The present application claims U.S. provisional application serial No. 61/973,193 filed 3/31/2014; U.S. provisional application serial No. 61/989,448 filed 5/6/2014; U.S. provisional application serial No. 62/073,873 filed on 31/10/2014; and U.S. provisional application serial No. 62/080,171 filed on 11/14/2014, each of which is hereby incorporated by reference in its entirety.
The contents of the following submissions on an ASCII text file are incorporated herein in their entirety by reference: sequence Listing in Computer Readable Form (CRF) (filename: 146392029140SEQLIST. txt, recording date: 2015, 3 months, 26 days, size: 143 KB).
Brief Description of Drawings
FIG. 1: humanized OX40 antibody variants were analyzed by FACS to assess binding of the antibody to huOX40 expressed on the surface of Hut78 cells.
FIG. 2: the OX40 agonist antibody 1a7.gr.1 binds human and cynomolgus monkey T cells with high affinity.
Fig. 3A and 3B: (FIG. 3A) monoclonal antibody 1A7.gr.1 had no effect on T cell proliferation in the absence of cross-linking. Increasing the concentration of mab 1a7.gr.1 co-stimulated CD4+ memory T cell proliferation in response to anti-CD 3 cross-linking. Calculation of co-stimulatory effect of mab 1a7.gr.1 EC50 was 9.96ng/mL (n ═ 2). (FIG. 3B) increasing the concentration of mAb 1A7.gr.1 co-stimulated interferon- γ production by CD4+ memory T cells in response to cross-linking with anti-CD 3.
FIG. 4A: the plate-bound mab 1a7 co-stimulated effector T cell proliferation in the presence of plate-bound anti-CD 3. In contrast, co-stimulatory activity was abolished when mab 1a7 was provided in soluble form in the presence of plate-bound anti-CD 3, at a level similar to that observed with plate-bound isotype control antibody in the presence of plate-bound anti-CD 3.
FIG. 4B: mab 1a7gr.1, containing the N297G mutation, failed to co-stimulate Teff cell proliferation. In contrast, co-stimulation of anti-CD 3 with wild-type (non-mutated) mab 1a7gr.1 induced Teff cell proliferation.
FIG. 5: OX40 agonist antibody treatment inhibited Treg cell-mediated suppression of naive CD4+ T cells. Naive CD4+ T cells (Tn) were suppressed when cultured alone by addition of Treg cells and isotype control antibody. Inhibition of Treg cell-mediated native CD4+ T cell proliferation was abolished in cultures containing anti-OX 40 antibody, mab 1a7. gr.1. Data represent the average of 3 independent experiments.
FIG. 6: mab 1a7.gr.1 treatment impaired suppressive function of Treg cells.
Fig. 7A and 7B: (FIG. 7A) monoclonal antibody 1A7.gr.1 treatment induced ADCC in OX40 expressing T cells. (FIG. 7B) treatment with mAb A7.gr.1(IgG1) induced greater ADCC in OX 40-expressing CD4+ T cells than the level of ADCC induced by mAb 1A7.gr.1(IgG 4).
Fig. 8A and 8B: the BT 474-human OX40 transgenic clone expressed varying levels of human OX 40. Figure 8A, BT474 cells under-expressing OX 40. FIG. 8B, BT474 cells highly expressing OX 40.
FIG. 9: OX40 agonist antibody treatment induces antibody-dependent cell-mediated phagocytosis of cell lines expressing human OX40, and the level of antibody-dependent cell-mediated phagocytosis is sensitive to the level of OX40 expression in target cells.
FIG. 10: OX40 agonist antibody 1a7.gr.1 treatment induced ADCC in OX40 expressing cells.
FIGS. 11A-I: amino acid sequence of the variable region of an anti-OX 40 antibody. Heavy chain HVR-H1, -H2, and-H3, and light chain HVR-L1, -L2, and-L3 sequences are indicated. Amino acid positions are numbered according to the Kabat numbering system described herein.
FIG. 12A: anti-human OX40 mab 1a7.gr.1 bound Hut78-hOX40 cells in a dose-dependent manner, with 70% of the maximum binding observed at about 200ng/mL antibody (indicated by dotted boxes).
FIG. 12B: OX40L-flag exhibited dose-dependent binding to Hut78-hOX40 cells.
FIG. 12C: binding of anti-human OX40 mab 1A7.gr.1 to Hut78-hOX40 cells decreased with increasing concentration of OX 40L-flag.
FIG. 12D: the presence of the control DR5-flag had no effect on monoclonal antibody 1A7.gr.1 binding.
FIG. 13: pharmacokinetics (PK) at 1mg/kg or 10mg/kg dosing in SCID mice.
Fig. 14A and 14B: administration of 1a7.gr.1 in cynomolgus monkeys resulted in minimal or transient elevation of c-reactive protein (CRP). (FIG. 14A) CRP levels over time observed in monkeys dosed with 0mg/kg or 0.01 mg/kg. (FIG. 14B) CRP levels over time observed in monkeys dosed with 0.3mg/kg or 10 mg/kg.
Fig. 15A and 15B: administration of 1a7.gr.1 in cynomolgus monkeys resulted in minimal or transient elevation of a mixed subset of cytokines. (FIG. 15A) levels of proinflammatory cytokines IL6 and MCP1 over time. (FIG. 15B) anti-inflammatory cytokine IL10 and IL1ra levels over time. In FIGS. 15A and 15B, monkey individuals exhibiting transiently elevated cytokine levels in the 10mg/kg dose group are marked with arrows.
Fig. 16A and 16B: exposure of cynomolgus monkeys to 1a7.gr.1 was confirmed by serum PK and peripheral receptor occupancy. (FIG. 16A) serum PK of monkeys was administered at 0.01, 0.3, or 10mg/kg1A7. gr.1. (FIG. 16B) OX40 receptor occupancy of peripheral CD4+ T cells over time in monkeys administered 0.01, 0.3, or 10mg/kg1A7. gr.1.
FIG. 17: pharmacokinetic (PK) doses at 0.5, 5, or 30mg/kg in cynomolgus monkeys 1a7. gr.1.
FIG. 18: OX40 receptor occupancy over time in monkeys administered 0, 0.5, 5, or 30mg/kg 1A7. gr.1. The arrow indicates the day the sample was obtained.
FIG. 19: MCP-1 levels over time in monkeys administered 0, 0.5, 5, or 30mg/kg 1A7. gr.1.
FIG. 20: no significant peripheral T cell activation or proliferation was observed in monkeys administered 0, 0.5, 5, or 30mg/kg 1a7. gr.1.
Detailed Description
I. Definition of
The term "dysfunction" in the context of immune dysfunction refers to a state of reduced immune responsiveness to antigenic stimulation.
As used herein, the term "dysfunction" also includes an inability to sense or respond to antigen recognition, in particular, an impaired ability to translate antigen recognition into downstream T cell effector functions, such as proliferation, cytokine production (e.g., gamma interferon) and/or target cell killing.
By "enhancing T cell function" is meant inducing, causing or stimulating effector or memory T cells with renewed, sustained or amplified biological function. Examples of enhancing T cell function include: elevated levels from CD8 relative to pre-intervention such levels+Gamma-interferon secretion from effector T cells, elevated levels from CD4+Elevated CD4 for gamma-interferon secretion by memory and/or effector T cells+Increased CD8 in response to and/or memory T cell proliferation+Effector T cells proliferate, increased antigen responsiveness (e.g., clearance). In one embodiment, the level of enhancement is at least 50%, or 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. The manner of measuring this enhancement is known to those of ordinary skill in the art.
"tumor immunity" refers to the process by which a tumor evades immune recognition and clearance. As such, as a therapeutic concept, tumor immunity is "treated" when such evasion is diminished, and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage and tumor clearance.
"immunogenicity" refers to the ability of a particular substance to elicit an immune response. Tumors are immunogenic and enhancing tumor immunogenicity aids in the elimination of tumor cells by immune response.
For purposes herein, an "acceptor human framework" refers to a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework as defined below. An acceptor human framework "derived" from a human immunoglobulin framework or human consensus framework may comprise its identical amino acid sequence, or it may contain amino acid sequence variations. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence.
"affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including the methods described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.
As used herein, an "agonistic antibody" refers to an antibody that activates the biological activity of the antigen to which it binds.
"anti-angiogenic agent" refers to a compound that blocks or interferes to some extent with vascular development. The anti-angiogenic agent can be, for example, a small molecule or antibody that binds to a growth factor or growth factor receptor involved in promoting angiogenesis. In one embodiment, the anti-angiogenic agent is an antibody that binds Vascular Endothelial Growth Factor (VEGF), such as bevacizumab (AVASTIN).
"antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cytotoxic form in which secreted immunoglobulins that bind to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) enable these cytotoxic effector cells to specifically bind to antigen-bearing target cells, followed by killing of the target cells with cytotoxins. The main cell mediating ADCC, NK cells, expresses only Fc γ RIII, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. Ravech and Kinet, annu.rev.immunol.9:457-92(1991) page 464 summarizes FcR expression on hematopoietic cells. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay may be performed, such as described in U.S. Pat. No.5,500,362 or 5,821,337 or U.S. Pat. No.6,737,056 (Presta). Effector cells useful in such assays include PBMC and NK cells. Alternatively/additionally, the ADCC activity of a molecule of interest may be assessed in vivo, for example in animal models such as those disclosed in Clynes et al, PNAS (USA)95: 652-. An exemplary assay for assessing ADCC activity is provided in the examples herein.
The terms "anti-OX 40 antibody" and "antibody that binds OX 40" refer to an antibody that is capable of binding OX40 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent for targeting OX 40. In one embodiment, the anti-OX 40 antibody binds to an unrelated, non-OX 40 protein to less than about 10% of the binding of the antibody to OX40 as measured, for example, by Radioimmunoassay (RIA). In certain embodiments, an antibody that binds OX40 has a concentration of ≦ 1 μ M ≦ 100nM, ≦ 10nM, ≦ 1nM, ≦ 0.1nM, ≦ 0.01nM, or ≦ 0.001nM (e.g., 10 nM)-8M or less, e.g. 10-8M to 10-13M, e.g. 10-9M to 10-13M) dissociation constant (Kd). In certain embodiments, the anti-OX 40 antibody binds to an OX40 epitope that is conserved among OX40 from different species.
As used herein, the terms "bind," "specific binding," or "specific for … …" refer to a measurable and reproducible interaction, such as binding between a target and an antibody, that determines the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody that binds or specifically binds a target (which may be an epitope) is an antibody that binds this target with greater affinity, avidity, more readily, and/or for a greater duration than it binds other targets. In one embodiment, the extent to which the antibody binds to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by Radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds a target has a dissociation constant (Kd) of less than or equal to 1 μ M, less than or equal to 100nM, less than or equal to 10nM, less than or equal to 1nM, or less than or equal to 0.1 nM. In certain embodiments, the antibody specifically binds to an epitope on the protein that is conserved among proteins from different species. In another embodiment, specific binding may include, but need not be exclusive binding.
The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
An "antibody fragment" refers to a molecule distinct from an intact antibody that comprises a portion of an intact antibody and binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab ', Fab ' -SH, F (ab ')2(ii) a A diabody; a linear antibody; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.
An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks binding of the antibody to its antigen by 50% or more in a competition assay. Exemplary competition assays are provided herein.
The term "binding domain" refers to a region of a polypeptide that is capable of binding another molecule. In the case of an FcR, the binding domain may comprise the portion of its polypeptide chain (e.g., its alpha chain) responsible for Fc region binding. One useful binding domain is the extracellular domain of the FcR alpha chain.
A polypeptide having a variant IgG Fc with "altered" FcR, ADCC or phagocytic activity refers to a polypeptide having enhanced or reduced FcR binding activity (e.g., fcyr) and/or ADCC activity and/or phagocytic activity as compared to the parent polypeptide or to a polypeptide comprising a native sequence Fc region.
As used herein, the term "OX 40" refers to any native OX40 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full length," unprocessed OX40 as well as any form of OX40 that results from processing in a cell. The term also encompasses naturally occurring variants of OX40, such as splice variants or allelic variants. An exemplary amino acid sequence of human OX40 is shown in SEQ ID NO. 1.
"OX 40 activation" refers to the activation of the OX40 receptor. Generally, OX40 activation results in signal transduction.
The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, superficial spreading melanoma, lentigo malignant melanoma, acromegaly melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, Chronic Lymphocytic Leukemia (CLL), Acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with scarring nevus (phakomatases), edema (such as associated with brain tumors) and Meigs (Meigs) syndrome, brain tumors and cancers, as well as head and neck cancers, and associated metastases. In certain embodiments, cancers suitable for treatment by the antibodies of the invention include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-hodgkin's lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma (carcinoid carcinosa), head and neck cancer, ovarian cancer, mesothelioma, and multiple myeloma. In some embodiments, the cancer is selected from: non-small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast cancer (e.g., triple negative breast cancer), gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma. Also, in some embodiments, the cancer is selected from: non-small cell lung cancer, colorectal cancer, glioblastoma and breast cancer (e.g., triple negative breast cancer), including metastatic forms of those cancers.
The terms "cell proliferative disorder" and "proliferative disorder" refer to a disorder associated with a degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.
The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
The "class" of an antibody refers to the type of constant domain or constant region that its heavy chain possesses. There are 5 major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1，IgG2，IgG3，IgG4，IgA1And IgA2The constant domains of heavy chains corresponding to different classes of immunoglobulins are designated α,, γ, and μ, respectively.
"complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass) that has bound to its cognate antigen. To assess complement activation, CDC assays can be performed, for example as described in Gazzano-Santoro et al, J.Immunol.methods 202:163 (1996). Polypeptide variants having altered Fc region amino acid sequences (polypeptides having variant Fc regions) and increased or decreased C1q binding ability are described, for example, in U.S. patent No.6,194,551B1 and WO 1999/51642. See also, e.g., Idusogene et al, J.Immunol.164: 4178-.
The term "cytostatic agent" refers to a compound or composition that blocks cell growth in vitro or in vivo. As such, the cytostatic agent may be an agent that significantly reduces the percentage of cells in S phase. Other examples of cytostatics include agents that block cell cycle progression by inducing G0/G1 arrest or M-phase arrest. Humanized anti-Her 2 antibody trastuzumab (trastuzumab)Is an example of a cytostatic agent that induces G0/G1 arrest. Classical M-phase blockers include the vincas (vincristine) and vinblastine (vinblastine)), taxanes (taxanes), and topoisomerase II inhibitors such as doxorubicin (doxorubicin), epirubicin (epirubicin), daunorubicin (daunorubicin), etoposide (etoposide), and bleomycin (bleomycin). Certain agents that block G1 also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen (tamoxifen), prednisone (prednisone), dacarbazine (dacarbazine), mechloroethylmethylamine (mechloroethylamine), cisplatin (cissplatin), methotrexate (methotrexate), 5-fluorouracil (5-fluorouracil), and ara-C. For more information see, e.g., The eds of Mendelsohn and Israel, The Molecular Basis of Cancer, Chapter 1, entitled "cell regulation, oncogenes, and anticancer drugs", Murakami et al, W.B. Saunders, Philadelphia, 1995, e.g., page 13. Taxanes (paclitaxel and docetaxel) are anticancer drugs derived from the yew tree. Docetaxel derived from taxus baccata (c) Rhone-Poulenc Rorer is Parietai (R) ((R))Semi-synthetic analogs of Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, resulting in the inhibition of mitosis in cells.
As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to: radioisotope (e.g. At)211，I131，I125，Y90，Re186，Re188，Sm153，Bi212，P32，Pb212And radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate), doxorubicin (adriamicin), vinca alkaloids (vinca alkaloids) (vincristine), vinblastine (vinblastine), etoposide (etoposide)), doxorubicin (doxorubicin), melphalan (melphalan), mitomycin (mitomycin) C, chlorambucil (chlorembucil), daunorubicin (daunorubicin), or other intercalating agents); a growth inhibitor; enzymes and fragments thereof, such as nucleolytic enzymes; (ii) an antibiotic; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various antitumor or anticancer agents disclosed hereinafter.
"depleting anti-OX 40 antibody" refers to an anti-OX 40 antibody that kills or depletes OX40 expressing cells. Depleting OX 40-expressing cells can be accomplished by a variety of mechanisms, such as antibody-dependent cell-mediated cytotoxicity and/or phagocytosis. Depletion of OX 40-expressing cells can be assayed in vitro, and exemplary methods for in vitro ADCC and phagocytosis assays are provided herein. In some embodiments, the OX 40-expressing cell is a human CD4+ effector T cell. In some embodiments, the OX 40-expressing cell is a transgenic BT474 cell expressing human OX 40.
"Effector function" refers to those biological activities attributable to the Fc region of an antibody and which vary with the antibody isotype. Examples of antibody effector functions include: c1q binding and Complement Dependent Cytotoxicity (CDC); fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.
An "effective amount" of a pharmaceutical agent (e.g., a pharmaceutical formulation) refers to an amount effective to achieve the desired therapeutic or prophylactic result over the necessary dosage and period of time.
"Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR is one that binds an IgG antibody (gamma receptor), including receptors of the Fc γ RI, Fc γ RII, and Fc γ RIII subclasses, including allelic variants and alternatively spliced forms of those receptors. Fc γ RII receptors include Fc γ RIIA ("activating receptor") and Fc γ RIIB ("inhibiting receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor Fc γ RIIA comprises in its cytoplasmic domain an immunoreceptor tyrosine-based activation motif (ITAM). The inhibitory receptor Fc γ RIIB comprises in its cytoplasmic domain an immunoreceptor tyrosine-based inhibitory motif (ITIM) (see, e.g., for Annu.Rev.Immunol.15:203-234 (1997)). For reviews of FcRs see, for example, ravechand Kinet, Annu. Rev. Immunol.9:457-492 (1991); capel et al, immunolmethods 4:25-34 (1994); and de Haas et al, J.Lab.Clin.Med.126:330-41 (1995). The term "FcR" encompasses other fcrs herein, including those that will be identified in the future. The term "Fc receptor" or "FcR" also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al, j.immunol.117:587(1976) and Kim et al, j.immunol.24:249(1994)) and for the regulation of immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward., Immunol. today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology,15(7):637-640 (1997); Hinton et al., J.biol. chem.279(8):6213-6216 (2004); WO 2004/92219(Hinton et al.)). The in vivo binding and serum half-life of human FcRn high affinity binding polypeptides to human FcRn can be determined, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered with polypeptides having variant Fc regions. WO 2000/42072(Presta) describes antibody variants with increased or decreased binding to FcR. See also Such as Shields et al, J.biol.chem.9(2):6591-6604 (2001).
The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxy-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, published Health Service 5 th edition, National Institutes of Health, Bethesda, MD, 1991.
A "functional Fc region" possesses the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q combinations; CDC; fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors; BCR), and the like. Such effector functions generally require that the Fc region be associated with a binding domain (e.g., an antibody variable domain) and can be evaluated using a variety of assays, such as those disclosed in the definitions herein.
"human effector cells" refer to leukocytes which express one or more fcrs and which exert effector function. In certain embodiments, the cell expresses at least Fc γ RIII and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include Peripheral Blood Mononuclear Cells (PBMCs), Natural Killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. The effector cells may be isolated from their natural source, e.g., blood.
"framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. In general, the FRs of a variable domain consist of 4 FR domains: FR1, FR2, FR3, and FR 4. Thus, HVR and FR sequences typically occur in the following order in VH (or VL): FR1-H1(L1) -FR2-H2(L2) -FR3-H3(L3) -FR 4.
The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain comprising an Fc region as defined herein.
The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected in the originally transformed cell.
"human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source using a repertoire of human antibodies or other human antibody coding sequences. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues.
"human consensus framework" refers to a framework representing the amino acid residues most commonly found in the selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. Typically, the sequence subgroups are subgroups as in Kabat et al, Sequences of Proteins of Immunological Interest, fifth edition, NIHPublication 91-3242, Bethesda MD (1991), volumes 1-3. In one embodiment, for VL, the subgroup is as in Kabat et al, supra for subgroup kappa I. In one embodiment, for the VH, the subgroup is as in Kabat et al, supra, subgroup III.
A "humanized" antibody is a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise at least one, and typically two, substantially the entire variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. Optionally, the humanized antibody may comprise at least a portion of an antibody constant region derived from a human antibody. An antibody, e.g., a "humanized form" of a non-human antibody, refers to an antibody that has undergone humanization.
As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain which is hypervariable in sequence ("complementarity determining regions" or "CDRs") and/or which forms structurally defined loops ("hypervariable loops") and/or which contains antigen-contacting residues ("antigen contacts"). Generally, an antibody comprises 6 HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include:
(a) hypervariable loops present at amino acid residues 26-32(L1), 50-52(L2), 91-96(L3), 26-32(H1), 53-55(H2), and 96-101(H3) (Chothia and Lesk, J.mol.biol.196: 901-917 (1987));
(b) CDRs present at amino acid residues 24-34(L1), 50-56(L2), 89-97(L3), 31-35b (H1), 50-65(H2), and 95-102(H3) (Kabat et al, Sequences of Proteins of immunological interest,5th Ed. public Health Service, National Institutes of Health, Bethesda, MD (1991));
(c) antigen contacts, present at amino acid residues 27c-36(L1), 46-55(L2), 89-96(L3), 30-35b (H1), 47-58(H2), and 93-101(H3) (MacCallum et al.J.mol.biol.262: 732-745 (1996)); and
(d) a combination of (a), (b), and/or (c) comprising HVR amino acid residues 46-56(L2), 47-56(L2), 48-56(L2), 49-56(L2), 26-35(H1), 26-35b (H1), 49-65(H2), 93-102(H3), and 94-102 (H3).
Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al, supra.
In one embodiment, the HVR residues comprise those identified in FIGS. 11A-I or elsewhere in the specification.
An "immunoconjugate" refers to an antibody conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.
An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.
By "promoting cell growth or proliferation" is meant increasing the growth or proliferation of a cell by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%.
An "isolated" antibody refers to an antibody that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al, J.Chromatogr.B 848:79-87 (2007).
An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
"isolated nucleic acid encoding an anti-OX 40 antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, including such nucleic acid molecules in a single vector or in different vectors, and such nucleic acid molecules present at one or more locations in a host cell.
As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except, for example, for possible variant antibodies containing naturally occurring mutations or occurring during the production of a monoclonal antibody preparation, such variants are typically present in very small amounts. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be generated by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods that utilize transgenic animals containing all or part of a human immunoglobulin locus, such methods and other exemplary methods for generating monoclonal antibodies are described herein.
"naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radioactive label. The naked antibody may be present in a pharmaceutical formulation.
"Natural antibody" refers to a naturally occurring immunoglobulin molecule having a different structure. For example, a native IgG antibody is an heterotetrameric glycan protein of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains that are disulfide-bonded. From N to C-terminus, each heavy chain has one variable region (VH), also called variable or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH 3). Similarly, from N-to C-terminus, each light chain has a variable region (VL), also known as the variable light domain or light chain variable domain, followed by a Constant Light (CL) domain. Antibody light chains can be classified into one of two types, called kappa (κ) and lambda (λ), based on their constant domain amino acid sequences. A "native sequence Fc region" comprises an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-a and a allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and a native sequence human IgG4 Fc region; and naturally occurring variants thereof.
The term "package insert" is used to refer to instructions for use typically contained in commercial packages of therapeutic products that contain information regarding the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings relating to the use of such therapeutic products.
"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Comparison for the purpose of determining percent amino acid sequence identity can be performed in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or megalign (dnastar) software. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. However, for purposes of the present invention,% amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, inc and the source code has been submitted to the US Copyright Office (US Copyright Office, Washington d.c.,20559) along with the user document, where it is registered with US Copyright registration number TXU 510087. ALIGN-2 programs are publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from source code. The ALIGN2 program should be compiled for use on UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters were set by the ALIGN-2 program and were not changed.
In the case of employing ALIGN-2 to compare amino acid sequences, the% amino acid sequence identity of a given amino acid sequence a relative to (to), with (with), or against (against) a given amino acid sequence B (or may be stated as having or comprising a given amino acid sequence a relative to, with, or against a certain% amino acid sequence identity of a given amino acid sequence B) is calculated as follows:
fractional X/Y times 100
Wherein X is the number of amino acid residues scored as identical matches in the A and B alignments of the sequence alignment program by the program ALIGN-2, and wherein Y is the total number of amino acid residues in B. It will be appreciated that if the length of amino acid sequence a is not equal to the length of amino acid sequence B, then the% amino acid sequence identity of a relative to B will not equal the% amino acid sequence identity of B relative to a. Unless otherwise specifically indicated, all% amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.
The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective, and that is free of other components having unacceptable toxicity to a subject that will receive administration of the formulation.
"pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation that is different from the active ingredient and is not toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.
As used herein, "treatment" (and grammatical variations thereof, such as "treating" or "treatment") refers to clinical intervention in an attempt to alter the natural course of the treated individual, which may be for the purpose of prevention or in the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or palliating the disease state, and remission or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the onset/progression of disease, or to slow the progression of disease.
The term "tumor" refers to all neoplastic (neoplastic) cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein.
The term "variable region" or "variable domain" refers to a domain in an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The heavy and light chain variable domains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising 4 conserved Framework Regions (FR) and 3 hypervariable regions (HVRs). (see, e.g., Kindt et al, Kuby Immunology, 6 th edition, w.h.freeman and co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind a particular antigen can be isolated by screening libraries of complementary VL or VH domains using VH or VL domains, respectively, from antibodies that bind the antigen. See, e.g., Portolano et al, J.Immunol.150:880-887 (1993); clarkson et al, Nature 352: 624-.
A "variant Fc region" comprises an amino acid sequence that differs from a native sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution as compared to the native sequence Fc region or as compared to the Fc region of the parent polypeptide, e.g., from about 1 to about 10 amino acid substitutions, preferably from about 1 to about 5 amino acid substitutions, in the native sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein preferably shares at least about 80% homology with the native sequence Fc region and/or the Fc region of the parent polypeptide, most preferably at least about 90% homology therewith, and more preferably at least about 95% homology therewith.
As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors which are self-replicating nucleic acid structures and vectors which integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors".
The "VH subgroup III consensus framework" comprises the consensus sequence obtained from the amino acid sequences in variable heavy chain subgroup III of Kabat et al. In one embodiment, the VH subgroup III consensus framework amino acid sequence comprises at least a portion of, or all of: EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:185) -H1-WVRQAPGKGLEWV (SEQ ID NO:186) -H2-RFTISRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO:187) -H3-WGQGTLVTVSS (SEQ ID NO: 188).
The "VL subgroup I consensus framework" comprises the consensus sequence obtained from the amino acid sequences in variable light chain kappa subgroup I of Kabat et al. In one embodiment, the VL subgroup I consensus framework amino acid sequence comprises at least a portion of, or the entire of, each of the following sequences: DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:189) -L1-WYQQKPGKAPKLLIY (SEQ ID NO:190) -L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:191) -L3-FGQGTKVEIK (SEQ ID NO: 192).
As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to: radioisotope (e.g. At)211，I131，I125，Y90，Re186，Re188，Sm153，Bi212，P32，Pb212And radioactive isotopes of Lu); a chemotherapeutic agent; a growth inhibitor; enzymes and fragments thereof, such as nucleolytic enzymes; and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof. Exemplary cytotoxic agents may be selected from the group consisting of antimicrotubule agents, platinum coordination complexes, alkylating agents, antibiotic agents, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormone analogs, signal transduction pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, pro-apoptotic agents, inhibitors of LDH-a, inhibitors of fatty acid biosynthesis, inhibitors of cell cycle signaling, HDAC inhibitors, proteasome inhibitors, and inhibitors of cancer metabolism.
In one embodiment, the cytotoxic agent is selected from the group consisting of antimicrotubule agents, platinum coordination complexes, alkylating agents, antibiotic agents, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormone analogs, signal transduction pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, pro-apoptotic agents, inhibitors of LDH-a, inhibitors of fatty acid biosynthesis, inhibitors of cell cycle signaling, HDAC inhibitors, proteasome inhibitors, and inhibitors of cancer metabolism. In one embodiment, the cytotoxic agent is a taxane (taxane). In one embodiment, the taxane is paclitaxel (paclitaxel) or docetaxel (docetaxel). In one embodiment, the cytotoxic agent is a platinum agent. In one embodiment, the cytotoxic agent is an antagonist of EGFR. In one embodiment, the antagonist of EGFR is N- (3-ethynylphenyl) -6, 7-bis (2-methoxyethoxy) quinazolin-4-amine (e.g., erlotinib). In one embodiment, the cytotoxic agent is a RAF inhibitor. In one embodiment, the RAF inhibitor is a BRAF and/or CRAF inhibitor. In one embodiment, the RAF inhibitor is vemurafenib (vemurafenib). In one embodiment, the cytotoxic agent is a PI3K inhibitor.
"chemotherapeutic agents" include chemical compounds useful in the treatment of cancer. Examples of chemotherapeutic agents include erlotinib (erlotinib) (ll:)Genentech/OSI Pharm), bortezomib (bortezomib), (b), (dMillennium Pharm), disulfiram (disulphiram), epigallocatechin gallate (epigallocatechin gallate), salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol (radicol), lactate dehydrogenase A (LDH-A), fulvestrant (fulvestrant) ((r))AstraZeneca), sunitinib (sunitinb), (sunitinibPfizer/Sugen), letrozole (letrozole), (L-Toxole)Novartis), imatinib mesylate (imatinib mesylate), (I) a salt of N, NNovartis)，finasunate(Novartis), oxaliplatin (oxaliplatin) ((oxaliplatin)Sanofi), 5-FU (5-fluorouracil), leucovorin (leucovorin), Rapamycin (Rapamycin) (Sirolimus),wyeth), Lapatinib (Lapatinib), (Lapatinib)GSK572016, Glaxo Smith Kline), Lonafami (SCH66336), Sorafenib (sorafenib) (S) (G) (Bayer Labs), gefitinib (gefitinib) ((E) gefitinibAstraZeneca), AG1478, alkylating agents (alkylating agents), such as thiotepa and thiotepaCyclophosphamide (cyclophosphamide); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines (aziridines), such as benzotepa (benzodopa), carboquone (carboquone), metoclopramide (meteredopa), and uretepa (uredpa); ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoryl Amine (triethylenemhospernamide) and trimethymamine (trimethamelamine), annonaceous acetogenins (acetogenins) (especially bullatacin and bullatacin), camptothecin (camptothecin) (including topotecan and irinotecan), bryostatin (bryostatin), calalystatin, CC-1065 (including adrovasin (adozelessin), carmustine (carzelessin) and bizelesin (bizelesin) synthetic analogues), cryptophycins (cryptophycins) (especially cryptophycin 1 and cryptophycin 8), corticoids (adrenocorticoids) including prednisone (prednisolone) and prednisolone (kyphosantin), carmustine (carmustine hydrochloride), carmustine (carmustine) (especially carmustine), carmustine (carmustine) (especially carmustine) (including carmustine) (11), carmustine) (especially carmustine) (including carmustine) (aerosine), carmustine) (especially carmustine (carmustine) (aerosine), carmustine) (aerostatin), carmustine (carmustine) (including carmustine) (150), carmustine) (including carmustine) (carmustine), carmustine) (especially carmustine), carmustine (carmustine), carmustine) (including carmustine (carmustine), carmustine (carmustine), carmustine (carmustine), carmustine (carmustine), carmustine (carmustine), carmustine (carmustine), carmustine (carmustine), carmustine (carmustine), carmustine (carmustine), carmustine (carmustine), carmustine (carmustine), carmustine (carmustine), carmustine (carmusti Chromophorin enediyne antibiotic chromophores), aclacinomycins (aclacinomycins), actinomycins (actinomycin), anthranomycin (authramycin), azaserine (azaserine), bleomycin (bleomycin), actinomycin C (cactinomycin), carbacidin, carminomycin (caminomycin), carcinomycin (carzinophilin), tryptomycin (chromomycins), actinomycin D (dactinomycin), daunorubicin (daunorubicin), ditorexin (detorubicin), 6-diaza-5-oxo-L-norleucine,(doxorubicin)), morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolodoxorubicin and deoxydoxorubicin), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), marijumycin (marcelomycin), mitomycin (mitomycin) such as mitomycin C, mycophenolic acid (mycophenolic acid), norramycin (nogalamycin), olivomycin (olivomycin), pelomycin (polyplomycin), pofiomycin (porfiromycin), puromycin (puromycin), triiron doxorubicin (quelamycin), rodobicin (rodorubicin), streptonigrin (strepatorigrin), streptozocin (streptazocin), tubercidin (tubicin), wudimethicin (zionexen), zocin (zorubicin), zorubicin (zoxidin), zoxitin (zoxitin); antimetabolites such as methotrexate (methotrexate) and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteroyltriglutamic acid (pteropterin), trimetrexate; purine analogs such as fludarabine (fludarabine), 6-mercaptopurine (mercaptoprine), thiamiprine (thiamiprine), thioguanine (thioguanine); pyrimidine analogs such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine, carmofur (carmofur), cytarabine (cytarabine), dideoxyuridine (dideoxyuridine), deoxyfluorouridine (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine); androgens such as carotinone (calusterone), dromostanolone propionate, epitioandrostanol (epitiostanol), mepiquitane (mepiquitane), testolactone (testolactone); resist against Adrenals such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), trilostane (trilostane); folic acid supplements such as folinic acid (frilic acid); acetoglucurolactone (acegultone); an aldophosphamide glycoside (aldophosphamideglycoside); aminolevulinic acid (aminolevulinic acid); eniluracil (eniluracil); amsacrine (amsacrine); bestrabuucil; bisantrene; edatrexate (edatraxate); desphosphamide (defofamine); dimecorsine (demecolcine); diazaquinone (diaziqutone); elfosmithine; ammonium etitanium acetate; an epothilone; etoglut (etoglucid); gallium nitrate; hydroxyurea (hydroxyurea); lentinan (lentinan); lonidamine (lonidainine); maytansinoids (maytansinoids), such as maytansine (maytansine) and ansamitocins (ansamitocins); mitoguazone (mitoguzone); mitoxantrone (mitoxantrone); mopidanol (mopidamnol); diamine nitracridine (nitrarine); pentostatin (pentostatin); methionine mustard (phenamett); pirarubicin (pirarubicin); losoxantrone (losoxantrone); podophyllinic acid (podophyllic acid); 2-ethyl hydrazide (ethylhydrazide); procarbazine (procarbazine); Polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane (rizoxane); rhizomycin (rhizoxin); sisofilan (sizofuran); helical germanium (spirogermanium); tenuazonic acid (tenuazonic acid); triimine quinone (triaziquone); 2,2' -trichlorotriethylamine; trichothecenes (trichothecenes), especially the T-2 toxin, verrucin (verrucin) A, bacillocin (roridin) A and snakes (anguidine); urethane (urethan); vindesine (vindesine); dacarbazine (dacarbazine); mannitol mustard (mannomustine); dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromane (pipobroman); a polycytidysine; cytarabine (arabine) ("Ara-C"); cyclophosphamide (cyclophosphamide); thiotepa (thiotepa); taxols (taxoids), such as TAXOL (paclitaxel) (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J.),albumin-engineered nanoparticle dosage forms of paclitaxel (Cremophor-free), paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.) and taxotere (R.C.)(docetaxel, doxetaxel); Sanofi-Aventis); chlorambucil (chlorembucil); (gemcitabine); 6-thioguanine (thioguanine); mercaptopurine (mercaptoprine); methotrexate (methotrexate); platinum analogs such as cisplatin (cissplatin) and carboplatin (carboplatin); vinblastine (vinblastine); etoposide (VP-16); ifosfamide (ifosfamide); mitoxantrone (mitoxantrone); vincristine (vincristine);(vinorelbine); oncostatin (novantrone); teniposide (teniposide); edatrexate (edatrexate); daunomycin (daunomycin); aminopterin (aminopterin); capecitabine (capecitabine)Ibandronate (ibandronate); CPT-11; topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids (retinoids), such as retinoic acid (retinoic acid); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.
Chemotherapeutic agents also include (i) anti-hormonal agents that act to modulate or inhibit the action of hormones on tumors, such as anti-estrogens and Selective Estrogen Receptor Modulators (SERMs), including, for example, tamoxifen (includingTamoxifen citrate), raloxifene (raloxifene), droloxifene (droloxif)ene), iodoxyfen, 4-hydroxytamoxifen, trioxifene (trioxifene), naloxone (keoxifene), LY117018, onapristone (onapristone), and (toremifene citrate); (ii) aromatase inhibitors which inhibit aromatase which regulates estrogen production in the adrenal gland, such as, for example, 4(5) -imidazole, aminoglutethimide,(megestrol acetate),(exemestane); Pfizer), formestane (formestanie), fadrozole (fadrozole),(vorozole),(letrozole; Novartis), and(anastrozole); AstraZeneca; (iii) anti-androgens such as flutamide (flutamide), nilutamide (nilutamide), bicalutamide (bicalutamide), leuprolide (leuprolide) and goserelin (goserelin), buserelin (buserelin), triptorelin (triptorelin), medroxyprogesterone acetate (medroxyprogesterone acetate), diethylstilbestrol (diethltilbentrol), bimelin (premarin), fluoromethyltestosterone (fluoxymestrone), all-trans retinoic acid, fenretinide (fenretinide), and troxacitabine (troxacitabine) (1, 3-dioxolane nucleoside analogs); (PKC) protein kinase inhibitors (v) lipid kinase inhibitors (vi) antisense oligonucleotides, particularly oligonucleotides that inhibit the expression of genes such as H, α, such as, for example, through abnormal Ras-78; (vii) Ribozymes, such as VEGF expression inhibitors (e.g.) And inhibitors of HER2 expression; (viii) vaccines, such as gene therapy vaccines, e.g.AndrIL-2; topoisomerase 1 inhibitors, such asrmRH; and (ix) and pharmaceutically acceptable salts, acids and derivatives of any of the foregoing.
Chemotherapeutic agents also include antibodies such as alemtuzumab (Campath), bevacizumab (bevacizumab), (b) and (c)Genentech), cetuximab (cetuximab), (Imclone); panitumumab (panitumumab)Amgen), rituximab (rituximab), (b)Genentech/Biogen Idec), pertuzumab (pertuzumab) ((ii) Genentech/Biogen Idec), (ii) and (iii) pharmaceutically acceptable salts thereof2C4, Genentech), trastuzumab (Genentech), tositumomab (tositumomab) (Bexxar, Corixia), and antibody drug conjugates, gemtuzumab ozogamicin (gemtuzumab)ozogamicin)(Wyeth). Additional humanized monoclonal antibodies that have therapeutic potential as agents in combination with the compounds of the invention include: aprezumab (apilizumab), aselizumab (aselizumab), atlizumab (atlizumab), palivizumab (bapineuzumab), bivatuzumab mertansine, mocratizumab (cantuzumab), sijilizumab (cedelizumab), pemphilizumab (certolizumab pegol), cidfutuzumab (cidfutuzumab, cituzumab, daclizumab (daclizumab), eculizumab (eculizumab), efuzumab (efalizumab), epratuzumab (epratuzumab), erlizumab (erlizumab), non-veluzumab (feluzumab), trastuzumab (fontuzumab (fonticuzumab), trastuzumab (gemuzumab), geminuzumab (gemuzumab), trastuzumab (foslizumab), trastuzumab (gemuzumab), trastuzumab (gemtuzumab), trastuzumab (aspergizumab), trastuzumab (aspergizumab (zezumab), rit (zepinuzumab), trastuzumab (zezumab (zepinuzumab), trastuzumab (ze, pemphituzumab, pectuzumab, pexizumab (pexizumab), ralizumab, ranibizumab (ranibizumab), relivizumab, rayleigh mab (relizumab), resvizumab, rovizumab (rovelizumab), lucizumab (ruplizumab), sibrozumab (sibrozumab), sibilizumab (siplizumab), soltezumab (sontezumab), soltezumab (sontuzumab), tactuzumab (sotuzumab), tacatuzumab texazumab (tentuzumab), tacatuzumab (talizumab), tefibuzumab (tefibuzumab), tuzumab (tocuzumab), tolizumab (toralizumab), tutuzumab (tumomodulin), restituzumab (tebuclizumab), tusizumab (tuzumab), tusizumab (toralizumab), tusizumab (torat), tuzumab (toralizumab), tutututusizumab (tusizumab), tusizumab (tusizumab, tuzumab (tuzumab), tussivizumab (tuvituvizumab), tussivizumab (tuvituvituvizumab), tussivizumab (tuvikib), bezizumab (tuvituvizumab), tuvituvizumab (tuvikib), recombinant human, tuvizumab (abjejun (wut), recombinant human t-12, tuvituvituvit-12 (abjejun), recombinant human t-12, bexib), recombinant human antibody (abytuzumab) and recombinant human antibody (abutevituzumab).
Chemotherapeutic agents also include "EGFR inhibitors," which refer to compounds that bind to or otherwise interact directly with EGFR and prevent or reduce its signaling activity, which are otherwise also referred to as "EGFR antagonists. Examples of such agents include antibodies and small molecules that bind EGFR. Examples of EGFR-binding antibodies include MAb 579(ATCC CRL HB8506), MAb455(ATCC CRL HB8507), MAb 225(ATCC CRL 8508), MAb 528(ATCC CRL 8509) (see U.S. patent No.4,943,533, Mendelsohn et al) and variants thereof, such as chimeric 225(C225 or cetuximab;) And reconstituted human 225(H225) (see WO96/40210, Imclone Systems Inc.; IMC-11F 8), a fully human EGFR-targeting antibody (Imclone), antibodies that bind to type II mutant EGFR (U.S. Pat. No.5,212,290), humanized and chimeric antibodies that bind to EGFR as described in U.S. Pat. No.5,891,996, and human antibodies that bind to EGFR, such as ABX-EGF or Panitumumab (Panitumumab) (see WO98/50433, Abgenix/Ampen), EMD55900 (Straglitoto et al, Eur. J.Cancer 32A:636-640(1996)), EMD7200(matuzumab), a humanized antibody that is directed against EGFR and competes for binding with both EGFR and EGF (EMD/Merck), human antibodies, Huzhu zub, GmG 7263, humanized antibodies (EMD/Merck), WO99, WO 35,04168, WO 35,04135,221, WO 35,221,120,221, WO 35,221,120,120,120, WO 35,221,120,120,120,120, WO 35,120,120,120,120,221,150, and mAbs such as described in U.7,120,120,150, WO 35,150, WO 35,120,150, WO 35,150, WO 150, WO 48,150, 35,150, 150, WO 48,150, 35,150, 150, 35, WO 48,150, 150, WO 48,150, 150, WO 48, 150, and WO 48,150, 35,150, and WO 48,150, such as well as monoclonal antibodies that specifically bind to EGFR, 35, 35,150, 35, and similar to EGFR, and similar antibodies that specifically to EGFR, such as EGFR, 35, 35,150, 35, and similar antibodies, 35, and similar to EGFR, 35, 150, 35 Agents include OSI-774(CP-358774, erlotinib,Genentech/OSI Pharmaceuticals); PD 183805(CI 1033, 2-propenamide, N- [4- [ (3-chloro-4-fluorophenyl) amino)]-7- [3- (4-morpholinyl) propoxy]-6-quinazolinyl]Dihydrochloride, Pfizer Inc.); ZD1839, gefitinib (gefitinib)4- (3 '-chloro-4' -fluoroanilino) -7-methoxy-6- (3-morpholinopropoxy) quinazoline, AstraZeneca); ZM 105180 ((6-amino-4- (3-methylphenyl-amino) -quinazoline, Zeneca); BIBX-1382(N8- (3-chloro-4-fluoro-phenyl) -N2- (1-methyl-piperidin-4-yl) -pyrimido [5, 4-d)]Pyrimidine-2, 8-diamine, Boehringer Ingelheim); PKI-166((R) -4- [4- [ (1-phenylethyl) amino)]-1H-pyrrolo [2,3-d]Pyrimidin-6-yl]-phenol); (R) -6- (4-hydroxyphenyl) -4- [ (1-phenylethyl) amino group]-7H-pyrrolo [2,3-d]Pyrimidines); CL-387785(N- [4- [ (3-bromophenyl) amino)]-6-quinazolinyl]-2-butynylamide); EKB-569(N- [4- [ (3-chloro-4-fluorophenyl) amino group]-3-cyano-7-ethoxy-6-quinolinyl]-4- (dimethylamino) -2-butenamide) (Wyeth); AG1478 (Pfizer); AG1571(SU 5271; Pfizer); dual EGFR/HER2 tyrosine kinase inhibitors, such as lapatinib (lapatinib) ((R)) GSK572016 or N- [ 3-chloro-4- [ (3-fluorophenyl) methoxy group]Phenyl radical]-6[5[ [ [ 2-methylsulfonyl) ethyl group]Amino group]Methyl radical]-2-furyl radical]-4-quinazolinamines).
Chemotherapeutic agents also include "tyrosine kinase inhibitors" including the EGFR-targeting drugs mentioned in the preceding paragraph; small molecule HER2 tyrosine kinase inhibitors, such as TAK165 available from Takeda; CP-724,714, an oral ErbB2 receptor tyrosine kinase selective inhibitor (Pfizer and OSI); dual HER inhibitors that preferentially bind EGFR but inhibit both HER2 and EGFR-overexpressing cells, such as EKB-569 (available from Wyeth); lapatinib (GSK 572016; available from Glaxo-SmithKline), aOral HER2 and EGFR tyrosine kinase inhibitors; PKI-166 (available from Novartis); pan-HER inhibitors such as canertinib (CI-1033; Pharmacia); raf-1 inhibitors, such as antisense agents ISIS-5132, available from ISIS Pharmaceuticals, that inhibit Raf-1 signaling; non-HER targeted TK inhibitors, such as imatinib mesylate (i: (ii)Available from Glaxo SmithKline); multi-targeted tyrosine kinase inhibitors, such as sunitinib (sunitinib) ((iii))Available from Pfizer); VEGF receptor tyrosine kinase inhibitors such as vatalanib (PTK787/ZK222584, available from Novartis/Schering AG); MAPK extracellular regulated kinase I inhibitor CI-1040 (available from Pharmacia); quinazolines, such as PD 153035, 4- (3-chloroanilino) quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines, 4- (phenylamino) -7H-pyrrolo [2,3-d ]Pyrimidines; curcumin (diferuloylmethane, 4, 5-bis (4-fluoroanilino) -phthalimide); tyrphostine containing a nitrothiophene moiety; PD-0183805 (Warner-Lamber); antisense molecules (e.g., those that bind to a nucleic acid encoding HER); quinoxalines (U.S. patent No.5,804,396); trypostins (U.S. Pat. No.5,804,396); ZD6474 (AstraZeneca); PTK-787(Novartis/Schering AG); pan HER inhibitors such as CI-1033 (Pfizer); affinitac (ISIS 3521; ISIS/Lilly); imatinib mesylatePKI 166 (Novartis); GW2016(Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); semaxinib (pfizer); ZD6474 (AstraZeneca); PTK-787(Novartis/Schering AG); INC-1C11(Imclone), rapamycin (sirolimus,) (ii) a Or any of the followingDescribed in the patent publications: U.S. patent nos. 5,804,396; WO1999/09016(American Cyanamid); WO1998/43960(American Cyanamid); WO1997/38983(Warner Lambert); WO1999/06378(Warner Lambert); WO1999/06396 (WarnerLambert); WO1996/30347(Pfizer, Inc); WO1996/33978 (Zeneca); WO1996/3397(Zeneca) and WO1996/33980 (Zeneca).
Chemotherapeutic agents also include dexamethasone (dexamethasone), interferon, colchicine (colchicine), chlorphenamine (methaprine), cyclosporine (cyclosporine), amphotericin (amphotericin), metronidazole (metronidazole), alemtuzumab (alemtuzumab), alitretinoin (alitretinoin), allopurinol (allopurinol), amifostine (amifostine), arsenic trioxide (arsenical trioxide), asparaginase (asparagase), live BCG, bevacizumab (bevacizumab), bexarotene (bexarotene), cladribine (cladribine), ritaline (clofanabine), darbevacetin alfa, denbuzin (dexrazine), dexrazoxane (dexamectin), interferon alpha (interferon alpha-2), interferon alpha (interferon alpha, 2-acetate), nelarabine (nellabine), norflumumab (nofetumumab), opril (oprefletekin), palifermin (pamidronate), pemetrexed (peganurate), pegamphenzyme (pegademase), pemetrexed (pegaspartase), PEG filgrastim (pegfilgrastim), pemetrexed disodium (pemetrexed disodium), plicamycin (plicamycin), porfimer sodium (porfimer sodium), quinacrine (quinacrine), rasburiase (rasburicase), sargrastim (sargramostim), temozolomide (temozolomide), VM-26, 6-TG, toremifene (toremifene), tretinoin, ATRA, valrubicin (valrubicin), zoledronate (letronate), and zoledronate (zoledronate), and pharmaceutically acceptable salts thereof.
The chemotherapeutic agent further comprises hydrocortisone (hydrocortisone), hydrocortisone acetate (hydrocortisone acetate), hydrocortisone acetate (cortisone acetate), and tixocortol pivalate(tixocortol pivalate), triamcinolone acetonide (triamcinolone acetonide), triamcinolone alcohol (triamcinolone acetonide), mometasone (mometasone), amcinolone acetonide (amcinonide), budesonide (budesonide), desonide (desonide), fluoxinide, fluoxinone acetate, betamethasone (betamethasone), betamethasone sodium phosphate (betamethasone), dexamethasone (dexmethasone), dexamethasone sodium phosphate (dexamethosone), fluocortolone (fluxolone), hydrocortisone-17-butyrate (hydrocortisone-17-butyrate), hydrocortisone-17-valerate (hydrocortisone-17-valrate), acetominone (acetochlor-17-propionate), interferon (fluxolone-17-betaine), interferon (fluxolone-2-acetate), interferon (fluxolone-17-butyrate), interferon (fluxolone-17-valerate) (e.g), interferon-6-acetate), interferon-beta-arginine (fluxolone-17-acetate), interferon-2-acetate (fluxolone-17-D), interferon-2-acetate (fluxolone-17-D), interferon-2-D), interferon-2-acetate (fluxolone-2, beta-D), and a-D), such as a-2-D, a-2-D, a Interleukin-13 (IL-13) blockers such as lebrikizumab, interferon α (IFN) blockers such as Rontalizumab, β 7-integrin blockers such as rhuMAb Beta7, IgE pathway blockers such as anti-M1 prime, secreted homotrimeric LTa3 and membrane-bound heterotrimeric LTa1/β 2 blockers, such as antithrombin α (LTa), radioisotopes, e.g. At211，I131，I125，Y90，Re186，Re188，Sm153，Bi212，P32，Pb212And a Lu radioisotope; miscellaneous investigational agents, such as thioplatin, PS-341, phenylbutyrate, ET-18-OCH3Or farnesyltransferase inhibitors (L-739749, L-744832; polyphenols, such as quercetin (quercetin), resveratrol (resveratrol), piceatannol, epigallocatechin gallate (epigallocatechin gallate), theaflavin (theaflavin), flavanols (flavanols), procyanidins (procyanidins), betulinic acid (betulinic acid) and derivatives thereof; autophagy inhibitors, such as chloroquine, -9-tetrahydrocannabinol (dronol),) β -lapachone (lapachone), lapachol (lapachol), colchicines (colchicines), betulinic acid (betulinic acid), acetyl camptothecin, scopoletin (scopolectin), and 9-aminocamptothecin), podophyllotoxin (podophyllotoxin), tegafur (tegafur) Bexarotene (bexarotee)Diphosphonates (bisphosphates), such as clodronate (e.g. clodronate)Or) Etidronate sodium (etidronate)NE-58095, zoledronic acid/zoledronateAlendronate (alendronate)Pamidronate (pamidronate)Tiludronate (tirudronate)Or risedronate (risedronate)And epidermal growth factor receptor (EGF-R); vaccines, e.g.A vaccine; perifosine (perifosine), COX-2 inhibitors (e.g., celecoxib (celecoxib) or etoricoxib (etoricoxib)), proteosome inhibitors (e.g., PS 341); CCI-779; tipifarnib (R11577); orafenaib, ABT 510; bcl-2 inhibitors, such as oblimersen sodiumpixantrone; farnesyl transferase inhibitors, such as lonafarnib (SCH 6636, SARASAR)TM) (ii) a And pharmaceutically acceptable salts, acids or derivatives of any of the foregoing; and combinations of two or more of the above, such as CHOP (abbreviation for cyclophosphamide, doxorubicin, vincristine, and prednisolone combination therapy) and FOLFOX (oxaliplatin)TM) Abbreviation for treatment regimen combining 5-FU and folinic acid).
Chemotherapeutic agents also include nonsteroidal anti-inflammatory drugs having analgesic, antipyretic and anti-inflammatory effects. NSAIDs include non-selective inhibitors of the enzyme cyclooxygenase. Specific examples of NSAIDs include aspirin (aspirin), propionic acid derivatives such as ibuprofen (ibuprolofen), fenoprofen (fenoprofen), ketoprofen (ketoprofen), flurbiprofen (flurbiprofen), oxaprozin (oxazin) and naproxen (naproxen), acetic acid derivatives such as indomethacin (indomethacin), sulindac (sulindac), etodolac (etodolac), diclofenac (difofenac), enolic acid derivatives such as piroxicam (piroxicam), meloxicam (meloxicam), tenoxicam (tenoxicam), oxaxicam (droxicam), lornoxicam (lornoxicam) and isoxicam (isoxicam), fenamic acid derivatives such as mefenamic acid (mefenamic acid), meclofenamic acid (clofenamic acid), flufenamic acid (COX), fenamic acid (fenamic acid), fenamic acid (COX), fenamic acid (2-loxb), etoricoxib (etoricoxib), etoricoxib (loxacin (loxb), etoricoxib (loxacin), etoricoxib), rofecoxib (rofecoxib), and valdecoxib (valdecoxib). NSAIDs may be indicated for symptomatic relief of conditions such as rheumatoid arthritis, osteoarthritis, inflammatory joint disease, ankylosing spondylitis, psoriatic arthritis, reiter's syndrome, acute gout, dysmenorrhea, bone metastasis pain, headache and migraine, post-operative pain, mild to moderate pain due to inflammation and tissue injury, fever, ileus, and renal colic.
The term "cytokine" is a generic term for proteins released by one cell population that act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines; interleukins (IL), such as IL-1, IL-1 α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15; tumor necrosis factors such as TNF- α or TNF- β; and other polypeptide factors, including LIF and Kit Ligand (KL) and interferon gamma. As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the natural sequence cytokines, including small molecule entities produced by artificial synthesis, and pharmaceutically acceptable derivatives and salts thereof.
The term "PD-1 axis binding antagonist" is a molecule that inhibits the interaction of the PD-1 axis binding partner with one or more of its binding partners, thereby removing T cell dysfunction resulting from signaling on the PD-1 signaling axis-one outcome is restoration or enhancement of T cell function (e.g., proliferation, cytokine production, target cell killing). As used herein, PD-1 axis binding antagonists include PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists.
The term "PD-1 binding antagonist" is a molecule that reduces, blocks, inhibits, eliminates or interferes with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners (such as PD-L1, PD-L2). In some embodiments, the PD-1 binding antagonist is a molecule that inhibits binding of PD-1 to its binding partner. In a particular aspect, the PD-1 binding antagonist inhibits PD-1 from binding to PD-L1 and/or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signal transduction resulting from the interaction of PD-1 with PD-L1 and/or PD-L2. In one embodiment, the PD-1 binding antagonist reduces negative co-stimulatory signals (mediated signaling via PD-1) mediated by or via cell surface proteins expressed on T lymphocytes, thereby rendering dysfunctional T cells less dysfunctional (e.g., enhancing effector response to antigen recognition). In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In a particular aspect, the PD-1 binding antagonist is MDX-1106 as described herein. In another particular aspect, the PD-1 binding antagonist is Merck 3475 described herein. In another particular aspect, the PD-1 binding antagonist is CT-011 as described herein.
The term "PD-L1 binding antagonist" is a molecule that reduces, blocks, inhibits, eliminates or interferes with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners (such as PD-1, B7-1). In some embodiments, the PD-L1 binding antagonist is a molecule that inhibits binding of PD-L1 to its binding partner. In a particular aspect, the PD-L1 binding antagonist inhibits PD-L1 from binding to PD-1 and/or B7-1. In some embodiments, PD-L1 binding antagonists include anti-PD-L1 antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners (such as PD-1, B7-1). In one embodiment, the PD-L1 binding antagonist reduces negative co-stimulatory signals mediated by or via cell surface proteins expressed on T lymphocytes (signaling is mediated via PD-L1), thereby rendering dysfunctional T cells less dysfunctional (e.g., enhancing effector response to antigen recognition). In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In a particular aspect, the anti-PD-L1 antibody is yw243.55.s70 as described herein. In another specific aspect, the anti-PD-L1 antibody is MDX-1105 as described herein. In yet another specific aspect, the anti-PD-L1 antibody is MPDL3280A described herein.
The term "PD-L2 binding antagonist" is a molecule that reduces, blocks, inhibits, eliminates or interferes with signal transduction resulting from the interaction of PD-L2 with one or more of its binding partners (such as PD-1). In some embodiments, the PD-L2 binding antagonist is a molecule that inhibits binding of PD-L2 to its binding partner. In a particular aspect, the PD-L2 binding antagonist inhibits PD-L2 from binding to PD-1. In some embodiments, PD-L2 antagonists include anti-PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signal transduction resulting from the interaction of PD-L2 with one or more of its binding partners (such as PD-1). In one embodiment, the PD-L2 binding antagonist reduces negative co-stimulatory signals mediated by or via cell surface proteins expressed on T lymphocytes (signaling is mediated via PD-L2), thereby rendering dysfunctional T cells less dysfunctional (e.g., enhancing effector response to antigen recognition). In some embodiments, the PD-L2 binding antagonist is an immunoadhesin.
The term "phagocytosis" means the internalization of cellular or particulate matter by a cell. In some embodiments, the phagocytic cell or phagocytic cell is a macrophage or neutrophil. In some embodiments, the cell is a cell that expresses human OX 40. Methods for determining phagocytosis are known in the art and include the use of microscopy to detect the presence of another cell internalized within the cell. In other embodiments, FACS is used to detect phagocytosis, e.g., by detecting the presence of a detectably labeled cell (which may be detectably labeled, e.g., with a different label than the cells described below) within another cell.
As used herein, the phrase "does not possess substantial activity" or "substantially inactive" with respect to an antibody means that the antibody does not exhibit activity above background levels (in some embodiments, statistically significant above background levels). As used herein, the phrase "little to no activity" with respect to an antibody means that the antibody does not exhibit a biologically significant amount of function. The function can be measured or detected according to any assay or technique known in the art, including, for example, those described herein. In some embodiments, the antibody function is to stimulate effector T cell proliferation and/or cytokine secretion.
As used herein, the term "biomarker" or "marker" generally refers to a molecule, including a gene, mRNA, protein, carbohydrate structure, or glycolipid, whose expression or secretion in or on a tissue or cell can be detected by known methods (or methods disclosed herein) and is predictive of or useful in predicting (or aiding in predicting) the responsiveness of a cell, tissue, or patient to a therapeutic regimen.
"patient sample" refers to a collection of cells or fluids obtained from a cancer patient. The source of the tissue or cell sample may be a solid tissue, like from a fresh, frozen and/or preserved organ or tissue sample or biopsy sample or punch sample; blood or any blood component; body fluids such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from a subject at any time of pregnancy or development. Tissue samples may contain compounds that are not naturally intermixed with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. Examples of tumor samples herein include, but are not limited to, tumor biopsies, fine needle aspirates, bronchial lavage, pleural fluid (pleural fluid), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples, such as formalin-fixed, paraffin-embedded tumor samples or frozen tumor samples.
As used herein, the phrase "… … -based expression" means that information regarding the level of expression or the presence or absence (e.g., the presence or absence or prevalence (e.g., percentage of cells present)) of expression of one or more biomarkers herein (e.g., the presence or absence or amount or prevalence of FcR-expressing cells, or the presence or absence or amount or prevalence of, e.g., human effector cells) is used to inform treatment decisions, information provided on package inserts, or marketing/promotion instructions, etc.
A cancer or biological sample "having human effector cells" is a cancer or biological sample in which human effector cells (e.g., infiltrating human effector cells) are present in the sample in a diagnostic test.
A cancer or biological sample "having cells that express FcR" is a cancer or biological sample in which FcR expressing cells (e.g., cells that infiltrate FcR expressing cells) are present in the sample in a diagnostic test. In some embodiments, the FcR is an Fc γ R. In some embodiments, the FcR is an activating Fc γ R.
As used herein, the phrase "recommending treatment" refers to using information or data generated relating to the level or presence of c-met in a sample of a patient to identify the patient as suitable or unsuitable for treatment with a certain therapy. In some embodiments, the therapy may comprise a c-met antibody (e.g., onartuzumab) in some embodiments, the therapy may comprise a VEGF antagonist (e.g., bevacizumab). In some embodiments, the therapy may comprise an anti-human OX40 agonist antibody. The information or data may be in any form, written, verbal, or electronic. In some embodiments, using the generated information or data includes communicating, presenting, reporting, storing, sending, transferring, provisioning, transmitting, delivering, distributing, or a combination thereof. In some embodiments, the communicating, presenting, reporting, storing, sending, transferring, provisioning, transmitting, delivering, distributing, or a combination thereof, is performed by a computing device, an analysis unit, or a combination thereof. In some further embodiments, the communicating, presenting, reporting, storing, sending, transferring, supplying, transmitting, distributing, or a combination thereof, is performed by an individual (e.g., a laboratory or medical professional). In some embodiments, the information or data comprises a comparison of the amount or prevalence of FcR expressing cells to a reference level. In some embodiments, the information or data comprises a comparison of the amount or prevalence of human effector cells to a reference level. In some embodiments, the information or data comprises an indication of the presence or absence of human effector cells or FcR expressing cells in the sample. In some embodiments, the information or data includes an indication that FcR expressing cells and/or human effector cells are present in a particular percentage of cells (e.g., high prevalence). In some embodiments, the information or data comprises an indication that the patient is suitable or unsuitable for treatment with a therapy comprising an anti-human OX40 agonist antibody.
Compositions and methods
In one aspect, the invention is based, in part, on the identification of a plurality of OX40 binding agents. In certain embodiments, antibodies (e.g., agonist antibodies) that bind to human OX40 are provided. The antibodies of the invention are useful, for example, for the diagnosis or treatment of cancer and other disorders associated with OX40 expression and/or activity.
A. Exemplary anti-OX 40 antibodies
In one aspect, the invention provides isolated antibodies that bind to human OX 40.
In some embodiments, the anti-human OX40 agonist antibody binds human OX40 with an affinity of less than or equal to about 0.45 nM. In some embodiments, the anti-human OX40 antibody binds human OX40 with an affinity of less than or equal to about 0.4 nM. In some embodiments, the anti-human OX40 antibody binds human OX40 with an affinity of less than or equal to about 0.5 nM. In some embodiments, the binding affinity is determined using a radioimmunoassay.
In some embodiments, the anti-human OX40 agonist antibody binds to human OX40 and cynomolgus monkey OX 40. In some embodiments, binding is determined using FACS assays. In some embodiments, binding to human OX40 has an EC50 of about 0.2 ug/ml. In some embodiments, binding to human OX40 has an EC50 of about 0.3ug/ml or less. In some embodiments, the binding to cynomolgus OX40 has an EC50 of about 1.5 ug/ml. In some embodiments, the binding to cynomolgus OX40 has an EC50 of about 1.4 ug/ml.
In some embodiments, the anti-human OX40 agonist antibody does not bind to rat OX40 or mouse OX 40.
In some embodiments, the anti-human OX40 agonist antibody is a subtractive anti-human OX40 antibody (e.g., depletes cells expressing human OX 40). In some embodiments, the cell expressing human OX40 is a CD4+ effector T cell. In some embodiments, the human OX 40-expressing cell is a Treg cell. In some embodiments, the depleting is by ADCC and/or phagocytosis. In some embodiments, the antibody mediates ADCC by binding to Fc γ rs expressed by human effector cells and activating human effector cell function. In some embodiments, the antibody mediates phagocytosis by binding to Fc γ rs expressed by human effector cells and activating human effector cell function. Exemplary human effector cells include, for example, macrophages, Natural Killer (NK) cells, monocytes, neutrophils. In some embodiments, the human effector cell is a macrophage. In some embodiments, the human effector cell is an NK cell. In some embodiments, the depletion is not by apoptosis.
In some embodiments, the anti-human OX40 agonist antibody has a functional Fc region. In some embodiments, the effector function of the functional Fc region is ADCC. In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector functions of the functional Fc region are ADCC and phagocytosis. In some embodiments, the Fc region is human IgG 1. In some embodiments, the Fc region is human IgG 4.
In some embodiments, the anti-human OX40 agonist antibody does not induce apoptosis in OX 40-expressing cells (e.g., tregs). In some embodiments, apoptosis is determined using an antibody concentration of 30ug/ml, for example by using annexin V and propidium iodide stained tregs to determine whether apoptosis occurs.
In some embodiments, the anti-human OX40 agonist antibody enhances CD4+ effector T cell function, e.g., by increasing CD4+ effector T cell proliferation and/or increasing gamma interferon production of CD4+ effector T cells (e.g., as compared to proliferation and/or cytokine production prior to anti-human OX40 agonist antibody treatment). In some embodiments, the cytokine is interferon-gamma. In some embodiments, the anti-human OX40 agonist antibody increases the number of (infiltrating) CD4+ effector T cells (e.g., the total number of CD4+ effector T cells, or, e.g., the percentage of CD4+ cells in CD45+ cells) within the tumor, e.g., compared to the number of (infiltrating) CD4+ T cells within the tumor prior to treatment with the anti-human OX40 agonist antibody. In some embodiments, the anti-human OX40 agonist antibody increases the number of γ -interferon expressing intratumoral (infiltrating) CD4+ effector T cells (e.g., the total number of γ -interferon expressing CD4+ cells, or the percentage of γ -interferon expressing CD4+ cells, e.g., total CD4+ cells), e.g., as compared to the number of γ -interferon expressing intratumoral (infiltrating) CD4+ T cells prior to treatment with the anti-human OX40 agonist antibody.
In some embodiments, the anti-human OX40 agonist antibody increases the number of (infiltrating) CD8+ effector T cells within the tumor (e.g., the total number of CD8+ effector T cells, or, e.g., the percentage of CD8+ in CD45+ cells), e.g., as compared to the number of (infiltrating) CD8+ T effector cells within the tumor prior to treatment with the anti-human OX40 agonist antibody. In some embodiments, the anti-human OX40 agonist antibody increases the number of gamma-interferon expressing intratumoral (infiltrating) CD8+ effector T cells (e.g., the percentage of gamma-interferon expressing CD8+ cells in total CD8+ cells), e.g., as compared to the number of gamma-interferon expressing intratumoral (infiltrating) CD8+ T cells prior to treatment with the anti-human OX40 agonist antibody.
In some embodiments, the anti-human OX40 agonist antibody enhances memory T cell function, e.g., by increasing memory T cell proliferation and/or increasing cytokine production by memory cells. In some embodiments, the cytokine is interferon-gamma.
In some embodiments, the anti-human OX40 agonist antibody inhibits Treg function, for example by Treg suppression that reduces effector T cell function (e.g., effector T cell proliferation and/or effector T cell cytokine secretion). In some embodiments, the effector T cell is a CD4+ effector T cell. In some embodiments, the anti-human OX40 agonist antibody reduces the number of (infiltrating) tregs within a tumor (e.g., the total number of tregs or the percentage of Fox3p + cells among, e.g., CD4+ cells).
In some embodiments, the anti-human OX40 agonist antibody is engineered to increase effector function (e.g., as compared to effector function in wild-type IgG 1). In some embodiments, the antibody has increased binding to an Fc γ receptor. In some embodiments, the antibody lacks fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. In some embodiments, the Fc region comprises bisected oligosaccharides, e.g., wherein the biantennary oligosaccharides attached to the antibody Fc region are bisected by GlcNAc. In some embodiments, the antibody comprises an Fc region having one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and/or 334(EU residue numbering) of the Fc region.
In some embodiments, the anti-human OX40 agonist antibody increases OX40 signaling in an OX 40-expressing target cell. In some embodiments, OX40 signaling is detected by monitoring NFkB downstream signaling.
In some embodiments, the anti-human OX40 agonist antibody is stable after 2 weeks of treatment at 40 ℃.
In some embodiments, the anti-human OX40 agonist antibody binds to a human effector cell, e.g., binds to an Fc γ R (e.g., an activating Fc γ R) expressed by a human effector cell. In some embodiments, the human effector cell performs (is capable of performing) ADCC effector function. In some embodiments, the human effector cell performs (is capable of performing) phagocytic effector function.
In some embodiments, an anti-human OX40 agonist antibody comprising a variant IgG1 Fc polypeptide comprising a mutation that abrogates binding to human effector cells, e.g., a DANA mutation, has reduced activity (e.g., CD4+ effector T cell function, e.g., proliferation) relative to an anti-human OX40 agonist antibody comprising a native sequence IgG1 Fc portion. In some embodiments, an anti-human OX40 agonist antibody comprising a variant IgG1 Fc polypeptide comprising a mutation that abrogates binding to human effector cells, e.g., a DANA mutation, does not possess substantial activity (e.g., CD4+ effector T cell function, e.g., proliferation).
In some embodiments, antibody cross-linking is required for anti-human OX40 agonist antibody function. In some embodiments, the function is to stimulate CD4+ effector T cell proliferation. In some embodiments, antibody cross-linking is determined by providing an anti-human OX40 agonist antibody that adheres to a solid surface (e.g., a cell culture plate). In some embodiments, antibody cross-linking is determined by introducing mutations (e.g., DANA mutations) in the IgG1 Fc portion of the antibody and testing the mutant antibody for function.
In some embodiments, the anti-human OX40 agonist antibody competes with OX40L for binding to human OX 40. In some embodiments, addition of OX40L in an in vitro assay does not enhance anti-human OX40 antibody function.
According to another embodiment, the anti-human OX40 agonist antibody includes any one of the following properties, any combination of the following properties, or all of the following properties: (1) binds human OX40 with an affinity of less than or equal to about 0.45nM, in some embodiments, binds human OX40 with an affinity of less than or equal to about 0.4nM, in some embodiments, binds human OX40 with an affinity of less than or equal to about 0.5nM, in some embodiments, the binding affinity is determined using a radioimmunoassay; (2) binding to human OX40 and cynomolgus monkey OX40, in some embodiments binding is determined using FACS assays, (3) binding to human OX40 at about 0.2ug/ml of EC50, in some embodiments binding to human OX40 at about 0.3ug/ml or less of EC50, in some embodiments binding to cynomolgus monkey OX40 at about 1.5ug/ml of EC50, in some embodiments binding to cynomolgus monkey 40 at about 1.4ug/ml of EC50, (4) does not substantially bind to rat OX40 or mouse OX40, (6) is a depleting OX40 antibody (e.g., cells expressing human OX 40), in some embodiments the cells are CD4+ effector T cells and/or Treg cells, (7) enhances CD4+ effector T cell function, e.g., by increasing CD4+ effector T cells and/or increasing proliferation of human OX 4+ effector cells and/or anti-human gamma hormone receptor proliferation (pro-treatment with anti-human OX40 or anti-pro-interferon (e.g. anti-pro-treatment In contrast), (8) enhancing memory T cell function, e.g., by increasing memory T cell proliferation and/or increasing cytokine production by memory cells, (9) inhibiting Treg function, e.g., by Treg suppression that reduces effector T cell function (e.g., effector T cell proliferation and/or effector T cell cytokine secretion). In some embodiments, the effector T cell is a CD4+ effector T cell, (10) increases OX40 signaling in a target cell expressing OX40 (in some embodiments, OX40 signaling is detected by monitoring NFkB downstream signaling), (11) is stable after 2 weeks of treatment at 40 ℃, (12) binds a human effector cell, e.g., binds an fcyr expressed by a human effector cell, (13) an anti-human OX40 agonist antibody comprising a variant IgG1Fc polypeptide comprising a mutation that abrogates binding to a human effector cell, e.g., N297G, has reduced activity (e.g., CD4+ effector T cell function, e.g., proliferation) relative to an anti-human OX40 agonist antibody comprising a native sequence IgG1Fc portion, in some embodiments, an anti-human OX40 agonist antibody comprising a variant IgG1Fc polypeptide comprising a mutation that abrogates binding to a human effector cell, e.g., N297G, does not possess substantial activity (e.g., CD4+ effector cell function, e.g., proliferation), (14) anti-human OX40 agonist antibody function requires antibody cross-linking (e.g., via Fc receptor binding).
In one aspect, the invention provides anti-human OX40 agonist antibodies comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising amino acid sequence SEQ ID NO 2; (b) HVR-H2 comprising amino acid sequence SEQ ID NO 3; (c) HVR-H3 comprising amino acid sequence SEQ ID NO 4; (d) HVR-L1 comprising amino acid sequence SEQ ID NO 5; (e) HVR-L2 comprising amino acid sequence SEQ ID NO 6; and (f) HVR-L3 comprising amino acid sequence SEQ ID NO: 7.
In one aspect, the invention provides anti-human OX40 agonist antibodies comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 comprising amino acid sequence SEQ ID NO 2; (b) HVR-H2 comprising amino acid sequence SEQ ID NO 3; and (c) HVR-H3 comprising the amino acid sequence SEQ ID NO: 4. In one embodiment, the antibody comprises HVR-H3 comprising amino acid sequence SEQ ID NO 4. In another embodiment, the antibody comprises HVR-H3 comprising amino acid sequence SEQ ID NO. 4 and HVR-L3 comprising amino acid sequence SEQ ID NO. 7. In yet another embodiment, the antibody comprises HVR-H3 comprising amino acid sequence SEQ ID NO. 4, HVR-L3 comprising amino acid sequence SEQ ID NO. 7, and HVR-H2 comprising amino acid sequence SEQ ID NO. 3. In yet another embodiment, the antibody comprises (a) HVR-H1 comprising amino acid sequence SEQ ID NO 2; (b) HVR-H2 comprising amino acid sequence SEQ ID NO 3; and (c) HVR-H3 comprising the amino acid sequence SEQ ID NO: 4.
In another aspect, the invention provides anti-human OX40 agonist antibodies comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising amino acid sequence SEQ ID NO 5; (b) HVR-L2 comprising amino acid sequence SEQ ID NO 6; and (c) HVR-L3 comprising the amino acid sequence SEQ ID NO: 7. In one embodiment, the antibody comprises (a) HVR-L1 comprising amino acid sequence SEQ ID NO: 5; (b) HVR-L2 comprising amino acid sequence SEQ ID NO 6; and (c) HVR-L3 comprising the amino acid sequence SEQ ID NO: 7.
In another aspect, an anti-human OX40 agonist antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1 comprising amino acid sequence SEQ ID NO. 2, (ii) HVR-H2 comprising amino acid sequence SEQ ID NO. 3, and (iii) HVR-H3 comprising amino acid sequence SEQ ID NO. 4; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) HVR-L1 comprising amino acid sequence SEQ ID NO:5, (ii) HVR-L2 comprising amino acid sequence SEQ ID NO:6, and (c) HVR-L3 comprising amino acid sequence SEQ ID NO: 7.
In another aspect, the invention provides anti-human OX40 agonist antibodies comprising (a) HVR-H1 comprising the amino acid sequence SEQ id no: 2; (b) HVR-H2 comprising amino acid sequence SEQ ID NO 3; (c) HVR-H3 comprising amino acid sequence SEQ ID NO 4; (d) HVR-L1 comprising amino acid sequence SEQ ID NO 5; (e) HVR-L2 comprising amino acid sequence SEQ ID NO 6; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 7.
In one aspect, the invention provides anti-human OX40 agonist antibodies comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising amino acid sequence SEQ ID NO 2; (b) HVR-H2 comprising amino acid sequence SEQ ID NO 3; (c) HVR-H3 comprising amino acid sequence SEQ ID NO 4; (d) HVR-L1 comprising amino acid sequence SEQ ID NO 5; (e) HVR-L2 comprising amino acid sequence SEQ ID NO 6; and (f) HVR-L3 comprising amino acid sequence SEQ ID NO: 26.
In another embodiment, the antibody comprises HVR-H3 comprising amino acid sequence SEQ ID NO. 4 and HVR-L3 comprising amino acid sequence SEQ ID NO. 26. In yet another embodiment, the antibody comprises HVR-H3 comprising amino acid sequence SEQ ID NO. 4, HVR-L3 comprising amino acid sequence SEQ ID NO. 26, and HVR-H2 comprising amino acid sequence SEQ ID NO. 3.
In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1 comprising amino acid sequence SEQ ID NO. 2, (ii) HVR-H2 comprising amino acid sequence SEQ ID NO. 3, and (iii) HVR-H3 comprising amino acid sequence SEQ ID NO. 4; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) HVR-L1 comprising amino acid sequence SEQ ID NO. 5, (ii) HVR-L2 comprising amino acid sequence SEQ ID NO. 6, and (c) HVR-L3 comprising amino acid sequence SEQ ID NO. 26.
In another aspect, the invention provides an antibody comprising (a) HVR-H1 comprising amino acid sequence SEQ ID NO. 2; (b) HVR-H2 comprising amino acid sequence SEQ ID NO 3; (c) HVR-H3 comprising amino acid sequence SEQ ID NO 4; (d) HVR-L1 comprising amino acid sequence SEQ ID NO 5; (e) HVR-L2 comprising amino acid sequence SEQ ID NO 6; and (f) HVR-L3 comprising amino acid sequence SEQ ID NO: 26.
In one aspect, the invention provides anti-human OX40 agonist antibodies comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising amino acid sequence SEQ ID NO 2; (b) HVR-H2 comprising amino acid sequence SEQ ID NO 3; (c) HVR-H3 comprising amino acid sequence SEQ ID NO. 4; (d) HVR-L1 comprising amino acid sequence SEQ ID NO 5; (e) HVR-L2 comprising amino acid sequence SEQ ID NO 6; and (f) HVR-L3 comprising amino acid sequence SEQ ID NO: 27.
In another embodiment, the antibody comprises HVR-H3 comprising amino acid sequence SEQ ID NO. 4 and HVR-L3 comprising amino acid sequence SEQ ID NO. 27. In yet another embodiment, the antibody comprises HVR-H3 comprising amino acid sequence SEQ ID NO. 4, HVR-L3 comprising amino acid sequence SEQ ID NO. 27, and HVR-H2 comprising amino acid sequence SEQ ID NO. 3.
In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1 comprising amino acid sequence SEQ ID NO. 2, (ii) HVR-H2 comprising amino acid sequence SEQ ID NO. 3, and (iii) HVR-H3 comprising amino acid sequence SEQ ID NO. 4; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) HVR-L1 comprising amino acid sequence SEQ ID NO. 5, (ii) HVR-L2 comprising amino acid sequence SEQ ID NO. 6, and (c) HVR-L3 comprising amino acid sequence SEQ ID NO. 27.
In another aspect, the invention provides an antibody comprising (a) HVR-H1 comprising amino acid sequence SEQ ID NO. 2; (b) HVR-H2 comprising amino acid sequence SEQ ID NO 3; (c) HVR-H3 comprising amino acid sequence SEQ ID NO 4; (d) HVR-L1 comprising amino acid sequence SEQ ID NO 5; (e) HVR-L2 comprising amino acid sequence SEQ ID NO 6; and (f) HVR-L3 comprising amino acid sequence SEQ ID NO: 27.
In one aspect, the invention provides anti-human OX40 agonist antibodies comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising amino acid sequence SEQ ID NO 2, 8 or 9; (b) HVR-H2 comprising amino acid sequence SEQ ID NO 3, 10, 11, 12, 13 or 14; (c) HVR-H3 comprising amino acid sequence SEQ ID NO 4, 15, or 19; (d) HVR-L1 comprising amino acid sequence SEQ ID NO 5; (e) HVR-L2 comprising amino acid sequence SEQ ID NO 6; and (f) HVR-L3 comprising amino acid sequence SEQ ID NO 7, 22, 23, 24, 25, 26, 27, or 28.
In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 comprising amino acid sequence SEQ ID NO 2, 8 or 9; (b) HVR-H2 comprising amino acid sequence SEQ ID NO 3, 10, 11, 12, 13 or 14; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO 4, 15, or 19. In one embodiment, the antibody comprises HVR-H3 comprising amino acid sequence SEQ ID NO 4, 15, or 19. In another embodiment, the antibody comprises HVR-H3 comprising amino acid sequence SEQ ID NO 4, 15, or 19 and HVR-L3 comprising amino acid sequence SEQ ID NO 7, 22, 23, 24, 25, 26, 27, or 28. In yet another embodiment, the antibody comprises HVR-H3 comprising amino acid sequence SEQ ID NO 4, 15, or 19, HVR-L3 comprising amino acid sequence SEQ ID NO 7, 22, 23, 24, 25, 26, 27, or 28, and HVR-H2 comprising amino acid sequence SEQ ID NO 3, 10, 11, 12, 13, or 14. In yet another embodiment, the antibody comprises (a) HVR-H1 comprising amino acid sequence SEQ ID NO 2, 8 or 9; (b) HVR-H2 comprising amino acid sequence SEQ ID NO 3, 10, 11, 12, 13 or 14; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO 4, 15, or 19.
In another aspect, the invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising amino acid sequence SEQ ID NO 5; (b) HVR-L2 comprising amino acid sequence SEQ ID NO 6; and (c) HVR-L3 comprising amino acid sequence SEQ ID NO 7, 22, 23, 24, 25, 26, 27, or 28. In one embodiment, the antibody comprises (a) HVR-L1 comprising amino acid sequence SEQ ID NO: 5; (b) HVR-L2 comprising amino acid sequence SEQ ID NO 6; and (c) HVR-L3 comprising amino acid sequence SEQ ID NO 7, 22, 23, 24, 25, 26, 27, or 28.
In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1 comprising the amino acid sequence SEQ ID No. 2, 8 or 9, (ii) HVR-H2 comprising the amino acid sequence SEQ ID No. 3, 10, 11, 12, 13 or 14, and (iii) HVR-H3 comprising the amino acid sequence SEQ ID No. 4, 15 or 19; and (b) a VL domain comprising at least one, at least two, or all three VLHVR sequences selected from: (i) HVR-L1 comprising amino acid sequence SEQ ID NO. 5, (ii) HVR-L2 comprising amino acid sequence SEQ ID NO. 6, and (c) HVR-L3 comprising amino acid sequence SEQ ID NO. 7, 22, 23, 24, 25, 26, 27, or 28.
In another aspect, the invention provides an antibody comprising (a) HVR-H1 comprising amino acid sequence SEQ ID NO 2, 8 or 9; (b) HVR-H2 comprising amino acid sequence SEQ ID NO 3, 10, 11, 12, 13 or 14; (c) HVR-H3 comprising amino acid sequence SEQ ID NO 4, 15, or 19; (d) HVR-L1 comprising amino acid sequence SEQ ID NO 5; (e) HVR-L2 comprising amino acid sequence SEQ ID NO 6; and (f) HVR-L3 comprising amino acid sequence SEQ ID NO 7, 22, 23, 24, 25, 26, 27, or 28.
In one aspect, the invention provides anti-human OX40 agonist antibodies comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising amino acid sequence SEQ ID NO: 172; (b) HVR-H2 comprising amino acid sequence SEQ ID NO: 173; (c) HVR-H3 comprising amino acid sequence SEQ ID NO: 174; (d) HVR-L1 comprising amino acid sequence SEQ ID NO 5; (e) HVR-L2 comprising amino acid sequence SEQ ID NO 6; and (f) HVR-L3 comprising amino acid sequence SEQ ID NO: 175. In some embodiments, HVR-H2 is not DMYPDAAAASYNQKFRE (SEQ ID NO: 193). In some embodiments, HVR-H3 is not APRWAAAAA (SEQ ID NO: 194). In some embodiments, HVR-L3 is not QAAAAAAAT (SEQ ID NO: 195).
In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 comprising amino acid sequence SEQ ID NO: 172; (b) HVR-H2 comprising amino acid sequence SEQ ID NO: 173; and (c) HVR-H3 comprising amino acid sequence SEQ ID NO: 174. In one embodiment, the antibody comprises HVR-H3 comprising amino acid sequence SEQ ID NO: 174. In another embodiment, the antibody comprises HVR-H3 comprising amino acid sequence SEQ ID NO:174 and HVR-L3 comprising amino acid sequence SEQ ID NO: 175. In yet another embodiment, the antibody comprises HVR-H3 comprising amino acid sequence SEQ ID NO:174, HVR-L3 comprising amino acid sequence SEQ ID NO:175, and HVR-H2 comprising amino acid sequence SEQ ID NO: 173. In yet another embodiment, the antibody comprises (a) HVR-H1 comprising amino acid sequence SEQ ID NO: 172; (b) HVR-H2 comprising amino acid sequence SEQ ID NO: 173; and (c) HVR-H3 comprising amino acid sequence SEQ ID NO: 174. In some embodiments, HVR-H2 is not DMYPDAAAASYNQKFRE (SEQ ID NO: 193). In some embodiments, HVR-H3 is not APRWAAAAA (SEQ ID NO: 194). In some embodiments, HVR-L3 is not QAAAAAAAT (SEQ ID NO: 195).
In another aspect, the invention provides an antibody comprising (a) HVR-L1 comprising amino acid sequence SEQ ID NO: 5; (b) HVR-L2 comprising amino acid sequence SEQ ID NO 6; and (c) HVR-L3 comprising amino acid sequence SEQ ID NO: 175. In some embodiments, HVR-L3 is not QAAAAAAAT (SEQ ID NO: 195).
In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1 comprising amino acid sequence SEQ ID NO:172, (ii) HVR-H2 comprising amino acid sequence SEQ ID NO:173, and (iii) HVR-H3 comprising amino acid sequence SEQ ID NO: 174; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) HVR-L1 comprising amino acid sequence SEQ ID NO:5, (ii) HVR-L2 comprising amino acid sequence SEQ ID NO:6, and (c) HVR-L3 comprising amino acid sequence SEQ ID NO: 175.
In another aspect, the invention provides an antibody comprising (a) HVR-H1 comprising amino acid sequence SEQ ID NO: 172; (b) HVR-H2 comprising amino acid sequence SEQ ID NO: 173; (c) HVR-H3 comprising amino acid sequence SEQ ID NO: 174; (d) HVR-L1 comprising amino acid sequence SEQ ID NO 5; (e) HVR-L2 comprising amino acid sequence SEQ ID NO 6; and (f) HVR-L3 comprising amino acid sequence SEQ ID NO: 175. In some embodiments, HVR-H2 is not DMYPDAAAASYNQKFRE (SEQ ID NO: 193). In some embodiments, HVR-H3 is not APRWAAAAA (SEQ ID NO: 194). In some embodiments, HVR-L3 is not QAAAAAAAT (SEQ ID NO: 195).
The consensus sequences SEQ ID NO:172, 173, 174 and 175 cover all possible combinations of the above substitutions.
In one aspect, the invention provides anti-human OX40 agonist antibodies comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising amino acid sequence SEQ ID NO: 29; (b) HVR-H2 comprising amino acid sequence SEQ ID NO 30; (c) HVR-H3 comprising amino acid sequence SEQ ID NO 33; (d) HVR-L1 comprising amino acid sequence SEQ ID NO 37; (e) HVR-L2 comprising amino acid sequence SEQ ID NO: 39; and (f) HVR-L3 comprising amino acid sequence SEQ ID NO: 42.
In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 comprising amino acid sequence SEQ ID NO: 29; (b) HVR-H2 comprising amino acid sequence SEQ ID NO. 30; and (c) HVR-H3 comprising amino acid sequence SEQ ID NO: 33. In one embodiment, the antibody comprises HVR-H3 comprising amino acid sequence SEQ ID NO 33. In another embodiment, the antibody comprises HVR-H3 comprising amino acid sequence SEQ ID NO 33 and HVR-L3 comprising amino acid sequence SEQ ID NO 42. In yet another embodiment, the antibody comprises HVR-H3 comprising amino acid sequence SEQ ID NO 33, HVR-L3 comprising amino acid sequence SEQ ID NO 42, and HVR-H2 comprising amino acid sequence SEQ ID NO 30. In yet another embodiment, the antibody comprises (a) HVR-H1 comprising amino acid sequence SEQ ID NO: 29; (b) HVR-H2 comprising amino acid sequence SEQ ID NO 30; and (c) HVR-H3 comprising amino acid sequence SEQ ID NO: 33.
In another aspect, the invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising amino acid sequence SEQ ID NO 37; (b) HVR-L2 comprising amino acid sequence SEQ ID NO: 39; and (c) HVR-L3 comprising amino acid sequence SEQ ID NO: 42. In one embodiment, the antibody comprises (a) HVR-L1 comprising amino acid sequence SEQ ID NO 37; (b) HVR-L2 comprising amino acid sequence SEQ ID NO: 39; and (c) HVR-L3 comprising amino acid sequence SEQ ID NO: 42.
In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1 comprising amino acid sequence SEQ ID NO:29, (ii) HVR-H2 comprising amino acid sequence SEQ ID NO:30, and (iii) HVR-H3 comprising amino acid sequence SEQ ID NO: 33; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) HVR-L1 comprising amino acid sequence SEQ ID NO:37, (ii) HVR-L2 comprising amino acid sequence SEQ ID NO:39, and (c) HVR-L3 comprising amino acid sequence SEQ ID NO: 42.
In another aspect, the invention provides an antibody comprising (a) HVR-H1 comprising amino acid sequence SEQ ID NO: 29; (b) HVR-H2 comprising amino acid sequence SEQ ID NO 30; (c) HVR-H3 comprising amino acid sequence SEQ ID NO 33; (d) HVR-L1 comprising amino acid sequence SEQ ID NO 37; (e) HVR-L2 comprising amino acid sequence SEQ ID NO: 39; and (f) HVR-L3 comprising amino acid sequence SEQ ID NO: 42.
In one aspect, the invention provides anti-human OX40 agonist antibodies comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising amino acid sequence SEQ ID NO: 29; (b) HVR-H2 comprising amino acid sequence SEQ ID NO 30; (c) HVR-H3 comprising amino acid sequence SEQ ID NO 33; (d) HVR-L1 comprising amino acid sequence SEQ ID NO 37; (e) HVR-L2 comprising amino acid sequence SEQ ID NO 40; and (f) HVR-L3 comprising amino acid sequence SEQ ID NO: 42.
In another aspect, the invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising amino acid sequence SEQ ID NO 37; (b) HVR-L2 comprising amino acid sequence SEQ ID NO 40; and (c) HVR-L3 comprising amino acid sequence SEQ ID NO: 42. In one embodiment, the antibody comprises (a) HVR-L1 comprising amino acid sequence SEQ ID NO 37; (b) HVR-L2 comprising amino acid sequence SEQ ID NO 40; and (c) HVR-L3 comprising amino acid sequence SEQ ID NO: 42.
In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1 comprising amino acid sequence SEQ ID NO:29, (ii) HVR-H2 comprising amino acid sequence SEQ ID NO:30, and (iii) HVR-H3 comprising amino acid sequence SEQ ID NO: 33; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) HVR-L1 comprising amino acid sequence SEQ ID NO:37, (ii) HVR-L2 comprising amino acid sequence SEQ ID NO:40, and (c) HVR-L3 comprising amino acid sequence SEQ ID NO: 42.
In another aspect, the invention provides an antibody comprising (a) HVR-H1 comprising amino acid sequence SEQ ID NO: 29; (b) HVR-H2 comprising amino acid sequence SEQ ID NO 30; (c) HVR-H3 comprising amino acid sequence SEQ ID NO 33; (d) HVR-L1 comprising amino acid sequence SEQ ID NO 37; (e) HVR-L2 comprising amino acid sequence SEQ ID NO 40; and (f) HVR-L3 comprising amino acid sequence SEQ ID NO: 42.
In one aspect, the invention provides anti-human OX40 agonist antibodies comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising amino acid sequence SEQ ID NO: 29; (b) HVR-H2 comprising amino acid sequence SEQ ID NO 30, 31, or 32; (c) HVR-H3 comprising amino acid sequence SEQ ID NO 33; (d) HVR-L1 comprising amino acid sequence SEQ ID NO 37; (e) HVR-L2 comprising amino acid sequence SEQ ID NO 39, 40 or 41; and (f) HVR-L3 comprising amino acid sequence SEQ ID NO:42, 43, or 44.
In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 comprising amino acid sequence SEQ ID NO: 29; (b) HVR-H2 comprising amino acid sequence SEQ ID NO 30, 31, or 32; and (c) HVR-H3 comprising amino acid sequence SEQ ID NO: 33. In another embodiment, the antibody comprises HVR-H3 comprising amino acid sequence SEQ ID NO 33 and HVR-L3 comprising amino acid sequences SEQ ID NO 42, 43, or 44. In yet another embodiment, the antibody comprises HVR-H3 comprising amino acid sequence SEQ ID NO 33, HVR-L3 comprising amino acid sequence SEQ ID NO 42, 43, or 44, and HVR-H2 comprising amino acid sequence SEQ ID NO 39, 40, or 41. In yet another embodiment, the antibody comprises (a) HVR-H1 comprising amino acid sequence SEQ ID NO: 29; (b) HVR-H2 comprising amino acid sequence SEQ ID NO 30, 31, or 32; and (c) HVR-H3 comprising amino acid sequence SEQ ID NO: 33.
In another aspect, the invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising amino acid sequence SEQ ID NO 37; (b) HVR-L2 comprising amino acid sequence SEQ ID NO 39, 40 or 41; and (c) HVR-L3 comprising amino acid sequence SEQ ID NO:42, 43, or 44. In one embodiment, the antibody comprises (a) HVR-L1 comprising amino acid sequence SEQ ID NO 37; (b) HVR-L2 comprising amino acid sequence SEQ ID NO 39, 40 or 41; and (c) HVR-L3 comprising amino acid sequence SEQ ID NO:42, 43, or 44.
In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1 comprising amino acid sequence SEQ ID NO:29, (ii) HVR-H2 comprising amino acid sequence SEQ ID NO:30, 31, or 32, and (iii) HVR-H3 comprising amino acid sequence SEQ ID NO: 33; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) HVR-L1 comprising amino acid sequence SEQ ID NO:37, (ii) HVR-L2 comprising amino acid sequence SEQ ID NO:39, 40 or 41, and (c) HVR-L3 comprising amino acid sequence SEQ ID NO:42, 43, or 44.
In another aspect, the invention provides an antibody comprising (a) HVR-H1 comprising amino acid sequence SEQ ID NO: 29; (b) HVR-H2 comprising amino acid sequence SEQ ID NO 30, 31, or 32; (c) HVR-H3 comprising amino acid sequence SEQ ID NO 33; (d) HVR-L1 comprising amino acid sequence SEQ ID NO 37; (e) HVR-L2 comprising amino acid sequence SEQ ID NO 39, 40 or 41; and (f) HVR-L3 comprising amino acid sequence SEQ ID NO:42, 43, or 44.
In one aspect, the invention provides anti-human OX40 agonist antibodies comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising amino acid sequence SEQ ID NO: 29; (b) HVR-H2 comprising amino acid sequence SEQ ID NO: 175; (c) HVR-H3 comprising amino acid sequence SEQ ID NO 33; (d) HVR-L1 comprising amino acid sequence SEQ ID NO 37; (e) HVR-L2 comprising amino acid sequence SEQ ID NO: 177; and (f) HVR-L3 comprising amino acid sequence SEQ ID NO: 178.
In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 comprising amino acid sequence SEQ ID NO: 29; (b) HVR-H2 comprising amino acid sequence SEQ ID NO: 175; and (c) HVR-H3 comprising amino acid sequence SEQ ID NO: 33. In another embodiment, the antibody comprises HVR-H3 comprising amino acid sequence SEQ ID NO 33 and HVR-L3 comprising amino acid sequence SEQ ID NO 177. In yet another embodiment, the antibody comprises HVR-H3 comprising amino acid sequence SEQ ID NO 33, HVR-L3 comprising amino acid sequence SEQ ID NO 178, and HVR-H2 comprising amino acid sequence SEQ ID NO 176. In yet another embodiment, the antibody comprises (a) HVR-H1 comprising amino acid sequence SEQ ID NO: 29; (b) HVR-H2 comprising amino acid sequence SEQ ID NO: 176; and (c) HVR-H3 comprising amino acid sequence SEQ ID NO: 33.
In another aspect, the invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising amino acid sequence SEQ ID NO 37; (b) HVR-L2 comprising amino acid sequence SEQ ID NO: 177; and (c) HVR-L3 comprising amino acid sequence SEQ ID NO: 177. In one embodiment, the antibody comprises (a) HVR-L1 comprising amino acid sequence SEQ ID NO 37; (b) HVR-L2 comprising amino acid sequence SEQ ID NO: 177; and (c) HVR-L3 comprising amino acid sequence SEQ ID NO: 178.
In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1 comprising amino acid sequence SEQ ID NO:29, (ii) HVR-H2 comprising amino acid sequence SEQ ID NO:176, and (iii) HVR-H3 comprising amino acid sequence SEQ ID NO: 33; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) HVR-L1 comprising amino acid sequence SEQ ID NO:37, (ii) HVR-L2 comprising amino acid sequence SEQ ID NO:177, and (c) HVR-L3 comprising amino acid sequence SEQ ID NO: 178.
In another aspect, the invention provides an antibody comprising (a) HVR-H1 comprising amino acid sequence SEQ ID NO: 29; (b) HVR-H2 comprising amino acid sequence SEQ ID NO: 176; (c) HVR-H3 comprising amino acid sequence SEQ ID NO 33; (d) HVR-L1 comprising amino acid sequence SEQ ID NO 37; (e) HVR-L2 comprising amino acid sequence SEQ ID NO: 177; and (f) HVR-L3 comprising amino acid sequence SEQ ID NO: 178.
In any of the above embodiments, the anti-OX 40 agonist antibody is humanized. In one embodiment, the anti-OX 40 antibody comprises HVRs of any of the above embodiments or any of the embodiments in fig. 11A-I, and further comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework. In another embodiment, the anti-OX 40 antibody comprises HVRs of any of the above embodiments, and further comprises a VH and/or VL comprising FR sequences set forth in FIGS. 11A-I.
In another aspect, an anti-human OX40 agonist antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 108, 114, 116, 183, or 184. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but an anti-human OX40 agonist antibody comprising that sequence retains the ability to bind OX 40. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NOs 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 108, 114, 116, 183, or 184. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-human OX40 agonist antibody comprises the VH sequence of SEQ ID NO 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 108, 114, 116, 183, or 184, including post-translational modifications of the sequence. In certain embodiments, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1 comprising amino acid sequence SEQ ID NO. 2, (b) HVR-H2 comprising amino acid sequence SEQ ID NO. 3, and (c) HVR-H3 comprising amino acid sequence SEQ ID NO. 4.
In another aspect, anti-human OX40 agonist antibodies are provided, wherein the antibodies comprise a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to amino acid sequence SEQ ID NO:57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 109, 115, or 117. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-human OX40 agonist antibody comprising that sequence retains the ability to bind to OX 40. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 109, 115 or 117. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-human OX40 agonist antibody comprises the VL sequence of SEQ ID NO:57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 109, 115, or 117, including post-translational modifications of the sequence. In particular embodiments, the VL comprises one, two, or three HVRs selected from: (a) HVR-L1 comprising amino acid sequence SEQ ID NO 5; (b) HVR-L2 comprising amino acid sequence SEQ ID NO 6; and (c) HVR-L3 comprising the amino acid sequence SEQ ID NO: 7.
In another aspect, an anti-human OX40 agonist antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to amino acid sequence SEQ ID NO 56. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but an anti-human OX40 agonist antibody comprising that sequence retains the ability to bind OX 40. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO 56. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-human OX40 agonist antibody comprises the VH sequence in SEQ ID NO:56, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1 comprising amino acid sequence SEQ ID NO. 2, (b) HVR-H2 comprising amino acid sequence SEQ ID NO. 3, and (c) HVR-H3 comprising amino acid sequence SEQ ID NO. 4.
In another aspect, anti-human OX40 agonist antibodies are provided, wherein the antibodies comprise a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to amino acid sequence SEQ ID NO: 57. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-human OX40 agonist antibody comprising that sequence retains the ability to bind to OX 40. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO 57. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-human OX40 agonist antibody comprises the VL sequence of seq id No. 57, including post-translational modifications of the sequence. In particular embodiments, the VL comprises one, two, or three HVRs selected from: (a) HVR-L1 comprising amino acid sequence SEQ ID NO 5; (b) HVR-L2 comprising amino acid sequence SEQ ID NO 6; and (c) HVR-L3 comprising the amino acid sequence SEQ ID NO: 7.
In another aspect, an anti-human OX40 agonist antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence SEQ ID NO: 180. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but an anti-human OX40 agonist antibody comprising that sequence retains the ability to bind OX 40. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO 180. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-human OX40 agonist antibody comprises the VH sequence of SEQ ID NO 180, including post-translational modifications of the sequence. In certain embodiments, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1 comprising amino acid sequence SEQ ID NO. 2, (b) HVR-H2 comprising amino acid sequence SEQ ID NO. 3, and (c) HVR-H3 comprising amino acid sequence SEQ ID NO. 4.
In another aspect, anti-human OX40 agonist antibodies are provided, wherein the antibodies comprise a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to amino acid sequence SEQ ID NO: 179. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-human OX40 agonist antibody comprising that sequence retains the ability to bind to OX 40. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO 179. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-human OX40 agonist antibody comprises the VL sequence of seq id No. 179, including post-translational modifications of the sequence. In particular embodiments, the VL comprises one, two, or three HVRs selected from: (a) HVR-L1 comprising amino acid sequence SEQ ID NO 5; (b) HVR-L2 comprising amino acid sequence SEQ ID NO 6; and (c) HVR-L3 comprising the amino acid sequence SEQ ID NO: 7.
In another aspect, an anti-human OX40 agonist antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to amino acid sequence SEQ ID NO 94. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but an anti-human OX40 agonist antibody comprising that sequence retains the ability to bind OX 40. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO 94. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-human OX40 agonist antibody comprises the VH sequence in SEQ ID NO:94, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1 comprising amino acid sequence SEQ ID NO. 2, (b) HVR-H2 comprising amino acid sequence SEQ ID NO. 3, and (c) HVR-H3 comprising amino acid sequence SEQ ID NO. 4.
In another aspect, anti-human OX40 agonist antibodies are provided, wherein the antibodies comprise a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to amino acid sequence SEQ ID No. 95. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-human OX40 agonist antibody comprising that sequence retains the ability to bind to OX 40. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO 95. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-human OX40 agonist antibody comprises the VL sequence of seq id No. 95, including post-translational modifications of the sequence. In particular embodiments, the VL comprises one, two, or three HVRs selected from: (a) HVR-L1 comprising amino acid sequence SEQ ID NO 5; (b) HVR-L2 comprising amino acid sequence SEQ ID NO 6; and (c) HVR-L3 comprising amino acid sequence SEQ ID NO: 26.
In another aspect, an anti-human OX40 agonist antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to amino acid sequence SEQ ID NO 96. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but an anti-human OX40 agonist antibody comprising that sequence retains the ability to bind OX 40. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO 96. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-human OX40 agonist antibody comprises the VH sequence in SEQ ID NO:96, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1 comprising amino acid sequence SEQ ID NO. 2, (b) HVR-H2 comprising amino acid sequence SEQ ID NO. 3, and (c) HVR-H3 comprising amino acid sequence SEQ ID NO. 4.
In another aspect, anti-human OX40 agonist antibodies are provided, wherein the antibodies comprise a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to amino acid sequence SEQ ID NO: 97. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-human OX40 agonist antibody comprising that sequence retains the ability to bind to OX 40. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO: 97. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-human OX40 agonist antibody comprises the VL sequence of seq id No. 97, including post-translational modifications of the sequence. In particular embodiments, the VL comprises one, two, or three HVRs selected from: (a) HVR-L1 comprising amino acid sequence SEQ ID NO 5; (b) HVR-L2 comprising amino acid sequence SEQ ID NO 6; and (c) HVR-L3 comprising amino acid sequence SEQ ID NO: 27.
In another aspect, an anti-human OX40 agonist antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence SEQ ID NO 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but an anti-human OX40 agonist antibody comprising that sequence retains the ability to bind OX 40. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NOs 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-human OX40 agonist antibody comprises the VH sequence of SEQ ID NOs 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, including post-translational modifications of the sequence. In certain embodiments, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1 comprising amino acid sequence SEQ ID NO:29, (b) HVR-H2 comprising amino acid sequence SEQ ID NO:30, and (c) HVR-H3 comprising amino acid sequence SEQ ID NO: 33.
In another aspect, anti-human OX40 agonist antibodies are provided, wherein the antibodies comprise a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to amino acid sequences SEQ ID NOS 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-human OX40 agonist antibody comprising that sequence retains the ability to bind to OX 40. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NOs 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-human OX40 agonist antibody comprises the VL sequence of SEQ ID NOs 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, including post-translational modifications of the sequence. In particular embodiments, the VL comprises one, two, or three HVRs selected from: (a) HVR-L1 comprising amino acid sequence SEQ ID NO 37; (b) HVR-L2 comprising amino acid sequence SEQ ID NO: 39; and (c) HVR-L3 comprising amino acid sequence SEQ ID NO: 42.
In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:56 and SEQ ID NO:57, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:58 and SEQ ID NO:59, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:60 and SEQ ID NO:61, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:62 and SEQ ID NO:63, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:64 and SEQ ID NO:65, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:66 and SEQ ID NO:67, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:68 and SEQ ID NO:69, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:70 and SEQ ID NO:71, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:72 and SEQ ID NO:73, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:74 and SEQ ID NO:75, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:76 and SEQ ID NO:77, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:78 and SEQ ID NO:79, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:80 and SEQ ID NO:81, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:82 and SEQ ID NO:83, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:84 and SEQ ID NO:85, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:86 and SEQ ID NO:87, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:88 and SEQ ID NO:89, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:90 and SEQ ID NO:91, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:92 and SEQ ID NO:93, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:94 and SEQ ID NO:95, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:96 and SEQ ID NO:97, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:98 and SEQ ID NO:99, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:100 and SEQ ID NO:101, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:108 and SEQ ID NO:109, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:114 and SEQ ID NO:115, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:116 and SEQ ID NO:117, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:183 and SEQ ID NO:65, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:184 and SEQ ID NO:69, respectively, including post-translational modifications of those sequences.
In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:118 and SEQ ID NO:119, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:120 and SEQ ID NO:121, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:122 and SEQ ID NO:123, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:124 and SEQ ID NO:125, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:126 and SEQ ID NO:127, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:128 and SEQ ID NO:129, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO:130 and SEQ ID NO:131, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:132 and SEQ ID NO:133, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:134 and SEQ ID NO:135, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:136 and SEQ ID NO:137, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:138 and SEQ ID NO:139, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:140 and SEQ ID NO:141, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO:142 and SEQ ID NO:143, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:144 and SEQ ID NO:145, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:146 and SEQ ID NO:147, respectively, including post-translational modifications of those sequences.
In another aspect, an anti-human OX40 agonist antibody is provided, wherein the antibody comprises a VH in any of the embodiments provided above and a VL in any of the embodiments provided above.
In yet another aspect, the invention provides antibodies that bind to the same epitope as the anti-human OX40 antibodies provided herein. In some embodiments, the antibody is an anti-human OX40 agonist antibody.
In yet another aspect of the invention, the anti-OX 40 antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric, humanized or human antibody. In one embodiment, the anti-OX 40 antibody is an antibody fragment, such as an Fv, Fab, Fab ', scFv, diabody, or F (ab')2And (3) fragment. In another embodiment, the antibody is a full length antibody, e.g., whole IgG1 or other antibody classes or isotypes, as defined herein. In some embodiments, the antibody is a full length intact IgG4 antibody.
In yet another aspect, an anti-OX 40 antibody according to any of the above embodiments can incorporate any of the features described in sections 1-7 below, singly or in combination:
1. affinity of antibody
In certain embodiments, an antibody provided herein has a dissociation constant (Kd) (e.g., 10nM or less) of 1 μ M, 100nM or less, 10nM or less, 1nM or less, 0.1nM or less, 0.01nM or less, or 0.001nM or less (e.g., 10 nM)-8M or less, e.g. 10-8M to 10-13M, e.g. 10-9M to 10-13M)。
In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed with Fab versions of the antibody of interest and its antigen. For example, by using the minimum concentration of (in the presence of a titration series of unlabeled antigen)125I) The Fab is equilibrated with labeled antigen and then the solution binding affinity of the Fab for the antigen is measured by capturing the bound antigen with an anti-Fab antibody coated plate (see, e.g., Chen et al, J.mol.biol.293:865-881 (1999)). To establish the assay conditions, theMulti-well plates (Thermo Scientific) were coated with 5. mu.g/ml capture anti-Fab antibodies (Cappel Labs) in 50mM sodium carbonate (pH 9.6) overnight, followed by blocking with 2% (w/v) bovine serum albumin in PBS for 2-5 hours at room temperature (about 23 ℃). In the non-adsorption plate (Nunc #269620), 100pM or 26pM [ alpha ], [ beta ]125I]Antigen mixing with serial dilutions of Fab of interest (e.g.in agreement with the evaluation of anti-VEGF antibodies, Fab-12, by Presta et al, Cancer Res.57:4593-4599 (1997)). The Fab of interest was then incubated overnight; however, incubation may continue for longer periods of time (e.g., about 65 hours) to ensure equilibrium is reached. Thereafter, the mixture is transferred to a capture plate and incubated at room temperature (e.g., 1 hour). The solution was then removed and treated with 0.1% polysorbate 20 in PBS The plate was washed 8 times. After drying the plates, 150. mu.l/well scintillation fluid (MICROSCINT-20) was addedTM(ii) a Packard), thenIn TOPCOUNTTMPlates were counted on a gamma counter (Packard) for 10 minutes. The concentration at which each Fab gives less than or equal to 20% of the maximum binding is selected for use in competitive binding assays.
According to another embodiment, Kd is the useSurface plasmon resonance assay. For example, the immobilized antigen CM5 chip was used at about 10 Response Units (RU) at 25 deg.COr(BIAcore, inc., Piscataway, NJ) to perform the assay. In one embodiment, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen was diluted to 5. mu.g/ml (about 0.2. mu.M) with 10mM sodium acetate pH 4.8 and then injected at a flow rate of 5. mu.l/min to obtain about 10 Response Units (RU) of conjugated protein. After injection of the antigen, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, polysorbate 20 (TWEEN-20) was injected at 25 ℃ at a flow rate of about 25. mu.l/min at 0.05%TM) Two-fold serial dilutions of Fab (0.78nM to 500nM) in surfactant PBS (PBST). Using a simple one-to-one Langmuir (Langmuir) binding model ( Evaluation Software version 3.2) calculation of the Association Rate (k) by Simultaneous fitting of Association and dissociation sensorgramson) And dissociation rate (k)off). Equilibrium dissociation constant (Kd) in the ratio koff/konAnd (4) calculating. See, e.g., Chen et al, J.mol.biol.293:865-881 (1999). If the binding rate is more than 10 according to the above surface plasmon resonance assay6M-1S-1Then the speed of bondingThe rate can be determined using fluorescence quenching techniques, i.e., according to a spectrometer such as an Aviv Instruments spectrophotometer or 8000 series SLM-AMINCO equipped with a flow cutoff deviceTMMeasurement in a stirred cuvette in a spectrophotometer (ThermoSpectronic) measured the increase or decrease in fluorescence emission intensity (excitation 295 nM; emission 340nM, 16nM bandpass) of 20nM anti-antigen antibody (Fab form) in PBSpH 7.2 at 25 ℃ in the presence of increasing concentrations of antigen.
2. Antibody fragments
In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab ', Fab ' -SH, F (ab ')2For reviews of certain antibody fragments, see Hudson et al, nat. Med.9:129-134(2003) for reviews of scFv fragments, see for example Pluckth ü n, compiled by The Pharmacology of Monoclonal Antibodies, Vol.113, Rosenburg and Moore, (Springer-Verlag, New York), p.269-315 (1994), see also WO 93/16185, and U.S. Pat. Nos. 5,571,894 and 5,587,458 for Fab and F (ab') containing salvage receptor binding epitope residues and having an extended in vivo half-life2See U.S. Pat. No.5,869,046 for a discussion of fragments.
Diabodies are antibody fragments with two antigen binding sites, which may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993/01161; hudson et al, nat. Med.9: 129-; and Hollinger et al, Proc. Natl. Acad. Sci. USA 90: 6444-. Tri-and tetrabodies are also described in Hudson et al, nat. Med.9: 129-.
Single domain antibodies are antibody fragments that comprise all or part of the heavy chain variable domain or all or part of the light chain variable domain of the antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No.6,248,516B1).
Antibody fragments can be generated by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies and production of recombinant host cells (e.g., e.coli or phage), as described herein.
3. Chimeric and humanized antibodies
In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Pat. nos. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA,81: 6851-. In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In yet another example, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been altered from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.
In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and FRs (or portions thereof) are derived from a human antibody sequence. Optionally, the humanized antibody will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., an antibody from which HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.
Humanized antibodies and methods for their production are reviewed, for example, in Almagro and Fransson, front.biosci.13:1619-1633(2008), and further described, for example, in Riechmann et al, Nature 332:323-329 (1988); queen et al, Proc.nat' l Acad.Sci.USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337,7,527,791,6,982,321, and 7,087,409; kashmiri et al, Methods 36: 25-34(2005) (describes Specificity Determining Region (SDR) grafting); padlan, mol.Immunol.28: 489-498(1991) (described as "resurfacing"); dall' Acqua et al, Methods 36:43-60(2005) (describing "FR shuffling"); and Osbourn et al, Methods 36:61-68(2005) and Klimka et al, Br.J. cancer, 83: 252-.
Human framework regions that may be used for humanization include, but are not limited to: framework regions selected using the "best-fit" method (see, e.g., Sims et al, J.Immunol.151:2296 (1993)); framework regions derived from consensus sequences of a specific subset of human antibodies from the light or heavy chain variable regions (see, e.g., Carter et al, Proc. Natl. Acad. Sci. USA,89:4285 (1992); and Presta et al, J.Immunol.,151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, front.biosci.13:1619-1633 (2008)); and framework regions derived by screening FR libraries (see, e.g., Baca et al, J.biol.chem.272:10678-10684(1997) and Rosok et al, J.biol.chem.271:22611-22618 (1996)).
4. Human antibodies
In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be generated using a variety of techniques known in the art. In general, human antibodies are described in van Dijk and van de Winkel, Curr, Opin, Pharmacol.5:368-74(2001), and Lonberg, Curr, Opin, Immunol.20: 450-.
Human antibodies can be made by administering an immunogen to a transgenic animal that has been modified to produce fully human antibodies or fully antibodies with human variable regions in response to an antigenic challenge. Such animals typically contain all or part of a human immunoglobulin locus, which replaces an endogenous immunoglobulin locus, or which exists extrachromosomally or is randomly integrated into the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin locus has typically been inactivated. For an overview of the method of obtaining human antibodies from transgenic animals, see Lonberg, nat. Biotech.23:1117-1125 (2005). See also, for example, U.S. Pat. Nos. 6,075,181 and 6,150,584, which describe XENOMOUSETMA technique; U.S. Pat. No.5,770,429, which describesA technique; U.S. Pat. No.7,041,870, description thereofK-MTechnology, and U.S. patent application publication No. us 2007/0061900, which describesA technique). The human variable regions from the whole antibodies generated by such animals may be further modified, for example by combination with different human constant regions.
Human antibodies can also be generated by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the Production of human Monoclonal antibodies have been described (see, e.g., Kozbor J.Immunol.,133:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al, J.Immunol., 147:86 (1991)). Human antibodies generated via human B-cell hybridoma technology are also described in Li et al, Proc.Natl.Acad.Sci.USA,103:3557-3562 (2006). Other methods include those described, for example, in U.S. Pat. No.7,189,826, which describes the production of monoclonal human IgM antibodies from hybridoma cell lines, and Ni, Xiaondai Mianyixue,26(4):265-268(2006), which describes human-human hybridomas. The human hybridoma technique (Trioma technique) is also described in Vollmers and Brandlein, Histologyand Histopathlogy, 20(3): 927-.
Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.
5. Library-derived antibodies
Antibodies of the invention can be isolated by screening combinatorial libraries for antibodies having a desired activity or activities. For example, various methods for generating phage display libraries and screening such libraries for antibodies possessing desired binding characteristics are known in the art. Such Methods are reviewed, for example, in Hoogenboom et al, in Methods in molecular biology 178:1-37 (O' Brien et al, eds., Human Press, Totowa, NJ,2001), and further described, for example, in McCafferty et al, Nature 348: 552-; clackson et al, Nature 352: 624-; marks et al, J.mol.biol.222:581-597 (1992); marks and Bradbury, in Methods in Molecular Biology248:161-175(Lo eds., Human Press, Totowa, NJ, 2003); sidhu et al, J.mol.biol.338(2):299-310 (2004); lee et al, J.mol.biol.340(5):1073-1093 (2004); fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-; and Lee et al, J.Immunol.Methods284(1-2):119-132 (2004).
In some phage display methods, the repertoire of VH and VL genes, respectively, is cloned by Polymerase Chain Reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen-binding phages, as described in Winter et al, Ann. Rev. Immunol.,12:433-455 (1994). Phage typically display antibody fragments either as single chain fv (scfv) fragments or as Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, the natural repertoire can be cloned (e.g., from humans) to provide a single source of antibodies to a large panel of non-self and also self-antigens in the absence of any immunization, as described by Griffiths et al, EMBO J,12: 725-. Finally, non-rearranged V gene segments can also be synthesized by cloning non-rearranged V gene segments from stem cells and using PCR primers containing random sequences to encode the highly variable CDR3 regions and effecting rearrangement in vitro, as described by Hoogenboom and Winter, J.mol.biol.,227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No.5,750,373, and U.S. patent publication Nos. 2005/0079574,2005/0119455,2005/0266000,2007/0117126,2007/0160598,2007/0237764,2007/0292936 and 2009/0002360.
Antibodies or antibody fragments isolated from a human antibody library are considered to be human antibodies or human antibody fragments herein.
6. Multispecific antibodies
In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is directed to OX40 and the other is directed to any other antigen. In certain embodiments, a bispecific antibody can bind two different epitopes of OX 40. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing OX 40. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.
Techniques for generating multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537(1983)), WO 93/08829, and Traunecker et al, EMBO J.10:3655(1991)), and "node-in-hole" engineering (see, e.g., U.S. Pat. No.5,731,168). Effects can also be manipulated electrostatically by engineering the molecules for the generation of antibody Fc-heterodimers (WO 2009/089004a 1); crosslinking two or more antibodies or fragments (see, e.g., U.S. Pat. No.4,676,980, and Brennan et al, Science,229:81 (1985)); the use of leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al, J.Immunol.,148(5):1547-1553 (1992)); the "diabody" technique used to generate bispecific antibody fragments is used (see, e.g., Hollinger et al, Proc. Natl. Acad. Sci. USA, 90: 6444-; and the use of single chain fv (sFv) dimers (see, e.g., Gruber et al, J.Immunol.,152:5368 (1994)); and making a trispecific antibody to generate a multispecific antibody as described, for example, in Tutt et al, J.Immunol.147:60 (1991).
Also included herein are engineered antibodies having three or more functional antigen binding sites, including "octopus antibodies" (see, e.g., US 2006/0025576a 1).
Antibodies or fragments herein also include "dual action fabs" or "DAFs" comprising an antigen binding site that binds OX40 and another, different antigen (see, e.g., US 2008/0069820).
7. Antibody variants
In certain embodiments, amino acid sequence variants of the antibodies provided herein are encompassed. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, so long as the final construct possesses the desired characteristics, e.g., antigen binding.
a)Substitution, insertion, and deletion variants
In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in table a under the heading of "preferred substitutions". More substantial variations are provided in table a under the heading of "exemplary substitutions" and are described further below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest and the product screened for a desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.
TABLE A
According to common side chain properties, amino acids can be grouped as follows:
(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;
(2) neutral, hydrophilic: cys, Ser, Thr, Asn, Gln;
(3) acidic: asp, Glu;
(4) basic: his, Lys, Arg;
(5) residues that influence chain orientation: gly, Pro;
(6) aromatic: trp, Tyr, Phe.
Non-conservative substitutions may entail replacing one of these classes with a member of the other class.
One class of surrogate variants involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further study will have an alteration (e.g., an improvement) in certain biological properties (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody and/or will substantially retain certain biological properties of the parent antibody. Exemplary surrogate variants are affinity matured antibodies, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).
Changes (e.g., substitutions) can be made to HVRs, for example, to improve antibody affinity. Such changes can be made to HVR "hot spots", i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods mol. biol.207: 179. 196(2008)), and/or antigen-contacting residues, where the resulting variant VH or VL is tested for binding affinity. Affinity maturation by construction and re-selection of secondary libraries has been described, for example, in Hoogenboom et al, in Methods in Molecular Biology 178:1-37 (O' Brien et al, eds., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). Then, a secondary library is created. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves an HVR-directed method in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are frequently targeted.
In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, so long as such changes do not substantially reduce the ability of the antibody to bind antigen. For example, conservative changes (e.g., conservative substitutions, as provided herein) may be made to HVRs that do not substantially reduce binding affinity. For example, such changes may be outside of antigen-contacting residues in HVRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unaltered, or contains no more than 1, 2, or 3 amino acid substitutions.
One method that can be used to identify residues or regions of an antibody that can be targeted for mutagenesis is referred to as "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science,244: 1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions may be introduced at amino acid positions that indicate functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the contact points between the antibody and the antigen. As alternative candidates, such contact and adjacent residues may be targeted or eliminated. Variants can be screened to determine if they contain the desired property.
Amino acid sequence insertions include amino and/or carboxy-terminal fusions ranging in length from 1 residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include fusions of the N-or C-terminus of the antibody with an enzyme (e.g., for ADEPT) or a polypeptide that extends the serum half-life of the antibody.
b)Glycosylation variants
In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree of glycosylation of the antibody. Addition or deletion of glycosylation sites of an antibody can be conveniently achieved by altering the amino acid sequence such that one or more glycosylation sites are created or eliminated.
In the case of antibodies comprising an Fc region, the carbohydrate to which they are attached may be altered. Natural antibodies produced by mammalian cells typically comprise branched, bi-antennary oligosaccharides, which are typically N-linked to Asn297 of the CH2 domain attached to the Fc region. See, e.g., Wright et al, TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, for example, mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "backbone" of the bi-antennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the invention may be modified to create antibody variants with certain improved properties.
In one embodiment, antibody variants are provided that have a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all sugar structures (e.g. complexed, heterozygous and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, e.g. as described in WO 2008/077546. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody. Such fucosylated variants may have improved ADCC function. See, e.g., U.S. patent publication No. us 2003/0157108(Presta, L.); US 2004/0093621(Kyowa Hakko Kogyo co., Ltd). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; okazaki et al, J.mol.biol.336:1239-1249 (2004); Yamane-Ohnuki et al, Biotech.Bioeng.87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include protein fucosylation deficient Lec13CHO cells (Ripka et al, Arch. biochem. Biophys.249: 533-.
Further provided are antibody variants having bisected oligosaccharides, for example, wherein biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878(Jean-Mairet et al); U.S. Pat. No.6,602,684(Umana et al); and US 2005/0123546(Umana et al). Antibody variants having at least one galactose residue in an oligosaccharide attached to an Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087(Patel et al); WO 1998/58964(Raju, S.); and WO 1999/22764(Raju, S.).
c)Fc region variants
In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.
In certain embodiments, the invention encompasses antibody variants possessing some, but not all, effector functions that make them desirable candidates for applications where the in vivo half-life of the antibody is important, while certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and/or in vivo cytotoxicity assays may be performed to confirm the reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays may be performed to ensure that the antibody lacks fcyr binding (and therefore potentially lacks ADCC activity), but retains FcRn binding ability. The major cells mediating ADCC, NK cells, express Fc γ RIII only, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of ravatch and Kinet, Annu. Rev. Immunol.9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of molecules of interest are described in U.S. Pat. No.5,500,362 (see, e.g., Hellstrom, I.et al, Proc. nat' l Acad. Sci. USA 83: 7059-; 5,821,337 (see Bruggemann, M. et al, J.Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be employed (see, e.g., ACTI for flow cytometry)TMNon-radioactive cytotoxicity assays (Celltechnology, Inc. mountain View, CA; and CytoTox)Non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively/additionally, the ADCC activity of a molecule of interest can be assessed in vivo, for example in an animal model such as that disclosed in Clynes et al, Proc. nat' l Acad. Sci. USA95: 652-. A C1q binding assay may also be performed to confirm that the antibody is unable to bind C1q, and therefore lacks CDC activity. See, for example, WO2006/029879 and C1q and C3C in WO 2005/100402 bind to ELISA. To assess complement activation, CDC assays can be performed (see, e.g., Gazzano-Santoro et al, J.Immunol. methods 202:163 (1996); Cragg, M.S. et al, Blood 101: 1045-. FcRn binding and in vivo clearance/half-life assays can also be performed using methods known in the art (see, e.g., Petkova, s.b. et al, Int' l.immunol.18(12): 1759-.
Antibodies with reduced effector function include those having substitutions in one or more of residues 238,265,269,270,297,327 and 329 of the Fc region (U.S. Pat. No.6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265,269,270,297 and 327, including so-called "DANA" Fc mutants having substitutions of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).
Certain antibody variants with improved or reduced binding to FcR are described (see, e.g., U.S. Pat. No.6,737,056; WO 2004/056312, and Shields et al, J.biol.chem.9(2):6591-6604 (2001)).
In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 (EU numbering of residues) of the Fc region.
In some embodiments, alterations are made to the Fc region that result in altered (i.e., improved or reduced) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No.6,194,551, WO 99/51642, and Idusogene et al, J.Immunol.164: 4178-.
Antibodies with extended half-life and improved binding to neonatal Fc receptor (FcRn) responsible for the transfer of maternal IgG to the fetus are described in US2005/0014934A1(Hinton et al), the neonatal Fc receptor (FcRn) and are responsible for the transfer of maternal IgG to the fetus (Guyer et al, J.Immunol.117:587(1976) and Kim et al, J.Immunol.24:249 (1994)). Those antibodies comprise an Fc region having one or more substitutions therein that improve the binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of residues 238,256,265,272,286,303,305,307,311,312,317,340,356,360,362,376,378,380,382,413,424 or 434 of the Fc region, for example, at residue 434 of the Fc region (U.S. patent No.7,371,826).
Also found in Duncan and Winter, Nature 322:738-40 (1988); U.S. Pat. Nos. 5,648,260; U.S. Pat. Nos. 5,624,821; and WO 94/29351, which concerns other examples of Fc region variants.
d)Cysteine engineered antibody variants
In certain embodiments, it may be desirable to create cysteine engineered antibodies, e.g., "thiomabs," in which one or more residues of the antibody are replaced with cysteine residues. In particular embodiments, the substituted residues are present at accessible sites of the antibody. By replacing those residues with cysteine, the reactive thiol groups are thus localized at accessible sites of the antibody and can be used to conjugate the antibody with other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates, as further described herein. In certain embodiments, cysteine may be substituted for any one or more of the following residues: v205 of the light chain (Kabat numbering); a118 of the heavy chain (EU numbering); and S400 of the heavy chain Fc region (EU numbering). Cysteine engineered antibodies can be produced as described, for example, in U.S. patent No.7,521,541.
e)Antibody derivatives
In certain embodiments, the antibodies provided herein can be further modified to contain additional non-proteinaceous moieties known in the art and readily available. Suitable moieties for derivatization of the antibody include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in production due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative is to be used in a therapy under specified conditions, and the like.
In another embodiment, conjugates of an antibody and a non-proteinaceous moiety that can be selectively heated by exposure to radiation are provided. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al, Proc. Natl. Acad. Sci. USA 102: 11600-. The radiation can be of any wavelength and includes, but is not limited to, wavelengths that are not damaging to normal cells, but heat the non-proteinaceous moiety to a temperature at which cells in the vicinity of the antibody-non-proteinaceous moiety are killed.
B. Recombinant methods and compositions
Recombinant methods and compositions can be used to generate antibodies, for example, as described in U.S. Pat. No.4,816,567. In one embodiment, isolated nucleic acids encoding the anti-OX 40 antibodies described herein are provided. Such nucleic acids may encode an amino acid sequence comprising an antibody VL and/or an amino acid sequence comprising an antibody VH (e.g., the light and/or heavy chain of an antibody). In yet another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In yet another embodiment, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising nucleic acids encoding an amino acid sequence comprising a VL of an antibody and an amino acid sequence comprising a VH of an antibody, or (2) a first vector comprising nucleic acids encoding an amino acid sequence comprising a VL of an antibody and a second vector comprising nucleic acids encoding an amino acid sequence comprising a VH of an antibody. In one embodiment, the host cell is eukaryotic, such as a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of producing an anti-OX 40 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expression of the antibody, as provided above, and optionally, recovering the antibody from the host cell (or host cell culture broth).
For recombinant production of anti-OX 40 antibodies, nucleic acids encoding the antibodies (e.g., as described above) are isolated and inserted into one or more vectors for further cloning and/or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of an antibody).
Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237,5,789,199 and 5,840,523 (see also Charlton, Methods in Molecular Biology, Vol.248 (compiled by B.K.C.Lo., Humana Press, Totowa, NJ,2003), pp.245-254, which describes expression of antibody fragments in E.coli (E.coli)). After expression, the antibody can be isolated from the bacterial cell mass paste in a soluble fraction and can be further purified.
In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized" resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, nat. Biotech.22: 1409-.
Host cells suitable for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified which can be used with insect cells, particularly for transfecting Spodoptera frugiperda (Spodoptera frugiperda) cells.
Plant cell cultures may also be used as hosts. See, e.g., U.S. Pat. Nos. 5,959,177,6,040,498,6,420,548,7,125,978 and 6,417,429 (which describe PLANTIBODIIES for antibody production in transgenic plantsTMA technique).
Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney lines (293 or 293 cells as described, e.g., in Graham et al, J.Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli (sertoli) cells (TM4 cells, as described, for example, in Mather, biol. reprod.23:243-251 (1980)); monkey kidney cells (CV 1); VERO cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK; bovine (buffalo rate) hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, for example, in Mather et al, AnnalsN.Y.Acad.Sci.383:44-68 (1982); MRC 5 cells; and FS4 cells other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al, Proc. Natl. Acad. Sci. USA77:4216 (1980)); and myeloma cell lines such as Y0, NS0 and Sp 2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol.248 (edited by B.K.C.Lo, Humana Press, Totowa, NJ), pp.255-268 (2003).
C. Assay method
The anti-OX 40 antibodies provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art.
1. Binding assays and other assays
In one aspect, antibodies of the invention are tested for antigen binding activity, for example, by known methods such as ELISA, Western blot, and the like. OX40 binding can be determined using methods known in the art, exemplary methods being disclosed herein. In one embodiment, binding is measured using a radioimmunoassay. An exemplary radioimmunoassay is exemplified in the examples. The OX40 antibody was iodinated and a competition reaction mixture containing a fixed concentration of iodinated antibody and decreasing concentrations of serially diluted unlabeled OX40 antibody was prepared. OX40 expressing cells (e.g., BT474 cells stably transfected with human OX 40) are added to the reaction mixture. After incubation, the cells were washed to separate free iodinated OX40 antibody from OX40 antibody bound to the cells. The level of bound iodinated OX40 antibody is determined, e.g., by counting the radioactivity associated with the cells, and binding affinity is determined using standard methods. In another embodiment, the ability of an OX40 antibody to bind surface-expressed OX40 (e.g., on a subset of T cells) is assessed using flow cytometry. Peripheral white blood cells (e.g., from human, cynomolgus monkey, rat or mouse) are obtained and the cells are blocked with serum. Labeled OX40 antibody was added in serial dilutions and T cells were also stained to identify T cell subsets (using methods known in the art). After sample incubation and washing, the cells were sorted using a flow cytometer and the data analyzed using methods well known in the art. In another embodiment, surface plasmon resonance may be used to analyze OX40 binding. An exemplary surface plasmon resonance method is illustrated in the examples.
In another aspect, a competition assay can be used to identify antibodies that compete for binding to OX40 with any of the anti-OX 40 antibodies disclosed herein. In certain embodiments, such competitive antibodies bind to the same epitope (e.g., a linear or conformational epitope) as any of the anti-OX 40 antibodies disclosed herein bind to. A detailed exemplary method for locating epitopes bound by antibodies is described in Morris (1996) "Epitope Mapping Protocols", Methods in Molecular biology vol.66(Humana Press, Totowa, NJ). A competition assay is exemplified in the examples.
In one exemplary competition assay, immobilized OX40 is incubated in a solution comprising a first labeled antibody that binds OX, e.g., mab1a7.gr.1, mab 3c8.gr5, and a second unlabeled antibody to be tested for the ability to compete with the first antibody for binding to OX 40. The second antibody may be present in the hybridoma supernatant. As a control, immobilized OX40 was incubated in a solution containing the first labeled antibody but no second unlabeled antibody. After incubation under conditions that allow the primary antibody to bind to OX40, excess unbound antibody is removed and the amount of label associated with immobilized OX40 is measured. If the amount of marker associated with immobilized OX40 in the test sample is substantially reduced compared to the control sample, then this indicates that the second antibody competes with the first antibody for binding to OX 40. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14(Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
2. Activity assay
In one aspect, assays are provided for identifying anti-OX 40 antibodies that have biological activity. Biological activities may include, for example, binding to OX40 (e.g., binding to human and/or cynomolgus monkey OX40), increasing OX 40-mediated signal transduction (e.g., increasing NFkB-mediated transcription), depleting cells expressing human OX40 (e.g., T cells), depleting cells expressing human OX40 by ADCC and/or phagocytosis, enhancing T effector cell function (e.g., CD4+ effector T cells) (e.g., by increasing effector T cell proliferation and/or increasing cytokine production by effector T cells (e.g., gamma interferon)), enhancing memory T cell function (e.g., CD4+ memory T cells) (e.g., by increasing memory T cell proliferation and/or increasing cytokine production by memory T cells (e.g., gamma interferon)), inhibiting Treg that modulate T cell function (e.g., by decreasing effector T cell function (e.g., CD4+ effector T cell function)), binding human effector cells. Also provided are antibodies having such biological activity in vivo and/or in vitro.
In certain embodiments, antibodies of the invention are tested for such biological activity.
T cell co-stimulation can be determined using methods known in the art, and exemplary methods are disclosed herein. For example, T cells (e.g., memory or effector T cells) can be obtained from peripheral white blood cells (e.g., isolated from human whole blood using Ficoll gradient centrifugation). Memory T cells (e.g., CD4+ memory T cells) or effector T cells (e.g., CD4+ Teff cells) can be isolated from PBMCs using methods known in the art. For example, a Miltenyi CD4+ memory T cell isolation kit or a Miltenyi naive CD4+ T cell isolation kit can be used. Isolated T cells are cultured in the presence of antigen presenting cells (e.g., irradiated L cells expressing CD32 and CD 80) and activated by the addition of anti-CD 3 antibody in the presence or absence of OX40 agonist antibody. The effect of an agonist OX40 antibody on T cell proliferation can be measured using methods well known in the art. For example, the CellTiter Glo kit (Promega) can be used and the results read on a multi-marker reader (Perkin Elmer). The effect of agonist OX40 antibodies on T cell function can also be determined by analyzing cytokines produced by T cells. In one embodiment, CD4+ T cells are assayed for interferon gamma production, for example by measuring interferon gamma in the cell culture supernatant. Methods for measuring interferon gamma are well known in the art.
Treg cell function can be determined using methods known in the art, and exemplary methods are disclosed herein. In one example, the ability of tregs to suppress the proliferation of effector T cells is determined. T cells are isolated from human whole blood (e.g., memory T cells or naive T cells) using methods known in the art. The purified CD4+ naive T cells were labeled (e.g., with CFSE) and the purified Treg cells were labeled with different reagents. Irradiated antigen presenting cells (e.g., L cells expressing CD32 and CD 80) are co-cultured with labeled purified naive CD4+ T cells and purified tregs. Cocultures were activated using anti-CD 3 antibody and tested in the presence or absence of agonist OX40 antibody. After a suitable time (e.g., 6 days of co-culture), FACS analysis is used to track the level of CD4+ naive T cell proliferation by dye dilution in reduced marker staining (e.g., reduced CFSE marker staining).
OX40 signaling can be determined using methods well known in the art, and exemplary methods are disclosed herein. In one embodiment, transgenic cells are generated that express human OX40 and a reporter gene comprising an NFkB promoter fused to a reporter gene (e.g., β luciferase). Addition of OX40 agonist antibodies to cells resulted in increased transcription of NFkB, which was detected using an assay against a reporter gene.
Phagocytosis can be determined, for example, by using monocyte-derived macrophages or U937 cells, a human histiocytic lymphoma cell line with the morphology and characteristics of mature macrophages. OX40 expressing cells were added to monocyte derived macrophages or U937 cells in the presence or absence of anti-OX 40 agonist antibodies. After culturing the cells for a suitable period of time, the percent phagocytosis is determined by examining the percentage of cells double stained for the marker of 1) macrophages or U937 cells and 2) cells expressing OX40, and dividing this by the total number of cells displaying the marker of cells expressing OX40 (e.g., GFP). Analysis can be performed by flow cytometry. In another embodiment, the analysis may be performed by fluorescence microscopy analysis.
ADCC can be determined, for example, using methods well known in the art. Exemplary methods are described in the definitions section, and exemplary assays are disclosed in the examples. In some embodiments, the level of OX40 on OX40 expressing cells used for testing in an ADCC assay is characterized. Cells were stained with a detectably labeled anti-OX 40 antibody (e.g., PE labeled) and then fluorescence levels were determined using flow cytometry, with results presented as Median Fluorescence Intensity (MFI). In another embodiment, ADCC can be analyzed by the CellTiter Glo assay kit and cell viability/cytotoxicity can be determined by chemiluminescence.
The binding affinity of each antibody to Fc γ RIA, Fc γ RIIA, Fc γ RIIB, and two allotypes of Fc γ RIIIA (F158 and V158) can be measured in an ELISA-based ligand binding assay using the corresponding recombinant Fc γ receptor. The purified human Fc γ receptor was expressed as a fusion protein containing the extracellular domain of the gamma chain of the receptor linked to a C-terminal Gly/6 xHis/glutathione S-transferase (GST) polypeptide tag. The binding affinity of the antibodies to those human Fc γ receptors was determined as follows. For the low affinity receptors, two allotypes, F-158 and V-158, of Fc γ RIIA (CD32A), Fc γ RIIB (CD32B), and Fc γ RIIIA (CD16), two allotypes, F-158 and V-158, can be raised against human kappa chain F (ab')2Fragment (ICN biomedicalal; Irvine, CA) Cross-Linked (antibodies: cross-Linked with F (ab') in approximate molar ratio 1: 3)2) Antibodies were tested as multimers. Plates were coated with anti-GST antibody (Genentech) and blocked with Bovine Serum Albumin (BSA). Using Phosphate Buffered Saline (PBS) containing 0.05% Tween-20 and ELx405TMAfter washing with a plate washer (Biotek Instruments; Winooski, VT), Fc γ receptor was added to the plate at 25 ng/well and incubated for 1 hour at room temperature. After washing the plates, serial dilutions of the test antibody were added as multimeric complexes, and the plates were incubated for 2 hours at room temperature. After washing the plate to remove unbound antibody, goat anti-human F (ab') conjugated with horseradish peroxidase (HRP)2F (ab')2Fragments (Jackson ImmunoResearch Laboratories; WestGrove, Pa.) detect antibodies that bind to Fc γ receptors, followed by addition of the substrate, Tetramethylbenzidine (TMB) (Kirkegaardand Perry Laboratories; Gaithersburg, Md.). Depending on the Fc γ receptor tested, the plates were incubated at room temperature for 5-20 minutes to allow for color development. With 1M H3PO4The reaction is terminated and a microplate reader is used (190, Molecular Devices; sunnyvale, CA) measures the absorbance at 450 nm. Dose-response binding curves were generated by plotting the mean absorbance values from duplicate antibody dilutions against antibody concentration. Using SoftMax Pro (Molec)Tubular Devices) was fitted to the binding curve using a four parameter equation to determine the value of effective antibody concentration (EC) at which 50% of the maximal response from binding to Fc γ receptor was detected50)。
To select antibodies that induce cell death, loss of membrane integrity as indicated by, for example, Propidium Iodide (PI), trypan blue or 7AAD uptake can be assessed relative to controls. The PI uptake assay can be performed in the absence of complement and immune effector cells. OX 40-expressing cells are incubated in medium alone or in medium containing a suitable monoclonal antibody at a concentration of, for example, about 10. mu.g/ml. The cells are incubated for a certain period of time (e.g., 1 or 3 days). After each treatment, cells were washed and aliquoted. In some embodiments, cells are aliquoted into 35mm 12x 75 tubes (1 ml per tube, 3 tubes per treatment group) covered with a filter-mesh (filter-clamped) to remove cell clumps. PI (10. mu.g/ml) was then added to the tube. FACSCAN may be usedTMFlow cytometer and FACSCVERTTMThe samples were analyzed by CellQuest software (Becton Dickinson).
Cells for use in any of the above in vitro assays include cells or cell lines that naturally express OX40 or that are engineered to express OX 40. Such cells include activated T cells that naturally express OX40, Treg cells, and activated memory T cells. Such cells also include cell lines that express OX40 and cell lines that do not normally express OX40 but have been transfected with a nucleic acid encoding OX 40. Exemplary cell lines provided herein for use in any of the above in vitro assays include transgenic BT474 cells expressing human OX40 (a human breast cancer cell line).
It is understood that any of the above assays may be performed using the immunoconjugates of the invention in place of or in addition to an anti-OX 40 antibody.
It is understood that any of the above assays may be performed using anti-OX 40 antibodies and other therapeutic agents.
D. Immunoconjugates
The invention also provides immunoconjugates comprising an anti-OX 40 antibody herein conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant or animal origin, or a fragment thereof), or a radioisotope.
In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including but not limited to maytansinoids (see U.S. Pat. nos. 5,208,020, 5,416,064 and european patent EP 0425235B 1); auristatins such as monomethyl auristatin drug modules DE and DF (MMAE and MMAF) (see U.S. Pat. nos. 5,635,483 and 5,780,588 and 7,498,298); dolastatin (dolastatin); calicheamicin (calicheamicin) or a derivative thereof (see U.S. Pat. Nos. 5,712,374,5,714,586,5,739,116,5,767,285,5,770,701,5,770,710,5,773,001 and 5,877,296; Hinman et al, Cancer Res.53:3336-3342 (1993); and Lode et al, Cancer Res.58:2925-2928 (1998)); anthracyclines such as daunomycin (daunomycin) or doxorubicin (doxorubicin) (see Kratz et al, Current Med. chem.13: 477-; methotrexate; vindesine (vindesine); taxanes (taxanes) such as docetaxel (docetaxel), paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes (trichothecenes); and CC 1065.
In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin, or fragment thereof, including but not limited to diphtheria a chain, a non-binding active fragment of diphtheria toxin, exotoxin a chain (from Pseudomonas aeruginosa), ricin (ricin) a chain, abrin (abrin) a chain, modeccin (modeccin) a chain, α -fumagillin (sarcin), aleurites (aleurites fordii) toxic protein, dianthus caryophyllus (dianthin) toxic protein, phytolacca americana (phytolaccai americana) protein (papapi, PAPII and PAP-S), Momordica charantia (mordianica) localized inhibitor, curcin (curcin), crotin (crotin), saponaria officinalis (sapaonaris) inhibitor, resignathomycin (trichomycin), trichomycin (trichomycin), enomycin (enomycin) and trichothecenes (tricothecenes).
In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for use in generating radioconjugates. Examples include At211，I131，I125，Y90，Re186，Re188，Sm153，Bi212，P32，Pb212And radioactive isotopes of Lu. Where a radioconjugate is used for detection, it may contain a radioactive atom for scintigraphic studies, for example tc99m or I123, or a spin label for Nuclear Magnetic Resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as again iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
A variety of bifunctional protein coupling agents may be used to generate conjugates of the antibody and cytotoxic agent, such as N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), Iminothiolane (IT), imidoesters (such as dimethyl adipimidate hcl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis (p-diazoniumbenzoyl) -ethylenediamine), diisothiocyanates (such as toluene 2, 6-diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene) is used. For example, a ricin immunotoxin may be prepared as described in Vitetta et al, Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelator for conjugating radionucleotides to antibodies. See WO 94/11026. The linker may be a "cleavable linker" that facilitates release of the cytotoxic drug in the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al, Cancer Res 52: 127-.
Immunoconjugates or ADCs herein expressly encompass, but are not limited to, such conjugates prepared with crosslinking agents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl- (4-vinylsulfone) benzoate), which are commercially available (e.g., from Pierce Biotechnology, inc., Rockford, il., u.s.a.).
E. Methods and compositions for diagnosis and detection
In certain embodiments, any of the anti-OX 40 antibodies provided herein can be used to detect the presence of OX40 in a biological sample. As used herein, the term "detecting" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises a cell or tissue, such as a tumor (e.g., NSCLC or breast tumor) sample.
In one embodiment, anti-OX 40 antibodies are provided for use in a diagnostic or detection method. In yet another aspect, methods of detecting the presence of OX40 in a biological sample are provided. In certain embodiments, the methods comprise contacting the biological sample with an anti-OX 40 antibody under conditions that allow the anti-OX 40 antibody to bind to OX40, as described herein, and detecting whether a complex is formed between the anti-OX 40 antibody and OX 40. Such methods may be in vitro or in vivo. In one embodiment, an anti-OX 40 antibody is used to select subjects suitable for treatment with an anti-OX 40 antibody, e.g., where OX40 is a biomarker for selecting patients.
In some embodiments, the anti-OX 40 antibody for use in the diagnostic or detection method is an anti-human OX40 antibody comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising amino acid sequence SEQ ID NO. 2; (b) HVR-H2 comprising amino acid sequence SEQ ID NO 3; (c) HVR-H3 comprising amino acid sequence SEQ ID NO 4; (d) HVR-L1 comprising amino acid sequence SEQ ID NO 5; (e) HVR-L2 comprising amino acid sequence SEQ ID NO 6; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 7. In some embodiments, the anti-OX 40 antibody comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1 comprising amino acid sequence SEQ ID NO. 2, (ii) HVR-H2 comprising amino acid sequence SEQ ID NO. 3, and (iii) HVR-H3 comprising amino acid sequence SEQ ID NO. 4; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) HVR-L1 comprising amino acid sequence SEQ ID NO:5, (ii) HVR-L2 comprising amino acid sequence SEQ ID NO:6, and (c) HVR-L3 comprising amino acid sequence SEQ ID NO: 7. In some embodiments, the OX40 antibody comprises (a) HVR-H1 comprising amino acid sequence SEQ ID NO: 2; (b) HVR-H2 comprising amino acid sequence SEQ ID NO. 3; (c) HVR-H3 comprising amino acid sequence SEQ ID NO 4; (d) HVR-L1 comprising amino acid sequence SEQ ID NO 5; (e) HVR-L2 comprising amino acid sequence SEQ ID NO 6; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 7. In some embodiments, the antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence SEQ ID NO: 180. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but an anti-human OX40 agonist antibody comprising that sequence retains the ability to bind to human OX 40. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO 180. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-human OX40 agonist antibody comprises the VH sequence of SEQ id no:180, including post-translational modifications of the sequence. In a particular embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1 comprising amino acid sequence SEQ ID NO. 2, (b) HVR-H2 comprising amino acid sequence SEQ ID NO. 3, and (c) HVR-H3 comprising amino acid sequence SEQ ID NO. 4. In some embodiments, the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence SEQ ID NO 179. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-human OX40 agonist antibody comprising that sequence retains the ability to bind to human OX 40. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO 179. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-human OX40 agonist antibody comprises the VL sequence of SEQ ID NO 179, including post-translational modifications of the sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from: (a) HVR-L1 comprising amino acid sequence SEQ ID NO 5; (b) HVR-L2 comprising amino acid sequence SEQ ID NO 6; and (c) HVR-L3 comprising the amino acid sequence SEQ ID NO: 7.
In some embodiments, the anti-OX 40 antibody for use in the diagnostic or detection method is an anti-human OX40 antibody comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising amino acid sequence SEQ ID NO: 29; (b) HVR-H2 comprising amino acid sequence SEQ ID NO 30; (c) HVR-H3 comprising amino acid sequence SEQ ID NO: 31; (d) HVR-L1 comprising amino acid sequence SEQ ID NO 37; (e) HVR-L2 comprising amino acid sequence SEQ ID NO: 39; and (f) HVR-L3 comprising amino acid sequence SEQ ID NO: 42. In some embodiments, the anti-OX 40 antibody comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1 comprising amino acid sequence SEQ ID NO:29, (ii) HVR-H2 comprising amino acid sequence SEQ ID NO:30, and (iii) HVR-H3 comprising amino acid sequence SEQ ID NO: 31; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from: (i) HVR-L1 comprising amino acid sequence SEQ ID NO:37, (ii) HVR-L2 comprising amino acid sequence SEQ ID NO:39, and (c) HVR-L3 comprising amino acid sequence SEQ ID NO: 42. In some embodiments, the anti-OX 40 antibody comprises (a) HVR-H1 comprising amino acid sequence SEQ ID NO: 29; (b) HVR-H2 comprising amino acid sequence SEQ ID NO 30; (c) HVR-H3 comprising amino acid sequence SEQ ID NO 31; (d) HVR-L1 comprising amino acid sequence SEQ ID NO 37; (e) HVR-L2 comprising amino acid sequence SEQ ID NO: 39; and (f) HVR-L3 comprising amino acid sequence SEQ ID NO: 42. In some embodiments, the anti-OX 40 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to amino acid sequence SEQ ID NO: 182. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but an anti-human OX40 agonist antibody comprising that sequence retains the ability to bind OX 40. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO: 182. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-human OX40 agonist antibody comprises the VH sequence of SEQ ID NO:182, including post-translational modifications of that sequence. In a particular embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1 comprising amino acid sequence SEQ ID NO:29, (b) HVR-H2 comprising amino acid sequence SEQ ID NO:30, and (c) HVR-H3 comprising amino acid sequence SEQ ID NO: 31. In some embodiments, the anti-OX 40 antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to amino acid sequence SEQ ID NO: 181. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-human OX40 agonist antibody comprising that sequence retains the ability to bind to human OX 40. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO 181. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the anti-human OX40 agonist antibody comprises the VL sequence of SEQ ID NO:181, including post-translational modifications of the sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from: (a) HVR-L1 comprising amino acid sequence SEQ ID NO 37; (b) HVR-L2 comprising amino acid sequence SEQ ID NO: 39; and (c) HVR-L3 comprising amino acid sequence SEQ ID NO: 42.
In some embodiments, the anti-OX 40 antibody comprises the VH sequence of SEQ ID NO: 180. In some embodiments, the anti-OX 40 antibody comprises the VL sequence SEQ ID NO 179. In some embodiments, the anti-OX 40 antibody comprises a VH sequence of SEQ ID NO:180 and a VL sequence of SEQ ID NO: 179. In some embodiments, the anti-OX 40 antibody comprises the VH sequence of SEQ ID NO: 182. In some embodiments, the anti-OX 40 antibody comprises the VL sequence SEQ ID NO: 181. In some embodiments, the anti-OX 40 antibody comprises the VH sequence SEQ ID NO:182 and the VL sequence SEQ ID NO: 181.
Exemplary disorders that can be diagnosed using the antibodies of the invention include cancer.
In certain embodiments, labeled anti-OX 40 antibodies are provided. Labels include, but are not limited to, labels or moieties that are directly detectable (such as fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), and moieties that are indirectly detectable, such as enzymes or ligands, for example, via enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes32P，14C，125I，3H, and131fluorophores such as rare earth chelates or fluoresceins and the likeDerivatives, rhodamine (rhodamine) and its derivatives, dansyl, umbelliferone, luciferases, e.g., firefly luciferase and bacterial luciferase (U.S. Pat. No.4,737,456), luciferin, 2, 3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β -galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase (which are coupled with enzymes employing a hydrogen peroxide oxidation dye precursor such as HRP), lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, phage labels, stable free radicals, and the like.
In one aspect, the invention provides diagnostic methods, e.g., for identifying cancer patients who are likely to respond to treatment with anti-human OX40 agonist antibodies.
In some embodiments, methods are provided for identifying a patient likely to respond to anti-human OX40 agonist antibody therapy, the methods comprising (i) determining the presence or absence or amount of FcR expressing cells (e.g., number per given sample size) in a cancer sample from the patient, and (ii) identifying the patient as likely to respond if the sample comprises FcR expressing cells (e.g., a high number of FcR expressing cells). Methods for detecting FcR expressing cells are well known in the art, including, for example, by IHC. In some embodiments, the FcR is an Fc γ R. In some embodiments, the FcR is an activating Fc γ R. In some embodiments, the cancer is any cancer described herein. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast cancer (e.g., triple negative breast cancer), gastric cancer, colorectal cancer (CRC), or hepatocellular carcinoma. In some embodiments, the method is an in vitro method. In some embodiments, the method further comprises (iii) recommending anti-human OX40 agonist antibody (e.g., any anti-human OX40 agonist antibody described herein) treatment. In some embodiments, the method further comprises (iv) treating the patient with the anti-human OX40 agonist antibody.
In some embodiments, methods are provided for identifying a patient likely to respond to anti-human OX40 agonist antibody treatment, the methods comprising (i) determining the presence or absence or amount (e.g., number per given sample size) of human effector cells (e.g., infiltrating effector cells) in a cancer sample from the patient, and (ii) identifying the patient as likely to respond if the sample comprises effector cells (e.g., a high number of effector cells). Methods for detecting infiltrating human effector cells are well known in the art, including, for example, by IHC. In some embodiments, the human effector cell is one or more of an NK cell, a macrophage, a monocyte. In some embodiments, the effector cell expresses an activating Fc γ R. In some embodiments, the method is an in vitro method. In some embodiments, the cancer is any cancer described herein. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast cancer (e.g., triple negative breast cancer), gastric cancer, colorectal cancer (CRC), or hepatocellular carcinoma. In some embodiments, the method further comprises (iii) recommending anti-human OX40 agonist antibody (e.g., any anti-human OX40 agonist antibody described herein) treatment. In some embodiments, the method further comprises (iv) treating the patient with the anti-human OX40 agonist antibody.
Provided is a method of providing a cancer diagnosis, comprising: (i) measuring FcR expressing cells (e.g., level or presence or absence or prevalence of FcR (e.g., percentage of FcR expressing cells, e.g., by IHC)) in a sample from the patient; (ii) when the sample has FcR biomarker expression, the patient is diagnosed as having a cancer comprising an FcR biomarker (e.g., a high FcR biomarker). In some embodiments, the method further comprises (iii) selecting a therapy for the patient that (a) comprises an anti-human OX40 agonist antibody or (b) recommends a therapy that comprises an anti-human OX40 agonist antibody. In some embodiments, the method is an in vitro method.
Provided is a method of providing a cancer diagnosis, comprising: (i) measuring human effector cells (e.g., the level or presence or absence or prevalence of human effector cells (e.g., percentage of human effector cells)) in a sample from a patient; (ii) when the sample has a human effector cell biomarker, the patient is diagnosed as having a cancer comprising human effector cells (e.g., high human effector cells). In some embodiments, the method further comprises (iii) selecting a therapy for the patient that (a) comprises an anti-human OX40 agonist antibody or (b) recommends a therapy that comprises an anti-human OX40 agonist antibody. In some embodiments, the method is an in vitro method.
Provided is a method of recommending treatment for a cancer patient, comprising: (i) measuring FcR expressing cells (e.g., level or presence or absence or prevalence of FcR (e.g., percentage of FcR expressing cells)) in a sample from the patient; (ii) anti-human OX40 agonist antibody treatment is recommended when the sample has FcR expressing cells (in some embodiments, cells that are highly FcR expressing). In some embodiments, the method further comprises (iii) selecting a therapy for the patient comprising an anti-human OX40 agonist antibody. In some embodiments, the method is an in vitro method.
Provided are methods of recommending a treatment for a cancer patient, comprising (i) measuring human effector cells (e.g., the level or presence or absence or prevalence of human effector cells (e.g., percentage of human effector cells)) in a sample from the patient; (ii) when the sample has human effector cells (in some embodiments, high human effector cells), anti-human OX40 agonist antibody treatment is recommended. In some embodiments, the method further comprises (iii) selecting a therapy for the patient comprising an anti-human OX40 agonist antibody. In some embodiments, the method is an in vitro method.
In some embodiments of any of the inventions provided herein, the sample is obtained prior to treatment with an anti-human OX40 agonist antibody. In some embodiments, the sample is obtained prior to treatment with the cancer drug. In some embodiments, the sample is obtained after the cancer has metastasized. In some embodiments, the sample is formalin fixed, paraffin coated (FFPE). In some embodiments, the sample is a biopsy (e.g., core biopsy), a surgical specimen (e.g., a specimen from a surgical resection), or a fine needle aspirate.
F. Pharmaceutical formulations
Pharmaceutical formulations of anti-OX 40 antibodies as described herein are prepared by mixing such antibodies of the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16 th edition, Osol, a. eds. (1980)) in a lyophilized formulation or in an aqueous solution. Generally, pharmaceutically acceptable carriers are nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexane diamine chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; hydrocarbyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or a non-ionic surfactant, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further comprise an interstitial drug dispersant such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (r: (r)) Baxter International, Inc.). Certain exemplary shasegps and methods of use,including rHuPH20, are described in U.S. patent publication nos. 2005/0260186 and 2006/0104968. In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.
In some embodiments, a "histidine buffer" is a buffer comprising histidine ions. Examples of histidine buffers include histidine chloride, histidine acetate, histidine phosphate, histidine sulfate. The preferred histidine buffer identified in the examples herein was found to be histidine acetate. In a preferred embodiment, the histidine acetate buffer is prepared by titration of L-histidine (free base, solid) with acetic acid (liquid). In some embodiments, the histidine buffer or histidine-acetate buffer is at a pH of 5.0 to 6.0, in some embodiments, pH 5.3 to 5.8.
In some embodiments, "sugar" herein includes the general composition of (CH2O) n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and the like. Examples of sugars herein include glucose, sucrose, trehalose, lactose, fructose, maltose, dextran (dextran), erythritol, glycerol, arabitol, xylitol (sylitol), sorbitol, mannitol, melibiose (mellibiose), melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose (maltulose), glucitol, maltitol, lactitol, isomaltulose, and the like. In some embodiments, the sugar is a non-reducing disaccharide, such as trehalose or sucrose.
In some embodiments herein, "surfactant" refers to a surface active material, preferably a nonionic surfactant. Examples of surfactants herein include polysorbates (e.g., polysorbate 20 and polysorbate 80); poloxamers (e.g., poloxamer 188); triton; sodium Dodecyl Sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; lauryl, myristyl, linoleyl (linoleyl), or stearyl sulfobetaine; lauryl, myristyl, linoleyl or stearyl muscle(ii) an amino acid; linoleyl, myristyl, or cetyl betaine; lauramidopropyl, cocamido (cocamido) propyl, linoleamidopropyl, myristoamidopropyl, palmitamido (palmido) propyl, or isostearamidopropyl betaine (e.g. lauramidopropyl); myristamidopropyl, palmitamido (palmido) propyl, or isostearamidopropyl dimethylamine; sodium methyl cocoyl taurate or disodium methyl oleoyl taurate; and MONAQUATTMSeries (Mona Industries, inc., Paterson, New Jersey); polyethylene glycol, polypropylene glycol, and copolymers of ethylene glycol and propylene glycol (e.g., Pluronics, PF68, etc.); and the like. In some embodiments, the surfactant is polysorbate 20. In some embodiments, the surfactant is polysorbate 80.
An exemplary lyophilized antibody formulation is described in U.S. Pat. No.6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No.6,171,586 and WO2006/044908, the latter formulation comprising a histidine-acetate buffer.
The formulations herein may also contain more than one active ingredient necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide another medication (examples are provided herein). Suitably, such active ingredients are present in combination in an amount effective for the intended purpose.
The active ingredient may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16 th edition, Osol, A. eds (1980).
Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.
Formulations for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.
In some embodiments, provided herein are pharmaceutical formulations comprising: (a) any anti-human OX40 agonist antibody described herein; (b) histidine buffer at pH 5.0-6.0.
In some embodiments, provided herein are pharmaceutical formulations comprising: (a) any anti-human OX40 agonist antibody described herein; (b) histidine buffer at pH 5.0-6.0; (c) a sugar; and (d) a surfactant.
In some embodiments of any of the formulations, the anti-human OX40 agonist antibody is present at a concentration of between about 10mg/mL and about 100mg/mL (e.g., about 15mg/mL, 18mg/mL, 20mg/mL, 60mg/mL, and 75 mg/mL). In some embodiments, the anti-human OX40 agonist antibody is present at a concentration of about 20 mg/mL. In some embodiments, the anti-human OX40 agonist antibody is present at a concentration of about 50 mg/mL. In some embodiments, the anti-human OX40 agonist antibody is present at a concentration of about 60 mg/mL. In some embodiments, the anti-human OX40 agonist antibody is present at a concentration of about 70 mg/mL.
In some embodiments of any of the formulations, the sugar is present at a concentration of about 75mM to about 360mM (e.g., about 100mM, about 120mM, about 240mM, about 320mM to about 360 mM). In some embodiments, the sugar is present at a concentration of about 120 mM. In some embodiments, the sugar is present at a concentration of about 240 mM. In some embodiments, the sugar is present at a concentration of about 320 mM. In some embodiments, the saccharide is a disaccharide. In some embodiments, the disaccharide is trehalose. In some embodiments, the disaccharide is sucrose.
In some embodiments of any formulation, the histidine buffer is at a concentration of about 1mM to about 50mM (e.g., about 1mM to about 25 mM). In some embodiments, the histidine buffer is at a concentration of about 10 mM. In some embodiments, the histidine buffer is at a concentration of about 20 mM. In some embodiments, the histidine buffer is at a concentration of about 30 mM. In some embodiments, the histidine buffer is histidine acetate.
In some embodiments of any of the formulations, the surfactant is a polysorbate (e.g., polysorbate 20 or polysorbate 40), poloxamer (e.g., poloxamer 188); triton; sodium Dodecyl Sulfate (SDS); sodium lauryl sulfate; or sodium octyl glucoside.
In some embodiments of any of the formulations, the surfactant is a polysorbate. In some embodiments, the polysorbate is present at a concentration of about 0.005% to about 0.1%. In some embodiments, the polysorbate is present at a concentration of about 0.005%. In some embodiments, the polysorbate is present at a concentration of about 0.02%. In some embodiments, the polysorbate is present at a concentration of about 0.04%. In some embodiments, the polysorbate is present at a concentration of about 0.06%. In some embodiments, the polysorbate is polysorbate 20. In some embodiments, the polysorbate is polysorbate 80.
In some embodiments of any of the formulations, the formulation is diluted with a diluent (e.g., 0.9% NaCl). In some embodiments, the anti-human OX40 agonist antibody is present at a concentration of about 1 mg/mL.
In particular, provided herein are pharmaceutical formulations comprising (a) any of the anti-human OX40 agonist antibodies described herein; (b) a polysorbate, wherein the polysorbate concentration is about 0.005% to about 0.1%; and (c) a histidine buffer (e.g., a histidine buffer at pH 5.0 to 6.0).
In some embodiments, the pharmaceutical formulation comprises (a) any anti-human OX40 agonist antibody described herein (e.g., at a concentration between about 10mg/mL and about 100 mg/mL); (b) a polysorbate, wherein the polysorbate concentration is about 0.02% to about 0.06%; (c) histidine buffer (e.g., histidine buffer at pH 5.0 to 6.0); and a sugar, wherein the sugar concentration is about 120mM to about 320 mM. In some embodiments, the sugar is sucrose.
In some embodiments, the pharmaceutical formulation comprises (a) any anti-human OX40 agonist antibody described herein, at a concentration between about 10mg/mL and about 100 mg/mL; (b) a polysorbate, wherein the polysorbate concentration is about 0.02% to about 0.06%, wherein the polysorbate is polysorbate 20 or polysorbate 40; (c) histidine acetate buffer at pH 5.0 to 6.0; and a sugar (e.g., sucrose) at a concentration of about 120mM to about 320 mM.
In some embodiments, the pharmaceutical formulation comprises (a) any anti-human OX40 agonist antibody described herein; (b) polysorbate 20, wherein the polysorbate concentration is about 0.02% to about 0.06%; (c) a histidine acetate buffer (e.g., a histidine acetate buffer at a pH between 5.0 and 6.0); and (d) sucrose, wherein the sucrose concentration is about 120mM to about 320 mM.
In some embodiments, the pharmaceutical formulation comprises (a) any anti-human OX40 agonist antibody described herein; (b) polysorbate 40, wherein the polysorbate concentration is about 0.02% to about 0.06%; (c) a histidine acetate buffer (e.g., a histidine acetate buffer at a pH between 5.0 and 6.0); and sucrose, wherein the sucrose concentration is about 120mM to about 320 mM.
In some embodiments, the pharmaceutical formulation comprises (a) any anti-human OX40 agonist antibody described herein; (b) polysorbate 20, wherein the polysorbate concentration is about 0.02%; (c) histidine acetate buffer at ph 6.0; and (d) sucrose, wherein the sucrose concentration is about 320 mM.
In some embodiments, the pharmaceutical formulation comprises (a) any anti-human OX40 agonist antibody described herein; (b) polysorbate 20, wherein the polysorbate concentration is about 0.02%; (c) histidine acetate buffer at ph 5.5; and (d) sucrose, wherein the sucrose concentration is about 240 mM.
In some embodiments, the pharmaceutical formulation comprises (a) any anti-human OX40 agonist antibody described herein; (b) polysorbate 20, wherein the polysorbate concentration is about 0.04%; (c) histidine acetate buffer at ph 6.0; and (d) sucrose, wherein the sucrose concentration is about 120 mM.
In some embodiments, the pharmaceutical formulation comprises (a) any anti-human OX40 agonist antibody described herein; (b) polysorbate 40, wherein the polysorbate concentration is about 0.04%; (c) histidine acetate buffer at ph 5.0; and (d) sucrose, wherein the sucrose concentration is about 240 mM.
In some embodiments, the pharmaceutical formulation comprises (a) any anti-human OX40 agonist antibody described herein; (b) polysorbate 40, wherein the polysorbate concentration is about 0.04%; (c) histidine acetate buffer at ph 6.0; and (d) sucrose, wherein the sucrose concentration is about 120 mM.
In some embodiments, the pharmaceutical formulation is a liquid pharmaceutical formulation. In some embodiments, the pharmaceutical formulation is a stable pharmaceutical formulation. In some embodiments, the pharmaceutical formulation is a stable liquid pharmaceutical formulation.
In some embodiments of any of the pharmaceutical formulations described herein, the anti-human OX40 agonist antibody in the pharmaceutical formulation is present at a concentration between about 10mg/mL and about 100 mg/mL. In some embodiments, the concentration of the human OX40 agonist antibody is between any one of about 10mg/mL to 50mg/mL, 10mg/mL to 75mg/mL, 25mg/mL to 75mg/mL, 50mg/mL to 100mg/mL, 50mg/mL to 75mg/mL, and/or 75mg/mL to 100 mg/mL. In some embodiments, the concentration of the human OX40 agonist antibody is greater than any one of about 20mg/mL, 30mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, or 100 mg/mL.
The pharmaceutical formulation preferably comprises polysorbate. Polysorbates are generally included in an amount that reduces aggregate formation, such as occurs upon shaking or shipping. Examples of polysorbates include, but are not limited to, polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate), polysorbate 60 (polyoxyethylene (20) sorbitan monostearate), and/or polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In some embodiments, the polysorbate is polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate). In some embodiments of any of the pharmaceutical formulations described herein, the polysorbate concentration is sufficient to minimize aggregation and/or maintain stability upon long-term storage and/or during administration (e.g., after dilution in an IV bag). In some embodiments, the polysorbate concentration is about 0.005% w/v, about 0.02% w/v, about 0.04% w/v, and less than about 0.1% w/v. In some embodiments, the polysorbate concentration is greater than 0.01% w/v and less than about 0.1% w/v. In some embodiments, the polysorbate concentration is any one of about 0.005% w/v, about 0.02% w/v, 0.03% w/v, 0.04% w/v, or 0.05% w/v. In some embodiments, the polysorbate is present at a concentration of about 0.04% w/v. In some embodiments, the polysorbate is present at a concentration of about 0.02% w/v.
The pharmaceutical formulation preferably comprises a saccharide. Saccharides include monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and the like. Additional examples of saccharides include, but are not limited to, glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol (glycerol), dextran, erythritol, glycerol (glycerol), arabitol, xylitol (sylitol), sorbitol, mannitol, melibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, glucitol, maltitol, lactitol, isomaltulose, and the like. In some embodiments, the saccharide is a disaccharide. In some embodiments, the saccharide is a non-reducing disaccharide. In some embodiments, the saccharide is trehalose.
Sugars are typically included in amounts that reduce aggregate formation. In some embodiments of any of the pharmaceutical formulations described herein, the saccharide is present at a concentration between about any of 50mM to 250mM, 75mM to 200mM, 75mM to 150mM, 100mM to 150mM, 110mM to 130mM, 100mM to 320mM, 240mM to 320mM, or 240mM to 400 mM. In some embodiments, the saccharide is present at a concentration of greater than any of about 50mM, 75mM, 100mM, 110mM, or 115 mM. In some embodiments, the saccharide is present at a concentration of any of about 100mM, 110mM, 120mM, 130mM, or 140 mM. In some embodiments, the saccharide is present at a concentration of about 120 mM. In some embodiments of any of the formulations, the saccharide is present at a concentration of about 75mM to about 360mM (e.g., about 100mM, about 120mM, about 240mM, about 320mM to about 360 mM). In some embodiments, the saccharide is present at a concentration of about 240 mM. In some embodiments, the saccharide is present at a concentration of about 320 mM.
The pharmaceutical formulation preferably comprises a histidine buffer. Examples of histidine buffers include, but are not limited to, histidine chloride, histidine succinate, histidine acetate, histidine phosphate, histidine sulfate. In some embodiments, the histidine buffer is histidine acetate. In some embodiments of any of the pharmaceutical formulations described herein, the histidine buffer concentration is between any of about 1mM to 50mM, 1mM to 35mM, 1mM to 25mM, 1mM to 20mM, 7.5mM to 12.5mM, 5mM to 15mM, 20mM to 30mM, or 25mM to 35 mM. In some embodiments, the histidine buffer concentration is any one of about 5mM, 7.5mM, 10mM, 12.5mM, 15mM, 20mM, 25mM, 30mM, 35mM, or 40 mM. In some embodiments, the histidine buffer concentration is about 10 mM. In some embodiments, the histidine buffer concentration is about 20 mM. In some embodiments, the histidine buffer concentration is about 30 mM. In some embodiments, the histidine buffer concentration is about 40 mM. In some embodiments of any of the pharmaceutical formulations described herein, the histidine buffer is at a pH between pH5.0 and 6.0, e.g., about any of pH5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH5.8, pH5.9, or pH 6.0. In some embodiments, the pH is between pH 4.9 and pH 6.3.
The pharmaceutical formulations herein may also contain more than one active compound necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Suitably, such molecules are present in combination in an amount effective for the intended purpose.
Also, provided herein are vials containing the pharmaceutical formulations described herein and methods of filling the vials. In some embodiments, the pharmaceutical formulation is provided in a vial having a syringe pierceable stopper, preferably in aqueous form. It is desirable to store the vial for 24 hours at about 2-8 ℃ and up to 30 ℃ until it is administered to a subject in need thereof. The vial may be, for example, a 15cc vial (e.g., for a 200mg dose).
The pharmaceutical formulation for administration is preferably a liquid formulation (not lyophilized) and has not been previously lyophilized. Although the pharmaceutical formulation may be lyophilized, it is preferred that it is not lyophilized. In some embodiments of any of the pharmaceutical formulations, the pharmaceutical formulation is a lyophilized pharmaceutical formulation. In some embodiments, the pharmaceutical formulation is a liquid formulation. In some embodiments, the pharmaceutical formulation is free of a tonicity-changing (tonifying) amount of a salt such as sodium chloride. In some embodiments of any pharmaceutical formulation, the pharmaceutical formulation is diluted.
G. Therapeutic methods and compositions
Any of the anti-human OX40 antibodies provided herein can be used in a method of treatment.
In one aspect, anti-human OX40 agonist antibodies are provided for use as a medicament. In still further aspects, anti-human OX40 agonist antibodies are provided for use in treating cancer. In certain embodiments, anti-human OX40 agonist antibodies are provided for use in methods of treatment. In certain embodiments, anti-human OX40 agonist antibodies are provided, which are useful in methods of treating an individual having cancer comprising administering to the individual an effective amount of the anti-human OX40 agonist antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below.
In one aspect, provided is an anti-human OX40 agonist antibody for use in enhancing immune function (e.g., by upregulating a cell-mediated immune response) in an individual having cancer, comprising administering to the individual an effective amount of the anti-human OX40 agonist antibody. In one aspect, provided is an anti-human OX40 agonist antibody for use in enhancing T cell function in an individual with cancer, comprising administering to the individual an effective amount of the anti-human OX40 agonist antibody. In one aspect, provided is an anti-human OX40 agonist antibody for depleting human OX 40-expressing cells (e.g., OX 40-expressing T cells, e.g., OX 40-expressing tregs), comprising administering to the individual an effective amount of the anti-human OX40 agonist antibody. In some embodiments, the depleting is by ADCC. In some embodiments, the depleting is by phagocytosis. Provided are anti-human OX40 agonist antibodies for use in treating an individual having tumor immunity.
In yet further aspects, anti-human OX40 agonist antibodies are provided for use in treating infections (e.g., bacterial or viral or other pathogen infections). In certain embodiments, the invention provides anti-human OX40 agonist antibodies for use in a method of treating an individual having an infection, comprising administering to the individual an effective amount of the anti-human OX40 agonist antibody. In some embodiments, the infection is a viral and/or bacterial infection. In some embodiments, the infection is a pathogen infection.
In yet another aspect, the invention provides a use of an anti-OX 40 antibody for the manufacture or preparation of a medicament. In one embodiment, the medicament is for treating cancer. In yet another embodiment, the medicament is for use in a method of treating cancer, comprising administering to an individual having cancer an effective amount of the medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below.
In one aspect, the medicament is for enhancing immune function (e.g., by upregulating a cell-mediated immune response) in an individual having cancer, comprising administering to the individual an effective amount of the medicament. In one aspect, the medicament is for enhancing T cell function in an individual with cancer comprising administering to the individual an effective amount of the medicament. In some embodiments, the T cell dysfunctional disorder is cancer. In one aspect, the medicament is for depleting cells expressing human OX40 (e.g., cells expressing high OX40, such as T cells expressing OX 40), comprising administering to the individual an effective amount of the medicament. In some embodiments, the depleting is by ADCC. In some embodiments, the depleting is by phagocytosis. In one aspect, the medicament is for treating an individual having tumor immunity.
In yet other aspects, a medicament is provided for treating an infection (e.g., a bacterial or viral or other pathogen infection). In certain embodiments, the medicament is for use in a method of treating an individual having an infection comprising administering to the individual an effective amount of the medicament. In some embodiments, the infection is a viral and/or bacterial infection. In some embodiments, the infection is a pathogen infection.
In yet another aspect, the invention provides a method for treating cancer. In one embodiment, the method comprises administering to an individual having such cancer an effective amount of an anti-OX 40 antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. An "individual" according to any of the above embodiments may be a human.
In one aspect, provided are methods for enhancing immune function (e.g., by upregulating a cell-mediated immune response) in an individual having cancer, comprising administering to the individual an effective amount of the anti-human OX40 agonist antibody. In one aspect, provided is a method for enhancing T cell function in an individual with cancer comprising administering to the individual an effective amount of the anti-human OX40 agonist antibody. In one aspect, provided is a method for depleting cells that express human OX40 (e.g., cells that express high levels of OX40, such as OX 40-expressing T cells), comprising administering to the individual an effective amount of the anti-human OX40 agonist antibody. In some embodiments, the depleting is by ADCC. In some embodiments, the depleting is by phagocytosis. Provided are anti-human OX40 agonist antibodies for use in treating an individual having tumor immunity.
In some embodiments, examples of cancer further include, but are not limited to, B-cell lymphomas (including low grade/follicular non-Hodgkin's lymphoma (NHL), Small Lymphocytic (SL) NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL, high grade immunocytogenic NHL, high grade lymphoblastic NHL, high grade small non-nucleated NHL, storage disease (bulkydiaseNHL), mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's (Waldenstrom) macroglobulinemia), Chronic Lymphocytic Leukemia (CLL), Acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), and abnormal vascular proliferation associated with scarring nevus (phakomatoses), edema (such as associated with brain tumors), B cell proliferative disorders, and Meigs' syndrome. More specific examples include, but are not limited to, relapsed or refractory NHL, anterior (front line) low grade NHL, stage III/IVNHL, chemotherapy-resistant NHL, precursor B lymphoblastic leukemia and/or lymphoma, small lymphocytic lymphoma, B-cell chronic lymphocytic leukemia and/or prolymphocytic leukemia and/or small lymphocytic lymphoma, B-cell prolymphocytic lymphoma, immunocytoma and/or lymphoplasmacytic (lymphoplasmacytic) lymphoma, lymphoplasmacytic lymphoma, marginal zone B cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone (extranodal marginal zone) -MALT lymphoma, nodal marginal zone (nodal marginal zone) lymphoma, hairy cell leukemia, plasmacytoma and/or plasma cell myeloma, low grade/follicular lymphoma, intermediate grade/follicular NHL, mantle cell lymphoma, follicular central lymphoma (follicular), intermediate grade diffuse NHL, diffuse large B-cell lymphoma, aggressive (aggressive) NHL (including aggressive frontline NHL and aggressive relapsed NHL), relapsed or refractory NHL after autologous stem cell transplantation, primary mediastinal large B-cell lymphoma, primary effusion lymphoma, advanced immunoblastic NHL, advanced lymphoblastic NHL, advanced small anucleate NHL, deposit disease (bulky disease) NHL, Burkitt's (Burkitt) lymphoma, precursor (peripheral) large granular lymphocytic leukemia, mycosis fungoides and/or Sezary syndrome, cutaneous lymphoma, anaplastic large cell lymphoma, angiocentral lymphoma.
In some embodiments, examples of cancer further include, but are not limited to, B cell proliferative disorders, which further include, but are not limited to, lymphomas (e.g., B cell non-hodgkin's lymphoma (NHL)) and lymphocytic leukemias. Such lymphomas and lymphocytic leukemias include, for example, a) follicular Lymphoma, B) Small Non-nucleated lymphomas (Small Non-cleared Cell Lymphoma)/Burkitt's (Burkitt) lymphomas (including endemic Burkitt's Lymphoma, sporadic Burkitt's Lymphoma, and Non-Burkitt's Lymphoma), c) marginal zone lymphomas (including extranodal marginal zone B-Cell Lymphoma (mucosa-associated lymphoid tissue Lymphoma, MALT), nodal marginal zone B-Cell Lymphoma, and splenic marginal zone Lymphoma), d) Mantle Cell Lymphoma (MCL), e) large Cell Lymphoma (including B-Cell Diffuse Large Cell Lymphoma (DLCL), diffuse mixed Cell Lymphoma, immunoblastic Lymphoma, primary mediastinal B Cell Lymphoma, angiocentric Lymphoma-lung B Cell Lymphoma), f) hairy Cell leukemia, g) lymphocytic lymphoma, waldenstrom's macroglobulinemia, h) Acute Lymphocytic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL)/Small Lymphocytic Lymphoma (SLL), B-cell prolymphocytic leukemia, i) plasma cell neoplasms, plasma cell myeloma, multiple myeloma, plasmacytoma, and/or j) hodgkin's disease.
In some embodiments of any of the methods, the cancer is a B cell proliferative disorder. In some embodiments, the B cell proliferative disorder is lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed indolent NHL, refractory indolent NHL, Chronic Lymphocytic Leukemia (CLL), small lymphocytic lymphoma, leukemia, Hairy Cell Leukemia (HCL), Acute Lymphocytic Leukemia (ALL), or mantle cell lymphoma. In some embodiments, the B cell proliferative disorder is NHL, such as indolent NHL and/or aggressive NHL. In some embodiments, the B cell proliferative disorder is indolent follicular lymphoma or diffuse large B cell lymphoma.
In yet another aspect, the invention provides pharmaceutical formulations comprising any of the anti-OX 40 antibodies provided herein, e.g., for use in any of the above-described methods of treatment. In one embodiment, the pharmaceutical formulation comprises any of the anti-OX 40 antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any of the anti-OX 40 antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.
In some embodiments of any of the methods of the invention, the anti-human OX40 agonist antibody inhibits tumor immunity by inhibiting Treg function (e.g., inhibiting suppressive function of tregs), killing OX 40-expressing cells (e.g., high level OX 40-expressing cells), increasing effector T cell function, and/or increasing memory T cell function. In some embodiments of any of the methods of the invention, the anti-human OX40 agonist antibody treats cancer by inhibiting Treg function (e.g., inhibiting suppressive function of tregs), killing OX 40-expressing cells (e.g., high level OX 40-expressing cells), increasing effector T cell function, and/or increasing memory T cell function. In some embodiments of any of the methods of the invention, the anti-human OX40 agonist antibody enhances immune function by inhibiting Treg function (e.g., inhibiting suppressive function of tregs), killing OX 40-expressing cells (e.g., high level OX 40-expressing cells), increasing effector T cell function, and/or increasing memory T cell function. In some embodiments of any of the methods of the invention, the anti-human OX40 agonist antibody enhances T cell function by inhibiting Treg function (e.g., inhibiting suppressive function of tregs), killing OX 40-expressing cells (e.g., high level OX 40-expressing cells), increasing effector T cell function, and/or increasing memory T cell function.
In some embodiments of any of the methods, the anti-human OX40 agonist antibody is a subtractive anti-human OX40 agonist antibody. In some embodiments, the anti-human OX40 agonist antibody treatment results in cell depletion (e.g., depletion of cells expressing OX40, e.g., depletion of cells expressing high levels of OX 40). In some embodiments, the depleting is by ADCC. In some embodiments, the depleting is by phagocytosis.
In some embodiments of any of the methods, the anti-human OX40 agonist antibody inhibits Treg function, e.g., by inhibiting Treg suppression of effector and/or memory T cell function (in some embodiments, effector T cell and/or memory T cell proliferation and/or cytokine secretion) relative to Treg function prior to administration of the OX40 agonist antibody. In some embodiments of any of the methods, the anti-human OX40 agonist antibody increases effector T cell proliferation relative to effector T cell proliferation prior to administration of the OX40 agonist antibody. In some embodiments of any of the methods, the anti-human OX40 agonist antibody increases memory T cell proliferation relative to memory T cell proliferation prior to administration of the OX40 agonist antibody. In some embodiments of any of the methods, the anti-human OX40 agonist antibody increases effector T cell cytokine production (e.g., interferon-gamma production) relative to effector T cell cytokine production prior to administration of the OX40 agonist antibody. In some embodiments of any of the methods, the anti-human OX40 agonist antibody increases memory T cell cytokine production (e.g., interferon-gamma production) relative to memory T cell cytokine production prior to administration of the OX40 agonist antibody. In some embodiments of any of the methods, the anti-human OX40 agonist antibody increases CD4+ effector T cell proliferation and/or CD8+ effector T cell proliferation relative to CD4+ effector T cell proliferation and/or CD8+ effector T cell proliferation prior to administration of the OX40 agonist antibody. In some embodiments of any of the methods, the anti-human OX40 agonist antibody increases memory T cell proliferation (e.g., CD4+ memory T cell proliferation) relative to memory T cell proliferation prior to administration of the OX40 agonist antibody. In some embodiments, the CD4+ effector T cells in the individual have enhanced proliferation, cytokine secretion, and/or lytic activity relative to prior proliferation, cytokine secretion, and/or lytic activity of the administration of the anti-human OX40 agonist antibody.
In some embodiments of any of the methods of the invention, the number of CD4+ effector T cells is increased relative to prior to administration of the anti-human OX40 agonist antibody. In some embodiments, CD4+ effector T cell cytokine secretion is elevated relative to prior to administration of the anti-human OX40 agonist antibody. In some embodiments of any of the methods, the CD8+ effector T cells in the individual have enhanced proliferation, cytokine secretion, and/or lytic activity relative to prior to administration of the anti-human OX40 agonist antibody. In some embodiments, the number of CD8+ effector T cells is increased relative to prior to administration of the anti-human OX40 agonist antibody. In some embodiments, CD8+ effector T cell cytokine secretion is elevated relative to prior to administration of the anti-human OX40 agonist antibody.
In some embodiments of any of the methods of the invention, the anti-human OX40 agonist antibody binds to a human effector cell, e.g., binds to an Fc γ R expressed by a human effector cell. In some embodiments, the human effector cell performs ADCC effector function. In some embodiments, the human effector cell performs phagocytic effector function.
In some embodiments of any of the methods of the invention, an anti-human OX40 agonist antibody comprising a variant IgG1Fc polypeptide comprising a mutation that abrogates binding to human effector cells, e.g., a DANA or N297G mutation, has reduced activity (e.g., CD4+ effector T cell function, e.g., proliferation) relative to an anti-human OX40 agonist antibody comprising a native sequence IgG1Fc portion. In some embodiments, an anti-human OX40 agonist antibody comprising a variant IgG1Fc polypeptide comprising a mutation that abrogates binding to human effector cells, e.g., a DANA or N297G mutation, does not possess substantial activity (e.g., CD4+ effector T cell function, e.g., proliferation).
In some embodiments of any of the methods of the invention, antibody cross-linking is required for anti-human OX40 agonist antibody function. In some embodiments, the function is to stimulate CD4+ effector T cell proliferation. In some embodiments, antibody cross-linking is determined by providing an anti-human OX40 agonist antibody that adheres to a solid surface (e.g., a cell culture plate). In some embodiments, antibody cross-linking is determined by introducing a mutation (e.g., DANA or N297S mutation) in the IgG1Fc portion of the antibody and testing the function of the mutant antibody.
In some embodiments of any of the methods, the memory T cells in the individual have enhanced proliferation and/or cytokine secretion relative to prior to administration of the anti-human OX40 agonist antibody. In some embodiments, the number of memory T cells is increased relative to prior to administration of the anti-human OX40 agonist antibody. In some embodiments, memory T cell cytokine secretion (levels) are elevated relative to prior to administration of the anti-human OX40 agonist antibody. In some embodiments of any of the methods, the tregs in the individual have reduced suppression of effector T cell function (e.g., proliferation and/or cytokine secretion) relative to prior to administration of the anti-human OX40 agonist antibody. In some embodiments, the number of effector T cells is increased relative to prior to administration of the anti-human OX40 agonist antibody. In some embodiments, effector T cell cytokine secretion (levels) are increased relative to prior to administration of the anti-human OX40 agonist antibody.
In some embodiments of any of the methods of the invention, the number of (infiltrating) CD4+ effector T cells (e.g., the total number of CD4+ effector T cells, or, e.g., the percentage of CD4+ cells in CD45+ cells) within the tumor is increased relative to prior to administration of the anti-human OX40 agonist antibody. In some embodiments of any of the methods of the invention, the number of γ -interferon expressing intratumoral (infiltrating) CD4+ effector T cells (e.g., the percentage of γ -interferon expressing CD4+ cells in total, or e.g., total CD4+ cells expressing CD4+ cells) is increased relative to prior to administration of the anti-human OX40 agonist antibody.
In some embodiments of any of the methods of the invention, the number of (infiltrating) CD8+ effector T cells (e.g., the total number of CD8+ effector T cells, or, e.g., the percentage of CD8+ in CD45+ cells) within the tumor is increased relative to prior to administration of the anti-human OX40 agonist antibody. In some embodiments of any of the methods of the invention, the number of interferon-gamma expressing intratumoral (infiltrating) CD8+ effector T cells (e.g., the percentage of interferon-gamma expressing CD8+ cells in total CD8+ cells) is increased relative to prior to administration of the anti-human OX40 agonist antibody.
In some embodiments of any of the methods of the invention, the number of intratumoral (infiltrating) tregs (e.g., the total number of tregs or, e.g., the percentage of Fox3p + cells in CD4+ cells) is reduced relative to prior to administration of the anti-human OX40 agonist antibody.
In some embodiments of any of the methods of the invention, administration of an anti-human OX40 agonist antibody is combined with administration of a tumor antigen. In some embodiments, the tumor antigen comprises a protein. In some embodiments, the tumor antigen comprises a nucleic acid. In some embodiments, the tumor antigen is a tumor cell.
In some embodiments of any of the methods of the invention, the cancer is indicative of (e.g., is infiltrated by) human effector cells. Methods for detecting human effector cells are well known in the art and include, for example, by IHC. In some embodiments, the cancer exhibits high levels of human effector cells. In some embodiments, the human effector cell is one or more of an NK cell, a macrophage, a monocyte. In some embodiments, the cancer is any cancer described herein. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast cancer (e.g., triple negative breast cancer), gastric cancer, colorectal cancer (CRC), or hepatocellular carcinoma.
In some embodiments of any of the methods of the invention, the cancer displays FcR expressing cells (e.g., is infiltrated by FcR expressing cells). Methods for detecting FcR are well known in the art, including, for example, by IHC. In some embodiments, the cancer exhibits high levels of FcR expressing cells. In some embodiments, the FcR is an Fc γ R. In some embodiments, the FcR is an activating Fc γ R. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast cancer (e.g., triple negative breast cancer), gastric cancer, colorectal cancer (CRC), or hepatocellular carcinoma.
An "individual" according to any of the above embodiments is preferably a human.
The antibodies of the invention may be used alone or in combination with other agents in therapy. For example, an antibody of the invention can be co-administered with at least one additional therapeutic agent.
Such combination therapies noted above encompass both combined administration (where two or more therapeutic agents are contained in the same formulation or separate formulations), and separate administration, in which case administration of the antibody of the invention can occur prior to, concurrently with, and/or after administration of the other therapeutic agent and/or agents. In one embodiment, the administration of the anti-OX 40 antibody and the administration of the additional therapeutic agent occur within about one month, or within about one, two, or three weeks, or within about 1, 2, 3, 4, 5, or 6 days of each other. The antibodies of the invention may also be used in combination with radiotherapy.
In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with a chemotherapeutic or chemotherapeutic agent. In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with radiation or a radiation therapy agent. In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with a targeted therapy or targeted therapeutic. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an immunotherapy or immunotherapeutic agent, e.g., a monoclonal antibody.
In some embodiments, anti-human OX40 agonist antibodies may be administered in combination with PARP inhibitors (e.g., olaparib, Rucaparib, Niraparib, Cediranib, BMN673, Veliparib), Trabectedin, nab-paclitaxel (albumin-binding paclitaxel, ABRAXANE), Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluorouracil (e.g., FOLFOX, FOLFIRI), IFL, regorafenib, Reolysin, alima, Zykadia, Suzushi, Torisiel (temsolilimus), Inlyta (axitinib, Pfer), finitor (animals, novartists, Nexavar (sorafenib, Baysint, Bayside, Bayesian, Abies), Vezocaine-3514, Velcroidine, Vicker-0, Velcreamide, Velcroide, Toruloside, Velcroide, Toxil, Hazarine, Haemadine, Hazarine, Ha.
In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with a PD-1 axis binding antagonist. PD-1 axis binding antagonists include, but are not limited to, PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists. Alternative names for "PD-1" include CD279 and SLEB 2. Alternative names for "PD-L1" include B7-H1, B7-4, CD274, and B7-H. Alternative names for "PD-L2" include B7-DC, Btdc, and CD 273. In some embodiments, PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1, and PD-L2. In some embodiments, the PD-1 binding antagonist is a molecule that inhibits binding of PD-1 to its ligand binding partner. In a particular aspect, the PD-1 ligand binding partner is PD-L1 and/or PD-L2. In another embodiment, the PD-L1 binding antagonist is a molecule that inhibits PD-L1 from binding its binding partner. In a particular aspect, the PD-L1 binding partner is PD-1 and/or B7-1. In another embodiment, the PD-L2 binding antagonist is a molecule that inhibits PD-L2 from binding its binding partner. In a particular aspect, the PD-L2 binding partner is PD-1. The antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of: MDX-1106(nivolumab, OPDIVO), Merck 3475(MK-3475, pembrolizumab, KEYTRUDA) and CT-011 (Pidilizumab). In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 binding antagonist is AMP-224. In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In some embodiments, the anti-PD-Ll binding antagonist is selected from the group consisting of: YW243.55.S70, MPDL3280A, MEDI4736 and MDX-1105. MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody described in WO 2007/005874. Antibody YW243.55.S70 (heavy and light chain variable region sequences shown in SEQ ID Nos. 20 and 21, respectively) is anti-PD-L1 as described in WO 2010/077634A 1. MDX-1106, also known as MDX-1106-04, ONO-4538, BMS-936558 or nivolumab, is an anti-PD-1 antibody described in WO 2006/121168. Merck 3475, also known as MK-3475, SCH-900475 or pembrolizumab, is an anti-PD-1 antibody described in WO 2009/114335. CT-011, also known as hBAT, hBAT-1 or pidilizumab, is an anti-PD-1 antibody described in WO 2009/101611. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in WO2010/027827 and WO 2011/066342. In some embodiments, the anti-PD-1 antibody is MDX-1106. Alternative names for "MDX-1106" include MDX-1106-04, ONO-4538, BMS-936558, or nivolumab. In some embodiments, the anti-PD-1 antibody is nivolumab (CAS registry number 946414-94-4).
In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an agonist directed to an activating costimulatory molecule. In some embodiments, the activating costimulatory molecule can include CD40, CD226, CD28, GITR, CD137, CD27, HVEM, or CD 127. In some embodiments, the agonist to the activating co-stimulatory molecule is an agonistic antibody that binds CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD 127. In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with antagonists to inhibitory co-stimulatory molecules. In some embodiments, the inhibitory co-stimulatory molecule may comprise CTLA-4 (also known as CD152), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA/B, or arginase. In some embodiments, the antagonist against the inhibitory co-stimulatory molecule is an antagonistic antibody that binds CTLA-4, PD-1, TIM-3, BTLA, VISTA, LAG-3 (e.g., LAG-3-IgG fusion protein (IMP321)), B7-H3, B7-H4, IDO, TIGIT, MICA/B, or arginase.
In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with antagonists, e.g., blocking antibodies, against CTLA-4 (also known as CD 152). In some embodiments, an anti-human OX40 agonist antibody can be conjugated to ipilimumab (also known as MDX-010, MDX-101, or ) In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with tremellimumab (also known as ticilimumab or CP-675,206). in some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an antagonist, e.g., a blocking antibody, directed against B7-H3 (also known as CD 276). in some embodiments, an anti-human OX40 agonist antibody can be administered in combination with MGA 271.
In some embodiments, an anti-human OX40 agonist antibody can be administered in conjunction with a treatment comprising adoptive transfer of Chimeric Antigen Receptor (CAR) -expressing T cells (e.g., cytotoxic T cells or CTLs). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with UCART 19. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with WT128 z. In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with KTE-C19 (Kite). In some embodiments, an anti-human OX40 agonist antibody may be administered in combination with CTL019 (Novartis). In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with a therapy comprising adoptive transfer of T cells comprising a dominant negative TGF β receptor, e.g., a dominant negative TGF β type II receptor. In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with a treatment comprising a HERCREEM regimen (see, e.g., clinical trials. gov Identifier NCT 00889954).
In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an antagonist against CD 19. In some embodiments, an anti-human OX40 agonist antibody may be administered in combination with MOR 00208. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an antagonist against CD 38. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with daratumumab.
In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an agonist, e.g., an activating antibody, directed against CD137 (also known as TNFRSF9, 4-1BB, or ILA). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with urelumab (also known as BMS-663513). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an agonist, e.g., an activating antibody, directed to CD 40. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with CP-870893. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an agonist, e.g., an activating antibody, directed against OX40 (also known as CD 134). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with a different anti-OX 40 antibody (e.g., AgonOX). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an agonist, e.g., an activating antibody, directed to CD 27. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with CDX-1127. In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with antagonists against indoleamine-2, 3-dioxygenase (IDO). In some embodiments, the IDO antagonist is 1-methyl-D-tryptophan (also referred to as 1-D-MT).
In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an agonist, e.g., an activating antibody, directed against CD137 (also known as TNFRSF9, 4-1BB, or ILA). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with urelumab (also known as BMS-663513). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an agonist, e.g., an activating antibody, directed to CD 40. In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with CP-870893 or RO 7009789. In some embodiments, anti-human OX40 agonist antibodies can agonize against OX40 (also known as CD134)Agents, such as activating antibodies, are administered in combination. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an agonist, e.g., an activating antibody, directed to CD 27. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with CDX-1127 (also known as varliumab). In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with antagonists against indoleamine-2, 3-dioxygenase (IDO). In some embodiments, the IDO antagonist is 1-methyl-D-tryptophan (also referred to as 1-D-MT). In some embodiments, the IDO antagonist is an IDO antagonist as shown in WO2010/005958 (the contents of which are expressly incorporated by reference herein). In some embodiments, the IDO antagonist is 4- ({2- [ (aminosulfonyl) amino ]Ethyl } amino) -N- (3-bromo-4-fluorophenyl) -N' -hydroxy-1, 2, 5-oxadiazole-3-carboxamidine (e.g. as described in example 23 of WO 2010/005958). In some embodiments, the IDO antagonist is
In some embodiments, the IDO antagonist is INCB 24360. In some embodiments, the IDO antagonist is Indoximod (the D isomer of 1-methyl-tryptophan). In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with an antibody-drug conjugate. In some embodiments, the antibody-drug conjugate comprises mertansine or monomethyl auristatin e (mmae). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an anti-NaPi 2b antibody-MMAE conjugate (also known as DNIB0600A, RG7599, or lifastuzumab vedotin). In some embodiments, an anti-human OX40 agonist antibody can be conjugated to trastuzumab emtansine (also known as T-DM1, ado-trastuzumab emtansine, orGenentech). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an anti-MUC 16 antibody-MMAE conjugate, DMUC 5754A. In some embodiments, an anti-human OX40 agonist antibody can be conjugated to an anti-MUC 16 antibody-MMAE conjugate, DMUC4064 A is administered in combination. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an antibody-drug conjugate targeting endothelin B receptor (EDNBR), such as an antibody against EDNBR conjugated with MMAE. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an antibody-drug conjugate targeting lymphocyte antigen 6 complex, locus E (Ly6E), such as an MMAE conjugated antibody against Ly6E (also known as DLYE 5953A). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with polatuzumab vedotin. In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with antibody-drug conjugates targeting CD 30. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with ADCETRIS (also known as brentuximab vedotin). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with polatuzumab vedotin.
In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with an angiogenesis inhibitor. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an antibody directed against VEGF, e.g., VEGF-a. In some embodiments, anti-human OX40 agonist antibodies can be conjugated to bevacizumab (also known as bevacizumab) Genentech). In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with an antibody against angiopoietin 2 (also known as Ang 2). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with MEDI 3617. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an antibody directed against VEGFR 2. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with ramucirumab. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with a VEGF receptor fusion protein. In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with an aflibercept. In some embodiments, anti-human OX40 agonist antibodies can be conjugated to ziv-aflibercept (also known as VEGF trap or) The administration is combined. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with a bispecific antibody against VEGF and Ang 2. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with RG7221 (also known as vanucizumab). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an angiogenesis inhibitor and in combination with a PD-1 axis binding antagonist (e.g., a PD-1 binding antagonist such as an anti-PD-1 antibody, a PD-L1 binding antagonist such as an anti-PD-L1 antibody, and a PD-L2 binding antagonist such as an anti-PD-L2 antibody). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with bevacizumab and a PD-1 axis binding antagonist (e.g., a PD-1 binding antagonist such as an anti-PD-1 antibody, a PD-L1 binding antagonist such as an anti-PD-L1 antibody, and a PD-L2 binding antagonist such as an anti-PD-L2 antibody). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with bevacizumab and MDX-1106(nivolumab, OPDIVO). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with bevacizumab and Merck 3475(MK-3475, pembrolizumab, KEYTRUDA). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with bevacizumab and CT-011 (Pidilizumab). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with bevacizumab and yw243.55.s70. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with bevacizumab and MPDL 3280A. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with bevacizumab and MEDI 4736. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with bevacizumab and MDX-1105.
In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with an anti-neoplastic agent. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an agent that targets CSF-1R (also known as M-CSFR or CD 115). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an anti-CSF-1R antibody (also known as IMC-CS4 or LY 3022855). In some embodiments of the present invention, the substrate is,in some embodiments, the anti-human OX40 agonist antibody can be administered in combination with GM-CSF (also known as recombinant human granulocyte macrophage colony stimulating factor, rhu GM-CSF, sargramostim, or emactuzumab)) The administration is combined. In some embodiments, anti-human OX40 agonist antibodies can be conjugated to IL-2 (also known as aldesleukin or ) The administration is combined. In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with IL-12. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with IL 27. In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with IL-15. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with ALT-803. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an antibody targeting CD 20. In some embodiments, the antibody targeting CD20 is obinutuzumab (also known as GA101 or GA 101)) Or rituximab. In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with an antibody that targets GITR. In some embodiments, the antibody targeting GITR is TRX 518. In some embodiments, the antibody targeting GITR is MK04166 (Merck).
In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an inhibitor of Bruton's Tyrosine Kinase (BTK). In some embodiments, an anti-human OX40 agonist antibody may be administered in combination with ibrutinib. In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with an inhibitor of vaccine dehydrogenase 1(IDH1) and/or vaccine dehydrogenase 2(IDH 2). In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with AG-120 (Agios).
In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with obinutuzumab and a PD-1 axis binding antagonist (e.g., a PD-1 binding antagonist such as an anti-PD-1 antibody, a PD-L1 binding antagonist such as an anti-PD-L1 antibody, and a PD-L2 binding antagonist such as an anti-PD-L2 antibody).
In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with a cancer vaccine. In some embodiments, the cancer vaccine is a peptide cancer vaccine, which in some embodiments is a personalized peptide vaccine. In some embodiments, the peptide Cancer vaccine is a multivalent long peptide, a multiple peptide, a mixture of peptides, a hybrid peptide, or a peptide-pulsed dendritic cell vaccine (see, e.g., Yamada et al, Cancer Sci,104:14-21,2013). In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with an adjuvant. In some embodiments, an anti-human OX40 agonist antibody can be conjugated to a TLR agonist, such as Poly-ICLC (also known as Poly-ICLC)) In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with Tumor Necrosis Factor (TNF) α. in some embodiments, an anti-human OX40 agonist antibody can be administered in combination with IL-1. in some embodiments, an anti-human OX40 agonist antibody can be administered in combination with HMGB 1. in some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an IL-10 antagonist. in some embodiments, an anti-human OX 7 agonist antibody can be administered in combination with an IL-4 antagonist In embodiments, an anti-human OX40 agonist antibody can be administered in combination with an ICOS agonist (e.g., by administration of ICOS-L, or an agonist antibody to ICOS). In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with a treatment targeting CX3CL 1. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with a treatment targeting CXCL 9. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with a treatment targeting CXCL 10. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with a treatment that targets CCL 5. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an LFA-1 or ICAM1 agonist. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with a selectin agonist.
In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with an inhibitor of B-Raf. In some embodiments, an anti-human OX40 agonist antibody can be conjugated to vemurafenib (also known as vemurafenib)) The administration is combined. In some embodiments, anti-human OX40 agonist antibodies can be conjugated to dabrafenib (also known as dabrafenib)) The administration is combined. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with encorafenib (LGX 818).
In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an EGFR inhibitor. In some embodiments, anti-human OX40 agonist antibodies can be conjugated to erlotinib (also known as) The administration is combined. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an inhibitor of EGFR-T790M. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with gefitinib. In some embodiments, anti-human OX40 agonist antibodies can be conjugated to afatinibThe administration is combined. In some embodiments, anti-human OX40 agonist antibodies can be conjugated to cetuximab (also known as) The administration is combined. In some embodiments, an anti-human OX40 agonist antibody can be conjugated to panitumumab (also known as) The administration is combined. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with rociletinib. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with AZD 9291. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an inhibitor of MEK, such as MEK1 (also known as MAP2K1) and/or MEK2 (also known as MAP2K 2). In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with cobimetinib (also known as GDC-0973 or XL-518). In some embodiments, an anti-human OX40 agonist antibody can be conjugated to trametinib (also known as trametinib) ) The administration is combined. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with a binimetinib.
In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an inhibitor of B-Raf (e.g., vemurafenib or dabrafenib) and an inhibitor of MEK (e.g., MEK1 and/or MEK2) (e.g., cobimetinib or trametinb). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an inhibitor of ERK (e.g., ERK 1/2). In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with GDC-0994. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an inhibitor of B-Raf, an inhibitor of MEK, and an inhibitor of ERK 1/2. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an inhibitor of EGFR, an inhibitor of MEK, and an inhibitor of ERK 1/2. In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with one or more MAP kinase pathway inhibitors. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with CK 127. In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with an inhibitor of K-Ras.
In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an inhibitor of c-Met. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with onartuzumab (also known as MetMAb). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an inhibitor of Anaplacic Lymphoma Kinase (ALK). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with AF802 (also known as CH5424802 or alectinib). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with a crizotinib. In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with ceritinib. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an inhibitor of phosphatidylinositol 3-kinase (PI 3K). In some embodiments, anti-human OX40 agonist antibodies may be administered in combination with buparlisib (BKM-120). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with a pictiliib (also referred to as GDC-0941). In some embodiments, an anti-human OX40 agonist antibody may be administered in combination with buparlisib (also known as BKM-120). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with perifosine (also known as KRX-0401). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with a selective inhibitor of phosphatidylinositol 3-kinase (PI 3K). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with idelalisib (also known as GS-1101 or CAL-101). In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with taselisib (also known as GDC-0032). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with BYL-719. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an inhibitor of Akt. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with MK 2206. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with GSK 690693. In that In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with ipatasertib (also known as GDC-0068). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an inhibitor of mTOR. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with sirolimus (also known as rapamycins). In some embodiments, anti-human OX40 agonist antibodies can be conjugated to temsirolimus (also known as CCI-779 or) The administration is combined. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with everolimus (also known as RAD 001). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with ridaforolimus (also known as AP-23573, MK-8669, or defoliomus). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with OSI-027. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with AZD 8055. In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with INK 128. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with a dual PI3K/mTOR inhibitor. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with XL 765. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with GDC-0980. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with BEZ235 (also known as NVP-BEZ 235). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with BGT 226. In some embodiments, an anti-human OX40 agonist antibody may be administered in combination with GSK 2126458. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with PF-04691502. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with PF-05212384 (also known as PKI-587).
In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with agents that selectively degrade estrogen receptors. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with GDC-0927. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an inhibitor of HER 3. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with duligotuzumab. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an inhibitor of LSD 1. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an inhibitor of MDM 2. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an inhibitor of BCL 2. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with a venetocalax. In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with an inhibitor of CHK 1. In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with GDC-0575. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an inhibitor of the activated hedgehog signaling pathway. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with erivcedge.
In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with radiation therapy. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with gemcitabine. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with nab-paclitaxel (abraxane). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with trastuzumab. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with TVEC. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with IL 27. In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with cyclophosphamide. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an agent that recruits T cells to a tumor. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with lirilumab (IPH 2102/BMS-986015). In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with Idelalisib. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an antibody targeting CD3 and CD 20. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with REGN 1979. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an antibody targeting CD3 and CD 19. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with blinatumomab.
In some embodiments, anti-human OX40 agonist antibodies can be administered in combination with an oncolytic virus. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with carboplatin and nab-paclitaxel. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with carboplatin and paclitaxel. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with cisplatin and pemetrexed. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with cisplatin and gemcitabine. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with FOLFOX. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with FOLFIRI.
Such combination therapies noted above encompass both combined administration (where two or more therapeutic agents are contained in the same formulation or separate formulations), and separate administration, in which case administration of the antibody of the invention can occur prior to, concurrently with, and/or after administration of the additional therapeutic agent and/or adjuvant. The antibodies of the invention may also be used in combination with radiotherapy.
The antibodies of the invention (and any other therapeutic agent) may be administered by any suitable means, including parenterally, intrapulmonary, and intranasally, and intralesionally if desired for local treatment. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing may be by any suitable route (e.g., by injection, such as intravenous or subcutaneous injection), depending in part on whether administration is transient or chronic. Various dosing schedules are contemplated herein, including but not limited to a single administration or multiple administrations over multiple time points, bolus administration, and pulse infusion.
The antibodies of the present invention will be formulated, dosed and administered in a manner consistent with excellent medical practice. Factors considered in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of agent delivery, the method of administration, the schedule of administration and other factors known to medical practitioners. The antibody need not be, but is optionally, formulated with one or more agents currently used for the prevention or treatment of the disorder in question. The effective amount of such other drugs depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors described above. These are generally used at the same dosages and routes of administration described herein, or at about 1-99% of the dosages described herein, or at any dosage and any route empirically/clinically determined to be appropriate.
For the prevention or treatment of disease, the appropriate dosage of an antibody of the invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitable for administration to a patient in one or a series of treatments. Depending on the type and severity of the disease, about 1. mu.g/kg to 40mg/kg of antibody may be administered to the patient as an initial candidate dose, whether, for example, by one or more separate administrations or by continuous infusion. A typical daily dose may range from about 1. mu.g/kg to 100mg/kg or more, depending on the factors described above. For repeated administrations over several days or longer, depending on the condition, treatment will generally continue until the desired suppression of disease symptoms occurs. Such doses may be administered intermittently, such as weekly or every three weeks (e.g., such that the patient receives from about 2 to about 20 doses, or, for example, about 6 doses, of the antibody). A higher initial loading agent may be administered followed by a lower dose or doses. However, other dosage regimens may be useful. The progress of this therapy is readily monitored by conventional techniques and assays.
It is understood that any of the above formulations or therapeutic methods may be practiced using the immunoconjugates of the invention in place of or in addition to an anti-OX 40 antibody.
Article and kit
In another aspect of the invention, there is provided an article of manufacture containing materials useful in the treatment, prevention and/or diagnosis of the conditions described above. The article comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials such as glass or plastic. The container contains a composition effective, alone or in combination with another composition, in the treatment, prevention and/or diagnosis of a condition, and may have a sterile access port (e.g., the container may be a vial or intravenous solution bag having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates the use of the composition to treat the selected condition. In addition, the article of manufacture can comprise (a) a first container having a composition therein, wherein the composition comprises an antibody of the invention; and (b) a second container having a composition therein, wherein the composition comprises an additional cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the composition may be used to treat a particular condition. Alternatively, or in addition, the article of manufacture or kit may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
In some embodiments, provided herein is a kit comprising a medicament comprising an anti-human OX40 agonist antibody described herein and optionally a pharmaceutically acceptable carrier. In some embodiments, the kit further comprises instructions for administering the medicament to treat cancer.
It will be appreciated that any of the above preparations may include an immunoconjugate of the invention in place of or in addition to the anti-OX 40 antibody.
Sequence of