosis factor receptor superfamily member (i.e., 18). Provided compositions are useful in enhancing CD4+ and CD8+ T cell responses, and in the treatment, amelioration and prevention of diseases that can be counteracted with an augmented immune response, e.g., cancers. Also provided in the invention are polynucleotides and vectors that encode such molecules and host cells that harbor the polynucleotides or vectors; as well as pharmaceutical compositions that comprise such molecules and methods of use thereof.

Description

Composition and method for enhancing immune response, and cancer therapy

The present invention relates to antibodies, antibody fragments and antigen-binding molecules which bind to a tumor necrosis factor receptor superfamily member 18/glucocorticoid-induced TNFR-related protein ("GITR"), and more specifically an agonist Stimulate signaling through the receptor and/or modulate the immune response.

Glucocorticoid-induced TNFR-associated protein ("GITR") is a member of the tumor necrosis factor superfamily (TNFRSF), which includes more than 20 type I transmembrane proteins, several splicing variants, and several viral proteins, all of which have The cysteine-rich domain acts as a common structural feature. The extracellular domain (ECD) of GITR consists of three cysteine-rich domains (CRD), followed by the transmembrane domain (TM) and the intracellular domain (ICD).

Constitutive GITR expression was detected on murine and human CD4+CD25+ regulatory T cells, which may be further increased after activation. In contrast, CD4+CD25 effector T cells and CD8+CD25 effector T cells express GITR at levels as low as undetectable and rapidly upregulate after T cell receptor activation. GITR expression was also detected on activated NK cells, dendritic cells, and macrophages. Signal transduction pathways downstream of the GITR have been shown to include MAPK and the standard NFκB pathway. Various TRAF family members have been shown to act as signaling intermediates downstream of the GITR (Nocentini et al. (2005) Eur. J. Immunol., 35: 1016-1022).

Activation of cells via GITR is thought to be dependent on several cell functions and microenvironments, including but not limited to co-stimulation to enhance proliferation and effector function, inhibit inhibition of regulatory T cells, and avoid activation-induced cell death. (Shevach and Stephens (2006) Nat. Rev. Immunol., 6: 613-618). Ko et al. ((2005) J. Exp. Med., 202: 885-891) demonstrate for the first time that efficacious monoclonal antibodies against mouse GITR in mouse homologous tumor models effectively induce tumor-specific immunity and eradicate The tumor formed. Additionally and/or alternatively, anti-mGITRs with functional Fc effector activity have been shown to deplete regulatory T cells and enhance T effector cell proliferation and cytokine secretion in selected tumor settings in some preclinical models. These findings indicate that agonist antibodies to mGITR disrupt the balance of immune tolerance, which in turn allows T cells to fight tumor and persistent viral infections. However, research to date has focused primarily on the use of surrogate antibodies in rodent systems. Due to structural differences in mouse and human GITR, alternative studies in unknown mice see whether it can be converted to a change in human GITR function.

Antibodies that specifically bind to human glucocorticoid-induced tumor necrosis factor receptor superfamily member 18 ("GITR") have been identified, wherein the antibodies have in vitro hGITR agonist activity when in vivo, and wherein The antibody confers hGITR activity in vivo and induces an increased Teff:Treg ratio at the tumor site, thereby inhibiting tumor progression. Thus, the invention provides agonist antibodies, antibody fragments and antigen binding molecules that specifically bind and promote intracellular signaling and/or modulate immune responses via cells that target human GITR. In one aspect, the invention provides an isolated antibody, antibody fragment and antigen binding molecule that specifically binds to human GITR, wherein the antibody, antibody fragment or antigen binding molecule binds to a cysteine-rich domain 1 (" An epitope within CRD1"), and wherein the antibody, antibody fragment or antigen-binding molecule is an agonist of GITR, and wherein the antibody, antibody fragment or antigen-binding molecule optionally has a full or increased FcR effector function.

In some embodiments, an antibody, antibody fragment or antigen binding molecule binds to a human An epitope within residues 41-65 of GITR. In some embodiments, the antibody, antibody fragment or antigen binding molecule competes with an antibody or antibody fragment that binds to an epitope within residues 41-65 of human GITR. In some embodiments, the antibody, antibody fragment or antigen binding molecule binds to at least one amino acid residue within residues 41-65 of human GITR, eg, an antibody, antibody fragment or antigen binding molecule binds to a human GITR The epitope 41-65 overlaps the epitope.

In some embodiments, the antibody, antibody fragment or antigen binding molecule binds to SEQ ID NO: 4 and comprises (a) a heavy chain variable region comprising a human heavy chain, wherein: i) heavy chain CDR1 comprises SEQ ID NO: 22, and ii) the heavy chain CDR2 comprises a sequence selected from any one of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, and iii The heavy chain CDR3 comprises SEQ ID NO: 29 or SEQ ID NO: 109; and (b) a light chain variable region, wherein i) the light chain CDR1 comprises SEQ ID NO: 30 or SEQ ID NO: 31, and ii) The chain CDR2 comprises SEQ ID NO:33, and iii) the light chain CDR3 comprises SEQ ID NO:34.

With respect to other embodiments of antibodies, antibody fragments or antigen binding molecules, in some embodiments, the heavy chain variable region has at least 95%, 96%, 97%, 98%, 99 of the variable region of SEQ ID NO: % or 100% amino acid sequence identity, and the light chain variable region and the variable region of SEQ ID NO: 17 have at least 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence consistency. In a particular embodiment, the antibody, antibody fragment or antigen binding molecule comprises a heavy chain comprising SEQ ID NO: 16 and a light chain comprising SEQ ID NO: 17. In some embodiments, the antibody, antibody fragment or antigen binding molecule competes with an antibody comprising a heavy chain comprising SEQ ID NO: 16 and a light chain comprising SEQ ID NO: 17.

In some embodiments, the heavy chain FR4 is the human germline FR4. In a particular embodiment, the heavy chain FR4 is SEQ ID NO:42.

In some embodiments, the light chain FR4 is the human germline FR4. In a particular embodiment, the light chain FR4 is SEQ ID NO:50.

In some embodiments, an antibody, antibody fragment or antigen binding molecule is provided, wherein: i) the heavy chain CDR1 comprises SEQ ID NO: 22 or SEQ ID NO: 84; ii) the heavy chain CDR2 comprises SEQ ID NO: 28 or SEQ ID NO: 80; iii) heavy chain CDR3 comprises SEQ ID NO: 29 or SEQ ID NO: 109; iv) light chain CDR1 comprises SEQ ID NO: 30 or SEQ ID NO: 85; v) light chain CDR2 comprises SEQ ID NO :33 or SEQ ID NO:82, and vi) the light chain CDR3 comprises SEQ ID NO:34 or SEQ ID NO:83.

In some embodiments, an antibody, antibody fragment or antigen binding molecule is provided, wherein: i) heavy chain CDR1 comprises SEQ ID NO: 22; ii) heavy chain CDR2 comprises SEQ ID NO: 23; iii) heavy chain CDR3 comprises SEQ ID NO: 29; iv) light chain CDR1 comprises SEQ ID NO: 30; v) light chain CDR2 comprises SEQ ID NO: 33, and vi) light chain CDR3 comprises SEQ ID NO: 34.

In some embodiments, an antibody, antibody fragment or antigen binding molecule is provided, wherein: i) heavy chain CDR1 comprises SEQ ID NO:22; ii) heavy chain CDR2 comprises SEQ ID NO:24; iii) heavy chain CDR3 comprises SEQ ID NO: 29; iv) light chain CDR1 comprises SEQ ID NO: 31; v) light chain CDR2 comprises SEQ ID NO: 33, and vi) light chain CDR3 comprises SEQ ID NO: 34.

In some embodiments, an antibody, antibody fragment or antigen binding molecule is provided, wherein: i) heavy chain CDR1 comprises SEQ ID NO: 22; ii) heavy chain CDR2 comprises SEQ ID NO: 25; iii) heavy chain CDR3 comprises SEQ ID NO: 29; iv) light chain CDR1 comprises SEQ ID NO: 30; v) light chain CDR2 comprises SEQ ID NO: 33, and vi) light chain CDR3 comprises SEQ ID NO: 34.

In some embodiments, an antibody, antibody fragment or antigen binding molecule is provided, wherein: i) heavy chain CDR1 comprises SEQ ID NO:22; ii) heavy chain CDR2 comprises SEQ ID NO:26; iii) heavy chain CDR3 comprises SEQ ID NO: 29; iv) light chain CDR1 comprises SEQ ID NO: 30; v) light chain CDR2 comprises SEQ ID NO: 33, and vi) light chain CDR3 comprises SEQ ID NO: 34.

In some embodiments, an antibody, antibody fragment or antigen binding molecule is provided, wherein: i) heavy chain CDR1 comprises SEQ ID NO:22; ii) heavy chain CDR2 comprises SEQ ID NO:27; iii) heavy chain CDR3 comprises SEQ ID NO: 29; iv) light chain CDR1 comprises SEQ ID NO: 30; v) light chain CDR2 comprises SEQ ID NO: 33, and vi) light chain CDR3 comprises SEQ ID NO: 34.

In some embodiments, an antibody, antibody fragment or antigen binding molecule is provided, wherein: i) heavy chain CDR1 comprises SEQ ID NO: 22; ii) heavy chain CDR2 comprises SEQ ID NO: 25; iii) heavy chain CDR3 comprises SEQ ID NO: 109; iv) light chain CDR1 comprises SEQ ID NO: 30; v) light chain CDR2 comprises SEQ ID NO: 33, and vi) light chain CDR3 comprises SEQ ID NO: 34.

In another aspect, the invention provides an antibody, antibody fragment or antigen binding molecule that specifically binds to GITR, wherein the antibody or antibody fragment comprises a heavy chain variable region and a light chain variable region, wherein: i) a heavy chain CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 79 or SEQ ID NO: 84; ii) CIR2 of the heavy chain comprises SEQ ID NO: 23, SEQ ID NO: 24 , the amino acid sequence of the group consisting of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 62 and SEQ ID NO: 80; iii) the CDR3 of the heavy chain comprises SEQ ID NO: 29 or SEQ ID NO: 109; iv) CDR1 of the light chain comprising selected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 63, SEQ ID NO: 81, SEQ ID NO: 85, and SEQ ID NO : a group consisting of the amino acid sequence of the group; v) the CDR2 of the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 64 and SEQ ID NO: 82; and the light chain CDR3 comprises SEQ ID NO: 34 or SEQ ID NO:83.

In other embodiments of the antibody, antibody fragment or antigen binding molecule, the heavy chain variable region is selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO The variable region of the sequence of: 99 and SEQ ID NO: 105 has at least 90%, 93%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence Consistent, and the variable region of the light chain variable region and the sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 7 have at least 90%, 93%, 95%, 96%, 97%, 98 %, 99% or 100% amino acid sequence identity. In a particular embodiment, the isolated antibody, antibody fragment or antigen binding molecule comprises a molecule selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14. a heavy chain variable domain of the sequence of any one of SEQ ID NO: 99 and SEQ ID NO: 105; and the light chain variable domain comprises SEQ ID NO: 7 or SEQ ID NO: 9. In some embodiments, the isolated antibody, antibody fragment or antigen binding molecule comprises the heavy chain variable domain of SEQ ID NO: 6 and the light chain variable domain of SEQ ID NO: 7. In some embodiments, the isolated antibody or antibody fragment comprises a heavy chain variable domain comprising SEQ ID NO: 8 and a light chain variable domain comprising SEQ ID NO: 9. In other embodiments, the isolated antibody or antibody fragment comprises a heavy chain variable domain comprising SEQ ID NO: 10 and a light chain variable domain comprising SEQ ID NO: 7. In other embodiments, the isolated antibody or antibody fragment comprises a heavy chain variable domain comprising SEQ ID NO: 12 and a light chain variable domain comprising SEQ ID NO: 7. In other embodiments, the isolated antibody or antibody fragment comprises a heavy chain variable domain comprising SEQ ID NO: 14 and a light chain variable domain comprising SEQ ID NO: 7.

With respect to other embodiments of antibodies, antibody fragments or antigen binding molecules, in some embodiments, the heavy chain variable region has at least 90%, 93%, 95%, 96%, 97 of the variable region of SEQ ID NO:99 %, 98%, 99% or 100% amino acid sequence identity, and the light chain variable region and the variable region of SEQ ID NO: 7 have at least 90%, 93%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity. In some embodiments, the isolated antibody or antibody fragment comprises a heavy chain variable domain comprising SEQ ID NO: 99 and a light chain variable domain comprising SEQ ID NO: 7.

With respect to other embodiments of antibodies, antibody fragments or antigen binding molecules, in some embodiments, the heavy chain variable region has at least 90%, 93%, 95%, 96%, 97 of the variable region of SEQ ID NO:105 %, 98%, 99% or 100% amino acid sequence consistent and light The chain variable region has at least 90%, 93%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the variable region of SEQ ID NO: 7. In some embodiments, the isolated antibody or antibody fragment comprises a heavy chain variable domain comprising SEQ ID NO: 105 and a light chain variable domain comprising SEQ ID NO: 7.

In some embodiments, an antibody, antibody fragment or antigen binding molecule that binds to GITR is humanized. In certain embodiments, the antibody or antibody fragment comprises a human constant region.

In some embodiments, the antibody fragment is a Fab' fragment. In some embodiments, the antibody fragment is a single chain antibody (scFv). In some embodiments, the antibody fragment is a single domain antibody or a nanobody antibody.

In some embodiments, the antibody or antibody fragment is cross-linked to a second antibody or antibody fragment. In some embodiments, the antibody is glycosylated.

In some embodiments, the antibody, antibody fragment or antigen binding molecule is an IgG. In certain embodiments, the antibody, antibody fragment or antigen binding molecule comprises an IgG isotype antibody Fc region. In a particular embodiment, the antibody, antibody fragment or antigen binding molecule comprises an Fc region of an IgGl or IgG2 isotype antibody. In certain embodiments, the antibody, antibody fragment or antigen binding molecule is an IgGl or IgG2 antibody. In some embodiments, the antibody, antibody fragment or antigen binding molecule contains at least one mutation that modulates (ie, increases or decreases) the binding of the antibody or antibody fragment to the Fc receptor. In some embodiments, the antibody, antibody fragment or antigen binding molecule contains at least one mutation that modulates (ie, increases or decreases) an antibody, antibody fragment or antigen binding molecule that activates the Fc receptor. In a particular embodiment, the antibody, antibody fragment or antigen binding molecule contains at least one mutation that increases the binding of the antibody or antibody fragment to the Fc receptor. In certain embodiments, the antibody, antibody fragment or antigen binding molecule contains at least one mutation that increases an antibody, antibody fragment or antigen binding molecule that activates the Fc receptor.

In some embodiments, the antibody, antibody fragment or antigen binding molecule cross-reacts with human and non-human primate GITR. In some embodiments, the antibody, antibody fragment or antigen binding molecule does not cross the rodent GITR (eg, rat GITR or mouse GITR) reaction.

In a related aspect, the invention further provides a polynucleotide encoding an antibody, antibody fragment or antigen binding molecule of the invention as described herein. In some embodiments, the polynucleotide encoding the light chain variable region has at least 90%, 93%, 95% of the nucleic acid sequence selected from the group consisting of SEQ ID NO: 52, SEQ ID NO: 54 and SEQ ID NO: 102 , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity. In some embodiments, the polynucleotide encoding the heavy chain variable region is selected from the group consisting of SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, The nucleic acid sequences of SEQ ID NO: 101, SEQ ID NO: 107, and SEQ ID NO: 115 have at least 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity. In some embodiments, the polynucleotide encoding the light chain variable region has a nucleic acid sequence selected from the group consisting of SEQ ID NO:52, SEQ ID NO:54, and SEQ ID NO:102. In some embodiments, the polynucleotide encoding the heavy chain variable region is selected from the group consisting of SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57 Nucleic acid sequences of SEQ ID NO:101, SEQ ID NO:107 and SEQ ID NO:115.

In a related aspect, the invention further provides a composition comprising an antibody, antibody fragment or antigen binding molecule of the invention as described herein and a pharmaceutically acceptable carrier. In some embodiments, the invention provides a pharmaceutical composition comprising an antibody, antibody fragment or antigen binding molecule of the invention for administration to an individual.

In some embodiments, the composition further comprises a target antigen, such as a cancer associated antigen or a tumor associated antigen. In some embodiments, the target antigen is a viral antigen, a bacterial antigen, a fungal antigen, or a parasite antigen.

In some embodiments, the composition further comprises an antagonist of CTLA4. In some embodiments, the composition further comprises an inhibitor of PD-1/PD-L1 (eg, B7-H1 or an analog thereof, PD-1 antibody) interaction.

In another aspect, the invention further provides the invention comprising the invention as described herein A set of antibodies or antibody fragments.

In some embodiments, the kit further comprises a second agent for co-administering with the antibody. In some embodiments, the second agent is a target antigen, such as a cancer associated antigen or a tumor associated antigen. In some embodiments, the target antigen is a viral antigen, a bacterial antigen, a fungal antigen, or a parasite antigen.

In some embodiments, the second agent is an antagonist of CTLA4. In some embodiments, the second agent is an inhibitor of PD-1/PD-L1 (eg, B7-H1) interaction.

The antibody or antibody fragment and the second agent are provided as a mixture, as appropriate. The antibody or antibody fragment and the second agent are provided as separate formulations, as appropriate.

In another aspect, the invention provides a method of increasing a T cell response in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an anti-agonist antibody or antibody fragment of the invention as described herein. In another aspect, the invention provides an anti-GITR agonist antibody or antibody fragment of the invention for use in increasing a T cell response in an individual. In another aspect, the invention provides a composition comprising an antibody or antibody fragment of the invention for use in increasing a T cell response in an individual.

In another aspect, the invention provides a method of treating tumor growth of a cancer exhibiting a tumor-associated antigen in a subject in need thereof, comprising administering to the individual a therapeutically effective amount of an anti-GITR agonist of the invention as described herein. An antibody, antibody fragment or antigen binding molecule. The invention further provides an anti-GITR agonist antibody or antibody fragment of the invention for use in treating tumor growth in a cancer of an individual. The invention further provides a composition comprising an antibody or antibody fragment of the invention for use in reducing, inhibiting or preventing tumor growth in a cancer of a subject exhibiting a tumor associated antigen.

For embodiments of methods and medical uses, in some embodiments, an anti-GITR agonist antibody, antibody fragment or antigen binding molecule is co-administered with an antigen. In some embodiments, the antigen is a cancer associated antigen or a tumor associated antigen. In some embodiments, an anti-GITR agonist antibody or antibody fragment is co-administered with a cancer cell from a patient (ie, an autologous cancer cell) With the same investment.

In some embodiments, an anti-GITR agonist antibody, antibody fragment or antigen binding molecule is co-administered with an antagonist of CTLA4. In some embodiments, an anti-GITR agonist antibody or antibody fragment is co-administered with an inhibitor of PD-1/PD-L1 (eg, B7-H1) interaction.

In some embodiments, an anti-GITR agonist antibody, antibody fragment or antigen binding molecule is co-administered with a chemotherapeutic agent or a cytotoxin.

In some embodiments, the T cell response is a CD8+ cytotoxic T lymphocyte (CTL) T cell response. In some embodiments, the T cell response is a CD4+ helper T cell (Th) response.

In some embodiments, the patient has a cancer that exhibits a tumor associated antigen. In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, colorectal cancer, prostate cancer, non-small cell lung cancer (NSCLC), and breast cancer. In one embodiment, the cancer type is selected from the group consisting of pancreatic cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, gastric cancer, ovarian cancer, bladder cancer, brain cancer, or central nervous system. Cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterus or endometrial cancer, oral or pharyngeal cancer, head and neck squamous cell carcinoma (HNSCC), liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or Attachment cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma and blood tissue cancer.

In some embodiments, the patient has an infectious disease, such as a viral infection, a bacterial infection, a fungal antigen, or a parasitic antigen. In some embodiments, an anti-GITR agonist antibody is co-administered with a viral antigen (eg, from HCV, HSV, or HIV). In some embodiments, an anti-GITR agonist antibody is co-administered with a bacterial antigen. In some embodiments, an anti-GITR agonist antibody is co-administered with a fungal antigen. In some embodiments, an anti-GITR agonist antibody is co-administered with a parasitic antigen (eg, filariasis).

In other embodiments, an isolated antibody, antibody fragment or antibody for use in therapy is provided The original binding molecule. In certain embodiments, antibodies, antibody fragments or antigen binding molecules are provided for use in increasing T cell responses in an individual in need thereof. In certain embodiments, antibodies, antibody fragments or antigen binding molecules are provided for use in treating tumor growth for an individual in need thereof.

definition

"Antibody" refers to a polypeptide of the immunoglobulin family that is capable of non-covalent, reversible, and specific binding to a corresponding antigen. Exemplary antibody structural units comprise a tetramer. Each tetramer is composed of two identical polypeptide chain pairs, each pair having a "light" chain (about 25 kD) and a "heavy" chain (about 50-70 kD) linked via a disulfide bond. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes as well as various immunoglobulin variable region genes. The light chain is classified as κ or λ. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. The antibodies of the invention may have any isotype/category (e.g., IgG, IgM, IgA, IgD, and IgE) or any subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgA1, and IgA2). The N-terminus of each strand defines a variable region having about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (V _L) and variable heavy chain (V _H) refer to these regions of light and heavy chains. In addition to the V region, the heavy and light chains contain a constant (C) region or domain. The secretory form of the immunoglobulin C region consists of three C domains CH1, CH2, CH3, optionally present CH4 (Cμ) and a hinge region. The membrane-bound form of the immunoglobulin C region also has a membrane and an intracellular domain. Each light chain has a VL at the N-terminus and a constant domain (C) at its other end. VL is aligned with VH and CL is aligned with the first constant domain of the heavy chain. The VH and VL pairs together form a single antigen binding site. As used herein, "a conventional antibody" IgG immunoglobulin refers to an antibody that is in a naturally occurring configuration. Typically, conventional antibody IgGs have four chains, two identical heavy chains, and two identical light chains joined together via disulfide bonds. As used herein, "antibody" also encompasses antibody variants having a specific binding specificity (i.e., to GITR) and conventional antibody structures. Therefore, full-length antibodies, chimeric antibodies, and humanized antibodies having specific binding specificities for GITR fall within the scope of this concept.

The antibodies are present as intact immunoglobulins or as a plurality of well characterized fragments produced by digestion with various peptidases. Thus, for example, below the disulfide linkages in the hinge region pepsin digestion of the antibody to produce F (ab) _'2, i.e., a dimer of Fab which itself is joined by disulfide bonds to V _H -C _H 1 light chain. F(ab)' ₂ can be reduced under mild conditions to destroy the disulfide bonds in the hinge region, thereby converting the (Fab') ₂ dimer to a Fab' monomer. The Fab' monomer is essentially a Fab having a portion of the hinge region. Paul, Fundamental Immunology , 3rd edition (1993). While various antibody fragments are defined in terms of digesting intact antibodies, those skilled in the art will recognize that such fragments can be resynthesized chemically or by using recombinant DNA methods. As used herein, "antibody fragment" refers to one or more portions of an antibody that retains binding specificity and agonist activity to GITR by modification of whole antibody production or resynthesis using recombinant DNA methods. Examples of antibody fragments include Fv fragments having the same binding specificity, single chain antibodies (ScFv), Fab, Fab', Fd (Vh and CH1 domains), dAbs (Vh and isolated CDRs); and multimerization of such fragments Type (for example F(ab') ₂ ). Antibody fragments can also be incorporated into single domain antibodies, maximal antibodies, minibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, vNARs, double scFvs, and other variants of compounds such as antibodies to achieve the present invention. Binding specificity and activity.

The "Fab" domain used in the context of the present invention comprises a heavy chain variable domain, a constant region CH1 domain, a light chain variable domain, and a light chain constant region CL domain. The domain interaction is stabilized by a disulfide bond between the CH1 and CL domains. In some embodiments, the heavy chain domain of the Fab is in the order VH-CH from the N-terminus to the C-terminus, and the light-chain domain of the Fab is in the order VL-CL from the N-terminus to the C-terminus. In some embodiments, the heavy chain domain of the Fab is in the order CH-VH from the N-terminus to the C-terminus, and the light-chain domain of the Fab is in the order CL-VL. Although Fab has been identified by papain digestion of intact immunoglobulins, in the context of the present invention, "Fab" is typically produced recombinantly by any method. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen binding site.

The C-terminal portion of the immunoglobulin heavy chain comprising the CH2 and CH3 domains is the "Fc" domain. As used herein, "Fc region" refers to the constant region of an antibody, excluding the first constant region immunoglobulin domain. Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and IgE And the last three constant region immunoglobulin domains of IgM, and the flexible hinges at the N-terminus of these domains. For IgA and IgM, Fc can include a J chain. For IgG, Fc comprises the hinge between the immunoglobulin domains Cγ2 and Cγ3 and Cγ1 and Cγ. In the art, it will be appreciated that the boundaries of the Fc region may vary, however, the human IgG heavy chain Fc region is typically designated to contain residues C226 or P230 to its carboxy terminus, as used by Kabat et al. (1991, NIH Publication 91-3242). , the number of the EU index in National Technical Information Service, Springfield, Va.). "Fc region" may refer to this region in isolated form or in the case of antibodies or antibody fragments. The "Fc region" includes naturally occurring dual gene variants of the Fc region (eg, in the CH2 and CH3 regions) and modifications that modulate effector functions. The Fc region also includes variants that do not cause changes in biological function. For example, one or more amino acids can be deleted at the N-terminus or C-terminus of the immunoglobulin Fc region without substantial loss of biological function. For example, in certain embodiments, the C-terminal amide acid can be modified by displacement or removal. In a particular embodiment, one or more C-terminal residues in the Fc region are altered or removed. In certain embodiments, one or more C-terminal residues (eg, terminally deaminated with an acid) are deleted in the Fc. In certain other embodiments, one or more C-terminal residues in the Fc are substituted with an alternative amino acid (eg, the terminal is trans-amino acid substituted). Such variants can be selected to have minimal effect on activity according to the general rules known in the art (see, for example, Bowie et al, Science 247:306-1310, 1990). The Fc domain is a portion of Ig that is recognized by a cellular receptor (such as FcR) and binds to the complement activating protein C1q. The lower hinge region encoded in the 5' portion of the CH2 exon provides flexibility within the antibody to bind to the FcR receptor.

"Complementarity determining region" or "complementarity determining regions" ( "CDR") interchangeably refer to high V _L and V _H of the hypervariable region. A CDR is a target protein binding site with an antibody chain specific for such a target protein. There are three CDR (CDR1-3, numbered sequentially from the N-terminus), accounting for about 15-20% of each variable domain of a human V _L or V _H in. The CDRs are structurally complementary to the epitope of the target protein and thus contribute directly to the binding specificity. The amino acid sequence of the remaining stretched segment of V _L or V _H (the so-called framework region) exhibits less variation (Kuby, Immunology, 4th edition, Chapter 4, WH Freeman and Co., New York, 2000).

The location of the CDRs and framework regions can be determined using various well-known definitions in the art, such as Kabat, Chothia, International ImMunoGeneTics database (IMGT) (on imgt.cines.fr/ on the World Wide Web) And AbM (see, eg, Johnson et al, Nucleic Acids Res., 29: 205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987); Chothia et al, Nature, 342 : 877-883 (1989); Chothia et al, J. Mol. Biol., 227: 799-817 (1992); Al-Lazikani et al, J. Mol. Biol., 273: 927-748 (1997)) . The definition of antigen binding sites is also described in: Ruiz et al, Nucleic Acids Res., 28: 219-221 (2000); and Lefranc, MP, Nucleic Acids Res., 29: 207-209 (2001); MacCallum et al, J. Mol. Biol., 262: 732-745 (1996); and Martin et al, Proc. Natl. Acad. Sci. USA, 86: 9268-9272 (1989); Martin et al, Methods Enzymol 203: 121-153 (1991); and Rees et al, In Sternberg MJE (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996).

According to Kabat, the CDR amino acid residues in _VH are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3); and the number of CDR amino acid residues in _VL It is 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3). According to Chothia, the CDR amino acids in V _H are numbered 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and the amino acid residue in _VL is numbered 26- 32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3). By combining the CDR definitions of Kabat and Chothia, the CDRs are composed of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) in human VH and amino acid residues in human VL. The base consists of 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3).

The term "binding specificity determinant" or "BSD" interchangeably refers to the smallest contiguous or non-contiguous amino acid sequence required to determine the binding specificity of an antibody within a complementarity determining region. The minimal binding specificity determinant can be within one or more CDR sequences. In some embodiments, the minimum knot The specificity determinant is present in the heavy or light chain portion of the antibody or in the full length CDR3 sequence (ie, only by its decision).

As used herein, the "antibody light chain" or "heavy chain antibody" refers to a polypeptide comprising the V _H or V _L, respectively. The endogenous V _L is encoded by gene segments V (variable regions) and J (joining regions), and the endogenous V _H is encoded by V, D (diverse regions) and J. V _L or V _H each include a CDR and a framework region. In the present application, antibody light chains and/or antibody heavy chains may sometimes be referred to collectively as "antibody chains." As those skilled in the art will readily recognize, such term encompasses the mutation does not destroy the basic structure comprising a V _H or V _L chain of antibody.

As used herein, the term "valency" refers to the number of potential target binding sites in a polypeptide. Each target binding site specifically binds to a target molecule or a specific site on the target molecule. Where the polypeptide comprises more than one target binding site, each of the target binding sites can specifically bind to the same or different molecules (eg, can bind to different molecules, eg, different antigens, or different epitopes on the same molecule). Conventional antibodies have, for example, two binding sites and are bivalent. The antibody, antigen binding molecule and fragments thereof can be monovalent (ie, bound to a target molecule), bivalent or multivalent (ie, bound to more than one target molecule).

For the preparation of single or multiple antibodies, any technique known in the art can be used (see, for example, Kohler and Milstein, Nature 256:495-497 (1975); Kozbor et al, Immunology Today 4:72 (1983); Cole Et al., Monoclonal Antibodies and Cancer Therapy, pp. 77-96. Alan R. Liss, Inc. 1985). Techniques for making single-chain antibodies (U.S. Patent No. 4,946,778) may be suitable for the preparation of antibodies to the polypeptides of the invention. In addition, transgenic mice or other organisms, such as other mammals, can be used to express primatized or humanized antibodies. Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to a selected antigen (see, eg, McCafferty et al, supra; Marks et al, Biotechnology , 10:779-783, (1992)).

Methods for primatizing or humanizing non-human antibodies are well known in the art. Generally, primatized or humanized antibodies have one or more amino acid residues introduced therein from a source other than a non-primate or non-human. Such non-primate or non-human amino acid residues are often referred to as introduced residues, which are typically obtained from the introduction of variable domains. Humanization can basically follow the methods of Winter and collaborators (see, for example, Jones et al, Nature 321:522-525 (1986); Riechmann et al, Nature 332:323-327 (1988); Verhoeyen et al, Science 239: 1534-1536 (1988) and Presta, Curr. Op . Struct . Biol . 2:593-596 (1992)) were performed by replacing the corresponding sequences of human antibody sequences with rodent CDR or CDR sequences. Thus, such humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567) in which substantially less than the entire human variable domain has been substituted from the corresponding sequence of a non-human species. In practice, primatized or humanized antibodies are typically primate or human antibodies in which some of the complementarity determining region ("CDR") residues and possibly some framework ("FR") residues are passed through the starting species ( Residues of similar sites in, for example, rodent antibodies) are substituted to confer binding specificity.

The term "chimeric antibody" is an antibody molecule in which (a) a constant region or a portion thereof is altered, substituted or exchanged such that the antigen binding site (variable region) is linked to a class, effector function, and/or species are different or altered. Or a constant region of a completely different molecule, thereby conferring new properties to the chimeric antibody, such as enzymes, toxins, hormones, growth factors and drugs; or (b) the variable region or a portion thereof may have different or altered antigen specificity Variant changes, permutations, or exchanges.

The antibody or antigen binding molecule of the invention further comprises one or more immunoglobulin chains which chemically bind to other proteins or which together with other proteins behave as fusion proteins. It also includes bispecific antibodies. A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Other antigen-binding fragments or antibody portions of the invention include bivalent scFv (bifunctional antibodies), bispecific scFv antibodies (where antibody molecules recognize two different epitopes), single binding domains (dAbs), and minibodies.

The various antibodies or antigen-binding fragments described herein can be modified by enzymatic or chemical means. Whole antibody production is either re-synthesized using recombinant DNA methods (e.g., single-chain Fv) or using phage display library identification (see, e.g., McCafferty et al, Nature 348:552-554, 1990). For example, minibodies can be produced using methods described in the art, such as Vaughan and Sollazzo, Comb Chem High Throughput Screen. 4: 417-30 2001. Bispecific antibodies can be produced by a variety of methods including fusion of fusion tumors or ligation of Fab' fragments. See, for example, Songsivilai and Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al, J. Immunol. 148, 1547-1553 (1992). Single-chain antibodies can be identified using phage display libraries or ribosomal rendering libraries, and gene shuffling libraries. Such libraries can be constructed from synthetic, semi-synthetic or natural and immunologically active sources.

The term "antigen-binding molecule" or "non-antibody ligand" refers to an antibody mimetic using a non-immunoglobulin protein backbone, including but not limited to, fibronectin, high-affinity multimers, single-chain polypeptide-binding molecules, and A peptide mimetic such as an antibody.

The term "variable region" or "V region" interchangeably refers to a heavy or light chain comprising FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The endogenous variable region is encoded by an immunoglobulin heavy chain V-D-J gene or a light chain V-J gene. The V region can be naturally occurring, recombinant or synthetic.

As used herein, the term "variable region" or "V region" refers interchangeably to a subsequence of a variable region, including FR1-CDR1-FR2-CDR2-FR3. The endogenous V region is encoded by the immunoglobulin V gene. The V region can be naturally occurring, recombinant or synthetic.

As used herein, the term "J region" refers to a subsequence of the encoded variable region comprising the C-terminal portion of CDR3 and FR4. The endogenous J region is encoded by the immunoglobulin J gene. The J region can be naturally occurring, recombinant or synthetic.

A "humanized" antibody is an antibody that retains the reactivity (eg, binding specificity, activity) of a non-human antibody while being less immunogenic in humans. It can be obtained, for example, by retaining the non-human CDR regions and replacing the rest of the antibody with a human counterpart. See, for example, Morrison et al, Proc. Natl. Acad. Sci. USA , 81: 6851-6855 (1984); Morrison and Oi, Adv. Immunol. , 44: 65-92 (1988); Verhoeyen et al, Science , 239 : 1534-1536 (1988); Padlan, Molec. Immun. , 28: 489-498 (1991); Padlan, Molec. Immun. , 31(3): 169-217 (1994).

The term "corresponding human germline sequence" refers to a nucleic acid sequence encoding a human variable region amino acid sequence or subsequence as compared to all other known variable regions encoded by human germline immunoglobulin variable region sequences. An amino acid sequence which is identical to the highest amino acid sequence determined by the reference variable region amino acid sequence or subsequence. The corresponding human germline sequence may also refer to a human variable region having the highest amino acid sequence identity to a reference variable region amino acid sequence or subsequence compared to all other variable region amino acid sequences evaluated. Amino acid sequence or subsequence. Corresponding human germline sequences can be individual framework regions, individual complementarity determining regions, framework and complementarity determining regions, variable regions (as defined above), or other combinations of sequences or subsequences comprising the variable regions. Sequence identity can be determined using the methods described herein, for example, using BLAST, ALIGN, or another alignment algorithm known in the art to align two sequences. The corresponding human germline nucleic acid or amino acid sequence can have at least about 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 with the reference variable region nucleic acid or amino acid sequence. % or 100% sequence identity. The corresponding human germline sequence can be, for example, via the publicly available International Immunogenetics Database (IMGT) (on the World Wide Web at imgt.cines.fr/) and V-base (vbase.mrc-cpe on the World Wide Web) Determination on .cam.ac.uk).

The phrase "specifically binds" or "selectively binds" when used in the context of describing the interaction between an antigen (eg, a protein) and an antibody, antibody fragment, or antibody-derived binding agent, refers to a binding reaction that The presence of the antigen in a heterogeneous protein population and other biological agents, such as biological samples, such as blood, serum, plasma or tissue samples, is determined. Thus, under certain specified immunoassay conditions, an antibody or binding agent having a particular binding specificity binds to a particular antigen at least twice as background and does not substantially bind to other antigens present in the sample in significant amounts. In one embodiment, the binding of an antibody or binding agent having a particular binding specificity to a particular antigen under specified immunoassay conditions is At least ten (10) times the background and not substantially bound to other antigens present in the sample. Specific binding to an antibody or binding agent under such conditions may require the antibody or agent to be selected for its specificity for a particular protein, such as human GITR. As used herein, specific binding includes antibody fragments and binding molecules that selectively bind to human GITR, and does not include antibodies that cross-react with, for example, murine GITR molecules or other TNF receptor superfamily members. In some embodiments, an antibody or antibody fragment that cross-reacts with a non-human primate GITR (eg, macaque GITR) is selected.

A variety of immunoassay formats can be used to select antibodies that specifically immunoreact with a particular protein. For example, solid phase ELISA immunoassays are routinely used to select antibodies that specifically immunoreact with proteins (for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity, see, eg, Harlow and Lane, Using Antibodies , A Laboratory Manual (1998)). Typically, the signal produced by a specific or selective binding reaction is at least twice the background signal and more typically at least 10 to 100 times the background signal.

The term "equilibrium dissociation constant (Ko, M" refers to the dissociation rate constant (kd, time ^-1 ) divided by the association rate constant (k _a , time ^-1 , M ^-1 ). Any known in this technique can be used. The method measures equilibrium dissociation constants. The equilibrium dissociation constant of the antibodies of the invention is generally less than about 10 ^-7 or 10 ^-8 M, such as less than about 10 ^-9 M or 10 ^-10 M, and in some embodiments, less than about 10 ^-11. M, 10 ^-12 M or 10 ^-13 M. In some embodiments, the isolated antibody or antibody fragment binds to the human GITR with an equilibrium dissociation constant (K _D ) of about 1 nM or less. In some embodiments, the antibody or antibody fragment binding K _D of less than 1nM human GITR. in some embodiments, an antibody or antibody fragment K _D in the range of about 0.5nM to about 1.0nM of binding to human GITR.

As used herein, the term "antigen binding region" refers to a domain of the GITR binding molecule of the invention that is responsible for the specific binding between a molecule and GITR. The antigen binding region comprises at least one antibody heavy chain variable region and at least one antibody light chain variable region. Each GITR junction of the present invention At least one such antigen binding region is present in the conjugate molecule, and each antigen binding region may be identical or different from each other. In some embodiments, at least one of the antigen binding regions of the GITR binding molecules of the invention acts as an agonist of GITR.

The term "antibody agonist" or "agonist" refers interchangeably to an antibody that is capable of activating a receptor to induce a complete or partial receptor mediated response. For example, an agonist of GITR binds to GITR and induces GITR-mediated intracellular signaling (eg, increased NF-κB expression activation). Antibody agonists stimulate signaling via GITR similar to the natural ligand GITR-L. Binding of GITR-L to GITR induces NFκB activation due to degradation of IκB. In some embodiments, a GITR antibody agonist can be produced by binding to GITR and inducing T cell (eg, CD8 ⁺ CTL or CD4 ⁺ Th cells) proliferation, survival, cytolytic activity, and/or cytokine production (eg, IFNγ, IL) The ability of -10, IL-13, TNFα) is identified or identified as otherwise described herein.

The term "GITR" or "glucocorticoid-induced tumor necrosis factor receptor" or "tumor necrosis factor receptor superfamily member 18" or "TNFRSF18" is used interchangeably to refer to type I transmembrane as a member of the TNF receptor superfamily. protein. GITR is expressed at high levels on CD4 ⁺ CD25 ⁺ and activated effector CD4 ⁺ and CD8 ⁺ T cells. The nucleic acid and amino acid sequence of GITR are known and registered under GenBank accession number NM_004195.2→NP_004186.1 (Isoform 1 precursor; NM_148901.1→NP_683699.1 (Isoform 2 precursor); and NM_148902 .1→NP_683700.1 (the isoform 3 precursor) is disclosed. See also GenBank registration number NM_005092→NP_005083.2. Structurally, the GITR amino acid sequence is a type I transmembrane protein that is a member of the TNF receptor superfamily and has a signal peptide, an extracellular domain (ECD) comprising three cysteine-rich domains (CRD). And the full length thereof has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of Genbank accession number NP_004186.1, NP_683699.1, NP_005083.2 or NP_005083.2. , 97%, 98%, 99% or 100% sequence identity. Structurally, the full length of the GITR nucleic acid sequence has at least about 90%, 91%, 92%, 93% of the nucleic acid sequence of Genbank Accession Nos. NM_004195.2, NM_148901.1, NM_148902.1, NM_005092 or SEQ ID NO: 1-4. , 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. Functionally, the agonist effect of rodent GITR inhibits, at least transiently, the activity of CD25 ⁺ regulatory T cells (Treg). GITR agonism further enhances immune activity, such as proliferation, survival, cytokine production, and cytolytic activity of activated effector CD4 ⁺ and CD8 ⁺ T cells. See, for example, Nocentini et al, Eur J Immunol (2007) 37: 1165-1169; Expert Opin Ther Patents (2007) 17(5): 567-757; Shevach and Stephens, Nature Reviews Immunology (2006) 6: 613-618.

"Activity" of a polypeptide of the invention refers to the structural, regulatory or biochemical function of the polypeptide in its natural cells or tissues. Examples of polypeptide activity include direct activity and indirect activity. Exemplary activities of GITR agonism include intracellular signaling, which results in increased NF-κB activation, increased proliferation, survival, and cytokine production by activated effector CD4 ⁺ and CD8 ⁺ T cells (eg, IFNγ, IL-10) , IL-13, TNFα) and cytolytic activity. In therapeutic terms, the agonistic effect of GITR enhances anti-tumor and anti-viral T cell responses in vivo.

The term "isolated" when used in reference to a nucleic acid or protein means that the nucleic acid or protein is substantially free of other cellular components to which it is bound in its native state. It is preferably in a homogeneous state. It can be in the form of a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The protein which is the main substance present in the preparation is substantially purified. In particular, the isolated gene is isolated from an open reading frame flanked by the gene and encoding a protein other than the gene of interest. The term "purified" means that the nucleic acid or protein substantially produces a band in the electrophoresis gel. In particular, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure and optimally at least 99% pure.

The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (eg, degenerate codon substitutions), dual genes, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. In particular, degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is substituted with a mixed base and/or a deoxyinosine residue. (Batzer et al, Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al, J. Biol. Chem. 260: 2605-2608 (1985); and Rossolini et al, Mol. Cell. Probes 8: 91-98 (1994)).

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of an amino acid residue. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring Amino acid polymer.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are amino acids encoded by the genetic code, as well as late modified amino acids such as hydroxyproline, gamma-carboxy glutamic acid and O-phosphoric acid. An amino acid analog refers to a compound having the same basic chemical structure as the naturally occurring amino acid (ie, the alpha carbon is bonded to a hydrogen, a carboxyl group, an amine group, and an R group), such as homoserine, orthraenic acid, Amidoxime and methyl methionine. Such analogs have a modified R group (eg, orthanoic acid) or a modified peptide backbone, but retain the same basic chemical structure as the naturally occurring amino acid. Amino acid mimetic refers to a compound having a structure different from the general chemical structure of an amino acid, but functioning in a manner similar to a naturally occurring amino acid.

"Conservatively modified variants" apply to amino acids and nucleic acid sequences. With respect to a particular nucleic acid sequence, a conservatively modified variant system refers to those nucleic acids encoding a consensus or substantially identical amino acid sequence, or wherein the nucleic acid does not encode an amino acid sequence in accordance with a substantially identical sequence. Due to the degeneracy of the genetic code, multiple functionally identical nucleic acids encode any given egg White matter. For example, the codons GCA, GCC, GCG, and GCU all encode amino acid alanine. Thus, at each position where alanine is designated by a codon, the codon can become any of the corresponding codons without altering the encoded polypeptide. Such nucleic acid variations are "silent variations", which are a conservatively modified variation. Each of the nucleic acid sequences encoding a polypeptide herein also describes every possible silent variation of the nucleic acid. Those skilled in the art will recognize that each codon in a nucleic acid (except for the AUG, which is typically the only codon for methionine and the TGG, which is typically the only codon for tryptophan), can be modified to produce functionally identical The molecule. Thus, each silent variation of a nucleic acid encoding a polypeptide is implicit in each of said sequences.

With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or deletions of nucleic acid, peptide, polypeptide or protein sequences that alter, add or delete a single amino acid or a minor percentage of amino acid in the coding sequence are recognized. It is added as a "conservatively modified variant" in which the amino acid is substituted with a chemically similar amino acid. Conservative substitutions that provide functionally similar amino acids are well known in the art. Such conservatively modified variants additionally and do not exclude polymorphic variants, interspecies homologs and dual genes of the invention.

The following eight groups each contain a conservatively substituted amino acid: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) winter Indoleamine N), glutamic acid (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), proline (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); 8) Cysteine (C), methionine (M) (see for example Creighton, Proteins (1984)).

"Sequence Consistency Percent" is determined by comparing the sequences of the two optimal alignments in a comparison window, wherein the portion of the polynucleotide sequence in the comparison window is compared to the reference sequence used to optimally align the two sequences ( It does not contain additions or deletions (e.g., polypeptides of the invention) may include additions or deletions (i.e., voids). Calculate the percentage by determining the number of positions of identical nucleic acid bases or amino acid residues present in the two sequences, obtaining the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window and multiplying the result At 100, get the sequence consistency The ratio.

In the case of two or more nucleic acid or polypeptide sequences, the term "consistent" or "consistency" means that two or more sequences or subsequences are the same sequence. If one of the following sequence comparison algorithms is used or by manual alignment and visual inspection, when comparing and aligning in a comparison window or a specified area to obtain maximum consistency, if the two sequences have a specified percentage The same amino acid residue or nucleotide (ie, at a specified region, or at least when specified, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity), then the two sequences are "substantially consistent." The invention provides a polypeptide or polynucleotide, respectively, as exemplified herein (eg, the variable region exemplified in any one of SEQ ID NOs: 6-10, 12, 14, 59, and 61; SEQ ID NO: 16 The variable segment exemplified in any one of -17; the CDRs exemplified in any one of SEQ ID NOs: 22-34; exemplified in any one of SEQ ID NOs: 35-50 The FR; and the nucleic acid sequence exemplified in any one of SEQ ID NOS: 51-58 and 60) are substantially identical polypeptides or polynucleotides. Depending on the case, the identity is present over a region of at least about 15, 25 or 50 nucleotides in length, or more preferably in the region of 100 to 500 or 1000 or more nucleotides, or a reference sequence. Full length. With respect to amino acid sequences, uniformity or substantial identity may exist in at least 5, 10, 15 or 20 amino acids in length, optionally at least about 25, 30, 35, 40, 50, 75 or 100 in length. The amino acid, optionally in the region of at least about 150, 200 or 250 amino acids, or the entire length of the reference sequence. With respect to shorter amino acid sequences, such as amino acid sequences having 20 or fewer amino acids, substantial identity is conservatively substituted for one or two amino acid residues according to conservative substitutions as defined herein. It exists.

For sequence comparison, one sequence is typically used as a reference sequence for comparison to a test sequence. When using the sequence comparison algorithm, the test sequence and the reference sequence are entered into the computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are specified. Preset program parameters can be used, or alternate parameters can be specified. Based on program parameters, subsequent sequence comparison algorithm calculation The percent sequence identity of the trial sequence relative to the reference sequence.

As used herein, "comparison window" includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of 20 to 600, typically about 50 to about 200, and more typically about 100 to about 150, wherein After optimal alignment of the two sequences, the sequence can be compared to a reference sequence of the same number of adjacent positions. Sequence alignment methods for comparison are well known in the art. Optimal sequence alignment can be performed by comparison, for example, Smith and Waterman (1970) Adv. Appl. Math. 2: 482c local homology algorithm, Needleman and Wunsch (1970) J. Mol. Biol. 48 : 443 homologous alignment algorithm, Pearson and Lipman (1988) Proc. Nat'l . Acad. Sci. USA 85: 2444 similarity method search, computerized implementation of these algorithms (Wisconsin Genetics software suite, Genetics Computer Group, 575 Science Dr., GAP, BESTFIT, FASTA, and TFASTA in Madison, WI, or manual alignment and visual inspection (see, for example, Ausubel et al, Current Protocols in Molecular Biology (1995 Supplement)).

Two examples of algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402 and Altschul et al, respectively. (1990) J. Mol. Biol. 215: 403-410. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. The algorithm includes first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy certain positive values when aligned with words of the same length in the database sequence. Threshold score T. T is called the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating the search to find longer HSPs containing them. The word hits extend in two directions along each sequence as long as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty for mismatched residues; always < 0). For the amino acid sequence, the cumulative score is calculated using a scoring matrix. The extension of the word hit in all directions is interrupted when the cumulative comparison score is reduced from the maximum value X reached by it; the cumulative score becomes 0 or less due to the accumulation of one or more negative score residues. Or reach the end of either sequence. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses the following as a preset value: the word length (W) is 11, the expected value (E) is 10, M = 5, N = -4 and the two stocks are compared. For amino acid sequences, the BLASTP program uses the following as a preset: word length 3 and expected value (E) 10, and BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89 :10915) The alignment (B) is 50, the expected value (E) is 10, M=5, N=-4, and the two stocks are compared.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). A similarity measure obtained by the BLAST algorithm is the minimum total probability (P(N)), which provides an indication of the probability of random matching between two nucleotide or amino acid sequences. For example, a nucleic acid is considered to be similar to a reference sequence if the minimum overall probability of the test nucleic acid when compared to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

As described below, two nucleic acid sequences or polypeptides are substantially identical in that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the antibody raised against the polypeptide encoded by the second nucleic acid. Thus, a polypeptide will generally be substantially identical to a second polypeptide, for example, where two peptides differ only by conservative substitutions. As indicated below, another indication that two nucleic acid sequences are substantially identical is that two molecules or their complements hybridize to each other under stringent conditions. Another indication that two nucleic acid sequences are substantially identical is that the same primer can be used to amplify the sequence.

The term "ligation", when used to describe how an antigen binding region is ligated within a GITR binding molecule of the invention, encompasses all possible ways of physically joining the regions. Many antigen binding regions are frequently joined by chemical bonds (such as covalent bonds (such as peptide bonds or disulfide bonds) or non-covalent bonds), which can be direct bonds (ie, no connection between the two antigen binding regions) (a) or an indirect bond (ie, by means of at least one linker molecule between two or more antigen-binding regions).

The terms "subject/individual" and "patient" are used interchangeably to refer to a mammal, such as a human or non-human primate mammal. Mammals can also be laboratory mammals such as mice, rats, rabbits, hamsters. In some embodiments, the mammal can be an agricultural mammal (eg, a horse, a sheep, a cow, a pig, a camel) or a domesticated mammal (eg, a dog, a cat).

As used herein, the term "treating" any disease or condition, in one embodiment, refers to ameliorating a disease or condition (ie, slowing or suppressing or reducing the progression of a disease or at least one of its clinical symptoms). In another embodiment, "treating" refers to alleviating or ameliorating at least one physical parameter, including physical parameters that the patient may not recognize. In another embodiment, "treating" refers to modulating a disease or condition on the body (eg, a stable discernible symptom), physiologically (eg, stabilizing a body parameter), or both. In another embodiment, "treating" refers to preventing or delaying the onset or progression or progression of a disease or condition.

The terms "therapeutically acceptable amount" or "therapeutically effective dose" are used interchangeably to mean achieving a desired result (ie, reducing tumor size, inhibiting tumor growth, preventing cancer metastasis, inhibiting or preventing viral, bacterial, fungal or parasitic infections). The amount. In some embodiments, a therapeutically acceptable amount does not induce or cause adverse side effects. A therapeutically acceptable amount can be determined by first administering a low dose followed by incrementally increasing the dose until the desired effect is achieved. The "prophylactically effective dose" and the "therapeutically effective dose" of the GITR agonist antibody of the present invention can prevent or reduce the severity of the symptoms of the disease, including symptoms associated with cancer or infectious diseases, respectively.

The term "co-administered" refers to the simultaneous presence of two active agents in the blood of an individual. The co-administered active agents can be delivered simultaneously or sequentially.

As used herein, the phrase "consisting essentially of" means the genus or species of active agent included in the method or composition, and the intended purpose for the method or composition. Any inactive carrier or excipient. In some embodiments, the phrase "consisting essentially of" specifically excludes one or more active agents other than the agonistic anti-GITR antibodies of the invention. In some embodiments, the phrase "consisting essentially of" specifically excludes one or more active agents other than the agonistic anti-GITR antibodies of the invention and the second co-administered agent.

The term "cancer-associated antigen" or "tumor-associated antigen" or "tumor-specific marker" or "tumor marker" is used interchangeably to refer to a molecule that appears on the surface of cancer cells as compared to normal cells (usually protein, carbon water). A compound or lipid), and which is suitable for use in pharmacological agents to preferentially target cancer cells. Frequently, cancer-associated antigens are cell surface molecules that are overexpressed in cancer cells compared to normal cells, such as 1x overexpression, 2x overexpression, 3x or more overexpression than normal cells. Often, cancer-associated antigens are cell surface molecules that are inappropriately synthesized in cancer cells, such as molecules that contain deletions, additions, or mutations compared to molecules expressed on normal cells. Often, cancer-associated antigens are only expressed on the cell surface of cancer cells and are not synthesized or expressed on the surface of normal cells. Exemplary cell surface tumor markers include the protein c-erbB-2 and human epidermal growth factor receptor (HER) (breast cancer), PSMA (prostate cancer), and carbohydrate mucin (multiple cancers (including breast, ovarian, and colorectal) Cancer)).

As used herein, the terms "first," "second," "third," and "fourth" with respect to an antigen-binding portion (eg, Fab) are used to facilitate discrimination when more than one portion is present. The use of such terms is not intended to confer a particular order or orientation of the antibody unless otherwise stated.

Unless the context clearly indicates otherwise, the terms "a" and "the" include plural referents.

Agonistic anti-GITR antibody

The present invention provides antibodies, antibody fragments and antigen-binding molecules which bind to GITR and stimulate signaling via GITR and/or induce enhanced immunity in vivo. reaction. Antibodies, antibody fragments and antigen-binding molecules can be used to increase CD4+ T helper cells (Th) and/or CD8+ cell-soluble T lymphocyte (CTL) responses against target antigens, and can also be used for the treatment of the following diseases, the development of which can be Reversal or inhibition by an effective immune response, including cancer and infectious diseases.

The antibodies, antibody fragments and antigen-binding molecules of the invention exhibit suitable properties for use in human patients, for example, they have a low risk of immunogenicity for use in humans (except for binding specificity determining regions (BSD), especially at least CDR3 In addition, it is encoded by the human germline nucleic acid sequence; has high affinity for GITR (eg, K _D is at least less than 1 nM); does not cross-react with other members of the TNFR superfamily; cross-reacts with human and non-human primate GITR And promote GITR signaling at low doses (eg, at concentrations of less than 3 nM in an in vitro assay). Other activities and characteristics are also exhibited throughout the specification.

Accordingly, the present invention provides antibodies, antibody fragments and antigen-binding molecules that are agonists of GITR. The anti-GITR antibody, antibody fragment or antigen-binding molecule provided contains minimal binding sequences in the CDR3 of the heavy and light chains derived from the starting or reference monoclonal antibodies (eg, the antibodies described in Tables 1 and 2 below) Determinator (BSD). The remaining sequences (CDRs and FRs) of the heavy and light chain variable regions (e.g., V and J regions) are derived from the corresponding human germline and affinity mature amino acid sequences. The V region can be selected from a human V region library. Further sequence modification can be accomplished by affinity maturation or other methods known in the art to optimize binding activity or activity of an antibody, antibody fragment or antigen binding molecule of the invention.

In another embodiment, the heavy and light chain or antibody fragment of an anti-GITR antibody comprises, for example, a human V region (FR1-CDR1-FR2-CDR2-FR3) from a corresponding human germline sequence selected from a human V region library, And CDR3-FR4 sequence segments from the original monoclonal antibodies (eg, the antibodies described in Tables 1 and 2). The CDR3-FR4 sequence segments can be further improved by replacing sequence segments with corresponding human germline sequences and/or by affinity maturation. For example, the FR4 and/or CDR3 sequences surrounding the BSD can be replaced with corresponding human germline sequences while retaining the BSD from the CDR3 of the original monoclonal antibody.

In some embodiments, the corresponding human germline sequence of the heavy chain V region is VH3 3-13/30: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIRYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK (SEQ ID NO: 89). In one embodiment, the last amino acid of SEQ ID NO: 89 is substituted with arginine ("R"). In some embodiments, the corresponding human germline sequence of the heavy chain J region is JH4. In some embodiments, the heavy chain J region comprises the human germline JH4 partial sequence WGQGTLVTVSS (SEQ ID NO: 90). The full-length J region from human germline JH4 is YFDYWGQGTLVTVSS (SEQ ID NO: 91). The variable region genes are referred to according to the standard nomenclature of immunoglobulin variable region genes. Current immunoglobulin gene information is available on the World Wide Web, such as the ImMunoGeneTics (IMGT), V-base, and PubMed databases. See also Lefranc, Exp Clin Immunogenet. 2001; 18(2): 100-16; Lefranc, Exp Clin Immunogenet. 2001; 18(3): 161-74; Exp Clin Immunogenet. 2001; 18(4): 242-54 And Giudicelli et al., Nucleic Acids Res. January 1, 2005; 33 (Database Special): D256-61.

In some embodiments, the corresponding human germline sequence of the light chain V region is VKIII L16/A27: EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSP (SEQ ID NO: 92). In some embodiments, the corresponding human germline sequence of the light chain J region is JK2. In some embodiments, the light chain J region comprises the human germline Jk2 partial sequence FGQGTKLEIK (SEQ ID NO: 93). The full-length J region from human germline Jk2 is YTFGQGTKLEIK (SEQ ID NO: 94).

In some embodiments, the heavy chain V region has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence: (E/Q)VQLVESGGGLVQ(P/S) GGSLRLSCAASGFSLSSYGVDWVRQAPGKGLEW(L/V)GVIWGGGGTYY(A/T)(A/S)S(L/V)M(A/G)RFTISRDNSKNTLYLQMNSLRAEDTAVYYCA(K/R)(H/N)AYGHDGGFAMDYWGQ GTLVTVSS (SEQ ID NO: 16).

In some embodiments, the light chain V region has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence: EIVMTQSPATLSVSPGERATLSCRAS(E/Q)SVSSN(L/V AWYQQ(K/R)PGQAPRLLIYGASNRATGIP(D/A)RFSGSGSGTDFTLTISRLEPEDFAVYYCGQSYSYPFTFGQGTKLEIK (SEQ ID NO: 17).

In some embodiments, i) the heavy chain CDR3 comprises the amino acid sequence HAYGHDGGFAMDY (SEQ ID NO: 29) or NAYGHDGGFAMDY (SEQ ID NO: 109); and ii) the light chain CDR3 variable region comprises the amino acid sequence GQSYSYPFT ( SEQ ID NO: 34) or SYSYPF (SEQ ID NO: 83).

In some embodiments, an antibody or antibody fragment of the invention comprises a heavy chain variable region comprising CDR1 comprising the amino acid sequence SYGVD (SEQ ID NO: 22) or GFSLSSY (SEQ ID NO: 84); comprising an amine Acidic sequence VIWGGGGTYY(A/T)(A/S)S(L/V)M(A/G)(SEQ ID NO:28) or WG2 of WGGGG (SEQ ID NO:80); and HAYGHDGGFAMDY (SEQ ID NO: 29) or CDR3 of the amino acid sequence of NAYGHDGGFAMDY (SEQ ID NO: 109).

In some embodiments, an antibody or antibody fragment of the invention comprises a light chain variable region comprising an amino acid sequence RAS(E/Q)SVSSN(L/V)A(SEQ ID NO:32) or S (E/Q) CDR1 of SVSSN (SEQ ID NO: 87); CDR2 comprising amino acid sequence GASNRAT (SEQ ID NO: 33) or GAS (SEQ ID NO: 82); and GQSYSYPFT (SEQ ID NO: 34) Or CDR3 of the amino acid sequence of SYSYPF (SEQ ID NO: 83).

In some embodiments, an antibody or antibody fragment of the invention comprises a heavy chain variable region comprising CDR1 comprising the amino acid sequence SYGVD (SEQ ID NO: 22) or GFSLSSY (SEQ ID NO: 84); comprising an amine Acid sequence VIWGGGGTYY(A/T)(A/S)S(L/V)M(A/G)(SEQ ID NO:28) or WGGGG CDR2 of (SEQ ID NO: 80); and CDR3 comprising the amino acid sequence of HAYGHDGGFAMDY (SEQ ID NO: 29) or NAYGHDGGFAMDY (SEQ ID NO: 109). Such antibodies or antibody fragments of the invention further comprise a light chain variable region comprising an amino acid sequence comprising RAS(E/Q)SVSSN(L/V)A (SEQ ID NO: 32) or S (E/ Q) CDR1 of SVSSN (SEQ ID NO: 87); CDR2 comprising GASNRAT (SEQ ID NO: 33) or GAS (SEQ ID NO: 82); and comprising GQSYSYPFT (SEQ ID NO: 34) or SYSYPF (SEQ ID NO) : 83) The CDR3 of the amino acid sequence.

In some embodiments, an antibody or antibody fragment of the invention comprises a heavy chain variable region comprising a CDR1 comprising the amino acid sequence SYGVD (SEQ ID NO: 22) or GFSLRSY (SEQ ID NO: 79); comprising an amine CDR2 of the base acid sequence VIWGGGGTNYNSALMA (SEQ ID NO: 62) or WGGGG (SEQ ID NO: 80); and CDR3 comprising the amino acid sequence of HAYGHDGGFAMDY (SEQ ID NO: 29) or NAYGHDGGFAMDY (SEQ ID NO: 109). In some embodiments, the antibody or antibody fragment is humanized.

In some embodiments, an antibody or antibody fragment of the invention comprises a light chain variable region comprising CDR1 comprising the amino acid sequence KASENVDTFVS (SEQ ID NO: 63) or SENNDTF (SEQ ID NO: 81); comprising an amine CDR2 of the base acid sequence GASNRYT (SEQ ID NO: 64) or GAS (SEQ ID NO: 82); and CDR3 comprising the amino acid sequence of GQSYSYPFT (SEQ ID NO: 34) or SYSYPF (SEQ ID NO: 83). In some embodiments, the antibody or antibody fragment is humanized.

In some embodiments, an antibody or antibody fragment of the invention comprises a heavy chain variable region comprising a CDR1 comprising the amino acid sequence SYGVD (SEQ ID NO: 22) or GFSLRSY (SEQ ID NO: 79); comprising an amine CDR2 of the base acid sequence VIWGGGGTNYNSALMA (SEQ ID NO: 62) or WGGGG (SEQ ID NO: 80); and CDR3 comprising the amino acid sequence of HAYGHDGGFAMDY (SEQ ID NO: 29) or NAYGHDGGFAMDY (SEQ ID NO: 109). Such antibodies or antibody fragments further comprise a light chain a variable region comprising CDR1 comprising the amino acid sequence KASENVDTFVS (SEQ ID NO: 63) or SEINRDTF (SEQ ID NO: 81); comprising an amino acid sequence GASNRYT (SEQ ID NO: 64) or GAS (SEQ ID) CDR2 of NO: 82); and CDR3 comprising the amino acid sequence of GQSYSYPFT (SEQ ID NO: 34) or SYSYPF (SEQ ID NO: 83). In some embodiments, the antibody or antibody fragment is humanized.

In some embodiments, the heavy chain variable region comprises FR1 comprising the amino acid sequence of SEQ ID NO: 37; FR2 comprising the amino acid sequence of SEQ ID NO: 40; and the amino group comprising SEQ ID NO: 41 FR3 of the acid sequence; and FR4 comprising the amino acid sequence of SEQ ID NO:42. In some embodiments, the heavy chain variable region comprises FR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 35 and SEQ ID NO: 36; comprising a member selected from the group consisting of SEQ ID NO: 38 and SEQ ID NO: 39 FR2 of the amino acid sequence; FR3 comprising the amino acid sequence of SEQ ID NO: 41; and FR4 comprising the amino acid sequence of SEQ ID NO: 42. The identified amino acid sequence may have one or more substituted amino acids (e.g., from affinity maturation) or one or two conservatively substituted amino acids.

In some embodiments, the light chain variable region comprises FR1 comprising the amino acid sequence of SEQ ID NO: 43; FR2 comprising the amino acid sequence of SEQ ID NO: 46; and the amino group comprising SEQ ID NO: 49 FR3 of the acid sequence; and FR4 comprising the amino acid sequence of SEQ ID NO:50. In some embodiments, the light chain variable region comprises FR1 comprising the amino acid sequence of SEQ ID NO: 43; FR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 44 and SEQ ID NO: 45; FR3 selected from the amino acid sequences of SEQ ID NO: 47 and SEQ ID NO: 48; and FR4 comprising the amino acid sequence of SEQ ID NO: 50. The identified amino acid sequence may have one or more substituted amino acids (e.g., from affinity maturation) or one or two conservatively substituted amino acids.

The variable region of an anti-GITR antibody of the invention generally has at least about 85% (e.g., at least about 85%, 89%, 90%, 91%, 92%) over its entire length and corresponding human germline variable region amino acid sequence. , 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) A variable region (eg, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4) amino acid sequence identity. For example, the heavy chain of the anti-GITR antibody can be associated with the human germline variable region EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIRYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK-YFDYWGQGTLVTVSS (SEQ ID NO: 89 and 91) (VH3 3-13/30+CDR3+JH4, the hyphen indicates CDR3, the length can be Is variable) having at least about 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence consistency. In one embodiment, the last amino acid of SEQ ID NO: 89 is substituted with arginine (R) from the amino acid (K). The light chain of the anti-GITR antibody can be associated with the human germline variable region EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC-YTFGQGTKLEIK (SEQ ID NO: 98 and 94) (VKIII L16/A27+CDR3+JK2; the hyphen indicates that the CDR3 can be variable in length) At least about 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity. In some embodiments, only the amino acid in the framework region is added, deleted or substituted. In some embodiments, sequence identity comparisons exclude CDR3.

The CDRs of the antibodies listed in Table 1 can be determined by well-known numbering systems known in the art, including the numbering systems described herein. Table 2 lists the CDRs defined by the following methods: (1) using Kabat et al., (1991), "Sequences of Proteins of Immunological Interest," 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD ( "Kabat" numbering scheme), numbering system described in NIH Publication No. 91-3242; and (2) Chothia, see Al-Lazikani et al., (1997) "Standard conformations for the canonical structures of immunoglobulins," J. Mol .Biol. 273: 927-948.

In some embodiments, the anti-GITR antibody or antibody fragment of the invention, which binds to SEQ ID NO: 4, is selected from any of the following: i) an antibody, antibody fragment or antigen binding molecule, wherein: heavy chain CDR1 Included in SEQ ID NO: 22, heavy chain CDR2 comprises SEQ ID NO: 23, heavy chain CDR3 comprises SEQ ID NO: 29, light chain CDR1 comprises SEQ ID NO: 30, light chain CDR2 comprises SEQ ID NO: 33, and light chain CDR3 contains SEQ ID NO: 34; ii) an antibody, antibody fragment or antigen-binding molecule, wherein: heavy chain CDR1 comprises SEQ ID NO: 22, heavy chain CDR2 comprises SEQ ID NO: 24, heavy chain CDR3 comprises SEQ ID NO: 29, light chain CDR1 comprises SEQ ID NO: 31, light chain CDR2 comprises SEQ ID NO: 33, and light chain CDR3 comprises SEQ ID NO: 34; iii) an antibody, antibody fragment or antigen binding molecule, wherein: heavy chain CDR1 comprises SEQ ID NO :22, the heavy chain CDR2 comprises SEQ ID NO:25, the heavy chain CDR3 comprises SEQ ID NO:29, the light chain CDR1 comprises SEQ ID NO:30, the light chain CDR2 comprises SEQ ID NO:33, and the light chain CDR3 comprises SEQ ID NO: 34; iv) an antibody, antibody fragment or antigen-binding molecule, wherein: heavy chain CDR1 comprises SEQ ID NO: 22, heavy chain CDR2 comprises SEQ ID NO: 26, and heavy chain CDR3 comprises SEQ ID NO: 29, light chain CDR1 comprises SEQ ID NO:30, light chain CDR2 comprises SEQ ID NO:33, and light chain CDR3 comprises SEQ ID NO:34; v) an antibody, antibody fragment or antigen binding molecule, wherein: heavy chain CDR1 comprises SEQ ID NO :22, the heavy chain CDR2 comprises SEQ ID NO:27, the heavy chain CDR3 comprises SEQ ID NO:29, and the light chain CDR1 comprises SEQ ID NO:30, light The chain CDR2 comprises SEQ ID NO: 33 and the light chain CDR3 comprises SEQ ID NO: 34; and vi) an antibody, antibody fragment or antigen binding molecule, wherein: the heavy chain CDR1 comprises SEQ ID NO: 22 and the heavy chain CDR2 comprises SEQ ID NO: 25, heavy chain CDR3 comprises SEQ ID NO: 109, light chain CDR1 comprises SEQ ID NO:30, light chain CDR2 comprises SEQ ID NO:33, and light chain CDR3 comprises SEQ ID NO:34. In some embodiments, the antibody or antibody fragment is humanized. In a specific embodiment, the antibody or antibody fragment comprises a human constant region. In some embodiments, the antibody or antibody fragment comprises an IgG Fc region. In certain embodiments, the antibody or antigen-binding fragment is glycosylated. In some embodiments, the antibody or antibody fragment is modified or expressed in a modified cell, wherein such modification increases the FcR effector function of the antibody or antibody fragment. In certain embodiments, the antibody or antigenic fragment induces an increased Teff; Treg ratio in vivo. In some embodiments, the antibody or antibody fragment induces an enhanced immune response in vivo. In some embodiments, when the antibody or antibody fragment is cross-linked with a second antibody or antibody fragment, it is SEQ ID NO: 1, SEQ ID NO: 2 or an agonist of SEQ ID NO: 3.

In some embodiments, an anti-GITR antibody or antibody fragment of the invention comprises at least 95%, 96%, 97%, 98%, 99% or 100% amino acid with the heavy chain variable region of SEQ ID NO: Sequence-equilibrium heavy chain variable region, and comprising at least 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the light chain variable region of SEQ ID NO:17 Light chain variable region.

In some embodiments, an anti-GITR antibody or antibody fragment of the invention comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the heavy chain variable region of SEQ ID NO: a heavy chain variable region of 97%, 98%, 99% or 100% amino acid sequence identity, and comprising at least 90%, 91%, 92% of the light chain variable region of SEQ ID NO: Light chain variable region with 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity.

In some embodiments, an anti-GITR antibody or antibody fragment of the invention comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the heavy chain variable region of SEQ ID NO:8 a heavy chain variable region of 97%, 98%, 99% or 100% amino acid sequence identity, and comprising at least 90%, 91%, 92% of the light chain variable region of SEQ ID NO: Light chain variable region with 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity.

In some embodiments, an anti-GITR antibody or antibody fragment of the invention comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the heavy chain variable region of SEQ ID NO: a heavy chain variable region of 97%, 98%, 99% or 100% amino acid sequence identity, and comprising at least 90%, 91%, 92% of the light chain variable region of SEQ ID NO: Light chain variable region with 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity.

In some embodiments, an anti-GITR antibody or antibody fragment of the invention comprises at least 90%, 91%, 92%, 93%, 94% of the heavy chain variable region of SEQ ID NO: a heavy chain variable region of 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity and comprising at least 90%, 91 with the light chain variable region of SEQ ID NO: 7. Light chain variable regions of %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity.

In some embodiments, an anti-GITR antibody or antibody fragment of the invention comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the heavy chain variable region of SEQ ID NO: a heavy chain polypeptide of 97%, 98%, 99% or 100% amino acid sequence identity and comprising at least 90%, 91%, 92%, 93% of the light chain variable region of SEQ ID NO: 7. , 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence consistent light chain polypeptide.

In some embodiments, an anti-GITR antibody or antibody fragment of the invention comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the heavy chain variable region of SEQ ID NO:99 a heavy chain variable region of 97%, 98%, 99% or 100% amino acid sequence identity, and comprising at least 90%, 91%, 92% of the light chain variable region of SEQ ID NO: Light chain variable region with 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity.

In some embodiments, an anti-GITR antibody or antibody fragment of the invention comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the heavy chain variable region of SEQ ID NO:105 a heavy chain variable region of 97%, 98%, 99% or 100% amino acid sequence identity, and comprising at least 90%, 91%, 92% of the light chain variable region of SEQ ID NO: Light chain variable region with 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity.

In some embodiments, an anti-GITR antibody or antibody fragment of the invention comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of the heavy chain variable region of SEQ ID NO:61 a heavy chain polypeptide of 97%, 98%, 99% or 100% amino acid sequence identity and comprising at least 90%, 91% of the light chain variable region of SEQ ID NO: 59, A light chain polypeptide of 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity.

In some embodiments, such antibodies are human or humanized antibodies. The VH, VL, full length light chain and full length heavy chain sequences (amino acid sequences and nucleotide sequences encoding amino acid sequences) can be "mixed and matched" to produce additional antibodies of the invention that bind to GITR. Such "mixed and matched" antibodies that bind to GITR can be tested using binding assays known in the art (e.g., ELISA and other assays described in the Examples section) to confirm activity. When the strands are mixed and matched, a structurally similar VH sequence substitution is applied from a particular VH/VL paired VH sequence. Likewise, full length heavy chain sequences from a particular full length heavy chain/full length light chain pair should be replaced by structurally similar full length heavy chain sequences. Similarly, structurally similar VL sequence substitutions are applied from a particular VH/VL paired VL sequence. Likewise, structurally similar full length light chain sequence substitutions are applied from a full length heavy chain/full length light chain paired full length light chain sequence. Accordingly, in one aspect, the invention provides an isolated monoclonal antibody or antibody fragment having: an amine group comprising a population selected from the group consisting of SEQ ID NOs: 6, 8, 10, 12, 14, 99, and 105 a heavy chain variable region of an acid sequence; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 9; wherein the antibody specifically binds to GITR.

In some embodiments, an anti-GITR antibody or antibody fragment of the invention comprises and is selected from the group consisting of SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ The heavy chain sequence of any one of ID NO: 100 and SEQ ID NO: 106 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% Or a 100% amino acid sequence-equilibrium heavy chain polypeptide; and comprising at least 90%, 91%, 92%, 93%, 94%, 95% of the light chain of SEQ ID NO: 66 or SEQ ID NO: 70 , 96%, 97%, 98%, 99% or 100% amino acid sequence consistent light chain polypeptide. And X. Heavy chain of ID NO: 100 and SEQ ID NO: 106 a polypeptide; and comprising the light chain polypeptide of SEQ ID NO: 66 or SEQ ID NO: 70.

To identify amino acid sequences of less than 20 amino acids in length, one or two conservative amino acid residue substitutions may be tolerated while still retaining the desired specific binding and/or agonistic activity.

The anti-GITR antibodies and antibody fragments of the invention are typically, for example, less than about 10 ^-8 M or 10 ^-9 M or less than about 10 ^-10 M or 10 ^-11 M, and in some embodiments, less than about 10 ^-12 M or The equilibrium dissociation constant (K _D ) of 10 ^-13 M is combined with GITR, including isoform 1, isoform 2 and isoform 3.

Antibody that binds to the same epitope

The present invention provides antibodies and antibody fragments that bind to epitopes within residues 41-65 of human GITR, wherein such antibodies and antibody fragments are agonists of GITR. The present invention also provides antibodies and antibody fragments which bind to the same epitope as the antibody to GITR described in Table 1. Thus, other antibodies and antibody fragments can be identified based on their ability to cross-compete with other antibodies of the invention (e.g., competitively inhibit their binding in a statistically significant manner) in a GITR binding assay. The ability of a test antibody to inhibit binding of an antibody and antibody fragment of the invention to a GITR protein (eg, human GITR) demonstrates that the test antibody can compete with an antibody or antibody fragment that binds to GITR; such antibodies can bind to the GITR protein according to non-limiting theory. An epitope that is identical or related (eg, structurally similar or spatially close) to the antibody or antibody fragment to which it competes. In one embodiment, the antibody that binds to the same epitope on the GITR as the antibody or antibody fragment of the invention is a human or humanized monoclonal antibody. Such human or humanized monoclonal antibodies can be prepared and isolated as described herein.

Engineered and modified antibody

Furthermore, an antibody or antibody fragment of the invention can be prepared using an antibody having one or more of the CDRs and/or VH and/or VL sequences set forth herein (eg, Table 1) as a starting material to engineer the modified An antibody or antibody fragment that has altered properties compared to the starting antibody. An antibody or antibody fragment can be modified by one or two variable regions (ie, VH and/or VL) (eg, within one or more CDR regions and/or one or more framework regions) One or more residues are engineered. Additionally or alternatively, the antibody or antibody fragment can be engineered by modifying residues within the constant region to, for example, alter the effector function of the antibody.

One type of variable zone engineering that can be performed is CDR grafting. The antibody interacts with the target antigen primarily via amino acid residues located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within the CDRs are more diverse between individual antibodies than sequences outside the CDRs. Since the CDR sequences are responsible for most of the antibody-antigen interactions, recombinant antibodies can be expressed that mimic the properties of a particular antibody by constructing a vector comprising CDR sequences from a particular antibody that has been ligated from different traits. The framework sequences of the different antibodies (see, for example, Riechmann, L. et al, 1998 Nature 332: 323-327; Jones, P. et al, 1986 Nature 321: 522-525; Queen, C. et al, 1989 Proc. Natl. Acad., USA 86: 10029-10033; U.S. Patent No. 5,225,539 to Winter, and U.S. Patent No. 5,530,101 to Queen et al.; 5,585,089; 5,693,762 and 6,180,370.

Accordingly, another embodiment of the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a population selected from the group consisting of SEQ ID NOs: 22, 79 and 84, respectively. a CDR1 sequence of an amino acid sequence; a CDR2 sequence having an amino acid sequence selected from the group consisting of SEQ ID NOS: 23, 24, 25, 26, 27, 62 and 80; having a SEQ ID NO: 29, 34 And a CDR3 sequence of the amino acid sequence of the group consisting of 109; and a light chain variable region each comprising an amino acid sequence having a population selected from the group consisting of SEQ ID NOS: 30, 31, 63, 81, 85 and 86 a CDR1 sequence; a CDR2 sequence having an amino acid sequence selected from the group consisting of SEQ ID NOS: 33, 64 and 82; and a CDR3 consisting of an amino acid sequence selected from the group consisting of SEQ ID NOS: 34 and 83 sequence. Thus, such antibodies contain VH and VL CDR sequences of monoclonal antibodies, and may contain different framework sequences from such antibodies. In certain embodiments, the isolated antibody or antibody fragment comprises at least about 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of the corresponding sequence in this paragraph. , 97%, 98%, 99% or 100% amino acid sequence consistent Sexual sequence.

Such framework sequences can be obtained from public DNA databases or published literature, including germline antibody gene sequences. For example, the germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrc-cpe.cam.ac.uk/vbase Obtained) and Kabat, EA et al, 1991 Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, IM et al, 1992 J.fol. Biol. 227:776-798; and Cox, JPL et al, 1994 Eur. J Immunol. 24: 827-836.

Examples of framework sequences for use in the antibodies of the invention are framework sequences that are structurally similar to the framework sequences used in the antibodies of the invention (e.g., the common sequences and/or framework sequences used in the antibodies of the invention). The VH CDR1, 2 and 3 sequences and the VL CDR1, 2 and 3 sequences can be grafted onto framework regions having sequences consistent with sequences found in the germline immunoglobulin genes from which the framework sequences are obtained, or CDR sequences It can be transplanted into a framework region containing one or more mutations compared to the germline sequence. For example, it has been found that, in some cases, mutations in the framework regions are useful for maintaining or enhancing the antigen binding ability of the antibody (see, for example, U.S. Patent No. 5,530,101 to Queen et al; 5,585,089; 5,693,762 And No. 6,180,370).

Another type of variable region modification is to mutate an amino acid residue within the VH and/or VL CDR1, CDR2 and/or CDR3 regions to improve one or more binding properties (eg, affinity) of the antibody of interest, This is called "affinity maturity." Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce mutations, and can be assessed in vitro or in vivo assays as provided herein and in the Examples and/or alternatives or other assays known in the art. The effect on antibody binding or other functional properties of interest. Conservative modifications can be introduced. The mutation can be an amino acid substitution, addition or deletion. Furthermore, typically no more than one, two, three, four or five residues are altered within the CDR regions.

Engineered antibodies or antibody fragments of the invention include antibodies or antibody fragments in which the framework residues within VH and/or VL are modified, for example, to improve the properties of the antibody. Such framework modifications are typically performed to reduce the immunogenicity of the antibody. For example, one method is to "backmutate" one or more framework residues into the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain a framework residue different from the germline sequence from which the antibody is obtained. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibodies are obtained. In order to restore the framework region sequence to its germline configuration, somatic mutations can induce "backmutation" into a germline sequence by, for example, site-directed mutagenesis. The invention is also intended to encompass such "backmutation" antibodies.

Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This method is also referred to as "de-immunization" and is described in further detail in U.S. Patent Publication No. 20030153043 to Carr et al.

Where present, the constant region of an anti-GITR antibody or antibody fragment, if desired, can be of any type or subtype, and can be selected from a species of an individual treated by the methods of the invention (eg, human, non-human primate or Other mammals, such as agricultural mammals (e.g., horses, sheep, cattle, pigs, camels), domesticated mammals (e.g., dogs, cats) or rodents (e.g., rats, mice, hamsters, rabbits). In some embodiments, an anti-GITR antibody is engineered to produce a humanized or Humaneered® antibody. In some embodiments, the constant region is isotyped as an IgG, such as IgGl, IgG2, IgG3, IgG4. In certain embodiments, the constant region of isotype IgG _1.

In addition to or instead of modifications made in the framework or CDR regions, the antibodies or antibody fragments of the invention can be engineered to include modifications in the Fc region, typically to alter one or more of the functional properties of the antibody, such as serum half-life. , complement fixation, Fc receptor binding, and/or antigen-dependent cytotoxicity. Furthermore, an antibody or antibody fragment of the invention may be chemically modified (eg, one or more chemical moieties may be attached to the antibody) or modified to alter its glycosylation for further One or more of the functional properties of the antibody or antibody fragment are altered.

In one embodiment, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is altered, such as increased or decreased. This method is further described in U.S. Patent No. 5,677,425 to Bodmer et al. The number of cysteine residues in the hinge region of CH1 is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody or antibody fragment.

In another embodiment, the Fc hinge region of the antibody is mutated to alter the biological half life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc hinge fragment such that binding of the antibody to Staphylococcyl protein A (SpA) binds to the native Fc hinge domain SpA. Weakened. This method is described in further detail in U.S. Patent No. 6,165,745 to Ward et al.

In another embodiment, the antibody is modified to increase its biological half life. Various methods can be used. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F as described in U.S. Patent No. 6,277,375 to Ward. Alternatively, to extend the biological half-life, the antibody can be altered in the CH1 or CL region to contain a two-loop rescue receptor binding epitope derived from the CH2 domain of the Fc region of IgG, such as US Patent No. 5,869,046 to Presta et al. And described in 6,121,022.

In other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids can be replaced with different amino acid residues such that the antibody has altered affinity for the effector ligand, but retains the antigen binding ability of the parent antibody. An effector-modified effector ligand can be, for example, an Fc receptor (FcR) or a C1 component of complement. This method is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both toW.

In another embodiment, one or more amino acids selected from amino acid residues can be substituted with different amino acid residues such that the antibody has altered C1q binding and/or reduced or eliminated complement dependent Cytotoxicity (CDC). This method is described in further detail in Idusogie et al. U.S. Patent No. 6,194,551.

Antibodies containing such mutations mediate reduced or no antibody-dependent cellular cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). In some embodiments, the amino acid residues L234 and L235 of the IgGl constant region are substituted with Ala234 and Ala235. In some embodiments, the amino acid residue N267 of the IgGl constant region is substituted with Ala267.

In another embodiment, one or more amino acid residues are altered to thereby alter the ability of the antibody to fix complement. This method is further described in PCT Publication WO 94/29351 to Bodmer et al.

In another embodiment, the Fc region is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for the Fc gamma receptor by modifying one or more amino acids. This method is further described in PCT Publication WO 00/42072 to Presta. In addition, binding sites for FcγR1, FcγRII, FcγRIII, and FcRn on human IgG1 have been mapped and variants with improved binding have been described (see Shields, RL et al, 2001 J. Biol. Chen. 276: 6591-6604) .

In another embodiment, the glycosylation of the antibody is modified. For example, a deglycosylated antibody (ie, an antibody lacking glycosylation) can be prepared. Glycosylation can be altered to, for example, increase the affinity of the antibody for the "antigen." Such carbohydrate modification can be accomplished, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made to remove one or more variable region framework glycosylation sites, thereby removing glycosylation at that site. Such deglycosylation increases the affinity of the antibody for the antigen. No. 5,714,350 and 6,350,861 to Co. et al.

Additionally or alternatively, antibodies having altered types of glycosylation can be made, such as low fucosylated antibodies with reduced amounts of trehalose residues or antibodies with increased halved GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC ability of antibodies. Such carbohydrate modifications can be altered by, for example, a glycosylation mechanism The expression of antibodies in the main cells is completed. Cells having altered glycosylation machinery have been described in the art and can be used as host cells for the expression of recombinant antibodies of the invention to produce antibodies with altered glycosylation. For example, EP 1,176,195 to Hang et al. describes a cell line in which the FUT8 gene encoding a trehalyl transferase is functionally disrupted such that the antibodies expressed in such cell lines exhibit low haylation. PCT Publication WO 03/035835 to Presta describes a variant CHO cell line Lecl3 cell which reduces the ability of trehalose to attach to the carbohydrate associated with Asn (297) and also results in low seaweed production of antibodies present in the host cell. Glycosylation (see also Shields, RL et al, 2002 J. Biol. Chem. 277:26733-26740). PCT Publication WO 99/54342 to Umana et al. describes cells engineered to express glycoprotein-modified glycosyltransferases (e.g., β(1,4)-N-ethylglucanilide transferase III (GnTIII)) The strain, such that the antibody exhibited in the engineered cell line exhibits an increased dichotomous GlcNac structure, thereby increasing the ADCC activity of the antibody (see also Umana et al, 1999 Nat. Biotech. 17: 176-180).

The antigen binding domain is transplanted into an alternative framework or backbone

A variety of antibody/immunoglobulin frameworks or backbones can be used as long as the resulting polypeptide comprises at least one binding region that specifically binds to the GITR. Such frameworks or backbones include the five major individual genotypes of human immunoglobulins or fragments thereof, and include immunoglobulins of other animal species, which preferably have a humanized appearance. In this regard, a single heavy chain antibody, such as a single heavy chain antibody identified in camelids, has received much attention. Those skilled in the art will continue to discover and develop novel frameworks, skeletons, and fragments.

In one aspect, the invention relates to the production of non-immunoglobulin-based antibodies using non-immunoglobulin backbones from which the CDRs of the invention can be grafted. Known or future non-immunoglobulin frameworks and backbones can be used as long as they contain a binding region that is specific for the target GITR protein (eg, human and/or macaque GITR). Non-immunoglobulin frameworks or backbones are known to include, but are not limited to, Fibronectin (Compound Therapeutics, Inc., Waltham, MA), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd., Cambridge, MA and Ablynx nv, Zwijnaarde, Belgium), lipocalin (Pieris Proteolab AG, Freising, Germany), small module immunopharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, WA) ), maximal antibody (Avidia, Inc., Mountain View, CA), Protein A (Affibody AG, Sweden), and affilin (γ-crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany).

The fibronectin backbone is based on a fibronectin type III domain (eg, the tenth module of the fibronectin type III (10 Fn3 domain)). The fibronectin type III domain has 7 or 8 beta strands distributed between two beta sheets, which themselves are packed with each other to form the core of the protein, and further contain a loop that connects the beta strands to each other and to the solvent ( Similar to CDR). There are at least three such loops at each edge of the beta sheet sandwich structure, with the edges being the boundaries of the protein perpendicular to the beta strand direction (see US 6,818,418). These fibronectin based scaffolds are not immunoglobulins, however the overall folding is closely related to the folding of the minimal functional antibody fragment (heavy chain variable region) comprising the complete antigen recognition unit of camel and vicugna IgG. Due to this structure, non-immunoglobulin antibodies mimic antigen binding properties similar to antibodies in terms of properties and affinities. Similar to in vivo antibody affinity maturation methods, such scaffolds can be used in in vitro loop randomization and shuffling strategies. These fibronectin-based molecules can be used as backbones in which the loop regions of the molecule can be replaced with the CDRs of the invention using standard methods of selection.

The ankyrin technique is based on the use of a protein in which a repeating module derived from an ankyrin is used as a backbone for carrying a variable region that can be used to bind to different targets. The ankyrin repeat module is a 33 amino acid polypeptide consisting of two antiparallel alpha helices and a beta turn. The combination of variable regions is primarily optimized by the use of ribosome presentation.

High affinity multimers are derived from natural A-containing domain proteins such as LRP-1. These domains are essentially used for protein-protein interactions, and more than 250 proteins in humans are structurally based on the A domain. The high affinity polypolymer system consists of a plurality of different "A domain" monomers (2-10) linked via an amino acid linker. High affinity multimers that bind to the target antigen can It is produced by the methods described in, for example, U.S. Patent Application Publication Nos. 20040175756, No. 20050053973, No. 20050048512, and No. 20060008844.

The affinity antibody affinity ligand is a small, simple protein consisting of a triple helix bundle based on the backbone of one of the IgG binding domains of Protein A. Protein A is a surface protein from the bacterium Staphylococcus aureus. This backbone domain consists of 58 amino acids, 13 of which are randomized to produce a library of affinity antibodies with many ligand variants (see, for example, US 5,831,012). The affinity antibody molecule mimicking antibody has a molecular weight of 6 kDa compared to an antibody having a molecular weight of 150 kDa. Although the affinity antibody molecule has a small size, the binding site of the affinity antibody molecule is similar to the binding site of the antibody.

Anticalins is a product developed by Pieris ProteoLab AG. It is derived from lipocalins (a broad group of small stable proteins that typically involve the physiological transport or storage of chemically sensitive or insoluble compounds). Several natural lipocalins are found in human tissues or body fluids. The protein architecture is reminiscent of immunoglobulins, where the hypervariable loops are located on rigid frameworks. However, lipocalins consist of a single polypeptide chain having 160 to 180 amino acid residues, which is only slightly larger than a single immunoglobulin domain, as compared to an antibody or a recombinant fragment thereof. A set of four rings that make up the binding pocket exhibits significant structural plasticity and accommodates multiple side chains. Binding sites can therefore be reshaped by proprietary methods to recognize different target specific target molecules with high affinity and specificity. After the Pieris Brassicae, a bilin-binding protein (BBP), a protein in the lipocalin family, has been used to develop anti-carrier proteins by mutation induction of the four loops of this group. An example of a patent application describing an anti-carrier protein is found in PCT Publication No. WO 1999/16873.

Afilin molecules are small, non-immunoglobulin proteins that are designed to have specific affinities for proteins and small molecules. The new Affine molecule can be selected very quickly from two libraries, each based on a different human skeleton protein. Afilin molecules do not exhibit any structural homology to immunoglobulin proteins. Currently, two kinds of Affi structure are used, one of which is γ crystal The body (human structural eye lens protein), and the other is the "ubiquitin" superfamily protein. Both human skeletons are extremely small, exhibit high temperature stability and are almost resistant to pH changes and denaturants. This high stability is mainly due to the expansion of the beta sheet structure of the protein. Examples of proteins derived from gamma crystals are described in WO200104144 and examples of "ubiquitin-like" proteins are described in WO2004106368.

Protein epitope haplotype (PEM) is a medium size, circular, peptide-like molecule (MW 1-2 kDa) that mimics the beta hairpin secondary structure of a protein involved in the major secondary structure of protein-protein interactions.

Human or humanized antibody

The invention provides engineered human antibodies that specifically bind to a GITR protein, such as the human GITR. The antigenicity of the human GITR-binding antibody of the present invention is further reduced when administered to a human subject as compared to a chimeric, primatized or humanized antibody.

Human GITR binding antibodies can be produced using methods known in the art. For example, the use of Humaneered ^® technology platform (KaloBios, Sout San Francisco, CA ) will convert non-human antibody for the transformation of the human antibody engineered. U.S. Patent Publication No. 20050008625 describes an in vivo method of replacing a non-human antibody variable region in an antibody with a human variable region while maintaining the same or providing better binding characteristics relative to the non-human antibody. This method relies on epitope-directed replacement of the variable region of a non-human reference antibody with a fully human antibody. The resulting human antibody is generally not structurally related to a reference non-human antibody, but binds to the same epitope on the same antigen as the reference antibody.

The anti-GITR anti-system of the invention is based on engineered human antibodies in which the V region sequence shares substantial amino acid sequence identity with the human germline V region sequence while retaining the specificity and affinity of the reference antibody. See U.S. Patent Publication No. 2005/0255552 and U.S. Patent Publication No. 2006/0134098, each hereby incorporated hereinby reference. The improved method identifies minimal sequence information required to determine antigen binding specificity from a variable region of a reference antibody, and transmits information to a human partial V region gene sequence library to generate A pool of epitopes of the human antibody V region. Microbial-based secretion systems can be used to visualize members of the library as antibody Fab fragments, and community transfer binding assays can be used to screen antigen-binding Fabs such as libraries. See, for example, U.S. Patent Publication No. 2007/0020685. Positive pure lines can be further characterized to identify pure lines with the highest affinity. The resulting engineered human Fab retains the binding specificity of the parent reference anti-GITR antibody, typically having equal or higher affinity for the antigen compared to the parent antibody, and having a high sequence identity compared to the V region of the human germline antibody Sexual V zone.

The minimal binding specificity determinant (BSD) required to generate a pool of epitopes is typically represented by the sequence within the heavy chain CDR3 ("CDRH3") and the sequence within the light chain CDR3 ("CDRL3"). The BSD can comprise part or the entire length of the CDR3. The BSD can comprise contiguous or non-contiguous amino acid residues. In some cases, the epitope pool is constructed from a human V region sequence linked to a unique CDR3-FR4 region from a reference antibody comprising BSD and human germline J region sequences (see U.S. Patent Publication No. 2005/ 0255552). Alternatively, the human V-region library can be generated by a sequential cassette replacement in which only a portion of the antibody V region is initially replaced by a human sequence library. In the case of a residual reference antibody amino acid sequence, the subsequently identified human binding cassettes are then recombined in a second library screen to generate a full human V region (see U.S. Patent Publication No. 2006/0134098). .

In each case, the paired heavy and light chain CDR3 region, CDR3-FR4 region or J region containing a specific determinant from a reference antibody is used to limit binding specificity such that the antigen conjugate obtained from the library retains the reference antibody Epitope specificity. Other mature changes can be introduced into the CDR3 region of each strand during library construction to identify antibodies with optimal binding kinetics. The resulting engineered human antibody has a V region sequence derived from the human germline library, retains a short BSD sequence within the CDR3 region and has a human germline framework 4 (FR4) region.

Camel antibody

Member of the camel and dromedary (Camelus bactrianus and Calelus dromaderius) family members (including members of the New World, such as llama species) Antibody proteins such as Lama pacos, Lama glama, and Lama vicugna have been characterized in terms of size, structural complexity, and antigenicity to human individuals. Certain IgG antibodies from this mammalian family, as found in nature, lack a light chain and are therefore structurally distinct from other animal antibodies with a typical four-chain quaternary structure with two heavy chains and two light chains. See PCT/EP93/02214 (WO 94/04678, published March 3, 1994).

The region in which the camelid antibody is identified as VHH (small single variable domain) can be obtained by genetic engineering to produce a small protein with high affinity for the target, thereby producing a low molecular weight antibody-derived protein, called a camel. Nano antibodies". See U.S. Patent No. 5,759,808, issued June 2, 1998; also to Stijlemans, B. et al., 2004 J Biol Chem 279: 1256-1261; Dumoulin, M. et al., 2003 Nature 424: 783-788; Pleschberger, M. et al., 2003 Bioconjugate Chem 14: 440-448; Cortez-Retamozo, V. et al., 2002 Int J Cancer 89: 456-62; and Lauwereys, M. et al., 1998 EMBO J 17: 3512 3520. A library of engineered camel antibodies and antibody fragments is commercially available, for example, from Ablynx, Ghent, Belgium. Like other antibodies of non-human origin, the amino acid sequence of camelid antibodies can be recombinantly altered to yield sequences that are more closely analogous to human sequences, i.e., nano antibodies can be "humanized." Thereby, the natural low antigenicity of camelid antibodies to humans can be further reduced.

The molecular weight of the camelid nanobody is about one tenth of that of a human IgG molecule, and the physical diameter of the protein is only a few nanometers. One result of the small size is that the camelid antibody binds to an antigenic site that is functionally invisible to the larger antibody protein, ie, the camelid antibody is used as a reagent for detecting antigen (otherwise using classical immunological techniques, antigen Will be hidden), and possible therapeutic agents. Therefore, a further result of the small size is that the camelid nano-antibody can have an inhibitory effect due to binding to a specific site in the groove or narrow gap of the target protein, and thus can be more closely similar to the function of the classical antibody. The ability to function as a classic low molecular weight drug works.

The low molecular weight and compact size further provide Camel Nano antibodies with great thermal stability (stability for extreme pH and proteolytic digestion) and weak antigenicity. Another result is that camelid nano antibodies are susceptible to migration from the circulatory system into tissues and even across the blood brain barrier and can treat conditions affecting neural tissue. Nano-antibodies can further promote drug delivery across the blood-brain barrier. See U.S. Patent Application No. 20040161738, issued Aug. 19, 2004. These features, combined with low antigenicity to humans, indicate great therapeutic potential. In addition, such molecules can be expressed in prokaryotic cells such as E. coli and be expressed as a fusion protein with phage and are functional.

Therefore, a feature of the present invention is a camelid antibody or a nano-antibody having high affinity for GITR. In certain embodiments herein, a camelid antibody or a nanobody is naturally produced in a camelid animal, that is, a camelid animal is produced following immunization with GITR or a peptide fragment thereof using the techniques described herein for other antibodies. Alternatively, the GITR-binding camelid nanobody antibody is engineered, i.e., using GITR as a target, using a panning procedure, for example, from selection of a phage library that exhibits appropriately mutagenated camelid nanobody protein. The engineered nano-antibody can be further customized by genetic engineering to have a half-life of 45 minutes to 2 weeks in the recipient individual. In a specific embodiment, the camelid antibody or the nano-antibody system is obtained by grafting the CDR sequences of the heavy or light chain of the human antibody of the invention into a nanobody or single domain antibody framework sequence, such as PCT/EP93 As described in the example in /02214. In some embodiments, the invention provides a multivalent camelid antibody or a nanobody according to the methods described below.

Multivalent antibody

In another aspect, a multivalent molecule (monospecific, bispecific or multispecific) comprising a GITR binding antibody or fragment thereof of the invention is provided. An antibody or antigen binding region thereof of the invention may be derivatized or linked to another functional molecule, such as another peptide or protein (eg, another antibody or a ligand for a receptor) to produce binding to at least two different binding sites. A multivalent molecule (which may be the same or a different target site or molecule). In some embodiments, the invention An antibody is derivatized or functionally linked, for example, to more than one other functional molecule to produce a multivalent molecule that binds to two or more different binding sites, which are the same or different on the same target molecule Binding site. In certain embodiments, the multivalent binding sites are the same. In some embodiments, an antibody of the invention is derivatized or linked to more than one other functional molecule to produce a multispecific molecule that binds two or more different binding sites on at least two of the target molecules; as used herein, Such multispecific molecules are also intended to be encompassed by the terms "bispecific molecule" or "multispecificity". To produce a bispecific molecule of the invention, an antibody of the invention may be functionally linked to one or more other binding molecules (such as another antibody, antibody fragment, peptide or binding mimetic) (eg, by chemical coupling, inheritance) Fusion, non-covalent binding or otherwise linking) to produce multivalent molecules. The invention includes a bispecific molecule comprising at least one first binding specificity for GITR and a second binding specificity for a second target epitope. For example, the second target epitope is another epitope of the GITR that is different from the first target epitope. Additionally, for the invention in which the molecule is multispecific, in some embodiments, the molecule further comprises a third binding specificity in addition to the first and second target epitopes.

In one embodiment, the bispecific molecule of the invention comprises at least one antibody or antibody fragment thereof (including, for example, Fab, Fab', F(ab')2, Fv or single chain Fv) as binding specificity. The antibody may also be a light chain or a heavy chain dimer, or any minimally fragment thereof, such as an Fv or a single-stranded construct, as described in U.S. Patent No. 4,946,778 to Ladner et al.

Bifunctional antibodies are bivalent, bispecific molecules in which the VH and VL domains are expressed on a single polypeptide chain that is linked by a linker that is too short to allow pairing between the two domains on the same chain. The VH and VL domains are paired with complementary domains of another strand to create two antigen binding sites (see, eg, Holliger et al, 1993 Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al., 1994 Structure 2: 1121-1123). Bifunctional antibodies can be produced by expressing two polypeptide chains having the structural VHA-VLB and VHB-VLA (VH-VL configuration) or VLA-VHB and VLB-VHA (VL-VH configuration) in the same cell. Most of them are in bacteria It is expressed in a soluble form. Single-chain bifunctional antibodies (scDb) are produced by ligation of two polypeptide chains that form bifunctional antibodies by a linker of about 15 amino acid residues (see Holliger and Winter, 1997 Cancer Immunol. Immunother., 45 (3) -4): 128-30; Wu et al, 1996 Immunotechnology, 2(1): 21-36). scDb can be expressed in bacteria as a soluble, reactive monomer form (see Holliger and Winter, 1997 Cancer Immunol. Immunother., 45(34): 128-30; Wu et al, 1996 Immunotechnology, 2(1): 21-36; Pluckthun and Pack, 1997 Immunotechnology, 3(2): 83-105; Ridgway et al, 1996 Protein Eng., 9(7): 617-21). Bifunctional antibodies can be fused to Fc to produce "di-bifunctional antibodies" (see Lu et al, 2004 J. Biol. Chem., 279(4): 2856-65).

Other antibodies useful in the bispecific molecules of the invention are murine, chimeric and humanized monoclonal antibodies.

The bispecific and/or multivalent molecules of the invention can be prepared by using methods known in the art in combination with constitutive binding specificities, for example, the binding specificities of bispecific and/or multivalent molecules can be Generated separately and then combined with each other. When the binding specificity is a protein or peptide, a variety of coupling agents or cross-linking agents can be used for covalent bonding. Examples of the crosslinking agent include protein A, carbodiimide, N-butylenedimino-S-ethylidene-thioacetate (SATA), 5,5'-dithiobis(2-nitrate Benzoic acid) (DTNB), ortho-phenyl dimethyleneimine (oPDM), N-butylenedimino-3-(2-pyridyldithio)propionate (SPDP) And sulfobutanediamine 4-(N-methylene-2-imidazolylmethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, for example, Karpovsky et al., 1984 J. Exp. Med. 160: 1686; Liu, MA et al., 1985 Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described in Paulus, 1985 Behring Ins. Mitt. No. 78, 118-132; Brennan et al., 1985 Science 229: 81-83) and Glennie et al., 1987 J. Immunol. 139: 2367 -2375). The binders were SATA and sulfo-SMCC, all available from Pierce Chemical Co. (Rockford, IL).

When the binding specificity is an antibody, it can be bound by a sulfhydryl linkage of the constant domain hinge region of the two heavy chains. In a particular embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, such as one, prior to binding.

Alternatively, the binding specificity can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful where the bispecific and/or multivalent molecule is a mAb x mAb, mAb x Fab, Fab x F (ab') 2 or ligand x Fab fusion protein. The bispecific and/or multivalent molecule of the invention may be a single chain molecule comprising a single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants. The bispecific molecule can comprise at least two single chain molecules. The method of preparing the bispecific molecule is described, for example, in U.S. Patent No. 5,260,203, U.S. Patent No. 5,455,030, U.S. Patent No. 4,881,175, U.S. Patent No. 5,132,405, U.S. Patent No. 5,091,513, U.S. Patent No. 5,476,786, U.S. Patent No. 5,013,653 U.S. Patent No. 5,258,498 and U.S. Patent No. 5,482,858.

Bispecific and/or multivalent molecules can bind to their specific targets by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (REA), FACS analysis, bioanalysis (eg, growth inhibition), or Western blotting. Analysis to confirm. Each of these assays typically detects the presence of a protein-antibody complex of interest by using a labeled reagent (eg, an antibody) that is specific for the complex of interest. .

Half-life extended antibody

The present invention provides an antibody and an antibody fragment which specifically bind to a GITR protein, have an extended half-life in vivo.

Many factors can affect the in vivo half-life of a protein. For example, renal filtration, hepatic metabolism, degradation by proteolytic enzymes (protease), and immunogenic reactions (eg, proteins are neutralized by antibodies and absorbed by macrophages and dendritic cells). A variety of strategies are available to extend the half-life of the antibodies of the invention. For example, by chemical linkage to polyethylene glycol (PEG), reCODE PEG, antibody backbone, polysialic acid (PSA), hydroxyethyl starch (HES), a ligand binding to albumin and a carbohydrate mask; by genetic fusion with a protein that binds to serum proteins (such as albumin, IgG, FcRn) and by transfer; by binding to other binding proteins of serum proteins Coupling (such as nano-antibody, Fab, DARPin, high-affinity multimer, affinity antibody and anti-carrier protein) (genetic or chemical); by binding to rPEG, albumin, albumin domain, albumin and Fc genetic fusion; or by incorporation into a nanocarrier, sustained release formulation, or medical device.

In order to prolong the serum circulation of antibodies in vivo, an inert polymer molecule such as a high molecular weight PEG may specifically bind to the N-terminal or C-terminal site of the antibody via PEG or via an epsilon amine group present on the amino acid residue, The antibody or fragment thereof is ligated with or without a polyfunctional linker. To PEGylate an antibody, the antibody or fragment thereof is typically linked to a polyethylene glycol (PEG) (such as a reactive ester or aldehyde derivative of PEG) by attaching one or more PEG groups to the antibody or antibody fragment. The reaction under the conditions. PEGylation can be carried out by a deuteration reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any PEG form that has been used to derive other proteins, such as mono(C1-C10) alkoxy or aryloxy polyethylene glycols or polyethylene glycol-cis-butane. Alkene diimine. In certain embodiments, the antibody to be PEGylated is a deglycosylated antibody. Linear or branched polymer derivatization that minimizes loss of biological activity will be used. The degree of binding can be closely monitored by SDS-PAGE and mass spectrometry to ensure proper binding of the PEG molecule to the antibody. Unreacted PEG can be separated from the antibody-PEG conjugate by size exclusion or by ion exchange chromatography. Binding activity and in vivo efficacy of PEG-derivatized antibodies can be tested using methods well known to those skilled in the art, such as the immunoassays described herein. Protein pegylation methods are known in the art and are applicable to the antibodies of the invention. See, for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.

Other modified PEGylation techniques include re-establishing chemical orthogonal targeting engineering (ReCODE PEG) via a reconstitution system including tRNA synthetase and tRNA Chemically specific side chains are incorporated into the biosynthetic protein. This technology enables the incorporation of more than 30 neo-amino acids into synthetic proteins of bio-E. coli, yeast and mammalian cells. The tRNA incorporates the unnatural amino acid to any position where the amber codon is located, thereby converting the amber codon from the stop codon to a codon that conveys the combined signal of the chemically designated amino acid.

Recombinant PEGylation technology (rPEG) can also be used to extend serum half-life. This technique involves genetically fusing 300 to 600 amino acid unstructured protein tails to existing pharmaceutical proteins. Since the apparent molecular weight of such unstructured protein chains is about 15 times greater than its actual molecular weight, the serum half-life of the protein is greatly extended. This manufacturing process is greatly simplified and the product is homogeneous compared to conventional pegylation requiring chemical bonding and repurification.

Another technique is polysialylation, which uses natural polymer polysialic acid (PSA) to extend the useful life and improve the stability of therapeutic peptides and proteins. PSA is a sialic acid polymer (a sugar). When used to deliver proteins and therapeutic peptide drugs, polysialic acid provides a protective microenvironment for binding. This increases the useful life of the therapeutic protein in the circulation and prevents it from being recognized by the immune system. PSA polymers are naturally present in the human body. Some bacteria that have evolved over millions of years have covered their walls with PSA polymers. These natural polysialytic bacteria can then block the body's defense system by virtue of molecular mimicry. A large number of PSAs with a predetermined physical characteristic (natural ultimate stealing technique) can be easily produced in such bacteria. Since bacterial PSA is chemically identical to PSA in humans, bacterial PSA is completely non-immunogenic, even when coupled with proteins.

Another technique involves the use of a hydroxyethyl starch ("HES") derivative linked to an antibody. HES is a modified natural polymer derived from waxy corn starch and can be metabolized by enzymes of the body. HES solutions are usually administered to replace hypovolemia and to improve the rheological properties of the blood. Hydroxyethyl amylation of antibodies can increase biological activity by increasing molecular stability and by increasing renal half-life by reducing renal clearance. A wide range of HES antibody conjugates can be customized by varying different parameters, such as HES molecular weight.

An antibody having an extended half-life in vivo can also be produced by introducing one or more amino acid modifications (ie, substitutions, insertions or deletions) into an IgG constant domain or an FcRn binding fragment thereof (preferably an Fc or hinge Fc domain fragment). . See, for example, International Publication No. WO 98/23289; International Publication No. WO 97/34631; and U.S. Patent No. 6,277,375.

In addition, the antibody can bind to albumin to make the antibody or antibody fragment more stable in vivo or have a longer half-life in vivo. Such techniques are well known in the art, see, for example, International Publication No. WO 93/15199, No. WO 93/15200 and WO 01/77137, and European Patent No. EP 413,622.

Strategies for prolonging half-life are particularly useful for nanobodies, fibronectin-based binders, and other antibodies or proteins that require increased half-life in vivo.

Antibody conjugate

The invention provides an antibody or fragment thereof that specifically binds to a GITR protein, and a heterologous protein or polypeptide (or a fragment thereof, preferably at least 10, at least 20, at least 30, at least 40, at least 50, at least A fusion protein is produced by recombinant fusion or chemical binding (including covalent and non-covalent binding) of 60, at least 70, at least 80, at least 90 or at least 100 amino acid polypeptides. In particular, the invention provides fusion proteins comprising an antigen-binding fragment of an antibody described herein (eg, a Fab fragment, an Fd fragment, an Fv fragment, a F(ab)2 fragment, a VH domain, a VH CDR, a VL domain, or a VL CDR) And heterologous proteins, polypeptides or peptides. Methods of fusing or binding a protein, polypeptide or peptide to an antibody or antibody fragment are known in the art. See, for example, U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851 and 5,112,946; European Patent Nos. EP 307,434 and EP 367,166; International Publication No. WO 96/04388 and WO 91/06570; Ashkenazi et al, 1991, Proc. Natl. Acad. Sci. USA 88: 10535-10539; Zheng et al, 1995, J. Immunol. 154: 5590-5600; and Vil et al., 1992 , Proc. Natl. Acad. Sci. USA 89: 11337-11341.

Other fusion proteins can be produced by techniques of gene shuffling, motif shuffling, exon shuffling, and/or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling can be used to alter the activity of an antibody or fragment thereof of the invention (e.g., an antibody or fragment thereof having a higher affinity and a lower off rate). In general, see U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol .16(2): 76-82; Hansson et al., 1999, J. Mol. Biol. 287: 265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2): 308-313 (these patents and publications) Each case is hereby incorporated by reference in its entirety. The antibody or fragment thereof, or the encoded antibody or fragment thereof, can be altered prior to recombination by random mutation induction, random nucleotide insertion or other methods by error-prone PCR. A polynucleotide encoding an antibody or fragment thereof that specifically binds to a GITR protein can be recombined with one or more components, motifs, segments, portions, domains, fragments, and the like of one or more heterologous molecules.

In addition, antibodies or fragments thereof can be fused to a tag sequence such as a peptide to facilitate purification. In a preferred embodiment, the labeled amino acid sequence is a hexahistamine (SEQ ID NO: 11) peptide, particularly as provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311). Marks, many of which are commercially available. As described in Gentz et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824, for example, hexahistamine (SEQ ID NO: 11) facilitates purification of the fusion protein. Other peptide markers suitable for purification include, but are not limited to, a hemagglutinin ("HA") marker, which corresponds to an epitope derived from influenza red hemagglutinin protein (Wilson et al., 1984, Cell 37:767); And the "flag" tag.

In other embodiments, an antibody or fragment thereof of the invention is combined with a diagnostic or detection agent. As part of a clinical testing procedure, such antibodies can be adapted to monitor or prognose the onset, progression, progression, and/or severity of a disease or condition, such as determining the efficacy of a particular therapy. Such diagnosis and detection can be achieved by coupling antibodies to detectable substances and detecting Substances include, but are not limited to, different enzymes such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase or acetylcholinesterase; prosthetic groups such as, but not limited to Streptavidin/biotin and avidin/biotin; fluorescent substances such as, but not limited to, umbelliferone, luciferin, luciferin isothiocyanate, rhodamine, two Chlorotriazinylamine luciferin, dimethylamine sulfonaphthoquinone chloride or phycoerythrin; luminescent material such as, but not limited to, luminol; bioluminescent material such as, but not limited to, fluorescent Nuclease, luciferin and aequor; radioactive materials such as, but not limited to, iodine (131I, 125I, 123I and 121I), carbon (14C), sulfur (35S), antimony (3H), indium (115In, 113In , 112In and 111In), yttrium (99Tc), yttrium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), yttrium (133Xe), fluorine (18F), 153Sm, 177Lu, 159Gd, 149Pm , 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113Sn, and 117Tin; and using various positrons emission The positron-emitting metal of tomography, and non-radioactive paramagnetic metal ions.

Furthermore, the invention encompasses antibodies or fragments thereof that bind to a therapeutic moiety or a pharmaceutical moiety that modulates the intended biological action or reaction and use of the antibody or fragment thereof that binds to the therapeutic moiety. The therapeutic portion or drug portion should not be considered limited to classical chemotherapeutic agents. For example, the drug moiety can be a protein, peptide or polypeptide having the desired biological activity. Such proteins may include, for example, toxins such as acacia toxin, ricin A, Pseudomonas aeruginosa exotoxin, cholera toxin or diphtheria toxin; proteins such as tumor necrosis factor, alpha interferon, beta interferon, nerve growth factor, platelets Derived growth factor, tissue plasminogen activator; apoptosis agent; anti-angiogenic agent; or biological response modifier, such as lymphokine. The antibody or fragment thereof can bind to a therapeutic moiety (such as a cytotoxin, such as a cytostatic or cytocidal), a therapeutic agent, or a radioactive metal ion (eg, an alpha-emitter). Cytotoxins or cytotoxic agents include any agent that is detrimental to cells.

For example, an antibody can be combined with a therapeutic moiety, such as a radioactive metal ion A sub-ring, such as an alpha-emitter, such as 213Bi; or a macrocyclic chelating agent suitable for the binding of a metal ion, including but not limited to 131In, 131LU, 131Y, 131Ho, 131Sm, to a polypeptide. In certain embodiments, the macrocyclic chelating agent is 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (DOTA), which is connectable via The daughter molecule is attached to the antibody. Such linker molecules are generally known in the art and are described in Denardo et al, 1998, Clin Cancer Res. 4(10): 2482-90; Peterson et al, 1999, Bioconjug. Chem. 10(4): 553-7; and Zimmerman et al, 1999, Nucl. Med. Biol. 26(8): 943-50, each incorporated herein by reference in its entirety.

Techniques for binding a therapeutic moiety to an antibody are well known, see, for example, Arnon et al, "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", Controlled Drug Delivery (2nd Edition); Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987) Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy, Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985); and Thorpe et al., 1982, Immunol. Rev. :119-58.

The antibody may also be linked to a solid support, which is especially useful for immunoassays or purification of the target antigen. Such solid carriers include, but are not limited to, glass, cellulose, polypropylene decylamine, nylon, polystyrene, polyvinyl chloride or polypropylene.

Polynucleotide encoding an anti-GITR antibody

Anti-GITR antibodies, antigen binding molecules and fragments thereof can be prepared by any method known in the art including, but not limited to, recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers, while full-length monoclonal antibodies can be borrowed Obtained by, for example, fusion tumors or recombinant preparation. Recombinant expression can be from any suitable host cell known in the art, such as mammalian host cells, bacterial host cells, yeast host cells, insect host cells, and the like.

The invention further provides a polynucleotide encoding an antibody described herein, such as a polynucleotide encoding a heavy or light chain variable region or segment comprising a complementarity determining region as described herein. In some embodiments, the polynucleotide encoding the heavy chain variable region comprises and is selected from the group consisting of SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57 The polynucleotide of the group consisting of SEQ ID NO: 101, SEQ ID NO: 107 and SEQ ID NO: 115 has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95 Sequence of %, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity. In some embodiments, the polynucleotide encoding a light chain variable region comprises at least 85% of a polynucleotide selected from the group consisting of SEQ ID NO: 52, SEQ ID NO: 54 and SEQ ID NO: 102 , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence of nucleic acid sequence identity.

In some embodiments, the polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO:67. , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity. In some embodiments, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO:68. , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity.

In some embodiments, the polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO:72. , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity. In some embodiments, the polynucleotide encoding the light chain has at least one of the polynucleotides selected from SEQ ID NO: 73 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity.

In some embodiments, the polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO:74. , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity. In some embodiments, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO:68. , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity.

In some embodiments, the polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO:76. , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity. In some embodiments, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO:68. , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity.

In some embodiments, the polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO:78. , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity. In some embodiments, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO:68. , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity.

In some embodiments, the polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, and a polynucleotide selected from the group consisting of SEQ ID NO:103. 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity. In some embodiments, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO: 104 , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity.

In some embodiments, the polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO:108 , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity. In some embodiments, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO: 104 , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity.

In some embodiments, the polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO:116. , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity. In some embodiments, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO:68. , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity.

In some embodiments, the polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO: , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity. In some embodiments, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO:68. , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity.

In some embodiments, the polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO: 120 , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity. In some embodiments, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93% with a polynucleotide selected from the group consisting of SEQ ID NO:68. 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity.

In some embodiments, the polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO:122. , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity. In some embodiments, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO:68. , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity.

In some embodiments, the polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO: 124 , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity. In some embodiments, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO:68. , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity.

In some embodiments, the polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO:131 , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity. In some embodiments, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO:68. , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity.

In some embodiments, the polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO: , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity. In some embodiments, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO:68. , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity.

In some embodiments, the polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO:135 , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity. In some embodiments, the polynucleotide encoding the light chain has at least 85% of the polynucleotide of SEQ ID NO:68, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity.

In some embodiments, the polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO:137 , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity. In some embodiments, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO:68. , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity.

In some embodiments, the polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO:139. , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity. In some embodiments, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO:68. , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity.

In some embodiments, the polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO: 141 , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity. In some embodiments, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO:68. , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity.

In some embodiments, the polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO:143 , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity. In some embodiments, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO:68. , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity.

In some embodiments, the polynucleotide encoding the heavy chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO:60. , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity. In some embodiments, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% of the polynucleotide of SEQ ID NO:58. , 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity.

The polynucleotide of the present invention can only encode the variable region sequence of an anti-GITR antibody. It can also encode the variable and constant regions of an antibody. Some polynucleotide sequences encode polypeptides comprising the variable regions of the heavy and light chains of one of the exemplary mouse anti-GITR antibodies. Some other polynucleotides encode two polypeptide segments that are substantially identical to the variable regions of the heavy and light chains, respectively, of one of the mouse antibodies.

Polynucleotide sequences can be produced by re-solid phase DNA synthesis or by PCR mutation induction encoding an existing sequence of an anti-GITR antibody or binding fragment thereof, such as a sequence as described herein. Direct chemical synthesis of nucleic acids can be accomplished by methods known in the art, such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68:90; Brown et al., Meth. Enzymol. 68:109, 1979 Phosphate diester method; Beaucage et al., Tetra. Lett., 22: 1859, 1981 Aminophosphoric acid diethyl ester process; and U.S. Patent No. 4,458,066. Introduction of mutations into a polynucleotide sequence by PCR can be performed as described in the following literature: PCR Technology: Principles and Applications for DNA Amplification, HAErlich (ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (eds.), Academic Press, San Diego, CA, 1990; Mattila et al, Nucleic Acids Res. 19: 967, 1991; and Eckert et al., PCR Methods and Applications 1:17, 1991.

The present invention also provides expression vectors and host cells which produce the above anti-GITR antibodies. Different expression vectors can be used to express a polynucleus encoding an anti-GITR antibody chain, fragment or binding fragment Glycosylate. Both viral-based expression vectors and non-viral expression vectors can be used to produce antibodies in mammalian host cells. Non-viral vectors and systems include plastid, episomal vectors, which typically have a manifestation for expression of proteins or RNA and human artificial chromosomes (see, eg, Harrington et al, Nat Genet 15:345, 1997). For example, non-viral vectors suitable for displaying anti-GITR polynucleotides and polypeptides in mammalian (eg, human) cells include pThioHis A, B and C, pcDNA3.1/His, pEBVHis A, B, and C (Invitrogen) , San Diego, CA), MPSV vectors and many other vectors known in the art for expressing other proteins. Suitable viral vectors include vectors based on retrovirus, adenovirus, adeno-associated virus, herpes virus; based on SV40, papilloma virus, HBP Epstein Barr virus, vaccinia virus vector and victory base The carrier of the forest virus (Semliki Forest virus; SFV). See Brent et al., supra; Smith, Annu. Rev. Microbiol. 49: 807, 1995; and Rosenfeld et al, Cell 68: 143, 1992.

The choice of expression vector depends on the intended host cell of the expression vector. Typically, the expression vector contains a promoter and other regulatory sequences (e.g., enhancers) operably linked to a polynucleotide encoding an anti-GITR antibody chain or fragment. In some embodiments, an inducible promoter is used to prevent expression of the inserted sequence, except under induction conditions. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters or heat shock promoters. The transformed organism culture can be expanded under non-inducing conditions that render the population unbiased that the performance product is well tolerated by the host cell. In addition to the promoter, other regulatory elements may be necessary or required for the effective performance of the anti-GITR antibody chain or fragment. Such elements typically include an ATG start codon and an adjacent ribosome binding site or other sequence. In addition, performance efficiency can be enhanced by including enhancers suitable for the cell system used (see, for example, Scharf et al, Results Probl. Cell Differ. 20: 125, 1994; and Bittner et al, Meth. Enzymol., 153:516). , 1987). For example, an SV40 enhancer or CMV enhancer can be used to enhance expression in a mammalian host cell.

The expression vector can also provide a secretion signal sequence position to form a fusion protein with the polypeptide encoded by the inserted anti-GITR antibody sequence. More typically, the inserted anti-GITR antibody sequence is ligated to the signal sequence prior to inclusion in the vector. Vectors to be used to receive sequences encoding anti-GITR antibody light and heavy chain variable domains also sometimes encode a constant region or a portion thereof. Such vectors allow the variable region to be expressed as a fusion protein formed with the constant region, thereby producing an intact antibody or fragment thereof. Typically, such constant regions are human constant regions.

Host cells containing and expressing an anti-GITR antibody chain can be prokaryotic or eukaryotic. E. coli is a prokaryotic host suitable for the selection and expression of the polynucleotides of the present invention. Other microbial hosts suitable for use include bacilli (such as Bacillus subtilis), and other enterobacteriaceae (such as Salmonella, Serratia), and a variety of Pseudomonas species. In such prokaryotic hosts, expression vectors can also be produced which typically contain expression control sequences (e.g., origins of replication) that are compatible with the host cell. In addition, any number of well-known promoters will be present, such as a lactose promoter system, a tryptophan (trp) promoter system, a beta-endosinase promoter system, or a promoter system from phage lambda. Promoters typically control performance (controlling performance as appropriate with manipulation sequences) and have ribosome binding site sequences and similar sequences for initiation and completion of transcription and translation. Other microorganisms such as yeast can also be used to express the anti-GITR polypeptides of the invention. Combinations of insect cells and baculovirus vectors can also be used.

In some preferred embodiments, a mammalian host cell is used to express and produce an anti-GITR polypeptide of the invention. For example, it may be a fusion tumor cell line expressing an endogenous immunoglobulin gene (for example, a myeloma fusion tumor line as described in the Examples) or a mammalian cell line containing an exogenous expression vector (for example, exemplified below) SP2/0 myeloma cells). Such cells include any normal death or normal or abnormal immortal animal or human cell. For example, a variety of suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, transformed B cells, and fusions. tumor. Using mammalian tissue cell cultures for performance The use of polypeptides is generally discussed, for example, in Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y., 1987. Expression vectors for mammalian host cells can include expression control sequences such as origins of replication, promoters and enhancers (see, eg, Queen et al., Immunol. Rev. 89:49-68, 1986), and the necessary processing information sites (see, eg, Such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. Such expression vectors typically contain a promoter derived from a mammalian gene or derived from a mammalian virus. Suitable promoters can be constitutive, cell type specific, stage specific and/or regulatable or regulatable. Suitable promoters include, but are not limited to, metallothionein promoter, constitutive adenovirus major late promoter, dexamethasone-inducible MMTV promoter, SV40 promoter, MRP polIII promoter, constitutive MPSV promoter , a tetracycline-inducible CMV promoter (such as the human immediate early CMV promoter), a constitutive CMV promoter, and a promoter-enhancer combination known in the art.

The method of introducing a expression vector containing a polynucleotide sequence of interest varies depending on the type of the cell host. For example, calcium chloride transfection is typically used in prokaryotic cells, while calcium phosphate treatment or electroporation can be used in other cellular hosts. (See generally Sambrook et al., supra). Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, impact methods, viral particles, immunoliposomes, polycations: nucleic acid conjugates, naked DNA, artificial virions Fusion with herpesvirus structural protein VP22 (Elliot and O'Hare, Cell 88: 223, 1997), enhanced absorption of DNA agents, and ex vivo transduction. For long-term high yields of recombinant proteins, stable performance is usually required. For example, a cell line stably expressing an anti-GITR antibody chain or a binding fragment can be prepared using the expression vector of the present invention, and the expression vector of the present invention contains a viral origin of replication or an endogenous expression element and a selectable marker gene. After introduction of the vector, the cells can be allowed to grow for 1-2 days in a rich medium, followed by exchange of the rich medium with the selective medium. The purpose of the selectable marker is to confer resistance to selection and its presence allows for successful expression of the introduced sequence The cells are grown in selective medium. The stably transfected drug resistant cells can be propagated using tissue culture techniques appropriate to the cell type.

Identification of agonistic anti-GITR antibodies

Assays for identifying agonistic anti-GITR antibodies are known in the art and are described herein. The agonistic anti-GITR antibody binds to GITR and promotes, induces, stimulates intracellular signaling via GITR.

The binding of an anti-GITR antibody to GITR can be determined using any method known in the art. For example, binding to GITR can be determined using known techniques including, but not limited to, ELISA, Western blots, surface plasmonic resonance (eg, BIAcore), and flow cytometry.

Intracellular signaling via GITR can be measured using any method known in the art. For example, NFκB and MAPK signaling are promoted via GITR activation. Methods for measuring activation of NFκB and MAPK are standard methods in the art (eg, using reporter gene analysis, nuclear translocation of NFκB protein, phosphorylation status of MAPK protein). Activation via GITR is a costimulatory signal that promotes proliferation of activated CD4 ⁺ and CD8 ⁺ T cells in the presence of T cell receptor activation (eg, in the presence of primary or target antigens). Methods for measuring cell proliferation are standard methods in the art (e.g., ³ H-thymidine incorporation assay, CFSE labeling). Cytokines are also produced by co-stimulation of activated CD4 ⁺ and CD8 ⁺ T cells via GITR signaling in the presence of T cell receptor activation. Cytokines are also produced by stimulating activated NK cells via GITR signaling. The cytokine may be any one or two of Th1 type cytokines (such as interferon gamma, IL-2 and TNF) and Th2 type cytokines (such as IL-4, IL-5, IL-10 and IL-13). By. Methods for measuring cytokine production are well known in the art (e.g., ELISA assay, ELISpot assay). Apoptosis can also be induced by activation of GITR. Methods for measuring apoptosis are standard methods in the art (e.g., phospholipid binding protein V staining). In the in vitro assay, the test cells contacted with the agonistic anti-GITR antibody or the culture supernatant from the test cells can be cultured with control cells that have not been contacted with the agonistic anti-GITR antibody or culture supernatants from control cells. Liquid comparison.

The GITR agonizing function of the antibody of the present invention can also be measured in vivo. Preferred agonistic anti-GITR antibodies are capable of activating and expanding CD4 ⁺ and CD8 ⁺ T cells. In vivo activation and amplification of CD4 ⁺ and CD8 ⁺ T cells can be measured using any method known in the art (e.g., by flow cytometry). Preferred agonistic anti-GITR antibodies are therapeutically suitable for inhibiting tumor growth or promoting tumor shrinkage. Tumor growth or inhibition thereof can be measured using any method known in the art (e.g., visual inspection, caliper, weighing, imaging techniques, including MRI). Preferred agonistic anti-GITR antibodies are therapeutically useful for preventing, reducing, inhibiting or eliminating the cause of infectious diseases such as bacterial, fungal, viral or parasitic infections. The efficacy of an agonistic anti-GITR antibody in enhancing a T cell response or reducing the severity of a disease can be determined by administering to a subject a therapeutically effective amount of the antibody and comparing the individual before and after administration of the antibody. The efficacy of the agonistic anti-GITR antibody in enhancing the T cell response or reducing the severity of the disease can also be determined by administering an effective amount of the antibody to the test subject and comparing the test subject to a control individual to whom the antibody has not been administered.

Composition comprising an agonistic anti-GITR antibody

The present invention provides a pharmaceutical composition comprising an anti-GITR antibody or antigen-binding molecule of the invention formulated together with a pharmaceutically acceptable carrier, optionally containing one or more other compounds suitable for the treatment or prevention of a defined condition. Therapeutic agent. A pharmaceutically acceptable carrier enhances or stabilizes the composition or may facilitate the preparation of the composition. Pharmaceutically acceptable carriers include physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.

The pharmaceutical compositions of the invention can be administered by a variety of methods known in the art. The route and/or mode of administration will vary depending on the desired outcome. Preferably, the administration is administered intravenously, intramuscularly, intraperitoneally or subcutaneously or close to the target site. A pharmaceutically acceptable carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, intranasal, inhalation, spinal or epidermal administration (for example by injection or infusion). Depending on the route of administration, the active compound (eg the hair) An antibody or antigen-binding fragment or a multivalent molecule (eg, a monospecific, bispecific or multispecific molecule) can be coated in a material to protect the compound from the action of the acid and other compounds that render the compound inactive Natural conditions.

The antibody or fragment thereof, alone or in combination with other suitable components, can be formulated into an aerosol formulation (i.e., it can be "atomized") for administration via inhalation. Aerosol formulations can be placed in an acceptable pressurized propellant such as dichlorodifluoromethane, propane, nitrogen, and the like.

In some embodiments, the composition is sterile and fluid. For example, proper fluidity can be maintained by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it is preferred to include an isotonic agent, such as a sugar, a polyol (such as mannitol or sorbitol), and sodium chloride in the composition. Injectable compositions can be absorbed for a prolonged period of time by including agents which delay absorption, such as aluminum monostearate or gelatin. In certain embodiments, the compositions can be prepared for storage in lyophilized form using suitable excipients such as sucrose.

The pharmaceutical compositions of the present invention can be prepared according to methods well known and routinely practiced in the art. The pharmaceutically acceptable carrier moiety is determined by the particular composition being administered and by the particular method of administration of the composition. Thus, there are a variety of suitable formulations of the pharmaceutical compositions of the present invention. Suitable methods for formulating antibodies and determining appropriate dosing schedules can be found, for example, in Remington: The Science and Practice of Pharmacy , 21st Edition, University of the Sciences in Philadelphia, Lippincott Williams and Wilkins (2005); Martindale: The Complete Drug Reference , Sweetman, 2005, London: Pharmaceutical Press.; Martindale, Martindale: The Extra Pharmacopoeia , 31st edition, 1996, Amer Pharmaceutical Assn; and Sustained and Controlled Release Drug Delivery Systems, edited by JR Robinson, Marcel Dekker, Inc., New York In 1978, each of them is hereby incorporated by reference. The pharmaceutical composition is preferably manufactured under GMP conditions. Generally, a therapeutically effective or useful amount of an anti-GITR antibody is employed in the pharmaceutical compositions of the invention. Anti-GITR antibodies are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art. The dosage regimen is adjusted to achieve the desired response (eg, a therapeutic response). In determining a therapeutically or prophylactically effective dose, a low dose can be administered, followed by a gradual increase until the desired response is achieved with minimal or no adverse side effects. For example, as indicated by the state of emergency of the treatment situation, a single bolus administration may be administered, several fractional doses may be administered over time, or the dose may be reduced or increased proportionally. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to a physically discrete unit suitable as a single dosage for the subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the desired pharmaceutical carrier.

The actual dosage of the active ingredient in the pharmaceutical compositions of the present invention can be varied so that the amount of active ingredient that is effective to achieve the desired therapeutic response in a particular patient, composition, and mode of administration without toxic to the patient can be obtained. The selected dose will depend on a variety of pharmacokinetic factors, including the activity of the particular composition or its ester, salt or guanamine used in the present invention, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of treatment, and the particular Other drugs, compounds and/or substances used in combination with the composition, age, sex, weight, condition, general health and prior medical history of the patient being treated, and the like.

Co-distributed with the second agent

In some embodiments, the pharmacological composition comprises an anti-GITR antibody or a mixture of an antigen binding molecule and a second pharmacological agent. Exemplary second agents included in a mixture with an anti-GITR agonist antibody or antigen binding molecule of the invention include, but are not limited to, primary or target antigens, agents that increase the immunogenicity of tumor cells, inhibit or inhibit A drug that inhibits signals.

The anti-GITR antibodies or antigen binding molecules of the invention may be co-formulated with the primary or target antigen (i.e., provided as a mixture or as a mixture). Target antigen or vaccine It depends on the condition of the disease to be treated. For example, the target antigen can be from a tumor cell, a bacterial cell, a fungus, a virus, or a parasite. The target antigen may be in the form of a peptide, polypeptide, cell or polynucleotide.

In one embodiment, the target antigen is derived, for example, from a virus selected from the group consisting of hepatitis A, hepatitis B, hepatitis C, influenza virus, chickenpox, adenovirus, herpes simplex type I (HSV I), simple Herpes type II (HSV II), burdock, rhinovirus, echovirus, rotavirus, respiratory tract cell virus, papilloma virus, papovavirus, cytomegalovirus, ecinovirus ), arbovirus, hantavirus, coxsackie virus, mumps virus, measles virus, rubella virus, poliovirus, human immunodeficiency virus type I (HIV I) and human immunity Defective virus type II (HIV II), any small RNA virus family, enterovirus, calicivirus family, Norwalk group of viruses, chlamydia virus (such as alphavirus, flavivirus) , coronavirus, rabies virus, Marburg virus, ebola virus, parainfluenza virus, positive mucus virus, bunyavirus, ruby virus, reovirus, Rotavirus, ring disease Toxic, human T cell leukemia virus type I, human T cell leukemia virus type II, monkey immunodeficiency virus, lentivirus, polyoma virus, small virus, Epstein-Barr virus, human herpesvirus 6, monkey herpes virus 1 (B virus) and pox virus.

In one embodiment, the target antigen is derived, for example, from a bacterium selected from the group consisting of Neisseria spp, Streptococcus spp, S. mutans, Haemophilus Genus (Haemophilus spp.), Moraxella spp, Bordetella spp, Mycobacterium spp, Legionella spp, Escherichia spp ), Vibrio spp, Yersinia spp, Campylobacter spp, Salmonella spp, Listeria spp., Helicobacter Spp), Pseudomonas (Pseudomonas spp), Staphylococcus spp., Enterococcus spp, Clostridium spp., Bacillus spp, Corynebacterium spp., sparse Borrelia spp., Ehrlichia spp, Rickettsia spp, Chlamydia spp., Leptospira spp., Treponema sp. Treponema spp).

In some embodiments, the anti-GITR antibody or antigen binding molecule is co-formulated as a mixture with a tumor associated antigen (TAA). The TAA can be an isolated polypeptide or peptide that can be part of a whole cell or part of a tumor cell lysate. The TAA can be a polynucleotide, such as a naked body, or a viral vector comprising a polynucleotide encoding one or more TAAs. Examples of known TAAs include, but are not limited to, melanoma-associated antigens (MAGE-1, MAGE-3, TRP-2, melanosome membrane glycoproteins gp100, gp75, and MUC-1 associated with melanoma). 1)); CEA (carcinoembryonic antigen) which may, for example, be associated with ovarian, melanoma or colon cancer; folate receptor alpha expressed by ovarian cancer; free human manifested by a variety of different tumors including, but not limited to, myeloma Chorionic gonadotropin beta (hCGβ) subunit; HER-2/neu associated with breast cancer; encephalomyelitis antigen HuD associated with small cell lung cancer; tyrosine hydroxylase associated with neuroblastoma; Cancer-associated prostate-specific antigen (PSA); CA125 associated with ovarian cancer; and individual genotype determinants of B-cell lymphoma produce tumor-specific immunity (due to individual genotype-specific humoral immune responses). Furthermore, the antigen of human T cell leukemia virus type 1 has been shown to induce specific CTL responses and anti-tumor immunity against virus-induced human adult T cell leukemia (ATL). See, for example, Haupt et al, Experimental Biology and Medicine (2002) 227:227-237; Ohashi et al, Journal of Virology (2000) 74(20): 9610-9616. Other TAAs are known and are useful for co-provisioning with anti-GITR antibodies.

In some embodiments, an anti-GITR antibody or antigen binding molecule is co-formulated with an autologous tumor cell from a patient or an allogeneic tumor cell of the same tissue type from another patient. Tumor cells can be in the form of intact cells, tumor cell lysates, apoptotic tumor cells, or total tumor mRNA. Tumor cells can be transfected to express a polypeptide that enhances or enhances the immunogenicity of tumor cells in a patient, for example, transfected to express granulocyte colony stimulating factor (GM-CSF). Tumor cells can be derived from any cancerous tissue including, but not limited to, epithelial cancer or carcinoma, as well as sarcomas and lymphomas. In some embodiments, the cancer is melanoma, ovarian cancer, renal cancer, colorectal cancer, prostate cancer, lung cancer (including non-small cell lung cancer (NSCLC)), breast cancer, glioma, fibrosarcoma, blood cancer, or head and neck scales. Cellular carcinoma (HNSCC). See, for example, Pardee et al, Immunotherapy (2009) 1(2): 249-264 and the references discussed therein. In some embodiments, the tumor cells are from, for example, pancreatic cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer , esophageal cancer, cervical cancer, uterus or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or accessory cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, Cancer of chondrosarcoma and blood tissue.

In some embodiments, an anti-GITR antibody or antigen binding molecule is co-formulated with a cytotoxic agent. For example, an anti-GITR antibody or antigen binding molecule is co-formulated with an agonist antibody or antigen binding molecule that binds to and reduces or depletes CD4+CD25+ regulatory T cells (Treg). Exemplary Treg cells deplete antibodies or antigen binding molecules that bind to CD25 or CCR4. See Expert Opin Ther Patents (2007) 17(5): 567-575 and the references discussed therein.

In some embodiments, an anti-GITR antibody or antigen binding molecule is co-formulated with an inhibitor of a co-suppression signal. Exemplary inhibitors include inhibitors of CTLA-4 and inhibitors of PD-1/PD-L1 (eg, B7-H1) interactions. In some embodiments, an anti-GITR antibody is co-formulated with an antibody that binds to and inhibits CTLA-4. In some embodiments, an anti-GITR antibody is co-formulated with an antibody that binds to and inhibits PD-1. In some embodiments, an anti-GITR antibody is co-formulated with an antibody that binds to and inhibits B7-H1. See, for example, Expert Opin Ther Patents (2007) 17(5): 567-575; and Melero, et al, Clin Cancer Res (2009) 15(5): 1507-1509 and the references discussed therein. In certain embodiments, the formulation comprises a bispecific molecule comprising an anti-GITR antibody or antigen binding molecule and an inhibitor of a co-suppression signal. In some embodiments, the formulation comprises a bispecific molecule comprising an anti-GITR antibody or antigen binding molecule and an inhibitor of CTLA4. In some embodiments, the formulation comprises a bispecific molecule comprising an anti-GITR antibody or antigen binding molecule and an inhibitor of PD-1/PD-L1. In some embodiments, the formulation comprises a bispecific molecule comprising an anti-GITR antibody or antigen binding molecule and an inhibitor of B7H1.

Anti-GITR antibodies or antigen binding molecules can also be co-formulated with one or more immunostimulatory agents. For example, in some embodiments, an anti-GITR antibody is co-formulated with an immunostimulatory cytokine (eg, IL-7, IL-12, or IL-15). Alternatively, an anti-GITR antibody or antigen binding molecule can be co-formulated with a second immunostimulatory antibody. For example, an anti-GITR antibody or antigen binding molecule can also be co-formulated with an agonist antibody or antigen binding molecule of another member of the tumor necrosis factor receptor superfamily. Exemplary second immunostimulatory targets include, but are not limited to, TNFRSF4 tumor necrosis factor receptor superfamily member 4 (also known as OX40) or tumor necrosis factor receptor superfamily member 9 (also known as TNFRSF9, 4-1BB or CD137). See, for example, Expert Opin Ther Patents (2007) 17(5): 567-575; Pardee et al, Immunotherapy (2009) 1(2): 249-264; and Melero, et al, Clin Cancer Res (2009) 15 (5) ): 1507-1509 and the references discussed therein.

An anti-GITR antibody or antigen binding molecule can also be co-formulated with a chemotherapeutic agent. The agent selected depends on the condition to be treated, such as cancer or an infectious disease, such as a bacterial infection, a fungal infection, a viral infection, or a parasitic infection. Anti-GITR antibodies or antigen binding molecules can be co-formulated with chemotherapeutic agents known to those skilled in the art to treat the condition of the disease to be treated. For example, chemotherapeutic agents for the treatment of cancer and infectious diseases are known in the art and are described, for example, in the literature: Goodman and Gilman's The Pharmacological Basis of Therapeutics , 11th edition, edited by Brunton, Lazo and Parker, McGraw-Hill (2006); 2010 Physicians' Desk Reference (PDR) , 64th edition, Thomson PDR.

In some embodiments, an anti-GITR antibody or antigen binding molecule can be co-formulated with an anti-tumor agent. Exemplary antineoplastic agents useful in combination with an anti-GITR antibody include alkylating agents (e.g., nitrogen mustard, ethyleneimine, and methyl melamine, methyl anthracene derivatives, alkyl sulfonates, nitroso groups). Urea, triazene and platinum coordination complexes; antimetabolites (eg folic acid analogs, pyrimidine analogs, purine analogs); natural products (eg vinca alkaloids, taxanes, epipodophyllotoxins, Camptothecin, antibiotics and bismuth). In some embodiments, the anti-GITR antibody or antigen binding molecule is co-formulated with an antimetabolite antineoplastic agent, such as a folic acid analog (eg, methotrexate, pemetrexed, trimesat ( Trimetrexate)), pyrimidine analogs (eg, 5-fluorouracil, capecitabine, cytarabine, gemcitabine), purine analogs (eg, mercaptopurine, pentastatin ( Pentostatin), cladribine, fludarabine, or a mixture thereof. In some embodiments, the anti-GITR antibody or antigen binding molecule is co-formulated with an alkylating agent antineoplastic agent, such as nitrogen mustard (eg, nitrogen mustard, cyclophosphamide, ifosfamide, melphalan) , chlorambucil, ethimine (such as altretamine) and methyl melamine (such as thiotepa), methyl hydrazine derivatives (such as procarbazine) )), alkyl sulfonate (such as busulfan), nitrosourea (such as carmustine, streptozocin), triazene (such as dacarbazine) ), temozolomide, and a platinum coordination complex (eg, cisplatin, carboplatin, oxaliplatin).

In some embodiments, an anti-GITR antibody or antigen binding molecule can be co-formulated with an antiviral agent. Exemplary antiviral agents include, but are not limited to, anti-herpes virus agents (eg, acyclovir, cidofovir, famciclovir, foscarnet, thiovir) , fomivirsen, ganciclovir (ganciclovir), idoxuridine, penciclovir, trifluridine, valacyclovir, valgenciclovir, resiquimod ; anti-influenza agents (eg amantadine, oseltamivir, rimantadine, zanamivir, peramivir, E-118958); anti-influenza agents (amantadine, oseltamivir, zanamivir, zanamivir, peramivir, E-118958); Hepatitis agents (eg, adefovir dipivoxil, interferon alpha, lamivudine, entecavir, clevudine, emtricitabine, telbif Telbivudine, tenofovir, viramidine, BILN 2061, NM283, and other antiviral agents (eg ribavirin, imiquimod, maribavir) (maribavir), sICAM-1, pleconaril (pleconaril). The antiviral agent can be an antiretroviral agent. Exemplary antiretroviral agents include, but are not limited to, zidovudine, didanosine, stavudine, zalcitabine, lamivudine ( Lamivudine), abacavir, tenofavir, emtricitabine, nevirapine, efavirenz, delavirdine, saquina Saquinavir, indinavir, ritonavir, nelfinavir, amprenavir, lopinavir, atazanavir Atazanavir), fosamprenavir and enfuvirtide.

In some embodiments, an anti-GITR antibody or antigen binding molecule can be co-formulated with an antibacterial agent. Exemplary antibacterial agents include, but are not limited to, sulfonamides (eg, sulfanilamide, sulfadiazine, sulfamethoxazole, sulfisoxazole) , sulfacetamide, trimethoprim, quinolones (eg nalidixic acid, cinoxacin, norfloxacin, ciprofloxacin) Ciprofloxacin), ofloxacin, sparfloxacin, fleroxacin, perfloxacin, levofloxacin (levofloxacin), garenoxacin and gemifloxacin, methenamine, nitrofurantoin, penicillin (eg penicillin G, penicillin V, methicillin) ), oxacillin, cloxacillin, dicloxacillin, nafcilin, ampicillin, amoxicillin, carbencilin, Ticarcillin, mezlocillin and piperacillin, cephalosporin (eg cefazolin, cephalexin, cefdroxil, cephalosporin) Cefoxitin, cefaclor, cefprozil, cefuroxime, cefuroxime acetil, loracarbef, cefotetan, cefotetan Ceforanide, cefotaxime, cefpodoxime proxetil, cefbuten, cefdinir, cefditoren pivorxil, ceftizoxime (ceftizoxi Me), ceftrixone, cefoperazone, ceftazidime and cefepime, carbapenems (eg imipenem, aztreonam) And aminoglycosides (eg neomycin, kanamycin, streptomycin, gentamicin, tobramycin, netilmicin) ) and amikacin (amikacin).

In some embodiments, an anti-GITR antibody or antigen binding molecule can be co-formulated with an antiparasitic agent. Exemplary antiparasitic agents include, but are not limited to, anti-malarial agents (eg, quinoline, including (chloroquine), mefloquine, quinine, quinidine, and primaquine Diaminopyrimidines, including pyrimethamine, sulfadoxine, tetracycline, atovaquone, and proguanil; antiprotozoal agents, including amphotericin (amphotericin), chloroquine, eflornithine, emetine, fumagillin, 8-hydroxyquinoline, melarsoprol, metronidazole (metronidazole), miltefosine, nifurtimox, nitazoxanide, paromomycin, pentamidine, sodium gluconate and sulphate Lamin (suramin).

In some embodiments, an anti-GITR antibody or antigen binding molecule can be co-formulated with an antifungal agent. Exemplary antifungal agents include, but are not limited to, polyenes (eg, natamycin, rimoidin, filipin, nystatin, amphotericin B, kan Candicin and hamycin, imidazole (eg miconazole, ketoconazole, clotrimazole, econazole, bifonazole) Bifonazole), butoconazole, fenticonazole, isoconazole, oxiconazole, sertaconazole, sulconazole, thicon Tioconazole, triazole (eg fluconazole, itraconazole, isavuconazole, ravconazole, posaconazole, voriconazole) Voriconazole), terconazole, azole (eg abfungin), allylamine (eg terbinafine, amorolfine, naftifine, Butenafine, echinocandin (eg anidulafungin, caspofungin) ), micafungin, benzoic acid, ciclopirox, tolnaftate, undecylenic acid, flucytosine or 5-fluorocytosine, griseofulvin And haloprogin.

Set

The anti-GITR compositions of the present invention can be provided in a kit. Anti-GITR antibodies, antibody fragments or antigen binding molecules are typically in vials or containers. The antibody can be in a liquid or dry (e.g., lyophilized) form, as desired. The kit may comprise an anti-GITR antibody, antibody fragment or antigen binding molecule of the invention as described herein, and optionally a second or third agent. In some embodiments, the kit comprises an anti-GITR antibody, antibody fragment or antigen binding partner of the invention And a pharmaceutically acceptable diluent. The anti-GITR antibody, antibody fragment or antigen binding molecule can be provided in the kit together with the second or third agent in the same formulation or in separate formulations (eg, in the form of a mixture or in separate containers). The kit may contain aliquots of one or more doses of an anti-GITR antibody, antibody fragment or antigen binding molecule. If an aliquot of multiple administrations is provided, the dosage can be uniform or varied. Varying dosing regimens may be increased or decreased as needed. The dose of the anti-GITR antibody, antibody fragment or antigen binding molecule and the second agent can be independently identical or different.

In some embodiments, the kit can further comprise a target antigen. The target antigen or vaccine depends on the condition of the disease to be treated. For example, the target antigen can be from a tumor cell, a bacterial cell, a fungus, a parasite, or a virus. The target antigen may be in the form of a peptide, polypeptide, cell, polynucleotide (eg, a naked body or a viral vector). In some embodiments, the target antigen is a tumor associated antigen. Exemplary target antigens are as discussed herein; other target antigens known in the art can also be used.

In some embodiments, the kit further contains a cytotoxic agent. For example, a kit can contain an agonist antibody or antigen binding molecule that binds to and reduces or depletes CD4+CD25+ regulatory T cells (Tregs). Exemplary Treg cells deplete antibodies or antigen binding molecules that bind to CD25 or CCR4. See Expert Opin Ther Patents (2007) 17(5): 567-575 and the references discussed therein.

In some embodiments, the kit further comprises an inhibitor of a co-suppression signal. Exemplary inhibitors include inhibitors of CTLA-4 and inhibitors of PD-1/PD-L1 (eg, B7-H1) interactions. In some embodiments, the kit further comprises an antibody that binds to and inhibits CTLA-4. In some embodiments, the kit further comprises an antibody that binds to and inhibits PD-1. In some embodiments, the kit further comprises an antibody that binds to and inhibits B7-H1. See, for example, Expert Opin Ther Patents (2007) 17(5): 567-575; and Melero, et al, Clin Cancer Res (2009) 15(5): 1507-1509 and the references discussed therein.

In some embodiments, the kit further contains one or more immunostimulating agents. For example, in some embodiments, the kit contains an immunostimulatory cytokine, such as IL-7, IL-12 or IL-15. Alternatively, the kit can contain a second immunostimulatory antibody. For example, a kit can contain a agonist antibody or antigen binding molecule of another member of the tumor necrosis factor receptor superfamily. Exemplary second immunostimulatory targets include, but are not limited to, TNFRSF4 tumor necrosis factor receptor superfamily member 4 (also known as OX40) or tumor necrosis factor receptor superfamily member 9 (also known as TNFRSF9, 4-1BB or CD137). See, for example, Expert Opin Ther Patents (2007) 17(5): 567-575; Pardee et al, Immunotherapy (2009) 1(2): 249-264; and Melero et al, Clin Cancer Res (2009) 15(5) : 1507-1509 and the references discussed therein.

In some embodiments, the kit further contains a chemotherapeutic agent. The agent selected depends on the condition to be treated, such as cancer or an infectious disease, such as a bacterial infection, a fungal infection, a viral infection, or a parasitic infection. Exemplary chemotherapeutic agents include any of the anti-tumor, anti-viral, anti-bacterial, anti-parasitic, and anti-fungal agents known in the art and described herein.

Method for increasing T cell response Condition for treatment or prevention

The anti-GITR agonistic antibodies and antibody fragments of the invention can be used to enhance CD4 ⁺ T helper cells and CD8 ⁺ cell lytic T cell responses in patients in need thereof. Thus, antibodies can be used to increase or enhance a patient's T cell response, for example, to achieve a reduction, reversal, inhibition, or prevention of a disease that can be countered by an increased or enhanced immune response. In one aspect, the invention provides a method of increasing a T cell response in an individual in need thereof, comprising administering to the subject a therapeutically effective amount of an anti-GITR agonist antibody or antibody fragment of the invention as described herein. In one aspect, the invention also provides an anti-GITR agonist antibody or antibody fragment for use in increasing an individual's T cell response. In another aspect, the invention provides a composition comprising such an antibody or antibody fragment for use in increasing the T cell response of an individual.

The conditions for treatment include cancer and infectious diseases. For treatment purposes, patients may suffer There are cancers or tumors or infectious diseases such as bacteria, viruses, fungi or parasitic infections. For prophylactic purposes, the patient may be in the relief of cancer or may be expected to be exposed to bacterial, viral, fungal or parasitic infections. The antibody may also act as an adjuvant to enhance or promote or enhance an immune response against a primary antigen or a target antigen (eg, a vaccine).

In some embodiments, the patient has cancer, is suspected of having cancer, or is in the mitigation of cancer. Cancers treated with an anti-GITR antibody typically exhibit a tumor associated antigen (TAA) as described herein. Cancers to be treated include, but are not limited to, epithelial cancer or carcinoma, as well as sarcomas and lymphomas. In some embodiments, the cancer is melanoma, ovarian cancer, renal cancer, colorectal cancer, prostate cancer, lung cancer (including non-small cell lung cancer (NSCLC)), breast cancer, glioma, or fibrosarcoma. See, for example, Pardee et al, Immunotherapy (2009) 1(2): 249-264 and the references discussed therein. In some embodiments, the cancer type is selected from the group consisting of pancreatic cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, gastric cancer, ovarian cancer, bladder cancer, brain cancer, or central nervous system. Cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterus or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or accessory cancer, salivary gland cancer, thyroid cancer, Adrenal cancer, osteosarcoma, chondrosarcoma, blood tissue cancer, and head and neck squamous cell carcinoma (HNSCC).

In one aspect, the invention provides a method of treating tumor growth of a cancer exhibiting a tumor associated antigen in a subject in need thereof, comprising administering to the individual a therapeutically effective amount of an anti-GITR agonist antibody of the invention as described herein Or antibody fragments. The invention also provides an anti-GITR agonist antibody or antibody fragment of the invention for use in treating tumor growth in a subject exhibiting a tumor associated antigen. The invention further provides a composition comprising an antibody or antibody fragment of the invention for use in reducing, inhibiting or preventing tumor growth in a cancer of a subject exhibiting a tumor associated antigen.

In some embodiments, a method of promoting diagnosis or prognosis of an individual's cancer comprises detecting an individual's tumor or week using an anti-GITR agonist antibody or antibody fragment of the invention The performance of the GITR.

In some embodiments, the patient has an infectious disease, such as a bacterial, viral, fungal, or parasitic infection. Anti-GITR agonistic antibodies can be used to reduce, inhibit and/or prevent parasites such as filariasis and leishmaniasis.

In some embodiments, an anti-GITR agonistic antibody can be used to treat a viral infection, including but not limited to a hepatitis virus infection, such as a chronic hepatitis C (HCV) infection, a herpes simplex virus (HSV) infection, or a human immunodeficiency virus ( HIV) infection. In some embodiments, an anti-GITR agonistic antibody can be used to treat a viral infection selected from the group consisting of hepatitis A, hepatitis B, hepatitis C, influenza virus, chickenpox, adenovirus, herpes simplex type I (HSV) I), herpes simplex type II (HSV II), burdock, rhinovirus, echovirus, rotavirus, respiratory tract cell virus, papilloma virus, papovavirus, cytomegalovirus, october virus, Arbovirus, Hantavirus, Coxsackie virus, mumps virus, measles virus, rubella virus, poliovirus, human immunodeficiency virus type I (HIV I) and human immunodeficiency virus type II (HIV II), Any of the picornavirus, enterovirus, caloviridae, norwalk virus populations, chlamydiavirus (such as alphavirus, flavivirus), coronavirus, rabies virus, Marburg virus, Ebola virus , parainfluenza virus, positive mucus virus, bunia virus, ruby virus, reovirus, rotavirus, circovirus, human T cell leukemia virus type I, human T cell leukemia virus type II, monkey immunity Lack of virus, Virus, polyomavirus, a small virus, Epstein - Barr virus, human herpes virus 6, simian herpes virus 1 (B virus) and pox viruses.

In some embodiments, an anti-GITR agonistic antibody can be used to treat a bacterial infection, including but not limited to the following infections: Neisseria, Streptococcus, Streptococcus mutans, Haemophilus, Moraxella , Bacillus, Mycobacterium, Legionella, Escherichia, Vibrio, Yersinia, Aspergillus, Salmonella, Listeria, Helicobacter, Pseudomonas Genus, Staphylococcus, Enterococcus, Clostridium, Bacillus, Corynebacterium, Borrelia, Ehrlichia, Rickettsia, Chlamydia Genus, Leptospira, and Treponema.

Administration of anti-GITR antibodies

The physician or veterinarian can begin administering the antibody or antibody fragment of the invention for use in a pharmaceutical composition at a level below that required to achieve the desired therapeutic effect, and gradually increasing the dosage until the desired effect is achieved. In general, the effective dose of the compositions of the invention will vary depending on a number of different factors, including the particular disease or condition being treated, the method of administration, the target site, the physiological state of the patient, the patient being a human or animal, Other drugs and treatments are preventive or therapeutic. The therapeutic dose needs to be titrated to optimize safety and efficacy. For administration of the antibody, the dosage is in the range of from about 0.0001 to 100 mg per kg of host body weight, and more typically in the range of from 0.01 to 5 mg. For example, the dose can be 1 mg per kilogram of body weight or 10 mg per kilogram of body weight or within the range of 1-10 mg/kg. Administration may be daily, weekly, biweekly, monthly or more frequent or less frequent as needed or as appropriate. An exemplary treatment regimen requires administration once a week or once every two weeks or once every three to six months.

In some embodiments, a polynucleotide encoding an anti-GITR antibody, antibody fragment or antigen binding molecule of the invention is administered. In embodiments where the agent is a nucleic acid, a typical dosage may range from about 0.1 mg per kilogram of body weight to about 100 mg per kilogram of body weight (and including about 100 mg), such as between about 1 mg per kilogram of body weight to about 50 mg per kilogram of body weight. In some embodiments, about 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, or 50 mg per kilogram of body weight.

The antibody or antibody fragment can be administered in a single dose or in divided doses. Antibodies or antibody fragments are usually administered at multiple times. The time interval between single doses may be weekly, biweekly, monthly or yearly as needed or appropriate. The time interval can also be irregular, as indicated by measuring the blood content of the anti-GITR antibody or antibody fragment in the patient. In some methods, the dosage is adjusted to achieve a plasma antibody or antibody fragment concentration of 1-1000 [mu]g/ml, and in some methods 25-300 [mu]g/ml. Alternatively, the antibody or antibody fragment can be administered in the form of a sustained release formulation, in which case no frequent administration is required. Dose and frequency depending on the patient The half-life of the antibody or antibody fragment varies. In general, human antibodies display a half-life that is longer than the half-life of chimeric and non-human antibodies. The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are administered chronically at relatively infrequent intervals. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, it is sometimes desirable to administer relatively high doses at relatively short intervals until the progression of the disease is reduced or terminated, and preferably until the patient exhibits partial or complete improvement in the symptoms of the disease. Thereafter, a prophylactic regimen can be administered to the patient.

Co-administered with the second agent

In some embodiments, an anti-GITR antibody, antibody fragment or antigen binding molecule is co-administered with a second or third pharmacological agent. The anti-GITR antibody, antibody fragment or antigen binding molecule and the second or third agent can be administered as a mixture or as separate formulations. The anti-GITR antibody, antibody fragment or antigen binding molecule and the second or third agent can be administered simultaneously or sequentially. The anti-GITR antibody, antibody fragment or antigen binding molecule and the second or third agent may optionally be administered via the same route of administration or via different routes of administration. Exemplary second and third agents for co-administration with an anti-GITR agonist antibody, antibody fragment or antigen binding molecule of the invention include, but are not limited to, primary or target antigens, and increase immunogenicity of tumor cells An agent, an agent that inhibits or inhibits a co-suppression signal. Anti-GITR agonist antibodies, antibody fragments or antigen binding molecules can also be co-administered with a chemotherapeutic agent for treating the condition being treated, for example to increase the efficacy of the chemotherapeutic agent or to further increase the immune response against the target antigen. Anti-GITR agonist antibodies, antibody fragments or antigen binding molecules can also be used in combination therapy with existing procedures for treating a given disease condition, such as radiation or surgery.

The anti-GITR antibodies, antibody fragments or antigen binding molecules of the invention can be co-administered with a primary or target antigen. The target antigen or vaccine depends on the condition of the disease to be treated. For example, the target antigen can be from a tumor cell, a bacterial cell, a fungus, a virus, or a parasite. The target antigen may be in the form of a peptide, polypeptide, cell or polynucleotide.

In some embodiments, an anti-GITR antibody, antibody fragment or antigen binding molecule is co-administered with a target antigen from, for example, a virus selected from the group consisting of hepatitis A, hepatitis B, hepatitis C, influenza virus , varicella, adenovirus, herpes simplex type I (HSV I), herpes simplex type II (HSV II), burdock, rhinovirus, echovirus, rotavirus, respiratory tract cell virus, papilloma virus, milk lot Bubble virus, cytomegalovirus, ectroscopy virus, arbovirus, hantavirus, coxsackie virus, mumps virus, measles virus, rubella virus, poliovirus, human immunodeficiency virus type I (HIV I) And human immunodeficiency virus type II (HIV II), any small RNA virus family, enterovirus, calicivirus family, norovirus group, chlamy virus (such as alphavirus, flavivirus), coronavirus , rabies virus, Marburg virus, Ebola virus, parainfluenza virus, positive mucus virus, Bunia virus, sand granulosis virus, reovirus, rotavirus, circovirus, human T cell leukemia virus I Type, human T Type II cell leukemia virus, simian immunodeficiency virus, lentivirus, polyomavirus, a small virus, Epstein - Barr virus, human herpes virus 6, simian herpes virus 1 (B virus) and pox viruses.

In some embodiments, an anti-GITR antibody, antibody fragment or antigen binding molecule is co-administered with a target antigen from, for example, a bacterium selected from the group consisting of: Neisseria, Streptococcus, Streptococcus mutans, Haemophilus, Moraxella, Bacillus, Mycobacterium, Legionella, Escherichia, Vibrio, Yersinia, Aspergillus, Salmonella, Listeria Genus, Helicobacter, Pseudomonas, Staphylococcus, Enterococcus, Clostridium, Bacillus, Corynebacterium, Borrelia, Ehrlichia, Rickettsia, Chlamydia Genus, Leptospira, and Treponema.

In some embodiments, an anti-GITR antibody, antibody fragment or antigen binding molecule is co-administered with a tumor associated antigen (TAA). The TAA can be an isolated polypeptide or peptide that can be part of a whole cell or part of a tumor cell lysate. Exemplary TAAs are as described above; other TAAs known in the art can also be used.

In some embodiments, an anti-GITR antibody, antibody fragment or antigen binding molecule is co-administered with an autologous tumor cell from a patient or an allogeneic tumor cell of the same tissue type from another patient. Tumor cells can be in the form of intact cells, tumor cell lysates, apoptotic tumor cells, or total tumor mRNA. Tumor cells can be transfected to express a polypeptide that enhances or enhances the immunogenicity of tumor cells in a patient, for example, transfected to express granulocyte colony stimulating factor (GM-CSF). Tumor cells can be derived from any cancerous tissue including, but not limited to, epithelial cancer or carcinoma, as well as sarcomas and lymphomas. In some embodiments, the cancer is melanoma, ovarian cancer, renal cancer, colorectal cancer, prostate cancer, lung cancer (including non-small cell lung cancer (NSCLC)), breast cancer, glioma, or fibrosarcoma. See, for example, Pardee et al, Immunotherapy (2009) 1(2): 249-264 and the references discussed therein. In one embodiment, the tumor cells are derived, for example, from pancreatic cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, gastric cancer, ovarian cancer, bladder cancer, brain cancer or central nervous system cancer, peripheral nervous system Cancer, esophageal cancer, cervical cancer, uterus or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or accessory cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma , chondrosarcoma, blood tissue cancer and head and neck squamous cell carcinoma (HNSCC).

In some embodiments, an anti-GITR antibody, antibody fragment or antigen binding molecule is co-administered with a cytotoxic agent. For example, an anti-GITR antibody or antigen binding molecule is co-administered with an agonist antibody or antigen binding molecule that binds to and reduces or depletes CD4+CD25+ regulatory T cells (Treg). Exemplary Treg cells deplete antibodies or antigen binding molecules that bind to CD25 or CCR4. See Expert Opin Ther Patents (2007) 17(5): 567-575 and the references discussed therein.

In some embodiments, an anti-GITR antibody, antibody fragment or antigen binding molecule is co-administered with an inhibitor of a co-suppression signal. Exemplary inhibitors include inhibitors of CTLA-4 and inhibitors of PD-1/PD-L1 (eg, B7-H1) interactions. In some embodiments, an anti-GITR antibody is co-administered with an antibody that binds to and inhibits CTLA-4. In some embodiments, an anti-GITR antibody is co-administered with an antibody that binds to and inhibits PD-1. In some embodiments, an anti-GITR antibody is co-administered with an antibody that binds to and inhibits B7-H1. See, for example, Expert Opin Ther Patents (2007) 17(5): 567-575; and Melero et al, Clin Cancer Res (2009) 15(5): 1507-1509 and the references discussed therein.

Anti-GITR antibodies, antibody fragments or antigen binding molecules can also be co-administered with one or more immunostimulatory agents. For example, in some embodiments, an anti-GITR antibody or antibody fragment is co-administered with an immunostimulatory cytokine (eg, IL-7, IL-12, or IL-15). Alternatively, an anti-GITR antibody, antibody fragment or antigen binding molecule can be co-administered with a second immunostimulatory antibody. For example, an anti-GITR antibody, antibody fragment or antigen binding molecule can also be co-administered with an agonist antibody, antibody fragment or antigen binding molecule of another member of the tumor necrosis factor receptor superfamily. Exemplary second immunostimulatory targets include, but are not limited to, TNFRSF4 tumor necrosis factor receptor superfamily member 4 (also known as OX40) or tumor necrosis factor receptor superfamily member 9 (also known as TNFRSF9, 4-1BB or CD137). See, for example, Expert Opin Ther Patents (2007) 17(5): 567-575; Pardee et al, Immunotherapy (2009) 1(2): 249-264; and Melero et al, Clin Cancer Res (2009) 15(5) : 1507-1509 and the references discussed therein.

Anti-GITR antibodies, antibody fragments or antigen binding molecules can also be co-administered with a chemotherapeutic agent. The agent selected depends on the condition to be treated, such as cancer or an infectious disease, such as a bacterial infection, a fungal infection, a viral infection, or a parasitic infection. The anti-GITR antibody, antibody fragment or antigen binding molecule can be co-administered with a chemotherapeutic agent known to those skilled in the art to treat the condition being treated. Exemplary chemotherapeutic agents are as discussed herein; other chemotherapeutic agents known in the art can also be used.

Figures 1A-C illustrate epitope mapping of the GITR mAb of the invention. Figure 1A depicts the results of a hydrogen/deuterium exchange (HDXMS) analysis coupled to a mass spectrometer. The results indicate the CRD1 region of the ECD of the MAB1 (parent mAb) epitope (SEQ ID NO: 88). Figure IB depicts a schematic representation of an N-terminal deletion construct prepared using the extracellular domain of human GITR (hGITR ECD). Figure 1C depicts the results of binding of MAB to hGITR ECD constructs. The N-terminal deletion of the cysteine-rich domain 1 (CRD1) from the extracellular domain (ECD) of human GITR (hGITR) prevents MAB4 and MAB5 from binding to hGITR. Figure 1D depicts the results of alanine scanning mutation induction. In addition to the GITR mutant E78A, MAB7 binds to all mutant proteins. ForteBio ^TM binding assay performed, and the results also confirmed MAB7 not bind to a mutant protein hGITRE78A (not shown).

Figures 2A-2E depict the results of binding experiments with anti-GITR MAB antibodies. MAB4 and MAB5 specifically bind to GITR (A) from humans and macaques, but do not specifically bind to GITR (B) from rodents as determined by ELISA analysis; and MAB7 has a similar profile (C). MAB7 competes with the GITR ligand for competition (D) as determined by FACS competition analysis. ELISA analysis also showed that the anti-GITR antibodies of the invention do not bind to other members of the TNF receptor superfamily (TNFRSF) (E). Protagen ^(TM) wafer analysis also confirmed that these antibodies did not bind to other non-target proteins (not shown).

Figure 3 illustrates intracellular signaling in 293 cells engineered to express GITR. Figure 3A illustrates that intracellular signaling in 293 cells stably transfected to overexpress human GITR by recombinant human GITR ligand (GITR-L) activation. 3B illustrates MAB4 monoclonal antibodies when said antibodies and MAB5 crosslinking GITRL quite transfected by excessive activation of intracellular signaling performance 293 cells of the human GITR (GITRL the EC ₅₀ of approximately 65nM, with under ratio, in the presence of a crosslinker antibody agonist EC ₅₀ of about 2.5nM). Figure 3C illustrates intracellular signaling in activated cells of cross-linked MAB antibodies, as MAB7 and MAB8 also promoted stable transfection of human GITR and NFkB-luciferase reporter genes in 293 cells in a manner similar to cross-linking MAB4. NFκB activation. Figure 3D illustrates that cross-linked MAB4 and MAB5 promote NFκB activation in 293 cells stably transfected with cynomolgus GITR and NFκB-luciferase reporter genes. Cross-linked MAB7 showed similar activation (data not shown).

Figure 4 illustrates that the in vitro costimulatory activity of MAB7 on T cells depends on T cell activation. Anti-CD3 (OKT3), anti-CD28 (CD28.2) and anti-GITR mAbs were cross-linked on beads (at a ratio of 1:1:3) and subsequently incubated with PBMC. MAB7 is a co-activator of CD4+ T cells and stimulates T cell proliferation and cytokine production in CD4+ T cells (4A) and CD8+ T cells (4B) after TCR conjugation, such as IFNy production (4C). Similar results were observed for MAB 4 and 5 (data not shown).

Figures 5A-D illustrate in vitro ADCC activity of MAB7 in different levels of GITR-expressing cells. MAB7 is capable of inducing signaling via FcgRIIIa, with increased activity upon increased GITR signaling levels.

Figure 6 illustrates that GITR functions in the hGITR-hGITRL gene-embedded mouse. Splenocytes were isolated from hGITR-hGITRL gene-embedded mice and cultured for 48 hours without stimulation with or stimulation with CD3 and CD8 antibodies, followed by pulse treatment with different concentrations of control or MAB7 for 30 minutes, followed by fixation and binding of fluorophores The antibody was stained and analyzed by flow cytometry. Figure 6A depicts the results showing up-regulation of hGITR on stimulated CD8+ T cells. Figure 6B depicts the results of anti-hGITR antibodies binding to T cells by hFc staining, which demonstrates that MAB7 can bind to hGITR as expressed on mouse CD8+ T cells. Figures 6C and 6D depict that MAB7 binding to stimulated CD8+ T cells is associated with increased T cell activation, as shown by intracellular pIKK staining (6C) and T cell activation (6D). *p<0.05, **p<0.005.

Figures 7A-C illustrate that MAB7 functions in vivo. hGITR-hGITRL double gene-embedded mice with defined Colon26 tumors were treated with a single dose of vehicle (n=8/time point) or MAB7 (n=10/time point) antibody. 7A depicts twice weekly and tumor measured results using the equation (L × W ²⁾ / ² The tumor volume was calculated. The information shown is from the fifteen (15) day time point group. Figures 7B and 7C depict the results from whole blood, and Figures 7D and 7E depict results from tumors collected 3 days after dosing and analyzed for cell surface hGITR expression on immune cells by flow cytometry. (*p<0.05, ****p<0.00005).

Figure 8 illustrates that MAB7 elicits an anti-tumor immune response against Colon26 tumors in vivo. hGITR-hGITRL double gene-embedded mice with defined Colon26 tumors were treated with a single dose of vehicle (n=8/time point) or MAB7 (n=10/time point). Figure 8A depicts the results of T-regulated cells 3 days after administration. Figures 8B-8C depict the results of lymphocytes (8B) and activated CD8+ T cells (8C) present in the tumor site 15 days after administration according to the treatment level in the tumor. The absolute number of cells was normalized to tumor size to account for the significant difference in tumor size between the vehicle and the MAB7 treated group. Figure 8D depicts the treated animals obtained Teff / Treg ratio, which is CD8 + T cells and CD4 FOXP3 + Treg comparator to generate T _eff / T _reg + was determined by the ratio of the total tumor activated. Figure 8E depicts the results of spleen cell analysis of purified CD8+ T cells from ex vivo culture with Colon26 tumor cells and measuring CTL responses using IFNg ELISPOT assay. (*p<0.05, ***p<0.0005).

Production of GITR agonistic antibodies MAB2, MAB3, MAB4, MAB5, MAB6, MAB7 and MAB8

The human antibodies MAB2, MAB3, MAB4, MAB5, MAB6, MAB7 and MAB8 were produced by engineering a murine monoclonal antibody GITR agonistic antibody MAB1 with greater sequence homology to human germline antibodies. MAB2, MAB3, MAB4, MAB5, MAB6, MAB7 and MAB8 retained the epitope specificity, affinity and GITR cross-reactivity of the parental murine antibody MAB1. Compared to the original murine antibodies, MAB2, MAB3, MAB4, MAB5, MAB6, MAB7 and MAB8 have much higher homology to human germline sequences and should therefore be more tolerated by the human immune system.

(May)) is available on the WWW kalobios.com via KaloBios (South San Francisco, CA ( ) using Humaneered ^® technology platform will MAB1 mouse monoclonal protein sequences engineered to make it closer to human germline sequence and reduce Its immunogenicity. Humaneered ^® close proximity to human antibody antibody, having a human germline region sequence having high sequence homology to V, while still retaining the parent or reference antibody specificity and affinity (U.S. Patent Publication 2005/0255552 and 2006/0134098). The method first identifies the minimal antigen binding specificity determinant (BSD) in the heavy and light chain variable regions of the reference Fab (usually the sequences within the heavy chain CDR3 and light chain CDR3). Since these heavy and light chain libraries constructed BSD maintain all during the process, so that each of the epitopes is a centralized database repository, and the resulting antibody retains the antigen Humaneered ^® original mouse antibody decision Kit sex.

Subsequently, a complete strand library (where the entire light or heavy chain variable region is replaced by a library of human sequences) and/or a cassette library (where one of the heavy or light chain variable regions of the mouse Fab is subjected to human sequences) Library replacement). A bacterial-based secretion system can be used to visualize members of the library as antibody Fab fragments, and a library-bound antigen binding Fab using a community transfer binding assay (CLBA). Positive pure lines can be further characterized to identify pure lines with the highest affinity. In the case of a residual murine sequence, the identified human cassettes that support binding are combined in a final library screen to generate a fully human V region.

The resulting Humaneered ^® Fab antibodies having V region sequences derived from a human library, the sequence of the CDR3 BSD short retention zone identification purposes and having a human germline framework 4 region. The Fabs are converted to intact IgG by culturing the variable regions of the heavy and light chains in an IgG expression vector. Humaneered ^® antibodies of this process retain the binding specificity of the parent murine antibody, which compared to the parent antibody generally has an equal or higher affinity for the antigen, and having a protein level compared to the human germline antibody gene having A highly sequence-consistent V region.

method Production of murine anti-GITR mAb MAB1

The Bcl-2 transgenic mouse (C57BL/6-Tgn (bcl-) was used in the N-terminal region of human GITR (aa 26-161) using a procedure that requires multiple site repeated immunization (RIMMS) (McIntyre GD. Hybridoma 1997). 2) 22 wehi strain) immunization, which in turn produces fusion tumors from high-power mice. The sandwich ELISA and NFκB reporter gene assay for hGITR were used to identify and select fusion tumors secreting MAB1 to confirm hGITR binding and agonistic activity.

Colonization of the murine V region

The variable region from the murine monoculture MAB1 was amplified from the RNA obtained from the fusion tumor cell strain by RT-PCR using standard methods. The heavy chain variable region was amplified from MAB1 cDNA with HV3 (5'-GGGTCTAGACACCATGGCTGTCTTGGGGCTGCTCTTC-3' (SEQ ID NO: 95)) and HC constant (5'-GCGTCTAGAAYCTCCACACACAGGRRCCAGTGGATAGAC-3' (SEQ ID NO: 96)). The light chain variable region was amplified from the same cDNA with LV3 (5'-GGGTCTAGACACCATGGAGWCACAKWCTCAGGTCTTTRTA-3' (SEQ ID NO: 97)) and LC constant (5'-GCGTCTAGAACTGGATGGTGGGAAGATGG-3' (SEQ ID NO: 19)). The variable heavy and light chain products were inserted into the pcDNA3.1 vector and the sequences were verified. The heavy and light chain vectors were used as templates to PCR-incorporate restriction enzyme sites for selection in KaloBios vectors: Vh was cloned at Nco I (5') and Nhe I (3') at KB1292-His ( A modified version of KB1292 encoding a C-terminal flexible linker and a 6-His tag (SEQ ID NO: 11) of the amino acid sequence AAGASHHHHHH (SEQ ID NO: 13) on CH1; Vk at Nco I(5') and Bsi WI (3') were selected in KB1296. These individual heavy and light chain vectors are then combined into a single bicistronic KaloBios Fab expression vector by restriction digestion and ligation with BssHII and ClaI. The Fab fragment was expressed in E. coli from this vector. The hGITR-antigen binding of this Fab was tested and referred to as MAB1rFab.

Fab purification

Fab fragments are expressed by secretion from E. coli using the KaloBios expression vector. The cells were grown in 2 x YT medium to an OD500 of about 0.6. Performance was induced by the addition of 100 mM IPTG and shaking at 33 °C for 4 hours. The assembled Fab was obtained by osmotic dissolution and purification from the periplasmic fraction by affinity chromatography using a Ni-NTA column (HisTrap HP column; GE Healthcare Cat. No. 17-5247-01) according to standard methods. The Fab was dissolved in a buffer containing 500 mM imidazole and thoroughly dialyzed against PBS (pH 7.4) without calcium and magnesium.

Library construction

To limit the complexity of identifying complementary human CDRs that support BSD-FR4 in human GITR binding, the Karma method was used in which only one of the parental murine V regions was replaced by a human sequence library. The initial murine MAB1 Vk is closest to the human germline VkIII, so a mixture of two KaloBios libraries (KB1423 and KB1424) containing the VkIII germline was used in the preparation of the Vk cassette. The KaloBios library (KB1413, KB1414) containing the VH3 germline was used to construct the Vh cassette library. Two types of cassettes were constructed by overlapping PCR: using the above-mentioned germline restriction KaloBios library to amplify FR3 cards containing FR1, CDR1 and FR2 containing human sequences in the front end cassettes (8C1VK3FE-01 and MAB1VH3FE-01) and FR3 containing human sequences.匣 (MAB1VK3FR3-01 and MAB1VH3FR3-01). Each Vh cassette was selected in the vector KB1292-His at Nco I (5') and Kpn I (3'); each Vk cassette was stored in Nco I (5') and Hind III (3') The vector was cloned in the vector KB1296-B (a modified version of the KaloBios vector KB1296, which was added with a silent Hind III site in FR4). Subsequent digestion with Bss HII and Cla I followed by conjugation, combining the resulting Vh or Vk plastid library with the complementary strand from the optimized reference Fab (MAB1opVK or MAB1 opVH) (eg, the Vh front-end library and the most A reference to the Vk vector combination of Jiahua, to obtain a library of bicistronic vectors representing the complete Fab.

The VH3 front-end pure system combines the human GITR with high affinity. Therefore, the second VH3 front-end library (MAB1VH3FE-02) is constructed. This library contains human sequences in the FR1, FR2 and CDR2 populations, wherein the CDR2 population encodes a parent murine residue or a selected human germline residue at all positions. The FR3 region sequence of this library is derived from six pure lines selected from the VH3FR3 library (MAB1VH3FR3-01).

The final Vk complete strand library (MAB1VK3FCL-01) was constructed by combining the pure line and the VKFR3 cassette library from the VK front end, and the mutations in the VKFR3 cassette library induced the VK CDR2 to encode the parent mouse or selected at all positions. Human reproductive system residues. The resulting complete Vk library was cloned into KB1296b at the Nco I and Hind III sites. This VK complete strand library was paired with multiple selected VH3FR3 library lines to allow functional Fab expression and was screened by CLBS. Antigen-specific purity was confirmed by human GITR-specific ELISA and fractionated by antigen affinity titration ELISA. The VH3 complete strand library (MAB1VH3FcL-01) was generated using the selected pure line from the second VH3 front-end library (MAB1VH3FE-02), which has a CDR2 sequence group containing the parent mouse or human residue at each position. base. This VH complete strand library was cloned in KB1292-his at the Nco I and Kpn I sites. To generate the final full-stranded human Fab expression library, the selected VK intact chain pure line was combined with the VH full chain library at the Bss HII and Cla I sites.

Universal ELISA

Recombinant human GITR and human Fc fusion protein (hGITR-hFc) were used for all ELISA analyses. Typically, hGITR-hFc antigen diluted in PBS (pH 7.4) was bound to a 96-well microtiter plate at 200 ng per well by overnight incubation at 4 °C. After washing three times with PBST, the plate was blocked with 1% BSA in PBS for one hour at 37 ° C, followed by rinsing once with PBST. Fab-containing medium or diluted and purified Fab (50 μL) was then added to each well. After incubating for one hour at 37 ° C or overnight at 4 ° C, the plate was rinsed three times with PBST. An anti-human kappa chain HRP conjugate (Sigma #A7164) diluted 1:5000 in PBST (50 μL) was added to each well, and the plate was incubated at room temperature for 45 minutes. The plate was washed three times with PBST, then 100 μL of SureBlue TMB substrate (KPL #52-00-03) was added to each well, and the plate was incubated for about 10 minutes at room temperature. The plates were read at 650 nm in a spectrophotometer.

Affinity titration ELISA

To assess the antigen binding of the selected Fabs to produce pure lines, an affinity titration ELISA was developed. This assay combines two consecutive ELISA steps: the first, using a goat anti-human Fab (Jackson ImmunoResearch Lab #109-005-097) capture and goat anti-human kappa (Sigma #A7164) detector to measure cell culture Fab concentration was used to normalize the amount of Fab used in the second antigen titration ELISA; a second ELISA, a common antigen-specific ELISA, produced an antigen binding dilution curve with the same amount of starting Fab. High-affinity pure lines were identified by comparing dilution curves of different pure lines.

Community transfer combined with ELISA (CLBA)

A nitrocellulose fiber coated with hGITR-hFC in PBS (pH 7.4) at 2.0 μg/mL was used substantially as described in (U.S. Patent Publication Nos. 2005/0255552 and 2006/0134098). The membrane was subjected to screening of the Humaneered® antibody library of Fab fragments. The Fab bound to the antigen-coated filter was detected using a goat anti-human kappa chain HRP conjugate (Sigma #A7164) diluted 1:5000 in PBST, and the ECL plus Western blot detection system (GE Healthcare #RPN2132) Produce ink dots.

Removal of glycosylation sites in MAB4

Removal of the glycosylation site in the junction of FR3 and CDR3 of the MAB4 heavy chain by replacing the lysine with arginine or replacing the lysine with arginine and replacing histidine with aspartame "KH "." Transformation of KH to RH and KH to RN was accomplished by PCR-based mutation induction using p50H plastids as DNA templates. The reverse primer (TCTGGCGCAGTAATACACGGCC, SEQ ID NO: 110) incorporates arginine instead of lysine; the forward primer (NNKGCCTATGGCCATGATGGCG, SEQ ID NO: 111) has degenerate NNK trinucleotides at the histidine site. The PCR reaction was carried out in a 50 μl reaction volume using 100 ng of template, 0.2 μM of each primer, 200 μM dNTP, and 2.5 U of pfu UltraII DNA polymerase (Strategene). The PCR conditions were 94 ° C for 3 minutes, 1 cycle; 94 ° C for 15 seconds, 52 ° C for 20 seconds and 65 ° C for 5 minutes, 30 cycles; and finally at 72 ° C for 5 minutes, 1 cycle. DpnI (2 U) was added to the PCR reaction and incubated at 37 °C for 30 minutes to remove template p50H. The amplified MAB4 heavy chain variants were isolated by 1% SYBR gel and purified using the Qiagen Gel purification kit. The gel purified PCR product was treated with T4 DNA polynucleotide kinase, ligated and transformed into DH5α chemical competent cells (Invitrogen) under ampicillin selection.

The pure line with the MAB7 and MAB8 heavy chains was selected by community PCR using the forward (GCCTTTCTCTCCACAGG, SEQ ID NO: 112) and reverse (GGCAAACAACAGATGGCTGG, SEQ ID NO: 113) primers following the GoTaqClear protocol (Promega). The PCR conditions were 94 ° C for 3 minutes, 1 cycle; 94 ° C for 10 seconds, 55 ° C for 30 seconds, 72 ° C for 45 seconds, 25 times; and finally at 72 ° C for 5 minutes, 1 cycle. The PCR reaction was washed to incubate the sample for 30 minutes at 37 °C with exonuclease I and Shrimp alkaline phosphatase. And incubated at 80 ° C for 15 minutes for sequencing. The PCR samples were sequenced and the results were analyzed using Clone Manager software.

Antibody production and purification

The resulting antibodies MAB2, MAB3, MAB4, MAB5, MAB6, MAB7 and MAB8 (IgG1 kappa) were co-transfected into 293 Freestyle cells by using 293fectin transfection reagent (Invitrogen #51-0031) according to the manufacturer's protocol. produce.

MAB2-p35H+p35κ

MAB3-p38H+p38κ

MAB4-p50H+p35κ

MAB5-p51H+p35κ

MAB6-p56H+p35κ

MAB7-pMAB7H+p35κ

MAB8-pMAB8H+p35κ

The antibody was purified from 293 Freestyle cell supernatant using a 5-mL HiTrap Protein A HP column (GE Healthcare #17-0403-03). The antibody was lysed using IgG Dissolution Buffer (Pierce #21004) and the buffer was exchanged in PBS by dialysis. Protein A affinity chromatography was performed on an AKTA-FPLC liquid chromatography system (GE Healthcare).

Specific ELISA

For specific ELISA, crude cell lysates are made from bacteria that express members of the TNFRSF family. To prevent non-specific binding to the plates, 50 μL of 5% BSA was added per ml of bacterial lysate. A HisGrab 96-well nickel disk (Pierce #15142) was coated with TNFRSF-containing bacterial lysate in 100 μL of lysate/BSA per well and incubated for 1 hour at room temperature. The plate was then washed three times with PBST, then the MAB was diluted to 0.5 μg/mL with 10% FBS in PBS and 100 μL was added to each well. The plates were incubated for 1 hour at room temperature and then washed three times with PBST. Anti-human kappa antibody (Sigma #A7164) bound to HRP was diluted 1:5000 in 1:1 PBST: 10% FBS in PBS and 100 μL was added to each well. The plates were incubated for 1 hour at room temperature and then washed three times with PBST. 100 μL of SureBlue TMB substrate was added to each well, and the plate was incubated at room temperature for about 10 minutes, and then the reaction was terminated with 100 μL of 2N H ₂ SO ₄ per well. The plates were read at 450 nm in a spectrophotometer.

ELISA (GITR binding, species cross-reactivity, alanine scanning)

The binding of MAB to GITR, various alanine mutant constructs or GITR extracellular domains from various species was assessed using a 384-well plate using a rat, mouse, human or macaque GITR extracellular domain (ECD) coated at 50 ng per well. The wells were incubated overnight at 4 °C. The plates were blocked with 1% BSA in PBS for one hour at room temperature and then washed three times with PBST. The MAB was then diluted to 0.5 μg/mL or 1 μg/mL with PBS and 20 μL was added to each well. The plates were incubated for 1 hour at room temperature and then washed three times with PBST. Anti-human kappa antibody (Sigma #A7164), anti-human gamma antibody (Jackson Immunoresearch 109-036-098), goat anti-mouse antibody (Jackson ImmunoResearch 115-035-071) diluted 1:5000 in blocking buffer (25 μL) The conjugate was combined with HRP and added to each well, or HIS antibody (QIAGEN 1014992) in combination with hrp diluted 1:1000 in blocking buffer was added. The plates were incubated for 1 hour at room temperature and then washed six times with PBST. 25 μL of SureBlue TMB (KPL 52-00-02) substrate was added to each well and the plate was incubated for about 10 minutes at room temperature. The plates were read at 650 nm in a spectrophotometer.

Cell line, cell

To generate a 293-hGITR-NFκB reporter gene cell line, 293 cells were stably transfected with the NFκB-luciferase reporter gene and human GITR (or macaque GITR). Activation of the GITR signaling pathway in these cells was determined by measuring intracellularly induced luciferase levels after incubation with GITR-L or agonist antibodies for 24 hours. To assess the effect of cross-linked Ab, it was incubated with excess F(ab') ₂ goat anti-human Fc[gamma] fragment-specific antibody or protein A and subsequently used to report on gene analysis.

Produce a pure Daudi cell line, which can be expressed on human immune cells. See the amount of GITR.

Cynomolgus monkeys were prepared and GITR binding was determined using MAB. Briefly, macaque blood was transferred to a 50 mL conical tube (Falcon, #352098), followed by dilution and mixing with PBS (HyClone, #SH30256.01) 1:2. The diluted blood was carefully layered on 18 mL of 90% Ficoll Paque PLUS (GE Healthcare #17-1440-03, diluted with PBS) and at room temperature with 2,000 x g on a tabletop centrifuge without braking. Rotate the tube for 30 minutes. Carefully remove the plasma layer under the PBMC layer that does not interfere with the dispersion on the Ficoll. PBMC were then carefully collected and PBS was added to the isolated PBMC until the volume in the conical tube was 45 mL, mixed, and then spun at 300 x g in a tabletop centrifuge for 15 minutes at room temperature. The supernatant was carefully aspirated, 4 mL of 1 x BD dissolution solution (BD #555899) was added and the sample was gently vortexed. After incubating for 3 minutes at room temperature in the dark, 40 mL of PBS was added to each sample, and it was rotated at 200 x g in a tabletop centrifuge for 10 minutes at room temperature. The supernatant was carefully aspirated and the particles were washed twice with 45 mL PBS and then spun at 200 x g in a tabletop centrifuge for 10 minutes at room temperature. The resulting granules were filtered and resuspended at 1 × 10 ⁶ cells/ml in CTL test medium (CTLT-005) supplemented with penicillin/streptomycin/glutamic acid (Hyclone #SV30082.01). 100 μL of purified macaque PBMC was placed in a 96-well round chassis (Corning, #3799). To activate PBMC, 100 μL of M-280 toluenesulfonyl-activated dynabead (Life Technologies #142.04) bound to the SP34-2/CD28.2 antibody was added to each well. The plates were incubated for 48 hours in a tissue culture incubator at 37 ° C using a ratio of 3:1 CD3/CD28 beads to PBMC. For day 0 staining, 200 μL of PBMC was placed in a 96-well round chassis (Corning, #3799). For samples incubated for 48 hours, 100 [mu]L of supernatant was carefully removed and the remaining contents of the wells were carefully resuspended and 200 [mu]L was transferred to a FACS stained plate.

FACS

Plates were prepared from cells resuspended in 200 μL of cold PBS. The LIVE/DEAD fixable dye (Life Technologies #L23105) was reconstituted in 50 μL of DMSO, and 1 μL of the reconstituted dye was added per ml of cold PBS, and the cell pellet was immediately resuspended in 100 μL of LIVE/DEAD PBS solution to avoid on ice. The cells were incubated for 30 minutes, then washed and resuspended in 100 μL of cold FACS buffer containing 2 μg/mL MAB7 or homotype human IgG1 control antibody and incubated on ice for 30 minutes in the dark. Wash and resuspend in 100 μL of the following antibody mixture (PerCP Cy5.5 anti-human CD3 (BD #552852), Alexa Fluor 700 anti-human CD4 (BD #560836), V450 anti-human CD8 (BD #561426), PE-Cy7 anti-human In CD25 (BD #561405) and FACS buffer containing PE anti-human (Jackson Immuno #109-116-098), the plate was then incubated on ice for 30 minutes in the dark, followed by 3,200 RPM at 4 °C. Spin in a top centrifuge for 1 minute. The cells were washed with FACS buffer, then resuspended in 100 μL of BD CytoFix (BD #554655) and the plates were incubated for 15 minutes at room temperature in the dark, then washed twice and resuspended in 100 μL of FACS buffer. The plates were covered with foil (Beckman Coulter, #538619) and stored at 4 °C until ready for reading. Date read by FACS, rotating the culture plate for 1 minute to 3,200RPM in a desktop centrifuge, and the latex bead 50μL CML (Life Technologies # C37259) (per ml of FACS buffer and 4 × 10 ⁵ th) was added To each hole. Plates were read on a BD Fortessa flow cytometer and analyzed using FlowJo.

Transgenic mouse

The hGITR gene was inserted into the mouse by replacing the entire coding sequence (exons and introns) of the mouse GITR with the human GITR cDNA sequence. The untranslated sequence upstream of the initiation codon and downstream of the stop codon is derived from the mouse genome. Gene targeting is performed in BALB/c ES cells by standard techniques, wherein the targeting vector has a homologous group derived from BALB/c. Several ES cell lines were identified by PCR and confirmed by Southern blotting to contain precise human cDNA inserts. Following normal mouse embryology techniques, positive ES cells are injected purely into the blastocysts and transferred to a pseudopregnant recipient to nurture the maternal to produce chimeric offspring. Male chimeric mice were crossed with BALB/c female mice expressing Cre recombinase in the germ line to remove the neomycin-resistant cassette flanked by loxP. A pure line produces white offspring indicating target ES cells Can be transmitted by the reproductive system. The card for removing the lateral loxP was confirmed by PCR genotyping. The subsequent feeding step of BALB/c wt mice removes Cre recombinase.

The hGITRL gene was inserted into the mouse by replacing the coding portion of mouse exon 1 with a human GITRL cDNA sequence followed by a bovine growth hormone poly-A signal. All ES cell studies and mouse embryology were performed similarly to the above procedure. The hGITR-hGITRL double gene-embedded mouse was generated by heterologous mating of two basic lines for two generations to generate a homozygous double gene-embedded mouse.

Functional Analysis

The functional activity of MAB was tested in a stimulatory activity NFkB reporter gene assay. MAB was diluted to 6 μg/mL with PBS and incubated for 30 minutes at room temperature in the presence/absence of a 3-fold excess of F(ab') ₂ fragment goat anti-human Fcγ specific crosslinker. Alternatively, MAB was diluted to 6 μg/mL with PBS and incubated for 30 minutes at room temperature in the presence/absence of 2-fold excess protein A. Subsequently, 10 μL of the incubated MAB was added to a 384-well white clear bottom assay disk. The HEK-293 cell line stably transfected with the hGITR and NFκB reporter genes was diluted to 5 × 10 ⁵ cells/ml and 20 μL of the cell suspension was added to each well. The plates were incubated for 24 hours in a tissue culture incubator at 37 °C. 30 μL of Cell Bright Glo was added to each well and the luminescence of the culture plate was read on Acquest.

The ability of MAB to block ligand binding was assessed using HEK293 NFκB reporter parent cells, and hGITR stable cells were used for competitive binding assays and FACS analysis. Briefly, the collected cells were plated at 1 × 10 ⁶ cells per ml, 100 μL per well in a 96-well round bottom FACS plate (Corning), and then resuspended in 200 μL of cold FACS buffer per well (1 × PBS). +1% FBS-HI + 0.1% sodium azide). Human GITR ligand titration was performed at 270 nM to 1.52 pM in FACS buffer at 100 [mu]L per well. The plates were incubated on ice for 1 hour in the dark, the cells were washed, and then 4 nM isotype control was obtained, or MAB solution was prepared and added to appropriate wells at 100 μL per well and the plates were incubated on ice for 1 hour in the dark to wash the cells. Then, PE-conjugated goat anti-human detection antibody (Jackson ImmunoResearch) prepared by diluting 1:100 with FACS buffer was added at 100 μL per well, and the culture plate was incubated on ice for 30 minutes in the dark. The cells were washed with FACS buffer, and then the cells were fixed with 100 μL of BD CytoFix (BD Biosciences) per well and incubated on ice for 15 minutes in the dark. The fixed cells were washed twice, resuspended in a final volume of 150 μL of FACS buffer per well and analyzed on a BD Fortessa flow cytometer (BD Bioscience) within 1 week.

The stimulatory activity of MAB can also be found on primary T cells expressing endogenous levels of GITR via proliferation from primary T cells and cytokine secretion. MABs were bound to M-280 toluenesulfonyl activated beads (Invitrogen #142.04) according to the manufacturer's instructions. In some experiments, agonistic CD3 (OKT3) and CD28 (CD28.2) antibodies were also bound to the beads. 1 x 10 ⁵ freshly purified CFSE-labeled human PBMCs were plated in 10 μL of CTL test medium (CTL #CTLW-010) in a 96-well round bottom tissue culture dish. 100 μL of MAB-bound beads were then added at a ratio of 1 T cell: 1 bead. The plates were then incubated for 3 days in a tissue culture incubator at 37 °C. The amount of cytokine secreted in the medium was measured using MSD multiplex analysis according to the manufacturer's instructions. The cells were stained with anti-CD4, -CD8a, -CD25, -GITR antibodies and LIVE/DEAD dye. After staining, the cells were fixed and read on a flow cytometer. Proliferation of each CD4 and CD8 cells was assessed by CFSE staining and count beads were added followed by FACS reading so that the samples could be normalized.

The costimulatory activity of MAB on T cells was also measured using the ELISpot method to detect IFNg. Briefly, ELISPOT dishes (Millipore MSIPS 4510) were prepared overnight by coating with 70% ethanol for 2 minutes followed by PBS washing and incubation with 50 μg of IFNg monoclonal antibody in PBS (Mabtech 3321-3). Purified CD8+ T cells were isolated from the spleens of vehicle or MAB7 treated mice 15 days after dosing. T cells were plated at 0.25 x 10 ⁶ cells per well in CTL medium (CTL CTLT-005), 1 mM Hepes (Mediatech MT25-060-C1), 2 mM L-breast in coated ELISPOT dishes. Proline (Mediatech MT25-005-Cl), 1 mM sodium pyruvate (Mediatech MT25-000-Cl), 100 μM MEM non-essential amino acid (Mediatech MT25-025-Cl), 66 μM 2-mercaptoethanol (Gibco 21985- 023), 100 U/mL Pten/Strep (Gibco 10378016)). Colon26 cells were treated with 10% ConA sup (BD Biosciences 354115) for 48 hours at 37 ° C to upregulate MHC class II expression and washed with CTL medium and subsequently added to T cells. Colon26 tumor cells (20,000/well) were added to the CTL and incubated at 37 °C for 24-48 hours. The plates were then washed with 0.05% Tween-20/PBS, and 10 μg of biotinylated anti-IFNg Mab (Mabtech R4-6A2-biotin) was added to each well and incubated at 37 °C for 2 hours. The plates were then washed with 0.05% Tween-20/PBS, Vectastain ABC solution (Vector Labs PK6100) was added to each well and incubated for 1 hour at room temperature. The cells were subsequently washed with 0.05% Tween-20/PBS, AEC substrate (Sigma A6926) prepared according to the manufacturer's protocol was added to each well and incubated for 4 minutes at room temperature. The plates were then rinsed with tap water, dried and stored in the dark for 24 hours before being read.

The ability of MAB to induce ADCC was measured using reporter analysis. In a 96-well white plate (Perkin Elmer F6178), 2×10 ³ hGITR-Dodi cells and 4×10 ⁴ Jurkat-V158 cells (V158 variants stably expressing human FcgRIIIa and NFAT reporters) ) were incubated together in a ratio of 1 Doddy cell to 20 Jactech cells in 50 μL RPMI + 10% FBS. Equal volumes of MAB were added to the wells and the plates were incubated for 3 hours in a tissue culture incubator at 37 °C. After incubation, 60 μL of Bright Glo was added to each well and the plate was read on a luminometer.

In vitro spleen cell analysis was performed using spleens isolated from mice. Briefly, by using a gentleMACS Octo dissociator (Miltenyi Biotech 130-095-937) using a gentleMACS C tube (Miltenyi Biotech 130-096-334) in 5 mL of AutoMACS containing 5% BSA (Miltenyi Biotech 130-091-376) Automated homogenization in the rinsing solution (Miltenyi Biotech 130-091-222) dissociated the spleen from the mice. The homogenate was filtered through a 0.70 μM pore size cell strainer (Fisher Scientific 22363548) and washed with 10 mL of AutoMACS buffer. Resuspended spleen cells and coated with 100,000 cells per well RPMI (HyClone SH30027.02) + 10% human serum (Cellgro 35-060.C1) + 1 x Pen/Strep/L-Glut (Gibco 15 140-112) in a 96-well tissue culture dish (Costar 3799). For T cell stimulation, 0.4 μg/mL anti-mouse CD3 (eBioscience 16-0031-86) and 0.8 μg/mL anti-mouse CD28 (eBioscience 13-0281-86) antibody were added to appropriate wells. After 48 hours, cells were immediately assayed or pulsed with control or therapeutic antibodies for 30 minutes to 96 hours, stained with fluorophore-conjugated antibodies and analyzed by flow cytometry.

Flow cytometry: For surface markers, cells were treated with anti-CD19 (BD Biosciences 562291), anti-CD8 (Biolegend 100725), anti-CD4 (eBioscience 25-0041), anti-CD69 (BD Biosciences 561238), anti-resistant at 4 °C. hGITR (Miltenyi Biotech 130-092-895) and anti-hIgG (Life Sciences A-10631) antibody were stained for 30 minutes. For intracellular staining, after incubation with cell surface antibodies, the cells were washed, fixed and permeabilized with FOXP3 Fix/Perm buffer (Biolegend 421403) according to the manufacturer's protocol, and anti-phosphorylated IKKa/b antibody at 4 °C (Cell Signaling 2697) or anti-FOXP3 antibody (eBioscience 50-4774-42) was incubated for 30 minutes. Cells were read on BD LSRFortessa cell counters using BD FACSDiva software (BD Biosciences) and flow data was analyzed using FlowJo software (TreeStar Inc.).

In vivo tumor model. Murine Colon26 carcinoma cells were cultured in RPMI 1640 medium supplemented with 10% FBS (Gibco 10099-141), 10 mM HEPES (Gibco 15630-080) and 1 mM sodium pyruvate (Gibco 11360-070) ( HyClone SH30027.02). The 8-10 week old female hGITR-hGITRL gene was subcutaneously injected into the flank using 100 μL of RPMI containing 0.5×10 ⁶ Colon 26 cells into the mouse. Tumors were measured using a digital caliper gauge and tumor volume was calculated using the equation (L x W ² )/2. When tumors reached an average size of 180 mm ³ , mice were randomized and given a single intraperitoneal injection of vehicle (PBS) or 200 μL of therapeutic antibody (15 mg/kg) in PBS. Seven days after administration of the antibody, the mice were sacrificed and tumors were collected for analysis by flow cytometry. All animal experiments were performed in an AAALAC accreditation body using the IACUC approval program.

Statistical analysis was performed in the Prism software using a one-factor A's t-test with two 95% confidence intervals or a one-way ANOVA with Tukey correction.

result Murine and reference V-region amino acid sequence

RT-PCR products from hybridoma cells expressing MAB1 were sequenced and this sequence was subjected to a substantially (95% or greater than 95%) assay at the protein level using a ThermoElectron LTQ-Orbitrap mass spectrometer. The heavy and light chain variable regions of MAB1 were subsequently cloned into the KaloBios vector to generate a reference Fab MAB1rFab. The first amino acid in MAB1 must be changed from asparagine (N) to a glutamic acid (E) residue to enable colonization in the KaloBios vector to produce a reference Fab; thus, MAB1rFab is in the first VK position With glutamic acid. The Fab MAB1rFab has a complete murine V region from MAB1 fused to a human constant region. In addition to MAB1rFab, a optimized Fab, MAB1opFab, is constructed. Several of the framework amino acid residues in the MAB1rFab become human germline residues in the MAB1 opFab.

Reference and optimized reference Fab affinity analysis

The human germline residues in FR1 and FR3 incorporated into the optimized reference MAB1opFab are residues specified by PCR primers used to amplify the human V region lineage, and therefore are present in all members of the Humaneered® antibody V-region library. in. A optimized reference Fab was constructed to assess whether any changes to the human germline alter the properties of Fab binding. Affinity constants (Ka(1/Ms), Kd(1/s) and KD(M) of MAB1rFab, MAB1opFab were analyzed using a ForteBio Octed QK system and a Striptavidin high binding biosensor coated with biotinylated hGITR-hFc. Compared to MAB1rFab, MAB1opFab has a very similar Kd, but Ka is improved by a factor of five, indicating that the amino acid change in MAB1rFab is permissible.

Library Construction and Selection of Humaneered® Antibody Fab

Production of heavy and light chain front ends and FR3 cassettes (reproductive family limited to VH3 and VKIII) And filtered by CLBA. For VK, the identification of the VK front end (MAB1VK3FE-01) and FR3 (MAB1VK3FR3-01) card library is supported by the pure line of human GITR. For VH, the pure line that binds to human GITR is identified from the FR3 cassette library (MAB1VH3FR3-01) rather than from the VH3 front-end library (MAB1VH3FE-01). Because most of the members of the Vk front-end and FR3 cassettes are positive in CLBA, the entire lineage of the two libraries is used to encode the parental mouse or selected human germline (VKIII L-16) at all locations. Mutation of the residue induces VK CDR2 to perform overlapping PCR to construct the Vk complete strand library (MAB1VK3FcL-01). A variety of hGITR positive lines were identified from the VH3FR3 library (MAB1 VH3FR3-01) via CLBA and confirmed by human GITR specific ELISA. Six of these were paired with the VK Complete Chain Library (MAB1Vk3FcL-01) to enable functional Fab performance and screening of this library.

Since the pure line of hGITR is combined with high affinity from the VH front-end library (MAB1VH3FE-01), the second VH3 front-end library (MAB1VH3FE-02) is subsequently constructed. This library has a parental mouse or human germline (VH3 3-30) residue at each position from the CDR1 and FR3 sequences of the six selected VHFR3 pure lines. Multiple hGITR conjugates were identified from the VK Complete Chain Library (MAB1Vk3FcL-01) and the second VH Front End Library (MAB1VH3FE-02). These pure lines were confirmed by human GITR-specific ELISA analysis of Fab-containing cell supernatants and ranked by hGITR affinity titration ELISA.

Based on the hGITR affinity titration ELISA, four VK intact chain lines were selected from the VK complete chain library (MAB1VK3FcL01), and six pure lines were selected from the MAB1 VH3FE-02 library. The six VH pure lines were used as backbones to construct a complete VH library, where each position of CDR2 has a MAB1 murine or closest to the human germline (VH3 3-30) residue. This VH complete chain lineage was paired with four VK intact chain lines to form the final human full chain Fab library. CLBA identified a number of hGITR-binding homologues which were confirmed by ELISA using individual culture supernatants as Fab sources. Five human complete-chain Fab lines (MAB2, MAB3, MAB4, MAB5 and 5) were selected based on DNA sequence analysis and hGITR affinity titration ELISA results. MAB6).

Testing the affinity of the Humaneered® antibody Fab for GITR antigen using ForteBio Octet assay

Five human intact chain Fabs (MAB2, MAB3, MAB4, MAB5 and MAB6) were expressed and purified. Subsequently, the binding kinetics of these human Fabs were compared to the kinetics of the optimized reference Fab (MAB1opFab) using the ForteBio Octet system (numerical data are summarized in Table 3).

*a, b, and c indicate three separate Forte experiments. The results of the repeated experiments of the two samples were fitted as a whole.

The amino acid sequence of the antibodies MAB2, MAB3, MAB4, MAB5, MAB6 and the percentage of identity with the human germline sequence

The variable region amino acid sequences of the five Fabs (MAB2, MAB3, MAB4, MAB5, MAB6) are shown in Table 1. The percent identity of the five Fab and human germline sequences was determined by aligning the Vh and Vk amino acid sequences to the single germline sequence (VKIII L16/A27 and VH3 3-30, respectively; Table 4). The calculation of each strand omits the residues in CDRH3 and CDRL3.

Conservativeness of human GITR epitopes

The conservation of the epitope was assessed by indirect competition ELISA. Binding of all five Fab-blocking mouse antibody MAB1 to human GITR indicates that these human Fabs retain the epitope specificity of the original murine antibody.

Analysis of antigen specificity of MAB4 and MAB5 by ELISA

To test whether the antigen specificity of the parental mouse antibody MAB1 in IgG (i.e., MAB2, MAB3, MAB4, and MAB5) was retained, the binding of the antibody to a panel of human TNFR was tested in an ELISA assay. The results of one such assay with MAB4 and MAB5 (Fig. 2C) show that MAB4 and MAB5 are similar to the murine antibody MAB1 retaining high specificity for GITR. Similar results were obtained with MAB2, MAB3 and MAB6.

Antibody binding to human and macaque rather than rodent GITR proteins in ELISA

The parental mouse antibody MAB1 binds to human and macaque rather than rodent GITR proteins. 2A-B show that antibodies MAB4 and MAB5 are capable of binding human and macaque GITR rather than rodent GITR in a similar manner as MAB1. Similar results were found with MAB 6, 7, 8, 9, 10, 11, 12 and 13.

The binding affinities of the GITR agonistic antibodies MAB4 and MAB5 to human GITR (hGITR) and cynomolgus GITR (cGITR) were determined by Biacore analysis. See Table 5. The monoclonal antibodies MAB4 and MAB5 bind to human GITR with sub-nemo-binding affinity (KD). The antibodies MAB4 and MAB5 bind to the macaque GITR with a 2-3 fold lower binding affinity than the binding affinity for human GITR. In various biochemical analysis (including flow cytometry, ELISA, Biacore analysis and wafer Protagen ^TM), and anti-GITR agonist antibodies of the present invention selectively bind to human and cynomolgus GITR.

The monoclonal antibody MAB7 binds to human and macaque CD4+ T cells. FACS analysis of isolated macaques or human PBMCs revealed that MAB7 binds to isolated CD4+ T cells. In addition, FACS experiments revealed that GITR (due to MAB7 binding) and up-regulation of CD25 after CD3/CD28 activation of PBMC (CD4+ T cells). (data not shown)

Reporting the functional activity of antibodies in gene analysis and cell analysis

The functional activity of the antibodies was analyzed in a reporter gene assay (Figure 3). MAB4, MAB5, MAB7 and MAB8 IgG each induced NFκB activity at the level similar to the GITR ligand (GITR-L) at the time of cross-linking. See Figures 3A-D. Similar results were obtained with MAB2, MAB3 and MAB6 (data not shown).

MAB7 competes with human GITR ligands for binding to human GITR-expressing stable cell lines. Competition analysis was performed in three sets of values, and FACS competition analysis revealed inhibition of ligand binding. See Figure 2D.

To confirm functional activity on endogenous GITR, antibodies were conjugated to beads and incubated with purified CFSE-labeled human PBMC. MAB7 induced an increase in proliferation of CD4+ T cells (Fig. 4A) and CD8+ T cells (Fig. 4B) compared to isotype control antibodies. This increase in proliferation is also accompanied by increased secretion of several cytokines, including IFNy (Fig. 4C), TNF[alpha], IL-10 and IL-13 (not shown). Similar results were found with MAB 4, MAB5 (not shown). We were able to demonstrate that MAB7-induced proliferation and increased IFNy production depend on the presence of anti-CD3 and anti-CD28 agonist antibodies on the beads. Without such co-stimulatory antibodies, MAB has no agonistic effect on CD4+ or CD8+ T cells. Similar results were obtained with MAB2, MAB3, MAB4, MAB5 and MAB6.

It has also been found in in vitro assays that MAB7 displays the ability to induce FcgRIIIa signaling, which is shown to be associated with ADCC activity, in the presence of high levels of GITR. Dodge-hGITR cells were incubated with MAB7 or control Ab and the Jaccarat-V158 cell line was displayed MAB7 is able to induce FcgRIIIa signaling in an in vitro assay, and the ability of MAB7 to induce FcgRIIIa signaling is related to the level of receptors expressed on the surface of the Doddy cells (ie, higher receptor levels are equivalent to larger FcgRIIIa signaling). Induction). See Figure 5.

hGITR is expressed on T cells and functions in the hGITR-hGITRL gene-embedded mouse. Splenocytes were isolated from wild-type or hGITR-hGITRL gene-embedded mice and cultured for 24, 48, 72 or 96 hours without stimulation or with stimulation with CD3 and CD28 antibodies. Cells were subsequently stained with fluorophore-conjugated antibodies and analyzed by flow cytometry to demonstrate human GITR expression, and co-stimulation increased GITR expression profiles in wild-type or transgenic mice. Spleen cells isolated from the hGITR-hGITRL gene-inserted mouse showed induction of GITR expression in culture in response to co-stimulation (Fig. 6A). MAB7 efficiently binds to hGITR expressed on CD8+ cells (Fig. 6B); and as measured by pIKK staining (Fig. 6C) and T cell activation marker CD25+ (Fig. 6D), binding and increase of MAB7 to stimulated T cells It is related to T cell activation.

MAB7 functions in vivo. The hGITR-hGITRL double gene-embedded mice with defined Colon26 tumors were treated with a single dose of vehicle (n=8/time point) or MAB7 (n=10/time point) antibody as described above. Tumors were measured twice a week and tumor volume was calculated using the equation (L x W ² )/2. MAB treated animals exhibited a delay in colon26 tumor growth. Three days after treatment, whole blood (Fig. 7B-7C) and tumors (Fig. 7D-7E) were collected and cell surface hGITR expression on immune cells was analyzed by flow cytometry. The results indicated that hGITR occupied and fell off, thereby reducing hGITR from the treatment group for T-regulated cells and T helper cells in blood and tumors (*p<0.05, ****p<0.00005).

MAB7 elicits an anti-tumor immune response against Colon26 tumors in vivo. hGITR-hGITRL double gene-embedded mice with defined Colon26 tumors were treated with a single dose of vehicle (n=8/time point) or MAB7 (n=10/time point). Figure 8A depicts the results 3 days after dosing showing a decrease in Tregs in treated animals. Figures 8B-8C depict the results 15 days after dosing, showing increased lymphocytes (8B) and increased activated CD8+ T cells after treatment. (8C) is present in the tumor site. The absolute number of cells was normalized to tumor size to account for the significant difference in tumor size between the vehicle and the MAB7 treated group. MAB7 results indicate that treatment increases the Teff/Treg ratio in treated animals as determined by comparing total intratumoral activated CD8+ T cells to CD4+ FOXP3+ Treg. See Figure 8D. In addition, the results from spleen cell analysis in which purified CD8+ T cells were cultured ex vivo with Colon26 tumor cells and CTL responses were measured using IFNg ELISPOT assay indicated an increase in tumor-specific IFNg response in CD8+ T cells from MAB7 treated animals. (*p<0.05, ***p<0.0005). See Figure 8E.

It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications and variations are intended to be included within the spirit and scope of the application and the scope of the accompanying claims. Within the scope of this. All publications, serial accession numbers, patents, and patent applications cited herein are hereby incorporated by reference in their entirety in their entirety herein

SEQ ID NO: 1-GITR isoform 1 precursor (GenBank No. NP_004186.1)

SEQ ID NO: 2-GITR isoform 2 precursor (GenBank No. NP_683699.1)

SEQ ID NO: 3-GITR isoform 3 precursor (GenBank No. NP_683700.1)

SEQ ID NO: 4-GITR cysteine-rich domain 1 (CRD1), residues 34-65

SEQ ID NO: 5 - GITR antibody binding region, residues 41-65

SEQ ID NO: 6-Amino acid sequence of the heavy chain variable region (FR1 to FR4) of MAB2

SEQ ID NO: 7-MAB2, MAB4, MAB5, MAB6, MAB7 MAB8 and light chain variable regions (FR1 to FR4) amino acid sequence of

SEQ ID NO: 8-amino acid sequence of the heavy chain variable region (FR1 to FR4) of MAB3

SEQ ID NO: 9-amino acid sequence of the light chain variable region (FR1 to FR4) of MAB3

SEQ ID NO: 10 - Amino acid sequence of the heavy chain variable region (FR1 to FR4) of MAB4

SEQ ID NO: 11 - hexahistamine, 6-His marker

SEQ ID NO: 12 - amino acid sequence of the heavy chain variable region (FR1 to FR4) of MAB5

SEQ ID NO: 13-C-terminal flexible linker and 6-His marker

SEQ ID NO: 14 - Amino acid sequence of the heavy chain variable region (FR1 to FR4) of MAB6

SEQ ID NO: 15-5-aa Gly-Ser Linker

SEQ ID NO: 16 - Common heavy chain variable region (FR1 to FR4)

SEQ ID NO: 17 - Common light chain variable region (FR1 to FR4)

SEQ ID NO: 18-15-aa Gly-Ser Linker, (GGGGS) ₃

SEQ ID NO: 19 - Light chain variable region RT-PCR amplification primer LC constant region

SEQ ID NO: 20 - heavy chain IgG1 constant region, the crude Leu-Leu residue may also be Ala-Ala

SEQ ID NO: 21 - kappa chain

Amino acid sequence of CDRH1 of SEQ ID NO: 22-MAB1-8 (Kabat)

Amino acid sequence of CDRH2 of SEQ ID NO: 23-MAB2 (Kabat)

SEQ ID NO: 24-amino acid sequence of CDRH2 of MAB3 (Kabat)

Amino acid sequence of CDRH2 of SEQ ID NO: 25-MAB4 (Kabat)

Amino acid sequence of CDRH2 of SEQ ID NO:26-MAB5 (Kabat)

Amino acid sequence of CDRH2 of SEQ ID NO:27-MAB6 (Kabat)

SEQ ID NO:28 - Common Heavy Chain CDR2 (Kabat)

Amino acid sequence of CDRH3 of SEQ ID NO:29-MAB1-7 (Kabat/Chothia)

Amino acid sequence of CDR1 of SEQ ID NO: 30-MAB2, MAB4, MMABS, MAB6, MAB7 and MAB8 (Kabat)

SEQ ID NO: 31 - amino acid sequence of CDRL1 of MAB3 (Kabat)

SEQ ID NO: 32 - Common Light Chain CDR1 (Kabat)

SEQ ID NO: 33 - amino acid sequence of CDRL2 of MAB2-8 (Kabat)

Amino acid sequence of CDRs of SEQ ID NO: 34-MAB1-8 (Kabat)

SEQ ID NO: 35-Amino acid sequence of heavy chain FR1 of MAB2 and MAB3

Amino acid sequence of heavy chain FR1 of SEQ ID NO: 36-MAB4, MAB5, MAB6, MAB7 and MAB8

SEQ ID NO:37 - Amino acid sequence of the common heavy chain FR1

Amino acid sequence of heavy chain FR2 of SEQ ID NO: 38-MAB2, MAB4, MAB7 and MAB8

SEQ ID NO: 39 - amino acid sequence of heavy chain FR2 of MAB3, MAB5 and MAB6

SEQ ID NO: 40 - Common heavy chain FR2

SEQ ID NO: 41 - Amino acid sequence of heavy chain FR3

SEQ ID NO: 42 - amino acid sequence of heavy chain FR4

SEQ ID NO:43 - Amino acid sequence of light chain FR1

Amino acid sequence of light chain FR2 of SEQ ID NO: 44-MAB2, MAB4, MAB5, MAB6, MAB7 and MAB8

SEQ ID NO: 45 - amino acid sequence of light chain FR2 of MAB3

SEQ ID NO:46 - Common Light Chain FR2

SEQ ID NO: 47 - Amino acid sequence of light chain FR3 of MAB2, MAB4, MAB5, MAB6, MAB7 and MAB8

SEQ ID NO: 48-amino acid sequence of light chain FR3 of MAB3

SEQ ID NO: 49 - Common Light Chain FR3

SEQ ID NO: 50 - amino acid sequence of light chain FR4

SEQ ID NO: 51 - MAB2 VH nucleic acid sequence

Nucleic acid sequence of SEQ ID NO: 52-MAB2, MAB4, MAB5 and MAB6 VK

Nucleic acid sequence of SEQ ID NO: 53-MAB3 VH:

Nucleic acid sequence of SEQ ID NO: 54-MAB3 VK:

Nucleic acid sequence of SEQ ID NO: 55-MAB4 VH:

Nucleic acid sequence of SEQ ID NO: 56-MAB5 VH:

Nucleic acid sequence of SEQ ID NO: 57-MAB6 VH:

SEQ ID NO: 58 - Nucleic acid sequence of the variable region of parental mouse Vk

SEQ ID NO: 59 - Amino acid sequence of the variable region of parental mouse Vk

SEQ ID NO: 60 - Nucleic acid sequence of the variable region of the parental mouse VH

SEQ ID NO: 61 - Amino acid sequence of the variable region of parental mouse VH

Amino acid sequence of CDRH2 of SEQ ID NO: 62-MAB1 (Kabat)

SEQ ID NO: 63 - amino acid sequence of CDRL1 of MAB1 (Kabat)

SEQ ID NO: 64-amino acid sequence of CDRL2 of MAB1 (Kabat)

SEQ ID NO: 65-Amino acid sequence of the heavy chain of MAB2

Amino acid sequence of the light chain of SEQ ID NO: 66-MAB2, MAB4, MAB5, MAB6, MAB7 and MAB8

SEQ ID NO:67 - Nucleic acid sequence encoding the heavy chain of MAB2

SEQ ID NO:68 - Nucleic acid sequence encoding the light chain of MAB2, MAB4, MAB5 and MAB6

Amino acid sequence of the heavy chain of SEQ ID NO: 69-MAB3

Amino acid sequence of the light chain of SEQ ID NO: 70-MAB3

SEQ ID NO:71 - Nucleic acid sequence encoding the heavy chain of MAB3

SEQ ID NO:72 - Nucleic acid sequence encoding the light chain of MAB3

SEQ ID NO: 73-Amino acid sequence of the heavy chain of MAB4

SEQ ID NO:74 - Nucleic acid sequence encoding the heavy chain of MAB4

Amino acid sequence of the heavy chain of SEQ ID NO: 75-MAB5

SEQ ID NO:76 - Nucleic acid sequence encoding the heavy chain of MAB5

Amino acid sequence of the heavy chain of SEQ ID NO: 77-MAB6

SEQ ID NO:78 - Nucleic acid sequence encoding the heavy chain of MAB6

SEQ ID NO: 79 - amino acid sequence of CDRH1 of MAB1 (Chothia)

Amino acid sequence of CDRH2 of SEQ ID NO: 80-MAB1-8 (Chothia)

SEQ ID NO: 81 - amino acid sequence of CDRL1 of MAB1 (Chothia)

Amino acid sequence of CDR1 of SEQ ID NO: 82-MAB1-8 (Chothia)

SEQ ID NO: 83 - amino acid sequence of CDRL3 of MAB1-8 (Chothia)

Amino acid sequence of CDRH1 of SEQ ID NO: 84-MAB2-8 (Chothia)

Amino acid sequence of CDR1 of SEQ ID NO:85-MAB2, MAB4, MAB5, MAB6, MAB7 and MAB8 (Chothia)

SEQ ID NO: 86 - amino acid sequence of CDRL1 of MAB3 (Chothia)

SEQ ID NO:87 - Common amino acid sequence of CDRL1 (Chothia)

SEQ ID NO: 88 - cysteine-rich domain 1 (CRD1) region of the extracellular domain (ECD) of GITR

SEQ ID NO:89 - Human germline sequence of heavy chain V region (VH33-13/30)

SEQ ID NO: 90 - Human Germline JH4 Partial Sequence

SEQ ID NO:91 - Full length J region from human germline JH4

SEQ ID NO: 92 - Human germline sequence of the light chain V region (VKIII L16/A27)

SEQ ID NO:93 - Human germline Jk2 partial sequence

SEQ ID NO:94 - Full length J region from human germline Jk2

SEQ ID NO: 95 - Heavy chain variable region RT-PCR amplification primer HV3

SEQ ID NO:96-heavy chain variable region RT-PCR amplification primer HC constant region

SEQ ID NO:97-Light chain variable region RT-PCR amplification primer LV3

SEQ ID NO: 98 - Human Germline Variable Region (VKIII L16/A27)

Amino acid sequence of the heavy chain variable region of SEQ ID NO:99-MAB7

SEQ ID NO: 100-MAB7 heavy chain amino acid sequence

Nucleic acid sequence of SEQ ID NO: 101-MAB7 VH

Nucleic acid sequence of SEQ ID NO: 102-MAB7/MAB8 VL

SEQ ID NO: 103 - Nucleic acid sequence of MAB7 heavy chain

Nucleic acid sequence of SEQ ID NO: 104-MAB7/MAB8 light chain

SEQ ID NO: 105-MAB8 heavy chain variable region amino acid sequence

SEQ ID NO: 106 - Heavy chain amino acid sequence of MAB8

SEQ ID NO:107-MAB8 VH nucleic acid sequence

SEQ ID NO: 108 - Nucleic acid sequence of MAB8 heavy chain

Amino acid sequence of CDRH3 of SEQ ID NO: 109-MAB8

SEQ ID NO: 110 - Introduction

SEQ ID NO: 111 - Introduction

SEQ ID NO: 112 - Introduction

SEQ ID NO: 113 - Introduction

Claims (33)

An isolated antibody or antibody fragment or antigen binding molecule thereof, which binds to SEQ ID NO: 4, and wherein the antibody, antibody fragment or antigen binding molecule comprises (a) a heavy chain variable region, wherein: i) heavy chain CDR1 SEQ ID NO: 22 is included, and ii) the heavy chain CDR2 comprises any one selected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27. a sequence, and iii) the heavy chain CDR3 comprises SEQ ID NO: 29 or SEQ ID NO: 109; and (b) a light chain variable region, wherein i) the light chain CDR1 comprises SEQ ID NO: 30 or SEQ ID NO: 31 And ii) the light chain CDR2 comprises SEQ ID NO:33, and iii) the light chain CDR3 comprises SEQ ID NO:34. The antibody, antibody fragment or antigen-binding molecule thereof of claim 1, wherein the heavy chain variable region has at least 95% sequence identity to SEQ ID NO: 16, and wherein the light chain variable region is SEQ ID NO: Has at least 95% sequence identity. The antibody, antibody fragment or antigen-binding molecule of claim 1, wherein the heavy chain FR4 is human germline FR4 and/or the light chain FR4 is human germline FR4. The antibody, antibody fragment or antigen-binding molecule of claim 3, wherein the heavy chain FR4 is SEQ ID NO: 42 and/or the light chain FR4 is SEQ ID NO: 50. The antibody, antibody fragment or antigen-binding molecule of claim 1, wherein: A: i) the heavy chain CDR1 comprises SEQ ID NO: 22, ii) the heavy chain CDR2 comprises SEQ ID NO: 23, iii) the heavy chain CDR3 Included in SEQ ID NO:29, iv) the light chain CDR1 comprises SEQ ID NO:30, v) the light chain CDR2 comprises SEQ ID NO: 33, and vi) the light chain CDR3 comprises SEQ ID NO: 34; or B: i) the heavy chain CDR1 comprises SEQ ID NO: 22, ii) the heavy chain CDR2 comprises SEQ ID NO: 24, iii) the heavy chain CDR3 comprises SEQ ID NO: 29, iv) the light chain CDR1 comprises SEQ ID NO: 31, v) the light chain CDR2 comprises SEQ ID NO: 33, and vi) The chain CDR3 comprises SEQ ID NO: 34; or C: i) the heavy chain CDR1 comprises SEQ ID NO: 22, ii) the heavy chain CDR2 comprises SEQ ID NO: 25, iii) the heavy chain CDR3 comprises SEQ ID NO: 29 Iv) the light chain CDR1 comprises SEQ ID NO: 30, v) the light chain CDR2 comprises SEQ ID NO: 33, and vi) the light chain CDR3 comprises SEQ ID NO: 34; or D: i) the heavy chain CDR1 Included in SEQ ID NO: 22, ii) the heavy chain CDR2 comprises SEQ ID NO: 26, iii) the heavy chain CDR3 comprises SEQ ID NO: 29, iv) the light chain CDR1 comprises SEQ ID NO: 30, v) the light The chain CDR2 comprises SEQ ID NO: 33, and vi) the light chain CDR3 comprises SEQ ID NO: 34; or E: i) the heavy chain CDR1 comprises SEQ ID NO: 22, ii) the heavy chain CDR2 comprises SEQ ID NO: 27, iii) the heavy chain CDR3 comprises SEQ ID NO: 29, iv) the light chain CDR1 comprises SEQ ID NO: 30, v) the light chain CDR2 comprises SEQ ID NO: 33, and vi) the light chain CDR3 comprises SEQ ID NO: 34; or F: i) the heavy chain CDR1 comprises SEQ ID NO: 22, ii) the heavy chain CDR2 comprises SEQ ID NO: 25, iii) the heavy chain CDR3 comprises SEQ ID NO: 109, iv) the light chain CDR1 comprises SEQ ID NO: 30, v) the light chain CDR2 comprises SEQ ID NO: 33, and vi) the light chain CDR3 comprises SEQ ID NO: 34. The antibody, antibody fragment or antigen-binding molecule of claim 1 or 2, wherein the antibody or antibody fragment comprises an isolated antibody comprising the heavy chain of SEQ ID NO: 16 and a light chain comprising SEQ ID NO: 17 or an antigen thereof The fragment is either contiguous with an isolated antibody or antigen-binding fragment thereof comprising a heavy chain comprising SEQ ID NO: 16 and a light chain comprising SEQ ID NO: 17. The antibody, antibody fragment or antigen-binding molecule of claim 1, wherein the antibody, antibody fragment or antigen-binding molecule comprises a molecule selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO a heavy chain variable domain of the sequence of any one of SEQ ID NO: 14, SEQ ID NO: 99, and SEQ ID NO: 105; and a light chain comprising SEQ ID NO: 7 or SEQ ID NO: Variable domain. The antibody, antibody fragment or antigen-binding molecule of claim 7, wherein the antibody, antibody fragment or antigen-binding molecule comprises any one of the following: i) a heavy chain variable domain comprising SEQ ID NO: 6 and comprising the SEQ ID NO: a light chain variable domain of 7; ii) a heavy chain variable domain comprising SEQ ID NO: 8 and a light chain variable domain comprising SEQ ID NO: 9; iii) a heavy chain comprising SEQ ID NO: a variable domain and a light chain variable domain comprising SEQ ID NO: 7; iv) a heavy chain variable domain comprising SEQ ID NO: 12 and a light chain variable domain comprising SEQ ID NO: v) a heavy chain variable domain comprising SEQ ID NO: 14 and a light chain variable domain comprising SEQ ID NO: 7; vi) a heavy chain variable domain comprising SEQ ID NO: 99 and comprising SEQ ID NO: The light chain variable domain; and vii) comprises the heavy chain variable domain of SEQ ID NO: 105 and the light chain variable domain comprising SEQ ID NO: 7. The antibody, antibody fragment or antigen-binding molecule of claim 1, wherein the antibody or antibody fragment or antigen-binding molecule is humanized. An antibody, antibody fragment or antigen binding molecule according to claim 1, which comprises a Fab' fragment and/or a single chain antibody (scFv). The antibody, antibody fragment or antigen binding molecule of claim 1, wherein the antibody or antibody fragment comprises an IgG Fc and/or a human constant region. The antibody, antibody fragment or antigen-binding molecule of claim 11, wherein the antibody, antibody fragment or antigen-binding molecule comprises a molecule selected from the group consisting of SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO The heavy chain of the sequence of any one of SEQ ID NO: 77, SEQ ID NO: 100, and SEQ ID NO: 106; and the light chain comprising SEQ ID NO: 66 or SEQ ID NO: 70. The antibody, antibody fragment or antigen-binding molecule of claim 1, wherein the antibody or antibody fragment is cross-linked to a second antibody or antibody fragment, and the antibody, antibody fragment or antigen-binding molecule is SEQ ID NO: 1, SEQ ID NO: 2 or an agonist of SEQ ID NO: 3. An isolated antibody or antibody fragment or antigen binding molecule thereof, which binds to SEQ ID NO: 4, which is selected from any one of the following: A. an antibody, antibody fragment or antigen binding molecule, wherein: i) a heavy chain CDR1 comprises SEQ ID NO: 22, ii) heavy chain CDR2 comprises SEQ ID NO:23, Iii) heavy chain CDR3 comprises SEQ ID NO:29, iv) light chain CDR1 comprises SEQ ID NO:30, v) light chain CDR2 comprises SEQ ID NO:33, and vi) light chain CDR3 comprises SEQ ID NO:34; An antibody, antibody fragment or antigen binding molecule, wherein: i) the heavy chain CDR1 comprises SEQ ID NO: 22, ii) the heavy chain CDR2 comprises SEQ ID NO: 24, iii) the heavy chain CDR3 comprises SEQ ID NO: 29, iv The light chain CDR1 comprises SEQ ID NO: 31, v) the light chain CDR2 comprises SEQ ID NO: 33, and the vi) light chain CDR3 comprises SEQ ID NO: 34; C. an antibody, antibody fragment or antigen binding molecule, wherein: i) heavy chain CDR1 comprises SEQ ID NO: 22, ii) heavy chain CDR2 comprises SEQ ID NO: 25, iii) heavy chain CDR3 comprises SEQ ID NO: 29, iv) light chain CDR1 comprises SEQ ID NO: 30, v) The light chain CDR2 comprises SEQ ID NO: 33, and vi) the light chain CDR3 comprises SEQ ID NO: 34; D. an antibody, antibody fragment or antigen binding molecule, wherein: i) the heavy chain CDR1 comprises SEQ ID NO: 22, ii The heavy chain CDR2 comprises SEQ ID NO: 26, iii) the heavy chain CDR3 comprises SEQ ID NO: 29, iv) the light chain CDR1 comprises SEQ ID NO: 30, the v) light chain CDR2 comprises SEQ ID NO: 33, and vi) The light chain CDR3 comprises SEQ ID NO: 34; E. , Antibody fragment, or antigen-binding molecules, wherein: i) heavy chain CDR1 comprises SEQ ID NO: 22, ii) heavy chain CDR2 comprises SEQ ID NO: 27, iii) heavy chain CDR3 comprises SEQ ID NO: 29, iv) light chain CDR1 comprises SEQ ID NO: 30, v) The light chain CDR2 comprises SEQ ID NO: 33, and vi) the light chain CDR3 comprises SEQ ID NO: 34; and F. an antibody, antibody fragment or antigen binding molecule, wherein: ii) the heavy chain CDR1 comprises SEQ ID NO: Ii) heavy chain CDR2 comprises SEQ ID NO: 25, iii) heavy chain CDR3 comprises SEQ ID NO: 109, iv) light chain CDR1 comprises SEQ ID NO: 30, v) light chain CDR2 comprises SEQ ID NO: 33, and vi The light chain CDR3 comprises SEQ ID NO:34. The antibody, antibody fragment or antigen-binding molecule of claim 14, wherein the antibody or antibody fragment or antigen-binding molecule is humanized. An antibody, antibody fragment or antigen binding molecule according to claim 14, which comprises a Fab' fragment and/or a single chain antibody (scFv). The antibody, antibody fragment or antigen binding molecule of claim 14, wherein the antibody or antibody fragment comprises an IgG Fc and/or a human constant region. The antibody, antibody fragment or antigen-binding molecule of claim 17, wherein the antibody, antibody fragment or antigen-binding molecule comprises a molecule selected from the group consisting of SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO The heavy chain of the sequence of any one of SEQ ID NO: 77, SEQ ID NO: 100, and SEQ ID NO: 106; and the light chain comprising SEQ ID NO: 66 or SEQ ID NO: 70. An antibody, antibody fragment or antigen-binding molecule according to claim 14, wherein the antibody or antibody fragment is cross-linked with a second antibody or antibody fragment, the antibody, antibody fragment or antibody The pro-binding molecule is an agonist of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. The antibody, antibody fragment or antigen-binding molecule of claim 1 or 14, wherein the antibody or antibody fragment is glycosylated. The antibody, antibody fragment or antigen-binding molecule of claim 1 or 14, wherein the antibody is modified or expressed in a modified cell, wherein such modification increases the FcR effector function of the antibody, antibody fragment or antigen-binding molecule. The antibody, antibody fragment or antigen-binding molecule of any one of claims 1 to 21, wherein i) the antibody or antibody fragment induces an increased Teff:Treg ratio in vivo; ii) the antibody or antibody fragment is induced in vivo An enhanced immune response; and/or iii) the antibody cross-reacts with the non-human primate GITR and does not cross-react with the rodent GITR. A polynucleotide encoding the antibody, antibody fragment or antigen-binding molecule of any one of claims 1 to 21. A composition comprising the isolated antibody, antibody fragment or antigen-binding molecule of any one of claims 1 to 21, and a pharmaceutically acceptable carrier. The composition of claim 24, which further comprises a binding molecule that targets a tumor associated antigen, an antagonist of CTLA4, or an inhibitor of PD-1/PD-L1 interaction. A kit comprising the antibody, antibody fragment or antigen-binding molecule of any one of claims 1 to 21. Use of an isolated antibody, antibody fragment or antigen-binding molecule according to any one of claims 1 to 21 for the manufacture of a medicament for increasing a T cell response in an individual in need thereof. Use of an isolated antibody, antibody fragment or antigen-binding molecule according to any one of claims 1 to 21 for the manufacture of a tumor-producing tumor in a subject in need thereof A drug for tumor growth of an antigenic cancer. The use of claim 27 or 28, wherein the antibody, antibody fragment or antigen-binding molecule is an inhibitor of i) antigen, an antagonist of CTLA4 or PD-1/PD-L1, ii) a chemotherapeutic agent or Cytotoxins, iii) cancer cells from individuals and/or iv) tumor-associated antigens are co-administered. The use of claim 27 or 28, wherein the T cell response is a CD8+ cytotoxic T lymphocyte (CTL) T cell response or a CD4+ helper T cell (Th) response. The use of claim 30, wherein the cancer is selected from the group consisting of melanoma, ovarian cancer, colorectal cancer, prostate cancer, non-small cell lung cancer (NSCLC), breast cancer, and head and neck squamous cell carcinoma (HNSCC). The isolated antibody, antibody fragment or antigen-binding molecule of any one of claims 1 to 21 for use in increasing a T cell response in an individual in need thereof. The isolated antibody, antibody fragment or antigen binding molecule of any one of claims 1 to 21 for use in treating tumor growth in an individual in need thereof.