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WO2017021912A1 - Combined tlrs modulators with anti ox40 antibodies - Google Patents

Combined tlrs modulators with anti ox40 antibodies Download PDF

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Publication number
WO2017021912A1
WO2017021912A1 PCT/IB2016/054696 IB2016054696W WO2017021912A1 WO 2017021912 A1 WO2017021912 A1 WO 2017021912A1 IB 2016054696 W IB2016054696 W IB 2016054696W WO 2017021912 A1 WO2017021912 A1 WO 2017021912A1
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modulator
amino acid
seq
acid sequence
antibody
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PCT/IB2016/054696
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French (fr)
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Christopher John FRANCIS
Niranjan YANAMANDRA
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Glaxosmithkline Intellectual Property Development Limited
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Publication of WO2017021912A1 publication Critical patent/WO2017021912A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to a method of treating cancer in a mammal and to combinations useful in such treatment.
  • the present relates to combinations of anti-OX40 antigen binding proteins (ABPs) and one or more TLR7/8 modulators.
  • the present invention also relates to triple combinations of anti-OX40 ABPs and one or more TLR7/8 modulators and one or more TLR4 modulators.
  • cancer results from the deregulation of the normal processes that control cell division, differentiation and apoptotic cell death and is characterized by the proliferation of malignant cells which have the potential for unlimited growth, local expansion and systemic metastasis.
  • Deregulation of normal processes include abnormalities in signal transduction pathways and response to factors which differ from those found in normal cells.
  • Immunotherapies are one approach to treat hyperproliferative disorders.
  • a major hurdle that scientists and clinicians have encountered in the development of various types of cancer immunotherapies has been to break tolerance to self antigen (cancer) in order to mount a robust anti-tumor response leading to tumor regression.
  • cancer self antigen
  • immunotherapies target cells of the immune system that have the potential to generate a memory pool of effector cells to induce more durable effects and minimize recurrences.
  • OX40 is a costimulatory molecule involved in multiple processes of the immune system.
  • Antigen binding proteins and antibodies that bind OX-40 receptor and modulate OX40 signalling are known in the art and are disclosed as immunotherapy, for example for cancer.
  • Aminoalkyl glucosaminide phosphates are synthetic ligands of the Toll-like Receptor 4 (TLR4). AGPs are known to be useful as vaccine adjuvants and for stimulating cytokine production, activating macrophages, promoting innate immune response, and augmenting antibody production in immunized animals.
  • ABSPs anti-OX40 antigen binding proteins
  • TLR7/8 modulators are combinations of anti-OX40 antigen binding proteins (ABPs) and one or more TLR7/8 modulators.
  • compositions of the invention comprising administering to said subject an effective amount of the combinations, e.g., in one or more pharmaceutical compositions.
  • FIG. 1A is a graph showing dose-dependent anti-tumor activity (as measured by tumor volume over time) of TLR-4 agonist (CRX-527) in a CT-26 syngeneic mouse model of colon cancer. Results are the mean of 10 animals.
  • FIG. 1 B is a graph showing dose-dependent anti-tumor activity (as measured by tumor volume over time) of a mouse anti-mOX-40R antibody (OX-86) in a CT-26 syngeneic mouse model of colon cancer. Results are the mean of 10 animals; control treatments in Fig 1A are the same as those in Fig.1 B.
  • FIG. 1C is a graph showing dose-dependent anti-tumor activity (as measured by tumor volume over time) of TLR-7/8 agonist (CRX-649) in a CT-26 syngeneic mouse model of colon cancer. Results are the mean of 10 animals; control treatments in Fig 1AC are the same as those in Figs.1 A and 1 B.
  • Figure 2 is a graph showing dose-dependent anti-tumor activity (as measured by tumor volume over time) of a mouse anti-mOX-40R antibody (OX-86), 5 ug of TLR-4 agonist (CRX-527), and the combination of both in a CT-26 syngeneic mouse model of colon cancer. Results are the mean of 10 animals.
  • Figure 3 is a graph showing dose-dependent anti-tumor activity (as measured by tumor volume over time) of a mouse anti-mOX-40R antibody (OX-86), 25 ug of TLR-4 agonist (CRX-527), and the combination of both in a CT-26 syngeneic mouse model of colon cancer measured over 38 days. Results are the mean of 10 animals; control treatments in Figure 2 represent identical animals as those in Figure 3.
  • 4F are graphs showing dose-dependent anti-tumor activity (as measured by tumor volume over time) in individual mice of a control antibody (IgG), mouse anti- mOX-40R antibody (OX-86), 5 or 25 ug of TLR-4 agonist (CRX-527), and the combination of 0X86 and CRX-527 in a group of mice in a CT-26 syngeneic mouse model of colon cancer measured over 42 days.
  • Figure 5 is a graph showing dose-dependent anti-tumor activity (as measured by tumor volume over time) of a mouse anti-mOX-40R antibody (OX-86), 5 ug of TLR-7/8 agonist (CRX-649), and the combination of both in a CT-26 syngeneic mouse model of colon cancer. Results are the mean of 10 animals.
  • Figure 6 is a graph showing dose-dependent anti-tumor activity (as measured by tumor volume over time) of a mouse anti-mOX-40R antibody (OX-86), 25 ug of TLR-7/8 agonist (CRX-649), and the combination of both in a CT-26 syngeneic mouse model of colon cancer. Results are the mean of 10 animals; control treatments in Figure 5 represent identical animals as those in Figure 6.
  • FIG. 7A - FIG. 7F are graphs showing dose-dependent anti-tumor activity (as measured by tumor volume over time) in individual mice of a control antibody (IgG), mouse anti- mOX-40R antibody (OX-86), 5 or 25 ug of TLR-7/8 agonist (CRX-649and the combination of 0X86 and CRX-649 in a group of mice in a CT-26 syngeneic mouse model of colon cancer.
  • Figure 8 is a graph showing dose-dependent anti-tumor activity (as measured by tumor volume over time) of 4, 20, or 100 ug of TLR-4 agonist (CRX-601) in a CT-26 syngeneic mouse model of colon cancer.
  • Figures 9-14 show sequences of the ABPs and antibodies of the invention, e.g., CDRs and VH and VL sequences.
  • Figures 15-18 show mouse sequences of ABPs and antibodies of the invention, e.g., CDRs and VH and VL sequences that can comprise a humanized antibody.
  • Improved function of the immune system is a goal of immunotherapy for cancer. While not being bound by theory, it is thought that for the immune system to be activated and effectively cause regression or eliminate tumors, there must be efficient cross talk among the various compartments of the immune system as well at the at the tumor bed.
  • the tumoricidal effect is dependent on one or more steps, e.g. the uptake of antigen by immature dendritic cells and presentation of processed antigen via MHC I and II by mature dendritic cells to naive CD8 (cytotoxic) and CD4 (helper) lymphocytes, respectively, in the draining lymph nodes.
  • Naive T cells express molecules such as CTLA-4 and CD28 that engage with co-stimulatory molecules of the B7 family on antigen presenting cells (APCs) such as dendritic cells.
  • APCs antigen presenting cells
  • B7 on APCs preferentially binds to CTLA-4, an inhibitory molecule on T lymphocytes.
  • TCR T cell receptor
  • MHC Class I or II receptors MHC Class I or II receptors
  • the co-stimulatory molecule disengages from CTLA-4 and instead binds to the lower affinity stimulatory molecule CD28, causing T cell activation and proliferation.
  • This expanded population of primed T lymphocytes retains memory of the antigen that was presented to them as they traffic to distant tumor sites.
  • cytolytic mediators such as granzyme B and perforins.
  • OX40 T cell co-stimulatory molecules
  • OX40R a tumor necrosis factor receptor family member that is expressed, among other cells, on activated CD4 and CD8 T cells.
  • OX40L The ligand for OX40 (OX40L) is expressed by activated antigen-presenting cells.
  • the ABPs and antibodies of the invention modulate OX40 and promote growth and/or differentiation of T cells and increase long-term memory T-cell populations, e.g., in overlapping mechanisms as those of OX40L, by "engaging" OX40.
  • the ABPs and antibodies of the invention bind and engage OX40.
  • the ABPs and antibodies of the invention modulate OX40.
  • the ABPs and antibodies of the invention modulate OX40 by mimicking OX40L.
  • the ABPs and antibodies of the invention are agonist antibodies.
  • the ABPs and antibodies of the invention modulate OX40 and cause proliferation of T cells.
  • the ABPs and antibodies of the invention modulate OX40 and improve, augment, enhance, or increase proliferation of CD4 T cells.
  • the ABPs and antibodies of the invention improve, augment, enhance, or increase proliferation of CD8 T cells.
  • the ABPs and antibodies of the invention improve, augment, enhance, or increase proliferation of both CD4 and CD8 T cells.
  • the ABPs and antibodies of the invention enhance T cell function, e.g. of CD4 or CD8 T cells, or both CD4 and CD8 T cells.
  • the ABPs and antibodies of the invention enhance effector T cell function.
  • the ABPs and antibodies of the invention improve, augment, enhance, or increase long-term survival of CD8 T cells.
  • any of the preceding effects occur in a tumor micro-environment.
  • Tregs T regulatory cells
  • TGF- B Transforming Growth Factor
  • IL-10 interleukin-10
  • An important immune pathogenesis of cancer can be the involvement of Tregs that are found in tumor beds and sites of inflammation.
  • Treg cells occur naturally in circulation and help the immune system to return to a quiet, although vigilant state, after encountering and eliminating external pathogens. They help to maintain tolerance to self antigens and are naturally suppressive in function. They are phenotypically characterized as CD4+, CD25+, FOXP3+ cells.
  • one mode of therapy is to eliminate Tregs preferentially at tumor sites.
  • Targeting and eliminating Tregs leading to an antitumor response has been more successful in tumors that are immunogenic compared to those that are poorly immunogenic.
  • Many tumors secrete cytokines, e.g., TGF-B that may hamper the immune response by causing precursor CD4+25+ cells to acquire the FOXP3+ phenotype and function as Tregs.
  • Modulate as used herein, for example with regard to a receptor or other target means to change any natural or existing function of the receptor, for example it means affecting binding of natural or artificial ligands to the receptor or target; it includes initiating any partial or full conformational changes or signaling through the receptor or target, and also includes preventing partial or full binding of the receptor or target with its natural or artificial ligands. Also included in the case of membrane bound receptors or targets are any changes in the way the receptor or target interacts with other proteins or molecules in the membrane or change in any localization (or co-localization with other molecules) within membrane compartments as compared to its natural or unchanged state. Modulators are therefore compounds or ligands or molecules that modulate a target or receptor.
  • Modulate includes agonizing, e.g., signaling, as well as antagonizing, or blocking signaling or interactions with a ligand or compound or molecule that happen in the unchanged or unmodulated state.
  • modulators may be agonists or antagonists.
  • one of skill in the art will recognize that not all modulators will be have absolute selectivity for one target or receptor, but are still considered a modulator for that target or receptor; for example, a TLR4 modulator may also engage another TLR but still be considered a TLR4 modulator.
  • Other modulators are known to have multiple specificities, such as TLR7/8 modulators which modulate both TLR7 and TLR8.
  • a TLR7/8 modulator is a TLR7 modulator as used herein and likewise a TLR7/8 modulator is a TLR8 modulator, as used herein.
  • Antists of a target or receptor are molecules or compounds or ligands that mimic one or more functions of a natural ligand or molecule that interacts with the target or receptor and includes initiating one or more signaling events through the receptor, mimicking one or more functions of a natural ligand, initiating one or more partial or full conformational changes that are seen in known functioning or signaling through the receptor.
  • the OX40 ABP or antibody inhibits the suppressive effect of Treg cells on other T cells, e.g., within the tumor environment.
  • the OX40 ABPs or antibodies of the invention modulate OX40 to augment T effector number and function and inhibit Treg function. Enhancing, augmenting, improving, increasing, and otherwise changing the anti-tumor effect of OX40 is an object of the invention. Described herein are combinations of an anti- OX40 ABP or antibody of the invention and another compound, such as a TLR modulator described herein.
  • the term “combination of the invention” refers to a combination comprising an anti-OX40 ABP or antibody and a TLR7/8 modulator or a combination comprising an anti-OX40 ABP and a TLR7/8 modulator and a TLR4 modulator, where, in either combination, each component may be administered separately or simultaneously as described herein.
  • cancer As used herein, the terms “cancer,” “neoplasm,” and “tumor,” are used interchangeably and in either the singular or plural form, refer to cells that have undergone a malignant transformation or undergone cellular changes that result in aberrant or unregulated growth or hyperproliferation Such changes or malignant transformations usually make such cells pathological to the host organism, thus precancers or precancerous cells that are or could become pathological and require or could benefit from intervention are also intended to be included.
  • Primary cancer cells that is, cells obtained from near the site of malignant transformation
  • a cancer cell includes not only a primary cancer cell, but any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells.
  • a "clinically detectable" tumor is one that is detectable on the basis of tumor mass; e.g., by procedures such as CAT scan, MR imaging, X-ray, ultrasound or palpation, and/or which is detectable because of the expression of one or more cancer-specific antigens in a sample obtainable from a patient.
  • Tumors may be hematopoietic tumor, for example, tumors of blood cells or the like, meaning liquid tumors.
  • specific examples of clinical conditions based on such a tumor include leukemia such as chronic myelocytic leukemia or acute myelocytic leukemia; myeloma such as multiple myeloma; lymphoma and the like.
  • the term "agent” is understood to mean a substance that produces a desired effect in a tissue, system, animal, mammal, human, or other subject. Accordingly, the term “anti-neoplastic agent” is understood to mean a substance producing an antineoplastic effect in a tissue, system, animal, mammal, human, or other subject. It is also to be understood that an “agent” may be a single compound or a combination or composition of two or more compounds.
  • treating means: (1) to ameliorate the condition or one or more of the biological manifestations of the condition, (2) to interfere with (a) one or more points in the biological cascade that leads to or is responsible for the condition or (b) one or more of the biological manifestations of the condition (3) to alleviate one or more of the symptoms, effects or side effects associated with the condition or one or more of the symptoms, effects or side effects associated with the condition or treatment thereof, or (4) to slow the progression of the condition or one or more of the biological manifestations of the condition.
  • prevention is understood to refer to the prophylactic administration of a drug to substantially diminish the likelihood or severity of a condition or biological manifestation thereof, or to delay the onset of such condition or biological manifestation thereof. The skilled artisan will appreciate that “prevention” is not an absolute term.
  • Prophylactic therapy is appropriate, for example, when a subject is considered at high risk for developing cancer, such as when a subject has a strong family history of cancer or when a subject has been exposed to a carcinogen.
  • the term "effective amount" means that amount of a drug or
  • terapéuticaally effective amount means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder.
  • the term also includes within its scope amounts effective to enhance normal physiological function.
  • a therapeutically effective amount of the combinations of the invention are advantageous over the individual component compounds in that the combinations provide one or more of the following improved properties when compared to the individual administration of a therapeutically effective amount of a component compound: i) a greater anticancer effect than the most active single agent; ii) synergistic or highly synergistic anticancer activity; iii) a dosing protocol that provides enhanced anticancer activity with reduced side effect profile; iv) a reduction in the toxic effect profile; v) an increase in the therapeutic window; or vi) an increase in the bioavailability of one or both of the component compounds.
  • the invention further provides pharmaceutical compositions, which include one or more of the components herein, and one or more pharmaceutically acceptable carriers, diluents, or excipients.
  • the combination of the invention may comprise one pharmaceutical composition, or multiple pharmaceutical compositions comprising one or more of the components of the combination, in any groupings.
  • the combination of the invention may comprise two pharmaceutical compositions, for example one comprising an ABP or antibody of the invention, and the other comprising a TLR7/8 modulator, each of which may have the same or different carriers, diluents or excipients.
  • the combination of the invention may comprise three pharmaceutical compositions, one comprising an ABP or antibody of the invention, another comprising a TLR 7/8 modulator, and another comprising a TLR4 modulator, each of which may have the same or different carriers, diluents or excipients.
  • the carrier(s), diluent(s) or excipient(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation, capable of pharmaceutical formulation, and not deleterious to the recipient thereof.
  • the components of the combination of the invention, and pharmaceutical compositions comprising such components may be administered in any order, and in different routes; the components and pharmaceutical compositions comprising the same may be administered simultaneously.
  • a process for the preparation of a pharmaceutical composition including admixing a component of the combination of the invention and one or more pharmaceutically acceptable carriers, diluents or excipients.
  • the components of the invention may be administered by any appropriate route.
  • suitable routes include oral, rectal, nasal, topical (including buccal and sublingual), vaginal, and parenteral (including subcutaneous, intramuscular, intraveneous, intradermal, intrathecal, and epidural).
  • parenteral including subcutaneous, intramuscular, intraveneous, intradermal, intrathecal, and epidural.
  • the preferred route may vary with, for example, the condition of the recipient of the combination and the cancer to be treated.
  • each of the agents administered may be administered by the same or different routes and that the components may be administered.
  • one or more components of a combination of the invention are administered intravenously. In another embodiment, one or more components of a combination of the invention are administered intratumorally. In another embodiment, one or more components of a combination of the invention are administered systemically, e.g. intravenously, and one or more other components of a combination of the invention are administered intratumorally. In another embodiment, all of the components of a combination of the invention are administered systemically, e.g., intravenously. In an alternative embodiment, all of the components of the combination of the invention are administered intratumorally. In any of the embodiments, e.g., in this paragraph, the components of the invention are administered as one or more pharmaceutical
  • compositions comprising Antigen Binding Proteins and Antibodies that bind OX40
  • Antigen Binding Protein means a protein that binds an antigen, including antibodies or engineered molecules that function in similar ways to antibodies.
  • Such alternative antibody formats include triabody, tetrabody, miniantibody, and a minibody.
  • alternative scaffolds in which the one or more CDRs of any molecules in accordance with the disclosure can be arranged onto a suitable non-immunoglobulin protein scaffold or skeleton, such as an affibody, a SpA scaffold, an LDL receptor class A domain, an avimer (see, e.g., U.S. Patent Application Publication Nos. 2005/0053973, 2005/0089932, 2005/0164301) or an EGF domain.
  • An ABP also includes antigen binding fragments of such antibodies or other molecules.
  • an ABP may comprise the VH regions of the invention formatted into a full length antibody, a (Fab')2 fragment, a Fab fragment, a bi-specific or biparatopic molecule or equivalent thereof (such as scFV, bi- trior tetra-bodies, Tandabs, etc.), when paired with an appropriate light chain.
  • the ABP may comprise an antibody that is an lgG1 , lgG2, lgG3, or lgG4; or IgM; IgA, IgE or IgD or a modified variant thereof.
  • the constant domain of the antibody heavy chain may be selected accordingly.
  • the light chain constant domain may be a kappa or lambda constant domain.
  • the ABP may also be a chimeric antibody of the type described in WO86/01533 which comprises an antigen binding region and a non-immunoglobulin region.
  • an ABP of the invention or an anti-OX40 antigen binding protein is one that binds OX40, and does one or more of the following: modulate signaling through OX40; modulates the function of OX40; agonizes OX40 signalling; stimulated OX40 function; or co-stimulated OX40 signaling.
  • modulate signaling through OX40 modulates the function of OX40
  • agonizes OX40 signalling stimulated OX40 function
  • co-stimulated OX40 signaling One of skill in the art would readily recognize a variety of well known assays to establish such functions.
  • the term "antibody” as used herein refers to molecules with an antigen binding domain, and optionally an immunoglobulin-
  • an antibody included xenogeneic, allogeneic, syngeneic, or other modified forms thereof.
  • An antibody may be isolated or purified.
  • An antibody may also be recombinant, i.e., produced by recombinant means; for example, an antibody that is 90% identical to a reference antibody may be generated by mutagenesis of certain residues using recombinant molecular biology techniques known in the art.
  • the antibodies of the present invention may comprise heavy chain variable regions and light chain variable regions of the invention which may be formatted into the structure of a natural antibody or formatted into a full length recombinant antibody, a (Fab')2 fragment, a Fab fragment, a bi-specific or biparatopic molecule or equivalent thereof (such as scFV, bi- tri- or tetra-bodies, Tandabs etc.), when paired with an appropriate light chain.
  • the antibody may be an lgG1 , lgG2, lgG3, or lgG4 or a modified variant thereof.
  • the constant domain of the antibody heavy chain may be selected accordingly.
  • the light chain constant domain may be a kappa or lambda constant domain.
  • the antibody may also be a chimeric antibody of the type described in
  • WO86/01533 which comprises an antigen binding region and a non-immunoglobulin region.
  • ABPs and antibodies of the invention bind an epitope of OX40.
  • the epitope of an ABP is the region of its antigen to which the ABP binds.
  • Two ABPs bind to the same or overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen.
  • a 1x, 5x, 10x, 20x or 100x excess of one antibody inhibits binding of the other by at least 50% but preferably 75%, 90% or even 99% as measured in a competitive binding assay compared to a control lacking the competing antibody (see, e.g., Junghans, et al., Cancer Res. 50: 1495 (1990)).
  • two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.
  • the same epitope may include "overlapping epitopes", e.g., if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.
  • the strength of binding may be important in dosing and administration of an ABP or antibody of the invention.
  • the ABP or antibody of the invention binds to OX40, preferably human OX40, with high affinity.
  • OX40 preferably human OX40
  • the antibody binds to OX40, preferably human OX40, with an affinity of 1-
  • the antibody binds to OX40, preferably human OX40, when measured by BIACORE ® of between about 50nM and about 200nM or between about 50nM and about 150nM. In one aspect of the present invention the antibody binds OX40, preferably human OX40, with an affinity of less than 100nM.
  • binding is measured by BIACORE ® .
  • Affinity is the strength of binding of one molecule, e.g., an antibody of the invention, to another, e.g., its target antigen, at a single binding site.
  • the binding affinity of an antibody to its target may be determined by equilibrium methods (e.g., enzyme-linked immunoabsorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetics (e.g., BIACORE ® analysis).
  • ELISA enzyme-linked immunoabsorbent assay
  • RIA radioimmunoassay
  • kinetics e.g., BIACORE ® analysis
  • the BIACORE ® methods known in the art may be used to measure binding affinity.
  • the equilibrium dissociation constant (KD) of the ABP or antibody of the invention and OX40, preferably human OX40, interaction is 100 nM or less, 10 nM or less, 2 nM or less or 1 nM or less.
  • the KD may be between 5 and 10 nM; or between 1 and 2 nM.
  • the KD may be between 1 pM and 500 pM; or between 500 pM and 1 nM.
  • the reciprocal of KD i.e., 1/KD
  • KA equilibrium association constant
  • the dissociation rate constant (kd) or "off-rate” describes the stability of the complex of ABP or antibody on one hand and OX40, preferably human OX40 on the other hand, i.e. the fraction of complexes that decay per second. For example, a kd of 0.01 s-1 equates to 1 % of the complexes decaying per second.
  • the dissociation rate constant (kd) is 1x10-3 s-1 or less, 1x10-4 s-1 or less, 1x10-5 s-1 or less, or 1x10-6 s-1 or less.
  • the kd may be between 1x10-5 s-1 and 1x10-4 s-1 ; or between 1x10-4 s-1 and 1x10- 3 s-1.
  • Competition between an anti-OX40 ABP or antibody of the invention, and a reference antibody, e.g., for binding OX40, an epitope of OX40, or a fragment of the OX40 may be determined by competition ELISA, FMAT or BIACORE ® .
  • the competition assay is carried out by BIACORE ® .
  • the two proteins may bind to the same or overlapping epitopes, there may be steric inhibition of binding, or binding of the first protein may induce a conformational change in the antigen that prevents or reduces binding of the second protein.
  • Binding fragments as used herein means a portion or fragment of the ABPs or antibodies of the invention that include the antigen-binding site and are capable of binding OX40 as defined herein, e.g., but not limited to capable of binding to the same epitope of the parent or full length antibody.
  • binding fragments and “functional fragments” may be an Fab and F(ab')2 fragments that lack the Fc fragment of an intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl, et al., J. Nuc. Med. 24:316-325 (1983)). Also included are Fv fragments ⁇ Hochman, et al. (1973) Biochemistry 12:1 130-1135; Sharon, et a/.(1976) Biochemistry 15:1591-1594). These various fragments are produced using conventional techniques such as protease cleavage or chemical cleavage (see, e.g., Rousseaux et al., Meth. Enzymol., 121 :663-69 (1986)).
  • “Functional fragments” as used herein means a portion or fragment of the ABPs or antibodies of the invention that include the antigen-binding site and are capable of binding the same target as the parent ABP or antibody, e.g., but not limited to binding the same epitope, and that also retain one or more modulating or other functions described herein or known in the art.
  • ABPs and antibodies of the present invention may comprise heavy chain variable regions and light chain variable regions of the invention which may be formatted into the structure of a natural antibody, a functional fragment is one that retains binding or one or more functions of the full length ABP or antibody as described herein.
  • a binding fragment of an ABP or antibody of the invention may therefore comprise the VL or VH regions, a (Fab')2 fragment, a Fab fragment, a fragment of a bi-specific or biparatopic molecule or equivalent thereof (such as scFV, bi- tri- or tetra-bodies, Tandabs, etc.), when paired with an appropriate light chain.
  • CDR refers to the complementarity determining region amino acid sequences of an antigen binding protein. These are the hypervariable regions of immunoglobulin heavy and light chains. There are three heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. It will be apparent to those skilled in the art that there are various numbering conventions for CDR sequences; Chothia (Chothia, et al. (1989) Nature 342: 877-883), Kabat (Kabat, et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL I NTEREST, 4th Ed., U.S.
  • the minimum overlapping region using at least two of the Kabat, Chothia, AbM and contact methods can be determined to provide the "minimum binding unit".
  • the minimum binding unit may be a subportion of a CDR.
  • the structure and protein folding of the antibody may mean that other residues are considered part of the CDR sequence and would be understood to be so by a skilled person. It is noted that some of the CDR definitions may vary depending on the individual publication used.
  • references herein to "CDR”, “CDRL1”, “CDRL2”, “CDRL3”, “CDRH1”, “CDRH2”, “CDRH3” refer to amino acid sequences numbered according to any of the known conventions; alternatively, the CDRs are referred to as “CDR1 ,” “CDR2,” “CDR3” of the variable light chain and “CDR1 ,” “CDR2,” and “CDR3” of the variable heavy chain. In particular embodiments, the numbering convention is the Kabat convention.
  • CDR variant refers to a CDR that has been modified by at least one, for example 1 , 2 or 3, amino acid substitution(s), deletion(s) or addition(s), wherein the modified antigen binding protein comprising the CDR variant substantially retains the biological characteristics of the antigen binding protein pre-modification. It will be appreciated that each CDR that can be modified may be modified alone or in combination with another CDR. In one aspect, the modification is a substitution, particularly a conservative substitution, for example as shown in Table 1. Table 1
  • the amino acid residues of the minimum binding unit may remain the same, but the flanking residues that comprise the CDR as part of the Kabat or Chothia definition(s) may be substituted with a conservative amino acid residue.
  • Such antigen binding proteins comprising modified CDRs or minimum binding units as described above may be referred to herein as "functional CDR variants" or "functional binding unit variants”.
  • the antibody may be of any species, or modified to be suitable to administer to a cross species.
  • the CDRs from a mouse antibody may be humanized for administration to humans.
  • the antigen binding protein is optionally a humanized antibody.
  • a “humanized antibody” refers to a type of engineered antibody having its CDRs derived from a non-human donor immunoglobulin, the remaining immunoglobulin-derived parts of the molecule being derived from one (or more) human immunoglobulin(s).
  • framework support residues may be altered to preserve binding affinity (see, e.g., Queen et al., Proc. Natl Acad Sci USA, 86:10029-10032 (1989), Hodgson, et al., Bio/Technology, 9:421 (1991)).
  • a suitable human acceptor antibody may be one selected from a conventional database, e.g., the KABAT ® database, Los Alamos database, and Swiss Protein database, by homology to the nucleotide and amino acid sequences of the donor antibody.
  • a human antibody characterized by a homology to the framework regions of the donor antibody (on an amino acid basis) may be suitable to provide a heavy chain constant region and/or a heavy chain variable framework region for insertion of the donor CDRs.
  • a suitable acceptor antibody capable of donating light chain constant or variable framework regions may be selected in a similar manner. It should be noted that the acceptor antibody heavy and light chains are not required to originate from the same acceptor antibody.
  • the prior art describes several ways of producing such humanised antibodies - see, for example, EP-A-0239400 and EP-A-054951.
  • the humanized antibody has a human antibody constant region that is an IgG.
  • the IgG is a sequence as disclosed in any of the above references or patent publications.
  • nucleotide and amino acid sequences For nucleotide and amino acid sequences, the term “identical” or “identity” indicates the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate insertions or deletions.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below.
  • Percent identity between a query nucleic acid sequence and a subject nucleic acid sequence is the "Identities" value, expressed as a percentage, which is calculated by the BLASTN algorithm when a subject nucleic acid sequence has 100% query coverage with a query nucleic acid sequence after a pair-wise BLASTN alignment is performed.
  • Such pair- wise BLASTN alignments between a query nucleic acid sequence and a subject nucleic acid sequence are performed by using the default settings of the BLASTN algorithm available on the National Center for Biotechnology Institute's website with the filter for low complexity regions turned off.
  • a query nucleic acid sequence may be described by a nucleic acid sequence identified in one or more claims herein.
  • Percent identity between a query amino acid sequence and a subject amino acid sequence is the "Identities" value, expressed as a percentage, which is calculated by the BLASTP algorithm when a subject amino acid sequence has 100% query coverage with a query amino acid sequence after a pair-wise BLASTP alignment is performed.
  • Such pair- wise BLASTP alignments between a query amino acid sequence and a subject amino acid sequence are performed by using the default settings of the BLASTP algorithm available on the National Center for Biotechnology Institute's website with the filter for low complexity regions turned off.
  • a query amino acid sequence may be described by an amino acid sequence identified in one or more claims herein.
  • the ABP or antibody may have any one or all CDRs, VH, VL, with 99, 98, 97, 96, 95, 94, 93, 92, 91 , or 90 percent identity to the sequence shown or referenced, e.g., as defined by a SEQ ID NO.
  • ABPs and Antibodies of the Invention that bind OX-40 may have any one or all CDRs, VH, VL, with 99, 98, 97, 96, 95, 94, 93, 92, 91 , or 90 percent identity to the sequence shown or referenced, e.g., as defined by a SEQ ID NO.
  • ABPs and antibodies that bind human OX40 receptor are provided herein (i.e., an anti- OX40 ABP and an anti-human OX40 receptor (hOX-40R) antibody, sometimes referred to herein as an "anti-OX40 ABP or an anti- OX40 antibody” and/or other variations of the same).
  • These antibodies are useful in the treatment or prevention of acute or chronic diseases or conditions whose pathology involves OX40 signalling.
  • an antigen binding protein, or isolated human antibody or functional fragment of such protein or antibody, that binds to human OX40R and is effective as a cancer treatment or treatment against disease is described, for example in combination with another compound such as a TLR7/8 modulator or TLR7/8 agonist.
  • any of the antigen binding proteins or anti-OX40 antibodies disclosed herein may be used as a medicament. Any one or more of the antigen binding proteins or anti-OX40 antibodies may be used in the methods or compositions to treat cancer, e.g., those disclosed herein.
  • the isolated antibodies as described herein bind to OX40, and may bind to OX40 encoded from the following genes: NCBI Accession Number NP_003317, Genpept Accession Number P23510, or genes having 90 percent homology or 90 percent identity thereto.
  • the isolated antibody provided herein may further bind to the OX40 receptor having one of the following GenBank Accession Numbers: AAB39944, CAE11757, or AAI05071.
  • Antigen binding proteins and antibodies that bind and/or modulate OX-40 receptor are known in the art.
  • Exemplary ABPs and antibodies of the invention are disclosed, for example in International Publication No. WO2013/028231 (PCT/US2012/024570), international filing date 9 Feb. 2012, and WO2012/027328 (PCT/US201 1/048752), international filing date 23 August 2011.
  • the OX-40 antigen binding protein is the one disclosed in
  • the antigen binding protein comprises the CDRs of an antibody disclosed in WO2012/027328 (PCT/US2011/048752), international filing date 23 August 2011 , or CDRs with 90% identity to the disclosed CDR sequences.
  • the antigen binding protein comprises a VH, a VL, or both of an antibody disclosed in WO2012/027328 (PCT/US2011/048752), international filing date 23 August 2011 , or a VH or a VL with 90% identity to the disclosed VH or VL sequences.
  • the OX-40 antigen binding protein is one disclosed in
  • the antigen binding protein comprises the CDRs of an antibody disclosed in WO2013/028231 (PCT/US2012/024570), international filing date 9 Feb. 2012, or CDRs with 90% identity to the disclosed CDR sequences.
  • the antigen binding protein comprises a VH, a VL, or both of an antibody disclosed in WO2013/028231 (PCT/US2012/024570), international filing date 9 Feb. 2012, or a VH or a VL with 90% identity to the disclosed VH or VL sequences.
  • the anti-OX40 ABP or antibody of the invention comprises one or more of the CDRs or VH or VL sequences, or sequences with 90% identity thereto, shown in the Figures herein.
  • the ABP or antibody of the invention comprises the CDRs of the 106- 222 antibody, e.g., of Figures 6-7 herein, e.g. CDRH1 , CDRH2, and CDRH3 having the amino acid sequence as set forth in SEQ ID NOs 1 , 2, and 3, as disclosed in Figure 6, and e.g. CDRL1 , CDRL2, and CDRL3 having the sequences as set forth in SEQ ID NOs 7, 8, and 9 respectively.
  • the ABP or antibody of the invention comprises the CDRs of the 106-222, Hu106 or Hu106-222 antibody as disclosed in WO2012/027328 (PCT/US201 1/048752), international filing date 23 August 2011.
  • the anti-OX40 ABP or antibody of the invention comprises the VH and VL regions of the 106-222 antibody as shown in Figures 6-7 herein, e.g. a VH having an amino acid sequence as set forth in SEQ ID NO:4 and a VL as in Figure 7 having an amino acid sequence as set forth in SEQ ID NO: 10.
  • the ABP or antibody of the invention comprises a VH having an amino acid sequence as set forth in SEQ ID NO: 5 in Figure 6 herein, and a VL having an amino acid sequence as set forth in SEQ ID NO: 11 in Figure 7 herein.
  • the anti-OX40 ABP or antibody of the invention comprises the VH and VL regions of the Hu106-222 antibody or the 106-222 antibody or the Hu106 antibody as disclosed in WO2012/027328 (PCT/US201 1/048752), international filing date 23 August 2011.
  • the anti-OX40 ABP or antibody of the invention is 106-222, Hu106-222 or Hu106, e.g., as disclosed in
  • the ABP or antibody of the invention comprises CDRs or VH or VL or antibody sequences with 90% identity to the sequences in this paragraph.
  • the anti-OX40 ABP or antibody of the invention comprises the CDRs of the 1 19-122 antibody, e.g. of Figures 10-1 1 herein, e.g., CDRH1 , CDRH2, and CDRH3 having the amino acid sequence as set forth in SEQ ID NOs 13, 14, and 15 respectively.
  • the anti-OX40 ABP or antibody of the invention comprises the CDRs of the 1 19-122 or Hu119 or Hu1 19-222 antibody as disclosed in WO2012/027328 (PCT/US201 1/048752), international filing date 23 August 201 1.
  • the anti-OX40 ABP or antibody of the invention comprises a VH having an amino acid sequence as set forth in SEQ ID NO: 16 in Figure 10 herein, and a VL having the amino acid sequence as set forth in SEQ ID NO: 22 as shown in Figure 1 1 herein.
  • the anti-OX40 ABP or antibody of the invention comprises a VH having an amino acid sequence as set forth in SEQ ID NO: 17 and a VL having the amino acid sequence as set forth in SEQ ID NO: 23.
  • the anti- OX40 ABP or antibody of the invention comprises the VH and VL regions of the 1 19-122 or Hu1 19 or Hu119-222 antibody as disclosed in WO2012/027328 (PCT/US201 1/048752), international filing date 23 August 2011.
  • the ABP or antibody of the invention is 1 19-222 or Hu119 or Hu1 19-222 antibody, e.g. as disclosed in
  • the ABP or antibody of the invention comprises CDRs or VH or VL or antibody sequences with 90% identity to the sequences in this paragraph.
  • the anti-OX40 ABP or antibody of the invention comprises the CDRs of the 1 19-43-1 antibody, e.g., as shown in Figures 14-15 herein.
  • the anti-OX40 ABP or antibody of the invention comprises the CDRs of the 119-43-1 antibody as disclosed in WO2013/028231 (PCT/US2012/024570), international filing date 9 Feb. 2012.
  • the anti-OX40 ABP or antibody of the invention comprises one of the VH and one of the VL regions of the 119-43-1 antibody as shown in Figures 14-17.
  • the anti-OX40 ABP or antibody of the invention comprises the VH and VL regions of the 1 19-43-1 antibody as disclosed in WO2013/028231 (PCT/US2012/024570), international filing date 9 Feb. 2012.
  • the ABP or antibody of the invention is 119-43-1 or 1 19-43-1 chimeric as disclosed in Figures 14-17 herein.
  • any one of the ABPs or antibodies described in this paragraph are humanized. In further embodiments, any one of the any one of the ABPs or antibodies described in this paragraph are engineered to make a humanized antibody. In a further embodiment, the ABP or antibody of the invention comprises CDRs or VH or VL or antibody sequences with 90% identity to the sequences in this paragraph.
  • any mouse or chimeric sequences of any anti-OX40 ABP or antibody of the invention are engineered to make a humanized antibody.
  • the anti-OX40 ABP or antibody of the invention comprises: (a) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1 ; (b) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2; (c) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO. 3; (d) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO. 7; (e) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO. 8; and (f) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO. 9.
  • the anti-OX40 ABP or antibody of the invention comprises: (a) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 14; (c) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO. 15; (d) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO. 19; (e) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO. 20; and (f) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO. 21.
  • the anti-OX40 ABP or antibody of the invention comprises: a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 13; a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2 or 14; and/or a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3 or 15, or a heavy chain variable region CDR having 90 percent identity thereto.
  • the anti-OX40 ABP or antibody of the invention comprises: a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 7 or 19; a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 8 or 20 and/or a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 9 or 21 , or a heavy chain variable region having 90 percent identity thereto.
  • the anti-OX40 ABP or antibody of the invention comprises: a light chain variable region ("VL") comprising the amino acid sequence of SEQ ID NO: 10, 11 , 22 or 23, or an amino acid sequence with at least 90 percent identity to the amino acid sequences of SEQ ID NO: 10, 11 , 22 or 23.
  • VL light chain variable region
  • VH heavy chain variable region
  • the anti-OX40 ABP or antibody of the invention comprises a variable heavy sequence of SEQ ID NO:5 and a variable light sequence of SEQ ID NO: 11 , or a sequence having 90 percent identity thereto.
  • the anti-OX40 ABP or antibody of the invention comprises a variable heavy sequence of SEQ ID NO: 17 and a variable light sequence of SEQ ID NO: 23 or a sequence having 90 percent identity thereto.
  • the anti-OX40 ABP or antibody of the invention comprises a variable light chain encoded by the nucleic acid sequence of SEQ ID NO: 12, or 24, or a nucleic acid sequence with at least 90 percent identity to the nucleotide sequences of SEQ ID NO: 12 or 24.
  • the anti-OX40 ABP or antibody of the invention comprises a variable heavy chain encoded by a nucleic acid sequence of SEQ ID NO: 6 or 18, or a nucleic acid sequence with at least 90 percent identity to nucleotide sequences of SEQ ID NO: 6 or 18.
  • the monoclonal antibodies comprise a variable light chain comprising the amino acid sequence of SEQ ID NO: 10 or 22, or an amino acid sequence with at least 90 percent identity to the amino acid sequences of SEQ ID NO: 10 or 22.
  • monoclonal antibodies comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 4 or 16, or an amino acid sequence with at least 90 percent identity to the amino acid sequences of SEQ ID NO: 4 or 16.
  • Another embodiment of the invention includes CDRs, VH regions, and VL regions, and antibodies and nucleic acids encoding the same as disclosed in the below Sequence Listing. TLR-7/8 modulators
  • TLR7/8 modulators are known in the art. Such modulators bind TLR7/8. Some TLR7/8 modulators of the invention engage TLR7/8, optionally agonize TLR7/8, or initiate one or more molecular signaling events through TLR7/8.
  • a combination of the invention comprises an anti-OX40 ABP or antibody and a TLR7/8 modulator.
  • the TLR7/8 modulator is a TLR7/8 agonist.
  • the TLR7/8 modulator of the invention is selected from the group consisting of oxoadenines, lipidated oxoadenines, imidazoquinolines, and lipidated imadazoquinolines.
  • the TLR7/8 modulator is an oxoadenine compound, i.e., an oxoadenine compound of the invention.
  • Oxoadenine compounds are known in the art. Oxoadenines are disclosed in US Patent No. 8,563 717 and US Publication No. US201 10144136.
  • the TLR 7/8 modulator in a combination of the invention is an oxoadenine compound disclosed US Patent No. 8,563 717 and US Publication No. US201 10144136.
  • the TLR7/8 modulator is an oxoadenine a compound of formula (I):
  • R is Ci. 6 alkylamino, or Ci. 6 alkoxy
  • R 2 is a group having the structure:
  • n is an integer having a value of 1 to 6;
  • Het is a 6-membered saturated heterocycle containing one nitrogen atom wherein Het is attached to the -(CH 2 ) n - moiety at any carbon atom of the heterocycle;
  • R 3 is hydrogen, Ci_ 8 alkyl, or C 3 . 7 cycloalkylCo- 6 alkyl;
  • R is n-butyloxy. In another embodiment, R is (1 S)-1-methylbutoxy. In another embodiment, R is n-butylamino. In another embodiment, R is (1- methylethyl)oxy.
  • n is 1 . In another embodiment, n is 2. In another embodiment, n is 3. In another embodiment, n is 4. In another embodiment, n is 5. In another embodiment, n is 6. In another embodiment, n is 2, 3, or 4.
  • Het is attached to the -(CH 2 ) n - moiety at the 2-position of the heterocycle. In another embodiment, Het is attached to the -(CH 2 ) n - moiety at the 3- position of the heterocycle. In another embodiment, Het is attached to the -(CH 2 ) n - moiety at the 4-position of the heterocycle. In another embodiment, when Het is attached to the - (CH 2 ) n - moiety at the 3-position of the heterocycle, then the stereochemistry at the 3- position of the heterocycle is (R,S).
  • the stereochemistry at the 3- position of the heterocycle is (R).
  • the stereochemistry at the 3- position of the heterocycle is (S).
  • R 3 is ethyl. In another embodiment, R 3 is a hydrogen atom. In another embodiment, R 3 is n-propyl. In another embodiment, R 3 is 1 -methylethyl. In another embodiment, R 3 is n-butyl. In another embodiment, R 3 is 2-methylpropyl. In another embodiment, R 3 is 3-methylbutyl. In another embodiment, R 3 is cyclopentyl. In another embodiment, R 3 is cyclopentyl methyl. In another embodiment, R 3 is 2- cyclohexylethyl. In another embodiment, R 3 is 1-ethylpropyl. In another embodiment, R 3 is cyclohexyl. In a further aspect, there are provided compounds of formula ( ⁇ ):
  • R is Ci_ 6 alkylafnino, or d_ 6 alkoxy;
  • R 2 is a group having the structure:
  • n' is an integer having a value of 1 to 6;
  • Het' is a 6-membered saturated heterocycle containing one nitrogen atom wherein Het' is attached to the -(CH 2 ) n - moiety at any carbon atom of the heterocycle;
  • R 3 is hydrogen, Ci_ 8 alkyl, or C 3 - 7 cycloalkylCo- 6 alkyl;
  • R is n-butyloxy. In another embodiment, R is (1 S)-1-methylbutoxy. In another embodiment, R is n-butylamino. In another embodiment, R is (1- methylethyl)oxy.
  • n' is 1. In another embodiment, n' is 2. In another embodiment, n' is 3. In another embodiment, n' is 4. In another embodiment, n' is 5. In another
  • n' is 6. In another embodiment, n' is 2, 3, or 4. In one embodiment, Het' is attached to the -(CH 2 ) n - moiety at the 2-position of the heterocycle. In another embodiment, Het' is attached to the -(CH 2 ) n - moiety at the 3- position of the heterocycle. In another embodiment, Het' is attached to the -(CH 2 ) n - moiety at the 4-position of the heterocycle. In another embodiment, when Het' is attached to the - (CH 2 ) n - moiety at the 3-position of the heterocycle, then the stereochemistry at the 3- position of the heterocycle is (R,S).
  • the stereochemistry at the 3- position of the heterocycle is (R).
  • the stereochemistry at the 3- position of the heterocycle is (S).
  • R 3 is ethyl. In another embodiment, R 3 is a hydrogen atom. In another embodiment, R 3 is n-propyl. In another embodiment, R 3 is 1-methylethyl. In another embodiment, R 3 is n-butyl. In another embodiment, R 3 is 2-methylpropyl. In another embodiment, R 3 is 3-methylbutyl. In another embodiment, R 3 is cyclopentyl. In another embodiment, R 3 is cyclopentylmethyl. In another embodiment, R 3 is 2- cyclohexylethyl. In another embodiment, R 3 is 1 -ethyl propyl. In another embodiment, R 3 is cyclohexyl.
  • the TLR7/8 modulators, or salts thereof are selected from the group consisting of: 6-amino-2-butoxy-9-[(1-ethyl-3-piperidinyl)methyl]-7,9-dihydro-8/-/-purin-8-one;
  • references to 'alkyl' include references to both straight-chain and branched-chain aliphatic isomers of the corresponding alkyl containing up to eight carbon atoms, for example up to six carbon atoms, or up to four carbon atoms ,or up to two carbon atoms, or one carbon atom.
  • Such references to 'alkyl' are also applicable when an alkyl group is part of another group, for example an alkylamino or alkoxy group. Examples of such alkyl groups and groups containing alkyl groups are Ci_ 8 alkyl, Ci -6 alkyl, Ci. 6 alkylamino, and Ci. 6 alkoxy.
  • references to 'cycloalkyl' refer to monocyclic alkyl groups containing between three and seven carbon atoms, for example three carbon atoms, or five carbon atoms, or six carbon atoms. Examples of such cycloalkyl groups are cyclopropyl, cyclopentyl, and cyclohexyl.
  • references to 'heterocycle' or 'heterocyclyl' refer to a monocyclic saturated heterocyclic aliphatic ring containing 6 carbon atoms and one heteroatom, which heteroatom is nitrogen.
  • Such a heterocyclic ring is piperidine or piperidinyl.
  • references to 'halogen' refer to iodine, bromine, chlorine or fluorine, typically bromine, chlorine, or fluorine.
  • references to 'halo' refer to iodo, bromo, chloro or fluoro, typically bromo, chloro, or fluoro.
  • the TLR7/8 modulators have beneficial characteristics, e.g. inducing predominantly TLR7 activity. In other embodiments, the TLR7/8 modulators induce predominantly TLR8 activity. Beneficial cytokine profiles are described below.
  • the TLR7/8 modulator is CRX-692, having a formula shown below.
  • the TLR7/8 modulator is CRX-672, having a formula shown below.
  • the TLR7/8 modulator is CRX-677, having a formula shown below.
  • the TLR7/8 modulator is CRX-648, having a formula shown below.
  • the TLR7/8 modulator is CRX-649, having a formula shown below.
  • the TLR7/8 modulator is CRX-706, having a formula shown below.
  • the TLR7/8 modulator is CRX-705, having a formula shown below.
  • references herein to oxoadenine compounds of the invention mean a compound of formula (I) as the free base, or as a salt, for example a
  • TLR 7/8 modulators include oxoadenines that have been modified, specifically lipidated, e.g., in order to facilitate uptake into immune cells.
  • the TLR7/8 modulator in a combination of the invention is a compound disclosed in PCT Application No. US20120044708 filed August 6, 2010 and in US Application No. 13/389,307.
  • the TLR 7/8 modulator is a lipidated version of any of the above described oxoadenines.
  • the TLR7/8 modulator is a nitrogen heterocyclyl-substituted oxoadenine molecule (e.g. any of the above described nitrogen heterocyclyl-substituted oxoadenine molecules) covalently linked to a phospho- or phosphonolipid group.
  • the TLR7/8 modulator is a lipidated oxoadenine of Formula (II):
  • R 2 H or straight/branched/unsaturated C4-C24 alkyl or acyl
  • R 3 straightybranched/unsaturated C4-C24 alkyl or acyl or a pharmaceutically acceptable salt thereof.
  • R C1-6alkyl, C1-6alkylamino, C1-6alkoxy, C3-6cycloalkylC1-6alkyl, C3-6cycloalkylC1- 6alkylamino, C3-6cycloalkylC1-6alkoxy, C1-6alkoxyC1-6alkyl, C1-6alkoxyC1-6alkylamino, C1-6alkoxyC1-6alkoxy; branched or unbranched.
  • n 0-6
  • Z O, CH2, CF2 or covalent bond
  • R 2 H or straight/branched/unsaturated C 4 -C 2 4 alkyl or acyl
  • TLR7/8 modulator is a lipidated oxoadenine CRX-682 having the following structure:
  • the TLR7/8 modulator is a lipidated oxoadenine CRX-681 having the following structure:
  • Imadazoquinolines Imadazoquinolines (IQs). Imadazoquinolones are known in the art as TLR7/8 modulators, e.g. agonists. In one embodiment of the invention, the TLR7/8 modulator is an IQ compound. Known IQs include imiquimod, resiquimod, and gardiquimod, shown below. These and other substituted imadazoquinolines are shown in J. Med. Chem., 2014, 57 (2), pp 339-347. In another embodiment of the invention, the TLR 7/8 modulator is a 1 H-imidazo[4,5-c]quinolines. In a further embodiment, the 1 H-imidazo[4,5-c]quinolines is one disclosed in J. Med.
  • the TLR7/8 modulator is selected from the group consisting of imiquimod, resiquimod, and gardiquimod.
  • the TLR7/8 is a substituted imadazoquinoline, e.g. imiquimod, resiquimod, or gardiquimod, wherein such substitution results in a cytokine profile for said substituted imadazoquinoline with increased IL-12 production.
  • IQs Lipidated imadazoquinolines
  • the TLR7/8 modulator in a combination of the invention is a lipidated imadazoquinolone. Lipidated IQs are disclosed in US Patent No. 8,624,029, and US Publication No. US201 10282061 , and PCT Application PCT/US2009/61867 filed Oct. 23, 2009. In one embodiment, the TLR7/8 modulator in a combination of the invention is a compound disclosed in US Patent No. 8,624,029, US Publication No. US20110282061 , or PCT Application PCT/US2009/61867 filed Oct. 23, 2009.
  • the TLR 7/8 modulator is a lipidated version of any of the above described imadazoquinolones.
  • the TLR7/8 modulator is an imadazoquinolone covalently linked to a phospho- or phosphonolipid group.
  • the lipidated imadazoquinoline of the invention is a compound of Formula IV:
  • R 2 H or straight/branched/unsaturated C 4 -C 2 4 alkyl or acyl
  • R 3 straight/branched/unsaturated C 4 -C 24 alkyl or acyl (e.g. phosphatidyl,
  • the TLR7/8 modulator is a lipidated imadazoquinoline of Formula IV with a substitution shown in Table 2.
  • the lipidated imadazoquinoline of Formula IV with a substitution shown in Table 2.
  • imadazoquinoline of Formula IV is chosen from: CRX-664, CRX-650, CRX-652, CRX-651 , CRX-665, and CRX-658.
  • the combinations of the invention comprise TLR-4 modulators, that is molecules that modulate TLR4, for example by binding and initiating conformational changes or signaling by engaging TLR4.
  • the combinations comprise an anti-OX40 ABP, a TLR 7/8 modulator, and a TLR4 modulator.
  • the TLR-4 modulators are aminoalkyl glucosaminide phosphate compounds (AGPs).
  • AGPs aminoalkyl glucosaminide phosphate compounds
  • Toll-like receptor 4 recognizes bacterial LPS (lipopolysaccharide) and when activated initiates an innate immune response.
  • AGPs are a monosaccharide mimetic of the lipid A protein of bacterial LPS and have been developed with ether and ester linkages on the "acyl chains" of the compound. Processes for making these compounds are known and disclosed, for example, in WO 2006/016997, U.S. Patent Nos. 7,288,640 and 6, 113,918, and WO 01/90129.
  • Other AGPs and related processes are disclosed in U.S. Patent No. 7, 129,219, U.S.
  • AGPs with ether linkages on the acyl chains employed in the composition of the invention are known and disclosed in WO 2006/016997.
  • Of particular interest, are the aminoalkyl glucosaminide phosphate compounds set forth and described according to Formula (III) at paragraphs [0019] through [0021] in WO 2006/016997.
  • Aminoalkyl glucosaminide phosphate compounds employed in the present invention have the structure set forth in Formula 1 as follows:
  • n 0 to 6
  • n 0 to 4.
  • X is O or S, preferably O;
  • Y is O or NH
  • Z is O or H
  • each R1 , R2, R3 is selected independently from the group consisting of a C1-20 acyl and a C1-20 alkyl;
  • R4 is H or Me
  • R5 is selected independently from the group consisting of -H, -OH, -(CI-C4) alkoxy, -P03R8R9, -OP03R8R9, -S03R8, -OS03R8, -NR8R9, -SR8, -CN, -N02, -CHO, - C02R8, and -CONR8R9, wherein R8 and R9 are each independently selected from H and (CI-C4) alkyl; and
  • each R6 and R7 is independently H or P03H2.
  • the configuration of the 3' stereogenic centers to which the normal fatty acyl residues (that is, the secondary acyloxy or alkoxy residues, e.g., R10, R20, and R30) are attached is R or S, preferably R (as designated by Cahn-lngold-Prelog priority rules).
  • Configuration of aglycon stereogenic centers to which R4 and R5 are attached can be R or S. All stereoisomers, both enantiomers and diastereomers, and mixtures thereof, are considered to fall within the scope of the present invention.
  • the number of carbon atoms between heteroatom X and the aglycon nitrogen atom is determined by the variable "n", which can be an integer from 0 to 4, preferably an integer from 0 to 2.
  • the chain length of normal fatty acids R1 , R2, and R3 can be from about 6 to about 16 carbons, preferably from about 9 to about 14 carbons.
  • the chain lengths can be the same or different. Some embodiments include chain lengths where R1 , R2 and R3 are 6 or 10 or 12 or 14.
  • n is 0, R5 is C02H, R6 is P03H2, and R7 is H.
  • This AGP compound is set forth as the structure in Formula 1a as follows:
  • Formula 1a the configuration of the 3' stereogenic centers to which the normal fatty acyl residues (that is, the secondary acyloxy or alkoxy residues, e.g., R10, R20, and R30) are attached as R or S, preferably R (as designated by Cahn-lngold-Prelog priority rules).
  • Configuration of aglycon stereogenic centers to which R4 and C02H are attached can be R or S. All stereoisomers, both enantiomers and diastereomers, and mixtures thereof, are considered to fall within the scope of the present invention.
  • Formula 1a encompasses L/D-seryl, -threonyl, -cysteinyl ether or ester lipid AGPs, both agonists and antagonists.
  • CRX-601 Two compounds of Formula 1 are referred to as CRX-601 and CRX-527. Their structures are set forth as follows:
  • CRX-547 having the structure shown.
  • Still other embodiments include AGPs such as CRX-602 or CRX-526 providing increased stability to AGPs having shorter secondary acyl or alkyl chains.
  • the composition comprising a TLR4 modulator, preferably an AGP is buffered using a zwitterionoic buffer.
  • the zwitterionic buffer is an aminoalkanesulfonic acid or suitable salt. Examples of
  • amninoalkanesulfonic buffers include, but are not limlited to: HEPES, HEPPS/EPPS, MOPS, MOBS, and PIPES.
  • the buffer is a pharmaceutically acceptable buffer, suitable for use in humans, such as in for use in a commercial injection product.
  • the buffer is HEPES.
  • the present invention thus also provides a combination of the invention, for use in therapy, particularly in the treatment of disorders wherein the engagement of OX40 and/or TLR7/8, is beneficial, particularly cancer.
  • the present invention thus also provides a combination of the invention, for use in therapy, particularly in the treatment of disorders wherein the engagement of OX40 and TLR7/8 and TLR4, is beneficial, such as cancer.
  • a further aspect of the invention provides a method of treatment of a disorder wherein engagement of OX40 and/or TLR7/8 and/or TLR4 is beneficial, comprising administering a combination of the invention.
  • a further aspect of the present invention provides the use of a combination of the invention in the manufacture of a medicament for the treatment of a disorder engagement of OX40 and and/or TLR7/8 and/or TLR4 is beneficial.
  • the disorder is cancer.
  • cancers that are suitable for treatment with combination of the invention include, but are limited to, both primary and metastatic forms of head and neck, breast, lung, colon, ovary, and prostate cancers.
  • the cancer is selected from: brain (gliomas), glioblastomas, astrocytomas, glioblastoma multiforme, Bannayan-Zonana syndrome, Cowden disease, Lhermitte-Duclos disease, breast, inflammatory breast cancer, Wilm's tumor, Ewing's sarcoma, Rhabdomyosarcoma, ependymoma,
  • lymphoblastic T cell leukemia Chronic myelogenous leukemia, Chronic lymphocytic leukemia, Hairy-cell leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, AML, Chronic neutrophilic leukemia, Acute lymphoblastic T cell leukemia, plasmacytoma, Immunoblastic large cell leukemia, Mantle cell leukemia, Multiple myeloma Megakaryoblastic leukemia, multiple myeloma, acute megakaryocytic leukemia, promyelocytic leukemia, Erythroleukemia, malignant lymphoma, hodgkins lymphoma, non- hodgkins lymphoma, lymphoblastic T cell lymphoma, Burkitt
  • examples of a cancer to be treated include Barret's adenocarcinoma; billiary tract carcinomas; breast cancer; cervical cancer; cholangiocarcinoma; central nervous system tumors including primary CNS tumors such as glioblastomas, astrocytomas (e.g. , glioblastoma multiforme) and ependymomas, and secondary CNS tumors (i.e.
  • cancers including leukemias and lymphomas such as acute lymphoblastic leukemia, acute myelogenous leukemia (AML), myelodysplasia syndromes, chronic myelogenous leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, megakaryoblastic leukemia, multiple myeloma and erythroleukemia; hepatocellular carcinoma; lung cancer including small cell lung cancer and non-small cell lung cancer; ovarian cancer; endometrial cancer; pancreatic cancer; pituitary adenoma; prostate cancer; renal cancer; sarcoma; skin cancers including melanomas; and thyroid cancers.
  • leukemias and lymphomas such as acute lymphoblastic leukemia, acute myelogenous leukemia (AML), myelodysplasia syndromes, chronic myelogenous leukemia, Hodgkin's lymphoma, non-Hodgkin's lymph
  • the present invention relates to a method for treating or lessening the severity of a cancer selected from: brain (gliomas), glioblastomas, astrocytomas, glioblastoma multiforme, Bannayan-Zonana syndrome, Cowden disease, Lhermitte-Duclos disease, breast, colon, head and neck, kidney, lung, liver, melanoma, ovarian, pancreatic, prostate, sarcoma and thyroid.
  • a cancer selected from: brain (gliomas), glioblastomas, astrocytomas, glioblastoma multiforme, Bannayan-Zonana syndrome, Cowden disease, Lhermitte-Duclos disease, breast, colon, head and neck, kidney, lung, liver, melanoma, ovarian, pancreatic, prostate, sarcoma and thyroid.
  • the present invention relates to a method for treating or lessening the severity of a cancer selected from ovarian, breast, pancreatic and prostate.
  • the present invention relates to a method for treating or lessening the severity of pre-cancerous syndromes in a mammal, including a human, wherein the pre-cancerous syndrome is selected from: cervical intraepithelial neoplasia, monoclonal gammapathy of unknown significance (MGUS), myelodysplasia syndrome, aplastic anemia, cervical lesions, skin nevi (pre-melanoma), prostatic intraepithleial (intraductal) neoplasia (PIN), Ductal Carcinoma in situ (DCIS), colon polyps and severe hepatitis or cirrhosis.
  • MGUS monoclonal gammapathy of unknown significance
  • MUS monoclonal gammapathy of unknown significance
  • myelodysplasia syndrome aplastic anemia
  • cervical lesions aplastic anemia
  • cervical lesions skin nevi (pre-melanoma)
  • PIN prostatic intraepith
  • the combination of the invention may be used alone or in combination with one or more other therapeutic agents.
  • the invention thus provides in a further aspect a further combination comprising a combination of the invention with a further therapeutic agent or agents, compositions and medicaments comprising the combination and use of the further combination, compositions and medicaments in therapy, in particular in the treatment of diseases susceptible engagement of OX40 and/or and/or TLR7/8 and/or TLR4.
  • the combination of the invention may be employed with other therapeutic methods of cancer treatment.
  • combination therapy with other chemotherapeutic, hormonal, antibody agents as well as surgical and/or radiation treatments other than those mentioned above are envisaged.
  • Combination therapies according to the present invention thus include the administration of an anti-OX40 ABP or antibody of the invention and/or a TLR7/8 modulator and/or TLR4 modulator as well as optional use of other therapeutic agents including other anti- neoplastic agents.
  • Such combination of agents may be administered together or separately and, when administered separately this may occur simultaneously or sequentially in any order, both close and remote in time.
  • the pharmaceutical combination includes an anti-OX40 ABP or antibody of the invention and a TLR7/8 modulator and/or a TLR4 modulator, and optionally at least one additional antineoplastic agent.
  • the further anti-cancer therapy is surgical and/or radiotherapy.
  • the further anti-cancer therapy is at least one additional antineoplastic agent.
  • Any anti-neoplastic agent that has activity versus a susceptible tumor being treated may be utilized in the combination.
  • Typical anti-neoplastic agents useful include, but are not limited to: anti-microtubule agents such as diterpenoids and vinca alkaloids; platinum coordination complexes; alkylating agents, such as nitrogen mustards,
  • antibiotic agents such as anthracyclins, actinomycins and bleomycins
  • topoisomerase II inhibitors such as epipodophyllotoxins
  • antimetabolites such as purine and pyrimidine analogues and anti- folate compounds
  • topoisomerase I inhibitors such as camptothecins
  • hormones and hormonal analogues signal transduction pathway inhibitors
  • non-receptor tyrosine angiogenesis inhibitors immunotherapeutic agents
  • proapoptotic agents and cell cycle signaling inhibitors.
  • Anti-microtubule or anti-mitotic agents are phase-specific agents that are active against the microtubules of tumor cells during M or the mitosis phase of the cell cycle.
  • anti-microtubule agents include, but are not limited to, diterpenoids and vinca alkaloids.
  • Diterpenoids which are derived from natural sources, are phase-specific anti -cancer agents that operate at the G 2 /M phases of the cell cycle. It is believed that the
  • diterpenoids stabilize the ⁇ -tubulin subunit of the microtubules, by binding with this protein. Disassembly of the protein appears then to be inhibited with mitosis being arrested and cell death following.
  • diterpenoids include, but are not limited to, paclitaxel and its analog docetaxel.
  • Paclitaxel 5p,20-epoxy-1 ,2a,4,7p, 10p, 13a-hexa-hydroxytax-11-en-9-one 4,10-diacetate 2- benzoate 13-ester with (2R,3S)-N-benzoyl-3-phenylisoserine; is a natural diterpene product isolated from the Pacific yew tree Taxus brevifolia and is commercially available as an injectable solution TAXOL ® . It is a member of the taxane family of terpenes. Paclitaxel has been approved for clinical use in the treatment of refractory ovarian cancer in the United States (Markman, et al., Yale Journal of Biology and Medicine, 64:583, 1991 ;
  • Docetaxel (2R,3S)- N-carboxy-3-phenylisoserine,N-te/f-butyl ester, 13-ester with 5 ⁇ -20- epoxy-1 ,2a,4,7p, 10p, 13ot-hexahydroxytax-11-en-9-one 4-acetate 2-benzoate, trihydrate; is commercially available as an injectable solution as TAXOTERE ® .
  • Docetaxel is indicated for the treatment of breast cancer.
  • Docetaxel is a semi-synthetic derivative of paclitaxel q.v., prepared using a natural precursor, 10-deacetyl-baccatin III, extracted from the needle of the European Yew tree.
  • Vinca alkaloids are phase-specific anti-neoplastic agents derived from the periwinkle plant. Vinca alkaloids act at the M phase (mitosis) of the cell cycle by binding specifically to tubulin. Consequently, the bound tubulin molecule is unable to polymerize into
  • microtubules Mitosis is believed to be arrested in metaphase with cell death following.
  • Examples of vinca alkaloids include, but are not limited to, vinblastine, vincristine, and vinorelbine.
  • Vinblastine, vincaleukoblastine sulfate, is commercially available as VELBAN ® as an injectable solution.
  • VELBAN ® an injectable solution.
  • Myelosuppression is the dose-limiting side effect of vinblastine.
  • Vincristine vincaleukoblastine, 22-oxo-, sulfate
  • ONCOVIN ® an injectable solution.
  • Vincristine is indicated for the treatment of acute leukemias and has also found use in treatment regimens for Hodgkin's and non-Hodgkin's malignant lymphomas.
  • Alopecia and neurologic effects are the most common side effect of vincristine and to a lesser extent myelosupression and gastrointestinal mucositis effects occur.
  • Vinorelbine 3',4'-didehydro -4'-deoxy-C'-norvincaleukoblastine [R-(R*,R*)-2,3- dihydroxybutanedioate (1 :2)(salt)], commercially available as an injectable solution of vinorelbine tartrate (NAVELBINE ® ), is a semisynthetic vinca alkaloid.
  • Vinorelbine is indicated as a single agent or in combination with other chemotherapeutic agents, such as cisplatin, in the treatment of various solid tumors, particularly non-small cell lung, advanced breast, and hormone refractory prostate cancers. Myelosuppression is the most common dose-limiting side effect of vinorelbine.
  • Platinum coordination complexes are non-phase- specific anti-cancer agents, which are interactive with DNA. The platinum complexes enter tumor cells, undergo, aquation and form intra- and interstrand crosslinks with DNA causing adverse biological effects to the tumor. Examples of platinum coordination complexes include, but are not limited to, oxaliplatin, cisplatin and carboplatin.
  • Cisplatin cis-diamminedichloroplatinum
  • PLATINOL® an injectable solution.
  • Cisplatin is primarily indicated in the treatment of metastatic testicular and ovarian cancer and advanced bladder cancer.
  • Carboplatin platinum, diammine [1 ,1-cyclobutane-dicarboxylate(2-)-0,0'], is commercially available as PARAPLATIN ® as an injectable solution. Carboplatin is primarily indicated in the first and second line treatment of advanced ovarian carcinoma.
  • Alkylating agents Alkylating agents are non-phase anti-cancer specific agents and strong electrophiles. Typically, alkylating agents form covalent linkages, by alkylation, to DNA through nucleophilic moieties of the DNA molecule such as phosphate, amino, sulfhydryl, hydroxyl, carboxyl, and imidazole groups. Such alkylation disrupts nucleic acid function leading to cell death.
  • alkylating agents include, but are not limited to, nitrogen mustards such as cyclophosphamide, melphalan, and chlorambucil; alkyl sulfonates such as busulfan; nitrosoureas such as carmustine; and triazenes, such as dacarbazine.
  • Cyclophosphamide 2-[bis(2-chloroethyl)amino]tetrahydro-2H-1 ,3,2-oxazaphosphorine 2- oxide monohydrate, is commercially available as an injectable solution or tablets as CYTOXAN ® . Cyclophosphamide is indicated as a single agent or in combination with other chemotherapeutic agents, in the treatment of malignant lymphomas, multiple myeloma, and leukemias.
  • Melphalan 4-[bis(2-chloroethyl)amino]-L-phenylalanine, is commercially available as an injectable solution or tablets as ALKERAN ® .
  • Melphalan is indicated for the palliative treatment of multiple myeloma and non-resectable epithelial carcinoma of the ovary. Bone marrow suppression is the most common dose-limiting side effect of melphalan.
  • Chlorambucil 4-[bis(2-chloroethyl)amino]benzenebutanoic acid, is commercially available as LEUKERAN ® tablets. Chlorambucil is indicated for the palliative treatment of chronic lymphatic leukemia, and malignant lymphomas such as lymphosarcoma, giant follicular lymphoma, and Hodgkin's disease.
  • Busulfan 1 ,4-butanediol dimethanesulfonate, is commercially available as MYLERAN ® tablets. Busulfan is indicated for the palliative treatment of chronic myelogenous leukemia.
  • Carmustine 1 ,3-[bis(2-chloroethyl)-1-nitrosourea, is commercially available as single vials of lyophilized material as BiCNU ® .
  • Carmustine is indicated for the palliative treatment as a single agent or in combination with other agents for brain tumors, multiple myeloma, Hodgkin's disease, and non-Hodgkin's lymphomas.
  • dacarbazine 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide, is commercially available as single vials of material as DTIC-Dome ® .
  • dacarbazine is indicated for the treatment of metastatic malignant melanoma and in combination with other agents for the second line treatment of Hodgkin's Disease.
  • Antibiotic anti-neoplastics are non-phase-specific agents, which bind or intercalate with DNA. Typically, such action results in stable DNA complexes or strand breakage, which disrupts ordinary function of the nucleic acids leading to cell death.
  • antibiotic anti-neoplastic agents include, but are not limited to, actinomycins such as dactinomycin, anthrocyclins, such as daunorubicin and doxorubicin; and bleomycins.
  • Dactinomycin also know as Actinomycin D, is commercially available in injectable form as COSMEGEN ® . Dactinomycin is indicated for the treatment of Wilm's tumor and
  • Daunorubicin (8S-cis-)-8-acetyl-10-[(3-amino-2,3,6-trideoxy-a-L-lyxo-hexopyranosyl)oxy]- 7,8,9, 10-tetrahydro-6, 8,1 1-trihydroxy-1-methoxy-5, 12 naphthacenedione hydrochloride, is commercially available as a liposomal injectable form as DAUNOXOME ® or as an injectable as CERUBIDINE ® . Daunorubicin is indicated for remission induction in the treatment of acute nonlymphocytic leukemia and advanced HIV associated Kaposi's sarcoma.
  • Doxorubicin is primarily indicated for the treatment of acute lymphoblastic leukemia and acute myeloblastic leukemia, but is also a useful component in the treatment of some solid tumors and lymphomas.
  • Bleomycin a mixture of cytotoxic glycopeptide antibiotics isolated from a strain of
  • Topoisomerase II inhibitors include, but are not limited to, epipodophyllotoxins.
  • Epipodophyllotoxins are phase-specific anti-neoplastic agents derived from the mandrake plant. Epipodophyllotoxins typically affect cells in the S and G 2 phases of the cell cycle by forming a ternary complex with topoisomerase II and DNA causing DNA strand breaks. The strand breaks accumulate and cell death follows.
  • epipodophyllotoxins include, but are not limited to, etoposide and teniposide.
  • Etoposide 4'-demethyl-epipodophyllotoxin 9[4,6-0-(R )-ethylidene-p-D-glucopyranoside] is commercially available as an injectable solution or capsules as VePESID® and is commonly known as VP-16. Etoposide is indicated as a single agent or in combination with other chemotherapy agents in the treatment of testicular and non-small cell lung cancers.
  • Anti-metabolite neoplastic agents Anti-metabolite neoplastic agents are phase-specific anti-neoplastic agents that act at S phase (DNA synthesis) of the cell cycle by inhibiting DNA synthesis or by inhibiting purine or pyrimidine base synthesis and thereby limiting DNA synthesis. Consequently, S phase does not proceed and cell death follows.
  • antimetabolite anti-neoplastic agents include, but are not limited to, fluorouracil, methotrexate, cytarabine, mecaptopurine, thioguanine, and gemcitabine.
  • 5-fluorouracil 5-fluoro-2,4- (1 H,3H) pyrimidinedione
  • fluorouracil is commercially available as fluorouracil.
  • Administration of 5-fluorouracil leads to inhibition of thymidylate synthesis and is also incorporated into both RNA and DNA. The result typically is cell death.
  • 5- fluorouracil is indicated as a single agent or in combination with other chemotherapy agents in the treatment of carcinomas of the breast, colon, rectum, stomach and pancreas.
  • Other fluoropyrimidine analogs include 5-fluoro deoxyuridine (floxuridine) and 5- fluorodeoxyuridine monophosphate.
  • Cytarabine 4-amino-1-p-D-arabinofuranosyl-2 (I H)-pyrimidinone, is commercially available as CYTOSAR-U ® and is commonly known as Ara-C. It is believed that cytarabine exhibits cell phase-specificity at S-phase by inhibiting DNA chain elongation by terminal incorporation of cytarabine into the growing DNA chain. Cytarabine is indicated as a single agent or in combination with other chemotherapy agents in the treatment of acute leukemia. Other cytidine analogs include, but are not limited to, 5-azacytidine and 2',2'-difluorodeoxycytidine (gemcitabine).
  • Mercaptopurine 1 ,7-dihydro-6H-purine-6-thione monohydrate, is commercially available as PURINETHOL ® .
  • Mercaptopurine exhibits cell phase-specificity at S-phase by inhibiting DNA synthesis by an as of yet unspecified mechanism.
  • Mercaptopurine is indicated as a single agent or in combination with other chemotherapy agents in the treatment of acute leukemia.
  • a useful mercaptopurine analog is azathioprine.
  • Thioguanine 2-amino-1 ,7-dihydro-6H-purine-6-thione
  • TABLOID ® Thioguanine exhibits cell phase-specificity at S-phase by inhibiting DNA synthesis by an as of yet unspecified mechanism.
  • Thioguanine is indicated as a single agent or in combination with other chemotherapy agents in the treatment of acute leukemia.
  • Other purine analogs include pentostatin, erythrohydroxynonyladenine, fludarabine phosphate, and cladribine.
  • Gemcitabine 2'-deoxy-2', 2'-difluorocytidine monohydrochloride ( ⁇ -isomer), is
  • GEMZAR ® commercially available as GEMZAR ® .
  • Gemcitabine exhibits cell phase-specificity at S- phase and by blocking progression of cells through the G1/S boundary. Gemcitabine is indicated in combination with cisplatin in the treatment of locally advanced non-small cell lung cancer and alone in the treatment of locally advanced pancreatic cancer.
  • Methotrexate N-[4[[(2,4-diamino-6-pteridinyl) methyl]methylamino] benzoyl]-L-glutamic acid, is commercially available as methotrexate sodium. Methotrexate exhibits cell phase effects specifically at S-phase by inhibiting DNA synthesis, repair and/or replication through the inhibition of dyhydrofolic acid reductase which is required for synthesis of purine nucleotides and thymidylate.
  • Methotrexate is indicated as a single agent or in combination with other chemotherapy agents in the treatment of choriocarcinoma, meningeal leukemia, non-Hodgkin's lymphoma, and carcinomas of the breast, head, neck, ovary and bladder.
  • Topoisomerase I inhibitors Camptothecins, including, camptothecin and camptothecin derivatives are available or under development as Topoisomerase I inhibitors.
  • Camptothecins cytotoxic activity is believed to be related to its Topoisomerase I inhibitory activity.
  • camptothecins include, but are not limited to irinotecan, topotecan, and the various optical forms of 7-(4-methylpiperazino-methylene)-10, 11-ethylenedioxy-20- camptothecin described below.
  • Irinotecan is a derivative of camptothecin which binds, along with its active metabolite SN-38, to the topoisomerase I - DNA complex. It is believed that cytotoxicity occurs as a result of irreparable double strand breaks caused by interaction of the topoisomerase I: DNA:
  • Irinotecan is indicated for treatment of metastatic cancer of the colon or rectum.
  • Topotecan HCI (S)-10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-1 H- pyrano[3',4',6,7]indolizino[1 ,2-b]quinoline-3, 14-(4H, 12H)-dione monohydrochloride, is commercially available as the injectable solution HYCAMTIN ® .
  • Topotecan is a derivative of camptothecin which binds to the topoisomerase I - DNA complex and prevents religation of singles strand breaks caused by Topoisomerase I in response to torsional strain of the DNA molecule. Topotecan is indicated for second line treatment of metastatic carcinoma of the ovary and small cell lung cancer.
  • Hormones and hormonal analogues are useful compounds for treating cancers in which there is a relationship between the hormone(s) and growth and/or lack of growth of the cancer.
  • hormones and hormonal analogues useful in cancer treatment include, but are not limited to, adrenocorticosteroids such as prednisone and prednisolone which are useful in the treatment of malignant lymphoma and acute leukemia in children; aminoglutethimide and other aromatase inhibitors such as anastrozole, letrazole, vorazole, and exemestane useful in the treatment of adrenocortical carcinoma and hormone dependent breast carcinoma containing estrogen receptors; progestrins such as megestrol acetate useful in the treatment of hormone dependent breast cancer and endometrial carcinoma; estrogens, androgens, and anti-androgens such as flutamide, nilutamide, bicalutamide, cyproterone a
  • antagagonists such as goserelin acetate and luprolide.
  • Signal transduction pathway inhibitors are inhibitors that block or inhibit a chemical process which evokes an intracellular change. As used herein, this change is cell proliferation or differentiation.
  • Signal tranduction inhibitors useful in the present invention include inhibitors of receptor tyrosine kinases, non-receptor tyrosine kinases, SH2/SH3 domain blockers, serine/threonine kinases, phosphotidyl inositol-3 kinases, myo-inositol signaling, and Ras oncogenes.
  • Protein tyrosine kinases catalyse the phosphorylation of specific tyrosyl residues in various proteins involved in the regulation of cell growth.
  • Such protein tyrosine kinases can be broadly classified as receptor or non-receptor kinases.
  • Receptor tyrosine kinases are transmembrane proteins having an extracellular ligand binding domain, a transmembrane domain, and a tyrosine kinase domain.
  • Receptor tyrosine kinases are involved in the regulation of cell growth and are generally termed growth factor receptors.
  • Growth factor receptors include, for example, epidermal growth factor receptor (EGFr), platelet derived growth factor receptor (PDGFr), erbB2, erbB4, ret, vascular endothelial growth factor receptor (VEGFr), tyrosine kinase with immunoglobulin-like and epidermal growth factor identity domains (TIE-2), insulin growth factor -I (IGFI) receptor, macrophage colony stimulating factor (cfms), BTK, ckit, cmet, fibroblast growth factor (FGF) receptors, Trk receptors (TrkA, TrkB, and TrkC), ephrin (eph) receptors, and the RET protooncogene.
  • EGFr epidermal growth factor receptor
  • PDGFr platelet derived growth factor receptor
  • erbB2 erbB2
  • VEGFr vascular endothelial growth factor receptor
  • TIE-2 immunoglobulin-like and epidermal growth factor identity domain
  • inhibitors of growth receptors include ligand antagonists, antibodies, tyrosine kinase inhibitors and anti-sense oligonucleotides.
  • Growth factor receptors and agents that inhibit growth factor receptor function are described, for instance, in Kath, Exp. Opin. Ther. Patents (2000) 10(6):803-818; Shawver, et al DDT, Vol 2, No. 2 February 1997; and Lofts, et al, "Growth factor receptors as targets", NEW
  • Tyrosine kinases which are not growth factor receptor kinases, are termed non-receptor tyrosine kinases.
  • Non-receptor tyrosine kinases useful in the present invention include cSrc, Lck, Fyn, Yes, Jak, cAbl, FAK (Focal adhesion kinase), Brutons tyrosine kinase, and Bcr-Abl.
  • SH2/SH3 domain blockers are agents that disrupt SH2 or SH3 domain binding in a variety of enzymes or adaptor proteins including, PI3-K p85 subunit, Src family kinases, adaptor molecules (She, Crk, Nek, Grb2) and Ras-GAP.
  • SH2/SH3 domains as targets for anticancer drugs are discussed in Smithgall, et al. (1995), Journal of Pharmacological and Toxicological Methods 34(3) 125-32.
  • Inhibitors of Serine/Threonine Kinases including MAP kinase cascade blockers which include blockers of Raf kinases (rafk), Mitogen or Extracellular Regulated Kinase (MEKs), and Extracellular Regulated Kinases (ERKs); and Protein kinase C family member blockers including blockers of PKCs (alpha, beta, gamma, epsilon, mu, lambda, iota, zeta).
  • IkB kinase family IKKa, IKKb
  • PKB family kinases akt kinase family members
  • TGF beta receptor kinases TGF beta receptor kinases.
  • Inhibitors of Phosphotidyl inositol-3 Kinase family members including blockers of PI3- kinase, ATM, DNA-PK, and Ku are also useful in the present invention.
  • Such kinases are discussed in Abraham, R.T. (1996), Current Opinion in Immunology. 8 (3) 412-8; Canman, et al. (1998), Oncogene 17 (25) 3301-3308; Jackson, S.P. (1997), International Journal of Biochemistry and Cell Biology 29 (7):935-8; and Zhong, et al, Cancer Res, (2000) 60(6), 1541-1545.
  • Myo-inositol signaling inhibitors such as phospholipase C blockers and Myoinositol analogues.
  • signal inhibitors are described in Powis, et al., (1994) NEW MOLECULAR TARGETS FOR CANCER CHEMOTHERAPY ed., Paul Workman and David Kerr, CRC press 1994, London.
  • Another group of signal transduction pathway inhibitors are inhibitors of Ras Oncogene.
  • Such inhibitors include inhibitors of farnesyltransferase, geranyl-geranyl transferase, and CAAX proteases, as well as anti-sense oligonucleotides, ribozymes and immunotherapy.
  • antibody antagonists to receptor kinase ligand binding may also serve as signal transduction inhibitors.
  • This group of signal transduction pathway inhibitors includes the use of humanized antibodies to the extracellular ligand binding domain of receptor tyrosine kinases.
  • Imclone C225 EGFR specific antibody see Green, et al, Monoclonal Antibody Therapy for Solid Tumors, Cancer Treat.
  • HERCEPTIN ® erbB2 antibody see Tyrosine Kinase Signalling in Breast cancer.erbB Family Receptor Tyrosine Kinases, Breast cancer Res., 2000, 2(3), 176-183
  • 2CB VEGFR2 specific antibody see Brekken, et al, "Selective Inhibition of VEGFR2 Activity by a monoclonal Anti-VEGF antibody blocks tumor growth in mice", Cancer Res. (2000) 60, 51 17-5124).
  • Anti-angiogenic agents including non-receptorMEKngiogenesis inhibitors may alo be useful. Such anti-angiogenic agents such, may inhibit the effects of vascular edothelial growth factor, (for example the anti-vascular endothelial cell growth factor antibody bevacizumab [AvastinTM], and may include compounds that work by other mechanisms (for example linomide, inhibitors of integrin ⁇ 3 function, endostatin and angiostatin);
  • vascular edothelial growth factor for example the anti-vascular endothelial cell growth factor antibody bevacizumab [AvastinTM]
  • vastinTM anti-vascular endothelial cell growth factor antibody bevacizumab
  • linomide inhibitors of integrin ⁇ 3 function, endostatin and angiostatin
  • Immunotherapeutic agents Agents used in immunotherapeutic regimens may also be useful in combination with the compounds of formula (I).
  • Immunotherapy approaches including for example ex-vivo and in-vivo approaches to increase the immunogenecity of patient tumour cells, such as transfection with cytokines, such as interleukin 2, interleukin 4 or granulocyte-macrophage colony stimulating factor, approaches to decrease T-cell anergy, approaches using transfected immune cells such as cytokine-transfected dendritic cells, approaches using cytokine-transfected tumour cell lines and approaches using anti- idiotypic antibodies
  • cytokines such as interleukin 2, interleukin 4 or granulocyte-macrophage colony stimulating factor
  • Proapoptotoc agents Agents used in proapoptotic regimens (e.g., bcl-2 antisense oligonucleotides) may also be used in the combination of the present invention.
  • Cell cycle signalling inhibitors inhibit molecules involved in the control of the cell cycle.
  • a family of protein kinases called cyclin-dependent kinases (CDKs) and their interaction with a family of proteins termed cyclins controls progression through the eukaryotic cell cycle. The coordinate activation and inactivation of different cyclin/CDK complexes is necessary for normal progression through the cell cycle.
  • CDKs cyclin-dependent kinases
  • the combination of the present invention comprises an anti-OX40 ABP or antibody and a TLR7/8 modulator and/or a TLR4 modulator and at least one antineoplastic agent selected from: anti-microtubule agents, platinum coordination complexes, alkylating agents, antibiotic agents, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormonal analogues, signal transduction pathway inhibitors, non-receptor tyrosine MEKngiogenesis inhibitors, immunotherapeutic agents, proapoptotic agents, and cell cycle signaling inhibitors.
  • antineoplastic agent selected from: anti-microtubule agents, platinum coordination complexes, alkylating agents, antibiotic agents, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormonal analogues, signal transduction pathway inhibitors, non-receptor tyrosine MEKngiogenesis inhibitors, immunotherapeutic agents, proapoptotic agents, and cell cycle signaling inhibitor
  • the combination of the present invention comprises an anti-OX40 ABP or antibody and a TLR7/8 modulator and/or a TLR4 modulator and at least one antineoplastic agent that is an anti-microtubule agent selected from: diterpenoids and vinca alkaloids.
  • the at least one anti-neoplastic agent agent is a diterpenoid.
  • the at least one anti-neoplastic agent is a vinca alkaloid.
  • the combination of the present invention comprises an anti-OX40 ABP or antibody and a TLR7/8 modulator and/or a TLR4 modulator and at least one antineoplastic agent, which is a platinum coordination complex.
  • the at least one anti-neoplastic agent is paclitaxel, carboplatin, or vinorelbine.
  • the at least one anti-neoplastic agent is carboplatin.
  • the at least one anti-neoplastic agent is vinorelbine.
  • the at least one anti-neoplastic agent is paclitaxel.
  • the combination of the present invention comprises an anti-OX40 ABP or antibody and a TLR7/8 modulator and/or a TLR4 modulator and at least one antineoplastic agent which is a signal transduction pathway inhibitor.
  • the signal transduction pathway inhibitor is an inhibitor of a growth factor receptor kinase chosen from: VEGFR2, TIE2, PDGFR, BTK, erbB2, EGFr, IGFR-1 , TrkA, TrkB, TrkC, and c-fms.
  • a growth factor receptor kinase chosen from: VEGFR2, TIE2, PDGFR, BTK, erbB2, EGFr, IGFR-1 , TrkA, TrkB, TrkC, and c-fms.
  • the signal transduction pathway inhibitor is an inhibitor of a serine/threonine kinase rafk, akt, or PKC-zeta.
  • the signal transduction pathway inhibitor is an inhibitor of a nonreceptor tyrosine kinase selected from the src family of kinases. In a further embodiment the signal transduction pathway inhibitor is an inhibitor of c-src.
  • the signal transduction pathway inhibitor is an inhibitor of Ras oncogene selected from: inhibitors of farnesyl transferase and geranylgeranyl transferase.
  • the signal transduction pathway inhibitor is an inhibitor of a serine/threonine kinase selected from Pi3K.
  • the signal transduction pathway inhibitor is a dual EGFr/erbB2 inhibitor, for example, N- ⁇ 3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl ⁇ -6-[5-( ⁇ [2- (methanesulphonyl) ethyl]amino ⁇ methyl)-2-furyl]-4-quinazolinamine (structure below):
  • the combination of the present invention comprises a compound of formula I or a salt or solvate thereof and at least one anti-neoplastic agent which is a cell cycle signaling inhibitor.
  • the cell cycle signaling inhibitor is an inhibitor of CDK2, CDK4 or CDK6.
  • the mammal in the methods and uses of the present invention is a human.
  • therapeutically effective amounts of the combinations of the invention are administered to a human.
  • the therapeutically effective amount of the administered agents of the present invention will depend upon a number of factors including, for example, the age and weight of the subject, the precise condition requiring treatment, the severity of the condition, the nature of the formulation, and the route of administration. Ultimately, the therapeutically effective amount will be at the discretion of the attendant physician.
  • the following examples are intended for illustration only and are not intended to limit the scope of the invention in any way.
  • Example 1 Treatment of 0X86 Monotherapy in a CT-26 syngeneic mouse model for colon cancer
  • CT-26 mouse colon carcinoma (CT26.WT; ATCC #CRL-2638) cell line was obtained from ATCC. It is an N-nitroso-N-methylurethane-(NNMU) induced, undifferentiated colon carcinoma cell line known in the art. For example, it is described in: Wang M, et al. Active immunotherapy of cancer with a nonreplicating recombinant fowlpox virus encoding a model tumor-associated antigen. J. Immunol. 154: 4685-4692, 1995 (PubMed: 7722321). Rat lgG1 was obtained from Bioxcell.
  • 0X86 Hybridoma 134 cells were obtained from the European Cell Culture collection and manufactured by Harlan; 0X86 is the name for a tool anti-OX40 monoclonal antibody used in rodents; it is a rodent antibody that binds rodent OX40, e.g., mouse OX40 (receptor).
  • 0X86 and rat lgG1 are diluted in diluted DPBS.
  • a frozen (-140°C) vial of CT-26 (mouse colon carcinoma cells), from ATCC (cat# CRL-2638, lot# 59227052) are basic RPMI (with 10% FBS) media over the following week.
  • CT-26 cells (passage 12) are harvested from the flask in complete medium. Cells are centrifuged and resuspended in RPMI (without FBS), this step is repeated 3 times. Cell density and viability are checked via trypan blue exclusion. Cells are then diluted to desired density (5x10 5 cells per ml_) and kept on ice.
  • Escalating doses of OX40 monoclonal antibody (mAb) 0X86 are evaluated their efficacy in reducing tumor growth.
  • Animals are weighed and innoculated on the right hind quarter with 0.5x10 5 CT26 tumor cells per mouse on Day 0.
  • tumor growth and total body weight are measured 3 times a week for the duration of the study.
  • Randomization occurs on day 10 or 1 1 when the average tumor volume is approximately 100 mm 3 .
  • animals are dosed with 0X86 mAb or Rat lgG1 isotype i.p. biweekly, for a total of 6 doses.
  • Mice remain on study until tumors >2000 cu mm for two consecutive measurements, they are removed from study for other reasons (i.e. weight loss >20%, ulceration on tumor, etc.) or until the end of the study. After euthanization the tumors may be removed and subject to dissociation for flow analysis and/or FFPE for IHC analysis.
  • Group 1 0.5x10 5 cells per, Rat lgG1 400 ug per mouse 10-13
  • Group 2 0.5x10 5 cells per, 0X86 400 ug per mouse 10-13
  • Group 3 0.5x10 5 cells per, 0X86 200 ug per mouse 10-13
  • Group 4 0.5x10 5 cells per, 0X86 100 ug per mouse 10-13
  • Group 5 0.5x10 5 cells per, 0X86 50 ug per mouse 10-13
  • Days 1 , 4, 6, 8 Animals are weighed and checked for tumors and if present, tumors measured.
  • Randomization day (approx. day 10): Animals randomized and placed into cages representing appropriate groups
  • P values are calculated based on the following: P value tests the null hypothesis that the survival curves are identical in the overall populations. In other words, the null hypothesis is that the treatment did not change survival. Raw p-values adjusted for multiple comparisons via the Stepdown Bonferroni method
  • TLR4 modulators such as CRX-527
  • OX40 monotherapy treatment protocol can be used to study TLR4 monotherapy and the combination of anti- mOX40 immunotherapy with TLR4 modulators.
  • Group a 0.5x10 5 cells per, CRX-527; 4 ug 10-13
  • Group b 0.5x10 5 cells per, CRX-527; 20 ug 10-13
  • Group c 0.5x10 5 cells per, CRX-527; 100 ug 10-13 Day 0: sc innoculation with tumor cells
  • Days 1 , 4, 6, 8 Animals were weighed and checked for tumors and measured.
  • Randomization day (approx. day 10): Animals randomized and placed into cages representing appropriate groups
  • EXAMPLE 3 Combination Treatment with OX40 (i.e. OX-86, an antibody raised against rodent OX40 receptor) and CRX-527
  • Group 1 0.5x10 5 cells per, Rat lgG1 drug vehicle 10-13
  • Group 2 0.5x10 5 cells per, 0X86 50 ug drug vehicle 10-13
  • Group 3 0.5x10 5 cells per, RatlgGI CRX-527 5ug 10-13
  • Group 5 0.5x10 5 cells per, 0X86 50 ug CRX-527 5ug 10-13
  • Group 6 0.5x10 5 cells per, 0X86 50 ug CRX-527 25ug 10-13
  • Days 1 , 4, 6, 8 Animals checked for tumors and if present, tumors measured. Study enrollment day (approx. day 10): Animals randomized and received treatment 1.
  • mice received i.p. dose biweekly for a total 6 doses.
  • mice Triweekly through end of study: Animals weighed and tumors measured When 0X86 treatment was combined with TLR4 modulator treatment (CRX-527), mice exhibited a higher reduction in tumor burden and survived longer than either treatment alone.
  • EXAMPLE 3 Monotherapy and Combination Treatment with anti-mOX40R antibody and TLR-4 targeting molecules in a CT-26 model
  • Mice are administered OX-40 antibody; a compound of Formula 1 (including a compound of Formula la, CRX-527, CRX-547, and CRX-601 (TLR-4 agonists), or a combination of both. Each treatment has significant anti-tumor activity.
  • mice anti-OX40R or combination of anti-OX40 antibody and TLR4 agonist combination each delay the growth of established CT-26 tumors relative to an untreated control group.
  • mice significant antitumor effect is observed in TLR4 agonist and anti-OX40R antibody combinations as compared to monotherapy treatment.
  • Group 1 0.5x10 5 cells per, Rat lgG1 drug vehicle
  • Group 2 0.5x10 5 cells per, 0X86 50 ug drug vehicle
  • Group 3 0.5x10 5 cells per, RatlgGI CRX-527 5ug
  • Group 4 0.5x10 5 cells per, RatlgGI CRX-527 25ug
  • Group 5 0.5x10 5 cells per, RatlgGI CRX-649 5ug
  • Group 6 0.5x105 cells per, RatlgGI CRX-649 25ug
  • Group 7 0.5x105 cells per, 0X86 50 ug CRX-527 5ug
  • Group 8 0.5x105 cells per, 0X86 50 ug CRX-527 25ug
  • Group 9 0.5x105 cells per, 0X86 50 ug CRX-649 5ug
  • Group 10 0.5X105 cells per, 0X86 50 ug CRX-649 25ug Day 0: SC innoculation with tumor cells
  • Days 1 , 4, 6, 8 Animals were checked for tumors and if present, tumors measured.
  • mice received i.p. dose biweekly for a total 6 doses.
  • Combination dosing with 0X86 and either dose of CRX-649 resulted in some observable differences, but no significant difference in tumor growth at either day 22 or day 38 when compared to animals that were dosed with CRX-649/rat lgG1 , veh/OX86, or veh/rat lgG1.
  • Tumor bearing mice are administered OX-40 antibody; a compound of Formula 1
  • TLR-4 agonists include a compound of Formula la, CRX-527, CRX-547, and CRX-601 (TLR-4 agonists), or a TLR 7/8 compound, such as an oxoadenine, a lipidated oxoadenine, an
  • imadazoquinoline and a lipidated imadazoquinoline, or a combination of all three (i.e. an OX-40 antibody, a TLR4 agonist, and a TLR7/8 compound).
  • OX-40 antibody a TLR4 agonist
  • TLR7/8 compound a TLR7/8 compound
  • a compound of Formula 1 including a compound of Formula la, CRX-527, CRX-547, and CRX-601 (TLR-4 agonists), and/or a TLR 7/8 modulator, such as an oxoadenine, a lipidated oxoadenine, an imadazoquinoline, and a lipidated imadazoquinoline, or a combination of all three (i.e. an OX-40 antibody, a TLR4 agonist, and a TLR7/8 compound).
  • Anti-hOX40 antibody in combination with one or more TLR4 modulators and/or a TLR 7/8 compound has significant anti-tumor effect.
  • EXAMPLE 6 Combination Treatment with triple treatment combination of anti- mOX40R antibody and TLR4 and TLR7/8 in a CT-26 model.
  • Tumor bearing mice are administered OX-40 antibody; a compound of Formula 1
  • TLR-4 agonists include a compound of Formula la, CRX-527, CRX-547, and CRX-601 (TLR-4 agonists), or a TLR 7/8 compound, such as an oxoadenine, a lipidated oxoadenine, an
  • imadazoquinoline and a lipidated imadazoquinoline, or a combination of all three (i.e. an OX-40 antibody, a TLR4 agonist, and a TLR7/8 compound).
  • OX-40 antibody a TLR4 agonist
  • TLR7/8 compound a combination of all three
  • Trp lie Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys 1 5 10 15
  • Thr Val Lys lie Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
  • Gly Trp lie Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60
  • Gly Trp lie Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60
  • Leu Gin lie Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
  • Ala Ala lie Asn Ser Asp Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Met 50 55 60
  • Glu Arg Arg Phe lie lie Ser Arg Asp Asn Thr Lys Lys Thr Leu Tyr 65 70 75 80

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Abstract

The present diclosure relates to combinations of an OX40 modulator, preferably an OX40 antibody, and at least one TLR modulator chosen from: a TLR7/8 modulator and a TLR4 modulator ( TLRs are cell surface or intracellular receptors that recognize pathogen-associated molecular patterns commonly conserved in bacteria, viruses, and some fungi. Binding of these TLRs activates antigen-presenting cells, such as macrophages and dendritic cells, to commence both innate and adaptive immune responses). Pharmaceutical compositions thereof, uses thereof, and methods of treatment comprising administering said combination, including uses in cancer.

Description

COMBINED TLRS MODULATORS WITH ANTI OX40 ANTIBODIES
FIELD OF THE INVENTION
The present invention relates to a method of treating cancer in a mammal and to combinations useful in such treatment. In particular, the present relates to combinations of anti-OX40 antigen binding proteins (ABPs) and one or more TLR7/8 modulators. The present invention also relates to triple combinations of anti-OX40 ABPs and one or more TLR7/8 modulators and one or more TLR4 modulators.
BACKGROUND OF THE INVENTION
Effective treatment of hyperproliferative disorders including cancer is a continuing goal in the oncology field. Generally, cancer results from the deregulation of the normal processes that control cell division, differentiation and apoptotic cell death and is characterized by the proliferation of malignant cells which have the potential for unlimited growth, local expansion and systemic metastasis. Deregulation of normal processes include abnormalities in signal transduction pathways and response to factors which differ from those found in normal cells.
Immunotherapies are one approach to treat hyperproliferative disorders. A major hurdle that scientists and clinicians have encountered in the development of various types of cancer immunotherapies has been to break tolerance to self antigen (cancer) in order to mount a robust anti-tumor response leading to tumor regression. Unlike traditional development of small and large molecule agents that target the tumor, cancer
immunotherapies target cells of the immune system that have the potential to generate a memory pool of effector cells to induce more durable effects and minimize recurrences.
OX40 is a costimulatory molecule involved in multiple processes of the immune system. Antigen binding proteins and antibodies that bind OX-40 receptor and modulate OX40 signalling are known in the art and are disclosed as immunotherapy, for example for cancer.
Aminoalkyl glucosaminide phosphates (AGPs) are synthetic ligands of the Toll-like Receptor 4 (TLR4). AGPs are known to be useful as vaccine adjuvants and for stimulating cytokine production, activating macrophages, promoting innate immune response, and augmenting antibody production in immunized animals.
Though there have been many recent advances in the treatment of cancer, there remains a need for more effective and/or enhanced treatment of an individual suffering the effects of cancer. The combinations and methods herein that relate to combining therapeutic approaches for enhancing anti-tumor immunity address this need.
SUMMARY OF THE INVENTION
Provided herein are combinations of anti-OX40 antigen binding proteins (ABPs) and one or more TLR7/8 modulators.
Also provided are combinations of anti-OX40 AGPs, one or more TLR7/8 modulators and one or more TLR4 modulators
Further provided are methods of treating cancer in a human with the composition of the invention, and uses of the combinations for therapy, preferably for therapy for cancer. Further provided are methods for modulating the immune response of a subject in need of cancer treatment, preferably a human, comprising administering to said subject an effective amount of the combinations, e.g., in one or more pharmaceutical compositions.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A is a graph showing dose-dependent anti-tumor activity (as measured by tumor volume over time) of TLR-4 agonist (CRX-527) in a CT-26 syngeneic mouse model of colon cancer. Results are the mean of 10 animals.
FIG. 1 B is a graph showing dose-dependent anti-tumor activity (as measured by tumor volume over time) of a mouse anti-mOX-40R antibody (OX-86) in a CT-26 syngeneic mouse model of colon cancer. Results are the mean of 10 animals; control treatments in Fig 1A are the same as those in Fig.1 B.
FIG. 1C is a graph showing dose-dependent anti-tumor activity (as measured by tumor volume over time) of TLR-7/8 agonist (CRX-649) in a CT-26 syngeneic mouse model of colon cancer. Results are the mean of 10 animals; control treatments in Fig 1AC are the same as those in Figs.1 A and 1 B.
Figure 2 is a graph showing dose-dependent anti-tumor activity (as measured by tumor volume over time) of a mouse anti-mOX-40R antibody (OX-86), 5 ug of TLR-4 agonist (CRX-527), and the combination of both in a CT-26 syngeneic mouse model of colon cancer. Results are the mean of 10 animals.
Figure 3 is a graph showing dose-dependent anti-tumor activity (as measured by tumor volume over time) of a mouse anti-mOX-40R antibody (OX-86), 25 ug of TLR-4 agonist (CRX-527), and the combination of both in a CT-26 syngeneic mouse model of colon cancer measured over 38 days. Results are the mean of 10 animals; control treatments in Figure 2 represent identical animals as those in Figure 3. FIG. 4A - FIG. 4F are graphs showing dose-dependent anti-tumor activity (as measured by tumor volume over time) in individual mice of a control antibody (IgG), mouse anti- mOX-40R antibody (OX-86), 5 or 25 ug of TLR-4 agonist (CRX-527), and the combination of 0X86 and CRX-527 in a group of mice in a CT-26 syngeneic mouse model of colon cancer measured over 42 days. The mean results the 10 animals shown in FIGs 4A-4F were used to generate the plots in Figures 2-3.
Figure 5 is a graph showing dose-dependent anti-tumor activity (as measured by tumor volume over time) of a mouse anti-mOX-40R antibody (OX-86), 5 ug of TLR-7/8 agonist (CRX-649), and the combination of both in a CT-26 syngeneic mouse model of colon cancer. Results are the mean of 10 animals.
Figure 6 is a graph showing dose-dependent anti-tumor activity (as measured by tumor volume over time) of a mouse anti-mOX-40R antibody (OX-86), 25 ug of TLR-7/8 agonist (CRX-649), and the combination of both in a CT-26 syngeneic mouse model of colon cancer. Results are the mean of 10 animals; control treatments in Figure 5 represent identical animals as those in Figure 6.
FIG. 7A - FIG. 7F are graphs showing dose-dependent anti-tumor activity (as measured by tumor volume over time) in individual mice of a control antibody (IgG), mouse anti- mOX-40R antibody (OX-86), 5 or 25 ug of TLR-7/8 agonist (CRX-649and the combination of 0X86 and CRX-649 in a group of mice in a CT-26 syngeneic mouse model of colon cancer. The mean results the 10 animals shown in FIGs 7A-7F were used to generate the plots in Figures 5-6.
Figure 8 is a graph showing dose-dependent anti-tumor activity (as measured by tumor volume over time) of 4, 20, or 100 ug of TLR-4 agonist (CRX-601) in a CT-26 syngeneic mouse model of colon cancer.
Figures 9-14 show sequences of the ABPs and antibodies of the invention, e.g., CDRs and VH and VL sequences.
Figures 15-18 show mouse sequences of ABPs and antibodies of the invention, e.g., CDRs and VH and VL sequences that can comprise a humanized antibody.
DETAILED DESCRIPTION OF THE INVENTION
Compositions and Combinations
Improved function of the immune system is a goal of immunotherapy for cancer. While not being bound by theory, it is thought that for the immune system to be activated and effectively cause regression or eliminate tumors, there must be efficient cross talk among the various compartments of the immune system as well at the at the tumor bed. The tumoricidal effect is dependent on one or more steps, e.g. the uptake of antigen by immature dendritic cells and presentation of processed antigen via MHC I and II by mature dendritic cells to naive CD8 (cytotoxic) and CD4 (helper) lymphocytes, respectively, in the draining lymph nodes. Naive T cells express molecules such as CTLA-4 and CD28 that engage with co-stimulatory molecules of the B7 family on antigen presenting cells (APCs) such as dendritic cells. In order to keep T cells in check during immune surveillance, B7 on APCs preferentially binds to CTLA-4, an inhibitory molecule on T lymphocytes.
However, upon engagement of the T cell receptor (TCR) with MHC Class I or II receptors via cognate peptide presentation on APCs, the co-stimulatory molecule disengages from CTLA-4 and instead binds to the lower affinity stimulatory molecule CD28, causing T cell activation and proliferation. This expanded population of primed T lymphocytes retains memory of the antigen that was presented to them as they traffic to distant tumor sites. Upon encountering a tumor cell bearing the cognate antigen, they eliminate the tumor via cytolytic mediators such as granzyme B and perforins. This apparently simplistic sequence of events is highly dependent on several cytokines, co-stimulatory molecules and check point modulators to activate and differentiate these primed T lymphocytes to a memory pool of cells that can eliminate the tumor. Thus, an emerging immunotherapeutic strategy is to target T cell co-stimulatory molecules, e.g., OX40. OX40 (e.g., hOX40 or hOX40R) is a tumor necrosis factor receptor family member that is expressed, among other cells, on activated CD4 and CD8 T cells. One of its functions is in the differentiation and long-term survival of these cells. The ligand for OX40 (OX40L) is expressed by activated antigen-presenting cells. The ABPs and antibodies of the invention modulate OX40 and promote growth and/or differentiation of T cells and increase long-term memory T-cell populations, e.g., in overlapping mechanisms as those of OX40L, by "engaging" OX40. Thus, in one embodiment of the ABPs and antibodies of the invention bind and engage OX40. In another embodiment, the ABPs and antibodies of the invention modulate OX40. In a further embodiment, the ABPs and antibodies of the invention modulate OX40 by mimicking OX40L. In another embodiment, the ABPs and antibodies of the invention are agonist antibodies. In yet another embodiment, the ABPs and antibodies of the invention modulate OX40 and cause proliferation of T cells. In a further embodiment, the ABPs and antibodies of the invention modulate OX40 and improve, augment, enhance, or increase proliferation of CD4 T cells. In another embodiment, the ABPs and antibodies of the invention improve, augment, enhance, or increase proliferation of CD8 T cells. In further embodiment, the ABPs and antibodies of the invention improve, augment, enhance, or increase proliferation of both CD4 and CD8 T cells. In another embodiment, the ABPs and antibodies of the invention enhance T cell function, e.g. of CD4 or CD8 T cells, or both CD4 and CD8 T cells. In a further embodiment, the ABPs and antibodies of the invention enhance effector T cell function. In another embodiment, the ABPs and antibodies of the invention improve, augment, enhance, or increase long-term survival of CD8 T cells. In yet further embodiments, any of the preceding effects occur in a tumor micro-environment.
Not being bound by theory, of equal importance is the blockade of a potentially robust immunosuppressive response at the tumor site by mediators produced both by T regulatory cells (Tregs) as well as the tumor itself (e.g., Transforming Growth Factor (TGF- B) and interleukin-10 (IL-10)). An important immune pathogenesis of cancer can be the involvement of Tregs that are found in tumor beds and sites of inflammation. In general, Treg cells occur naturally in circulation and help the immune system to return to a quiet, although vigilant state, after encountering and eliminating external pathogens. They help to maintain tolerance to self antigens and are naturally suppressive in function. They are phenotypically characterized as CD4+, CD25+, FOXP3+ cells. In order to break tolerance to effectively treat certain cancers, one mode of therapy is to eliminate Tregs preferentially at tumor sites. Targeting and eliminating Tregs leading to an antitumor response has been more successful in tumors that are immunogenic compared to those that are poorly immunogenic. Many tumors secrete cytokines, e.g., TGF-B that may hamper the immune response by causing precursor CD4+25+ cells to acquire the FOXP3+ phenotype and function as Tregs.
"Modulate" as used herein, for example with regard to a receptor or other target means to change any natural or existing function of the receptor, for example it means affecting binding of natural or artificial ligands to the receptor or target; it includes initiating any partial or full conformational changes or signaling through the receptor or target, and also includes preventing partial or full binding of the receptor or target with its natural or artificial ligands. Also included in the case of membrane bound receptors or targets are any changes in the way the receptor or target interacts with other proteins or molecules in the membrane or change in any localization (or co-localization with other molecules) within membrane compartments as compared to its natural or unchanged state. Modulators are therefore compounds or ligands or molecules that modulate a target or receptor. Modulate includes agonizing, e.g., signaling, as well as antagonizing, or blocking signaling or interactions with a ligand or compound or molecule that happen in the unchanged or unmodulated state. Thus, modulators may be agonists or antagonists. Further, one of skill in the art will recognize that not all modulators will be have absolute selectivity for one target or receptor, but are still considered a modulator for that target or receptor; for example, a TLR4 modulator may also engage another TLR but still be considered a TLR4 modulator. Other modulators are known to have multiple specificities, such as TLR7/8 modulators which modulate both TLR7 and TLR8. Molecules with such known double or multiple specificities are considered a modulator of each of its target; that is, a TLR7/8 modulator is a TLR7 modulator as used herein and likewise a TLR7/8 modulator is a TLR8 modulator, as used herein.
"Agonists" of a target or receptor are molecules or compounds or ligands that mimic one or more functions of a natural ligand or molecule that interacts with the target or receptor and includes initiating one or more signaling events through the receptor, mimicking one or more functions of a natural ligand, initiating one or more partial or full conformational changes that are seen in known functioning or signaling through the receptor.
Thus, in one embodiment, the OX40 ABP or antibody inhibits the suppressive effect of Treg cells on other T cells, e.g., within the tumor environment.
Accumulating evidence suggests that the ratio of Tregs to T effector cells in the tumor correlates with anti tumor response. Therefore, in one embodiment, the OX40 ABPs or antibodies of the invention modulate OX40 to augment T effector number and function and inhibit Treg function. Enhancing, augmenting, improving, increasing, and otherwise changing the anti-tumor effect of OX40 is an object of the invention. Described herein are combinations of an anti- OX40 ABP or antibody of the invention and another compound, such as a TLR modulator described herein.
Thus, as used herein the term "combination of the invention" refers to a combination comprising an anti-OX40 ABP or antibody and a TLR7/8 modulator or a combination comprising an anti-OX40 ABP and a TLR7/8 modulator and a TLR4 modulator, where, in either combination, each component may be administered separately or simultaneously as described herein.
As used herein, the terms "cancer," "neoplasm," and "tumor," are used interchangeably and in either the singular or plural form, refer to cells that have undergone a malignant transformation or undergone cellular changes that result in aberrant or unregulated growth or hyperproliferation Such changes or malignant transformations usually make such cells pathological to the host organism, thus precancers or precancerous cells that are or could become pathological and require or could benefit from intervention are also intended to be included. Primary cancer cells (that is, cells obtained from near the site of malignant transformation) can be readily distinguished from non-cancerous cells by well-established techniques, particularly histological examination. The definition of a cancer cell, as used herein, includes not only a primary cancer cell, but any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells. When referring to a type of cancer that normally manifests as a solid tumor, a "clinically detectable" tumor is one that is detectable on the basis of tumor mass; e.g., by procedures such as CAT scan, MR imaging, X-ray, ultrasound or palpation, and/or which is detectable because of the expression of one or more cancer-specific antigens in a sample obtainable from a patient. In other words, the terms herein include cells, neoplasms, cancers, and tumors of any stage, including what a clinician refers to as precancer, tumors, in situ growths, as well as late stage metastatic growths, Tumors may be hematopoietic tumor, for example, tumors of blood cells or the like, meaning liquid tumors. Specific examples of clinical conditions based on such a tumor include leukemia such as chronic myelocytic leukemia or acute myelocytic leukemia; myeloma such as multiple myeloma; lymphoma and the like.
As used herein the term "agent" is understood to mean a substance that produces a desired effect in a tissue, system, animal, mammal, human, or other subject. Accordingly, the term "anti-neoplastic agent" is understood to mean a substance producing an antineoplastic effect in a tissue, system, animal, mammal, human, or other subject. It is also to be understood that an "agent" may be a single compound or a combination or composition of two or more compounds.
By the term "treating" and derivatives thereof as used herein, is meant therapeutic therapy. In reference to a particular condition, treating means: (1) to ameliorate the condition or one or more of the biological manifestations of the condition, (2) to interfere with (a) one or more points in the biological cascade that leads to or is responsible for the condition or (b) one or more of the biological manifestations of the condition (3) to alleviate one or more of the symptoms, effects or side effects associated with the condition or one or more of the symptoms, effects or side effects associated with the condition or treatment thereof, or (4) to slow the progression of the condition or one or more of the biological manifestations of the condition.
As used herein, "prevention" is understood to refer to the prophylactic administration of a drug to substantially diminish the likelihood or severity of a condition or biological manifestation thereof, or to delay the onset of such condition or biological manifestation thereof. The skilled artisan will appreciate that "prevention" is not an absolute term.
Prophylactic therapy is appropriate, for example, when a subject is considered at high risk for developing cancer, such as when a subject has a strong family history of cancer or when a subject has been exposed to a carcinogen. As used herein, the term "effective amount" means that amount of a drug or
pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought, for instance, by a researcher or clinician.
Furthermore, the term "therapeutically effective amount" means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal physiological function.
The administration of a therapeutically effective amount of the combinations of the invention (or therapeutically effective amounts of each of the components of the combination) are advantageous over the individual component compounds in that the combinations provide one or more of the following improved properties when compared to the individual administration of a therapeutically effective amount of a component compound: i) a greater anticancer effect than the most active single agent; ii) synergistic or highly synergistic anticancer activity; iii) a dosing protocol that provides enhanced anticancer activity with reduced side effect profile; iv) a reduction in the toxic effect profile; v) an increase in the therapeutic window; or vi) an increase in the bioavailability of one or both of the component compounds.
The invention further provides pharmaceutical compositions, which include one or more of the components herein, and one or more pharmaceutically acceptable carriers, diluents, or excipients. The combination of the invention may comprise one pharmaceutical composition, or multiple pharmaceutical compositions comprising one or more of the components of the combination, in any groupings. The combination of the invention may comprise two pharmaceutical compositions, for example one comprising an ABP or antibody of the invention, and the other comprising a TLR7/8 modulator, each of which may have the same or different carriers, diluents or excipients. The combination of the invention may comprise three pharmaceutical compositions, one comprising an ABP or antibody of the invention, another comprising a TLR 7/8 modulator, and another comprising a TLR4 modulator, each of which may have the same or different carriers, diluents or excipients. The carrier(s), diluent(s) or excipient(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation, capable of pharmaceutical formulation, and not deleterious to the recipient thereof.
The components of the combination of the invention, and pharmaceutical compositions comprising such components may be administered in any order, and in different routes; the components and pharmaceutical compositions comprising the same may be administered simultaneously.
In accordance with another aspect of the invention there is also provided a process for the preparation of a pharmaceutical composition including admixing a component of the combination of the invention and one or more pharmaceutically acceptable carriers, diluents or excipients.
The components of the invention may be administered by any appropriate route. For some components, suitable routes include oral, rectal, nasal, topical (including buccal and sublingual), vaginal, and parenteral (including subcutaneous, intramuscular, intraveneous, intradermal, intrathecal, and epidural). It will be appreciated that the preferred route may vary with, for example, the condition of the recipient of the combination and the cancer to be treated. It will also be appreciated that each of the agents administered may be administered by the same or different routes and that the components may be
compounded together or in separate pharmaceutical compositions.
In one embodiment, one or more components of a combination of the invention are administered intravenously. In another embodiment, one or more components of a combination of the invention are administered intratumorally. In another embodiment, one or more components of a combination of the invention are administered systemically, e.g. intravenously, and one or more other components of a combination of the invention are administered intratumorally. In another embodiment, all of the components of a combination of the invention are administered systemically, e.g., intravenously. In an alternative embodiment, all of the components of the combination of the invention are administered intratumorally. In any of the embodiments, e.g., in this paragraph, the components of the invention are administered as one or more pharmaceutical
compositions. Antigen Binding Proteins and Antibodies that bind OX40
"Antigen Binding Protein (ABP)" means a protein that binds an antigen, including antibodies or engineered molecules that function in similar ways to antibodies. Such alternative antibody formats include triabody, tetrabody, miniantibody, and a minibody, Also included are alternative scaffolds in which the one or more CDRs of any molecules in accordance with the disclosure can be arranged onto a suitable non-immunoglobulin protein scaffold or skeleton, such as an affibody, a SpA scaffold, an LDL receptor class A domain, an avimer (see, e.g., U.S. Patent Application Publication Nos. 2005/0053973, 2005/0089932, 2005/0164301) or an EGF domain. An ABP also includes antigen binding fragments of such antibodies or other molecules. Further, an ABP may comprise the VH regions of the invention formatted into a full length antibody, a (Fab')2 fragment, a Fab fragment, a bi-specific or biparatopic molecule or equivalent thereof (such as scFV, bi- trior tetra-bodies, Tandabs, etc.), when paired with an appropriate light chain. The ABP may comprise an antibody that is an lgG1 , lgG2, lgG3, or lgG4; or IgM; IgA, IgE or IgD or a modified variant thereof. The constant domain of the antibody heavy chain may be selected accordingly. The light chain constant domain may be a kappa or lambda constant domain. The ABP may also be a chimeric antibody of the type described in WO86/01533 which comprises an antigen binding region and a non-immunoglobulin region. Thus, herein, an ABP of the invention or an anti-OX40 antigen binding protein is one that binds OX40, and does one or more of the following: modulate signaling through OX40; modulates the function of OX40; agonizes OX40 signalling; stimulated OX40 function; or co-stimulated OX40 signaling. One of skill in the art would readily recognize a variety of well known assays to establish such functions. The term "antibody" as used herein refers to molecules with an antigen binding domain, and optionally an immunoglobulin-like domain or fragment thereof and includes
monoclonal (for example IgG, IgM, IgA, IgD or IgE and modified variants thereof), recombinant, polyclonal, chimeric, humanized, biparatopic, bispecific, and heteroconjugate antibodies, or a closed conformation multispecific antibody. An "antibody" included xenogeneic, allogeneic, syngeneic, or other modified forms thereof. An antibody may be isolated or purified. An antibody may also be recombinant, i.e., produced by recombinant means; for example, an antibody that is 90% identical to a reference antibody may be generated by mutagenesis of certain residues using recombinant molecular biology techniques known in the art. Thus, the antibodies of the present invention may comprise heavy chain variable regions and light chain variable regions of the invention which may be formatted into the structure of a natural antibody or formatted into a full length recombinant antibody, a (Fab')2 fragment, a Fab fragment, a bi-specific or biparatopic molecule or equivalent thereof (such as scFV, bi- tri- or tetra-bodies, Tandabs etc.), when paired with an appropriate light chain. The antibody may be an lgG1 , lgG2, lgG3, or lgG4 or a modified variant thereof. The constant domain of the antibody heavy chain may be selected accordingly. The light chain constant domain may be a kappa or lambda constant domain. The antibody may also be a chimeric antibody of the type described in
WO86/01533, which comprises an antigen binding region and a non-immunoglobulin region. One of skill in the art will recognize that the ABPs and antibodies of the invention bind an epitope of OX40. The epitope of an ABP is the region of its antigen to which the ABP binds. Two ABPs bind to the same or overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a 1x, 5x, 10x, 20x or 100x excess of one antibody inhibits binding of the other by at least 50% but preferably 75%, 90% or even 99% as measured in a competitive binding assay compared to a control lacking the competing antibody (see, e.g., Junghans, et al., Cancer Res. 50: 1495 (1990)).
Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Also the same epitope may include "overlapping epitopes", e.g., if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.
The strength of binding may be important in dosing and administration of an ABP or antibody of the invention. In one embodiment, the ABP or antibody of the invention binds to OX40, preferably human OX40, with high affinity. For example, when measured by BIACORE®, the antibody binds to OX40, preferably human OX40, with an affinity of 1-
1000nM or 500nM or less or an affinity of 200nM or less or an affinity of 100nM or less or an affinity of 50 nM or less or an affinity of 500pM or less or an affinity of 400pM or less, or 300pM or less. In a further aspect the antibody binds to OX40, preferably human OX40, when measured by BIACORE® of between about 50nM and about 200nM or between about 50nM and about 150nM. In one aspect of the present invention the antibody binds OX40, preferably human OX40, with an affinity of less than 100nM.
In a further embodiment, binding is measured by BIACORE®. Affinity is the strength of binding of one molecule, e.g., an antibody of the invention, to another, e.g., its target antigen, at a single binding site. The binding affinity of an antibody to its target may be determined by equilibrium methods (e.g., enzyme-linked immunoabsorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetics (e.g., BIACORE® analysis). For example, the BIACORE® methods known in the art may be used to measure binding affinity.
Avidity is the sum total of the strength of binding of two molecules to one another at multiple sites, e.g., taking into account the valency of the interaction. In an aspect, the equilibrium dissociation constant (KD) of the ABP or antibody of the invention and OX40, preferably human OX40, interaction is 100 nM or less, 10 nM or less, 2 nM or less or 1 nM or less. Alternatively, the KD may be between 5 and 10 nM; or between 1 and 2 nM. The KD may be between 1 pM and 500 pM; or between 500 pM and 1 nM. A skilled person will appreciate that the smaller the KD numerical value, the stronger the binding. The reciprocal of KD (i.e., 1/KD) is the equilibrium association constant (KA) having units M-1. A skilled person will appreciate that the larger the KA numerical value, the stronger the binding.
The dissociation rate constant (kd) or "off-rate" describes the stability of the complex of ABP or antibody on one hand and OX40, preferably human OX40 on the other hand, i.e. the fraction of complexes that decay per second. For example, a kd of 0.01 s-1 equates to 1 % of the complexes decaying per second. In an embodiment, the dissociation rate constant (kd) is 1x10-3 s-1 or less, 1x10-4 s-1 or less, 1x10-5 s-1 or less, or 1x10-6 s-1 or less. The kd may be between 1x10-5 s-1 and 1x10-4 s-1 ; or between 1x10-4 s-1 and 1x10- 3 s-1.
Competition between an anti-OX40 ABP or antibody of the invention, and a reference antibody, e.g., for binding OX40, an epitope of OX40, or a fragment of the OX40, may be determined by competition ELISA, FMAT or BIACORE®. In one aspect, the competition assay is carried out by BIACORE®. There are several possible reasons for this
competition: the two proteins may bind to the same or overlapping epitopes, there may be steric inhibition of binding, or binding of the first protein may induce a conformational change in the antigen that prevents or reduces binding of the second protein.
"Binding fragments" as used herein means a portion or fragment of the ABPs or antibodies of the invention that include the antigen-binding site and are capable of binding OX40 as defined herein, e.g., but not limited to capable of binding to the same epitope of the parent or full length antibody.
Functional fragments of the ABPs and antibodies of the invention are contemplated herein.
Thus, "binding fragments" and "functional fragments" may be an Fab and F(ab')2 fragments that lack the Fc fragment of an intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl, et al., J. Nuc. Med. 24:316-325 (1983)). Also included are Fv fragments {Hochman, et al. (1973) Biochemistry 12:1 130-1135; Sharon, et a/.(1976) Biochemistry 15:1591-1594). These various fragments are produced using conventional techniques such as protease cleavage or chemical cleavage (see, e.g., Rousseaux et al., Meth. Enzymol., 121 :663-69 (1986)).
"Functional fragments" as used herein means a portion or fragment of the ABPs or antibodies of the invention that include the antigen-binding site and are capable of binding the same target as the parent ABP or antibody, e.g., but not limited to binding the same epitope, and that also retain one or more modulating or other functions described herein or known in the art.
As the ABPs and antibodies of the present invention may comprise heavy chain variable regions and light chain variable regions of the invention which may be formatted into the structure of a natural antibody, a functional fragment is one that retains binding or one or more functions of the full length ABP or antibody as described herein. A binding fragment of an ABP or antibody of the invention may therefore comprise the VL or VH regions, a (Fab')2 fragment, a Fab fragment, a fragment of a bi-specific or biparatopic molecule or equivalent thereof (such as scFV, bi- tri- or tetra-bodies, Tandabs, etc.), when paired with an appropriate light chain.
The term, "CDR", as used herein, refers to the complementarity determining region amino acid sequences of an antigen binding protein. These are the hypervariable regions of immunoglobulin heavy and light chains. There are three heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. It will be apparent to those skilled in the art that there are various numbering conventions for CDR sequences; Chothia (Chothia, et al. (1989) Nature 342: 877-883), Kabat (Kabat, et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL I NTEREST, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath) and Contact (University College London). The minimum overlapping region using at least two of the Kabat, Chothia, AbM and contact methods can be determined to provide the "minimum binding unit". The minimum binding unit may be a subportion of a CDR. The structure and protein folding of the antibody may mean that other residues are considered part of the CDR sequence and would be understood to be so by a skilled person. It is noted that some of the CDR definitions may vary depending on the individual publication used.
Unless otherwise stated and/or in absence of a specifically identified sequence, references herein to "CDR", "CDRL1", "CDRL2", "CDRL3", "CDRH1", "CDRH2", "CDRH3" refer to amino acid sequences numbered according to any of the known conventions; alternatively, the CDRs are referred to as "CDR1 ," "CDR2," "CDR3" of the variable light chain and "CDR1 ," "CDR2," and "CDR3" of the variable heavy chain. In particular embodiments, the numbering convention is the Kabat convention.
The term "CDR variant" as used herein, refers to a CDR that has been modified by at least one, for example 1 , 2 or 3, amino acid substitution(s), deletion(s) or addition(s), wherein the modified antigen binding protein comprising the CDR variant substantially retains the biological characteristics of the antigen binding protein pre-modification. It will be appreciated that each CDR that can be modified may be modified alone or in combination with another CDR. In one aspect, the modification is a substitution, particularly a conservative substitution, for example as shown in Table 1. Table 1
Figure imgf000015_0001
For example, in a variant CDR, the amino acid residues of the minimum binding unit may remain the same, but the flanking residues that comprise the CDR as part of the Kabat or Chothia definition(s) may be substituted with a conservative amino acid residue. Such antigen binding proteins comprising modified CDRs or minimum binding units as described above may be referred to herein as "functional CDR variants" or "functional binding unit variants".
The antibody may be of any species, or modified to be suitable to administer to a cross species. For example the CDRs from a mouse antibody may be humanized for administration to humans. In any embodiment, the antigen binding protein is optionally a humanized antibody.
A "humanized antibody" refers to a type of engineered antibody having its CDRs derived from a non-human donor immunoglobulin, the remaining immunoglobulin-derived parts of the molecule being derived from one (or more) human immunoglobulin(s). In addition, framework support residues may be altered to preserve binding affinity (see, e.g., Queen et al., Proc. Natl Acad Sci USA, 86:10029-10032 (1989), Hodgson, et al., Bio/Technology, 9:421 (1991)). A suitable human acceptor antibody may be one selected from a conventional database, e.g., the KABAT® database, Los Alamos database, and Swiss Protein database, by homology to the nucleotide and amino acid sequences of the donor antibody. A human antibody characterized by a homology to the framework regions of the donor antibody (on an amino acid basis) may be suitable to provide a heavy chain constant region and/or a heavy chain variable framework region for insertion of the donor CDRs. A suitable acceptor antibody capable of donating light chain constant or variable framework regions may be selected in a similar manner. It should be noted that the acceptor antibody heavy and light chains are not required to originate from the same acceptor antibody. The prior art describes several ways of producing such humanised antibodies - see, for example, EP-A-0239400 and EP-A-054951.
In yet a further embodiment, the humanized antibody has a human antibody constant region that is an IgG. In another embodiment, the IgG is a sequence as disclosed in any of the above references or patent publications.
For nucleotide and amino acid sequences, the term "identical" or "identity" indicates the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate insertions or deletions.
The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions/total number of positions multiplied by 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below.
Percent identity between a query nucleic acid sequence and a subject nucleic acid sequence is the "Identities" value, expressed as a percentage, which is calculated by the BLASTN algorithm when a subject nucleic acid sequence has 100% query coverage with a query nucleic acid sequence after a pair-wise BLASTN alignment is performed. Such pair- wise BLASTN alignments between a query nucleic acid sequence and a subject nucleic acid sequence are performed by using the default settings of the BLASTN algorithm available on the National Center for Biotechnology Institute's website with the filter for low complexity regions turned off. Importantly, a query nucleic acid sequence may be described by a nucleic acid sequence identified in one or more claims herein.
Percent identity between a query amino acid sequence and a subject amino acid sequence is the "Identities" value, expressed as a percentage, which is calculated by the BLASTP algorithm when a subject amino acid sequence has 100% query coverage with a query amino acid sequence after a pair-wise BLASTP alignment is performed. Such pair- wise BLASTP alignments between a query amino acid sequence and a subject amino acid sequence are performed by using the default settings of the BLASTP algorithm available on the National Center for Biotechnology Institute's website with the filter for low complexity regions turned off. Importantly, a query amino acid sequence may be described by an amino acid sequence identified in one or more claims herein.
In any embodiment of the invention herein, the ABP or antibody may have any one or all CDRs, VH, VL, with 99, 98, 97, 96, 95, 94, 93, 92, 91 , or 90 percent identity to the sequence shown or referenced, e.g., as defined by a SEQ ID NO. ABPs and Antibodies of the Invention that bind OX-40
ABPs and antibodies that bind human OX40 receptor are provided herein (i.e., an anti- OX40 ABP and an anti-human OX40 receptor (hOX-40R) antibody, sometimes referred to herein as an "anti-OX40 ABP or an anti- OX40 antibody" and/or other variations of the same). These antibodies are useful in the treatment or prevention of acute or chronic diseases or conditions whose pathology involves OX40 signalling. In one aspect, an antigen binding protein, or isolated human antibody or functional fragment of such protein or antibody, that binds to human OX40R and is effective as a cancer treatment or treatment against disease is described, for example in combination with another compound such as a TLR7/8 modulator or TLR7/8 agonist. Any of the antigen binding proteins or anti-OX40 antibodies disclosed herein may be used as a medicament. Any one or more of the antigen binding proteins or anti-OX40 antibodies may be used in the methods or compositions to treat cancer, e.g., those disclosed herein.
The isolated antibodies as described herein bind to OX40, and may bind to OX40 encoded from the following genes: NCBI Accession Number NP_003317, Genpept Accession Number P23510, or genes having 90 percent homology or 90 percent identity thereto.
The isolated antibody provided herein may further bind to the OX40 receptor having one of the following GenBank Accession Numbers: AAB39944, CAE11757, or AAI05071.
Antigen binding proteins and antibodies that bind and/or modulate OX-40 receptor are known in the art. Exemplary ABPs and antibodies of the invention are disclosed, for example in International Publication No. WO2013/028231 (PCT/US2012/024570), international filing date 9 Feb. 2012, and WO2012/027328 (PCT/US201 1/048752), international filing date 23 August 2011. In one embodiment, the OX-40 antigen binding protein is the one disclosed in
WO2012/027328 (PCT/US201 1/048752), international filing date 23 August 201 1. In another embodiment, the antigen binding protein comprises the CDRs of an antibody disclosed in WO2012/027328 (PCT/US2011/048752), international filing date 23 August 2011 , or CDRs with 90% identity to the disclosed CDR sequences. In a further
embodiment the antigen binding protein comprises a VH, a VL, or both of an antibody disclosed in WO2012/027328 (PCT/US2011/048752), international filing date 23 August 2011 , or a VH or a VL with 90% identity to the disclosed VH or VL sequences.
In another embodiment, the OX-40 antigen binding protein is one disclosed in
WO2013/028231 (PCT/US2012/024570), international filing date 9 Feb. 2012. In another embodiment, the antigen binding protein comprises the CDRs of an antibody disclosed in WO2013/028231 (PCT/US2012/024570), international filing date 9 Feb. 2012, or CDRs with 90% identity to the disclosed CDR sequences. In a further embodiment the antigen binding protein comprises a VH, a VL, or both of an antibody disclosed in WO2013/028231 (PCT/US2012/024570), international filing date 9 Feb. 2012, or a VH or a VL with 90% identity to the disclosed VH or VL sequences.
In another embodiment, the anti-OX40 ABP or antibody of the invention comprises one or more of the CDRs or VH or VL sequences, or sequences with 90% identity thereto, shown in the Figures herein. In one embodiment, the ABP or antibody of the invention comprises the CDRs of the 106- 222 antibody, e.g., of Figures 6-7 herein, e.g. CDRH1 , CDRH2, and CDRH3 having the amino acid sequence as set forth in SEQ ID NOs 1 , 2, and 3, as disclosed in Figure 6, and e.g. CDRL1 , CDRL2, and CDRL3 having the sequences as set forth in SEQ ID NOs 7, 8, and 9 respectively. In one embodiment, the ABP or antibody of the invention comprises the CDRs of the 106-222, Hu106 or Hu106-222 antibody as disclosed in WO2012/027328 (PCT/US201 1/048752), international filing date 23 August 2011. In a further embodiment, the anti-OX40 ABP or antibody of the invention comprises the VH and VL regions of the 106-222 antibody as shown in Figures 6-7 herein, e.g. a VH having an amino acid sequence as set forth in SEQ ID NO:4 and a VL as in Figure 7 having an amino acid sequence as set forth in SEQ ID NO: 10. In another embodiment, the ABP or antibody of the invention comprises a VH having an amino acid sequence as set forth in SEQ ID NO: 5 in Figure 6 herein, and a VL having an amino acid sequence as set forth in SEQ ID NO: 11 in Figure 7 herein. In a further embodiment, the anti-OX40 ABP or antibody of the invention comprises the VH and VL regions of the Hu106-222 antibody or the 106-222 antibody or the Hu106 antibody as disclosed in WO2012/027328 (PCT/US201 1/048752), international filing date 23 August 2011. In a further embodiment, the anti-OX40 ABP or antibody of the invention is 106-222, Hu106-222 or Hu106, e.g., as disclosed in
WO2012/027328 (PCT/US201 1/048752), international filing date 23 August 201 1. In a further embodiment, the ABP or antibody of the invention comprises CDRs or VH or VL or antibody sequences with 90% identity to the sequences in this paragraph.
In another embodiment, the anti-OX40 ABP or antibody of the invention comprises the CDRs of the 1 19-122 antibody, e.g. of Figures 10-1 1 herein, e.g., CDRH1 , CDRH2, and CDRH3 having the amino acid sequence as set forth in SEQ ID NOs 13, 14, and 15 respectively. In another embodiment, the anti-OX40 ABP or antibody of the invention comprises the CDRs of the 1 19-122 or Hu119 or Hu1 19-222 antibody as disclosed in WO2012/027328 (PCT/US201 1/048752), international filing date 23 August 201 1. In a further embodiment, the anti-OX40 ABP or antibody of the invention comprises a VH having an amino acid sequence as set forth in SEQ ID NO: 16 in Figure 10 herein, and a VL having the amino acid sequence as set forth in SEQ ID NO: 22 as shown in Figure 1 1 herein. In another embodiment, the anti-OX40 ABP or antibody of the invention comprises a VH having an amino acid sequence as set forth in SEQ ID NO: 17 and a VL having the amino acid sequence as set forth in SEQ ID NO: 23. In a further embodiment, the anti- OX40 ABP or antibody of the invention comprises the VH and VL regions of the 1 19-122 or Hu1 19 or Hu119-222 antibody as disclosed in WO2012/027328 (PCT/US201 1/048752), international filing date 23 August 2011. In a further embodiment, the ABP or antibody of the invention is 1 19-222 or Hu119 or Hu1 19-222 antibody, e.g. as disclosed in
WO2012/027328 (PCT/US201 1/048752), international filing date 23 August 201 1. In a further embodiment, the ABP or antibody of the invention comprises CDRs or VH or VL or antibody sequences with 90% identity to the sequences in this paragraph. In another embodiment, the anti-OX40 ABP or antibody of the invention comprises the CDRs of the 1 19-43-1 antibody, e.g., as shown in Figures 14-15 herein. In another embodiment, the anti-OX40 ABP or antibody of the invention comprises the CDRs of the 119-43-1 antibody as disclosed in WO2013/028231 (PCT/US2012/024570), international filing date 9 Feb. 2012. In a further embodiment, the anti-OX40 ABP or antibody of the invention comprises one of the VH and one of the VL regions of the 119-43-1 antibody as shown in Figures 14-17. In a further embodiment, the anti-OX40 ABP or antibody of the invention comprises the VH and VL regions of the 1 19-43-1 antibody as disclosed in WO2013/028231 (PCT/US2012/024570), international filing date 9 Feb. 2012. In a further embodiment, the ABP or antibody of the invention is 119-43-1 or 1 19-43-1 chimeric as disclosed in Figures 14-17 herein. In a further embodiment, the ABP or antibody of the invention as disclosed in WO2013/028231 (PCT/US2012/024570), international filing date 9 Feb. 2012. In further embodiments, any one of the ABPs or antibodies described in this paragraph are humanized. In further embodiments, any one of the any one of the ABPs or antibodies described in this paragraph are engineered to make a humanized antibody. In a further embodiment, the ABP or antibody of the invention comprises CDRs or VH or VL or antibody sequences with 90% identity to the sequences in this paragraph.
In another embodiment, further embodiment, any mouse or chimeric sequences of any anti-OX40 ABP or antibody of the invention are engineered to make a humanized antibody. In one embodiment, the anti-OX40 ABP or antibody of the invention comprises: (a) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1 ; (b) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2; (c) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO. 3; (d) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO. 7; (e) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO. 8; and (f) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO. 9.
In another embodiment, the anti-OX40 ABP or antibody of the invention comprises: (a) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 14; (c) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO. 15; (d) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO. 19; (e) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO. 20; and (f) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO. 21.
In another embodiment, the anti-OX40 ABP or antibody of the invention comprises: a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 13; a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2 or 14; and/or a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3 or 15, or a heavy chain variable region CDR having 90 percent identity thereto.
In another embodiment, the anti-OX40 ABP or antibody of the invention comprises: a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 7 or 19; a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 8 or 20 and/or a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 9 or 21 , or a heavy chain variable region having 90 percent identity thereto.
In another embodiment, the anti-OX40 ABP or antibody of the invention comprises: a light chain variable region ("VL") comprising the amino acid sequence of SEQ ID NO: 10, 11 , 22 or 23, or an amino acid sequence with at least 90 percent identity to the amino acid sequences of SEQ ID NO: 10, 11 , 22 or 23. In another embodiment, the anti-OX40 ABP or antibody of the invention comprises a heavy chain variable region ("VH") comprising the amino acid sequence of SEQ ID NO: 4, 5, 16 and 17, or an amino acid sequence with at least 90 percent identity to the amino acid sequences of SEQ ID NO: 4, 5, 16 and 17. In another embodiment, the anti-OX40 ABP or antibody of the invention comprises a variable heavy sequence of SEQ ID NO:5 and a variable light sequence of SEQ ID NO: 11 , or a sequence having 90 percent identity thereto. In another embodiment, the anti-OX40 ABP or antibody of the invention comprises a variable heavy sequence of SEQ ID NO: 17 and a variable light sequence of SEQ ID NO: 23 or a sequence having 90 percent identity thereto.
In another embodiment, the anti-OX40 ABP or antibody of the invention comprises a variable light chain encoded by the nucleic acid sequence of SEQ ID NO: 12, or 24, or a nucleic acid sequence with at least 90 percent identity to the nucleotide sequences of SEQ ID NO: 12 or 24. In another embodiment, the anti-OX40 ABP or antibody of the invention comprises a variable heavy chain encoded by a nucleic acid sequence of SEQ ID NO: 6 or 18, or a nucleic acid sequence with at least 90 percent identity to nucleotide sequences of SEQ ID NO: 6 or 18.
Also provided herein are monoclonal antibodies. In one embodiment, the monoclonal antibodies comprise a variable light chain comprising the amino acid sequence of SEQ ID NO: 10 or 22, or an amino acid sequence with at least 90 percent identity to the amino acid sequences of SEQ ID NO: 10 or 22. Further provided are monoclonal antibodies comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 4 or 16, or an amino acid sequence with at least 90 percent identity to the amino acid sequences of SEQ ID NO: 4 or 16. Another embodiment of the invention includes CDRs, VH regions, and VL regions, and antibodies and nucleic acids encoding the same as disclosed in the below Sequence Listing. TLR-7/8 modulators
TLR7/8 modulators are known in the art. Such modulators bind TLR7/8. Some TLR7/8 modulators of the invention engage TLR7/8, optionally agonize TLR7/8, or initiate one or more molecular signaling events through TLR7/8. In one embodiment, a combination of the invention comprises an anti-OX40 ABP or antibody and a TLR7/8 modulator. In a further embodiment, the TLR7/8 modulator is a TLR7/8 agonist. In a further embodiment, the TLR7/8 modulator of the invention is selected from the group consisting of oxoadenines, lipidated oxoadenines, imidazoquinolines, and lipidated imadazoquinolines.
Oxoadenines In one embodiment, the TLR7/8 modulator is an oxoadenine compound, i.e., an oxoadenine compound of the invention. Oxoadenine compounds are known in the art. Oxoadenines are disclosed in US Patent No. 8,563 717 and US Publication No. US201 10144136. In certain embodiments, the TLR 7/8 modulator in a combination of the invention is an oxoadenine compound disclosed US Patent No. 8,563 717 and US Publication No. US201 10144136.
In one embodiment, the TLR7/8 modulator is an oxoadenine a compound of formula (I):
Figure imgf000022_0001
R is Ci.6alkylamino, or Ci.6alkoxy;
R2 is a group having the structure:
Figure imgf000022_0002
n is an integer having a value of 1 to 6; Het is a 6-membered saturated heterocycle containing one nitrogen atom wherein Het is attached to the -(CH2)n- moiety at any carbon atom of the heterocycle;
R3 is hydrogen, Ci_8alkyl, or C3.7cycloalkylCo-6alkyl;
or a pharmaceutically acceptable salt thereof.
In one embodiment, R is n-butyloxy. In another embodiment, R is (1 S)-1-methylbutoxy. In another embodiment, R is n-butylamino. In another embodiment, R is (1- methylethyl)oxy.
In one embodiment, n is 1 . In another embodiment, n is 2. In another embodiment, n is 3. In another embodiment, n is 4. In another embodiment, n is 5. In another embodiment, n is 6. In another embodiment, n is 2, 3, or 4.
In one embodiment, Het is attached to the -(CH2)n- moiety at the 2-position of the heterocycle. In another embodiment, Het is attached to the -(CH2)n- moiety at the 3- position of the heterocycle. In another embodiment, Het is attached to the -(CH2)n- moiety at the 4-position of the heterocycle. In another embodiment, when Het is attached to the - (CH2)n- moiety at the 3-position of the heterocycle, then the stereochemistry at the 3- position of the heterocycle is (R,S). In another embodiment, when Het is attached to the - (CH2)n- moiety at the 3-position of the heterocycle, then the stereochemistry at the 3- position of the heterocycle is (R). In another embodiment, when Het is attached to the - (CH2)n- moiety at the 3-position of the heterocycle, then the stereochemistry at the 3- position of the heterocycle is (S).
In a one embodiment, R3 is ethyl. In another embodiment, R3 is a hydrogen atom. In another embodiment, R3 is n-propyl. In another embodiment, R3 is 1 -methylethyl. In another embodiment, R3 is n-butyl. In another embodiment, R3 is 2-methylpropyl. In another embodiment, R3 is 3-methylbutyl. In another embodiment, R3 is cyclopentyl. In another embodiment, R3 is cyclopentyl methyl. In another embodiment, R3 is 2- cyclohexylethyl. In another embodiment, R3 is 1-ethylpropyl. In another embodiment, R3 is cyclohexyl. In a further aspect, there are provided compounds of formula (Γ):
Figure imgf000024_0001
wherein;
R is Ci_6alkylafnino, or d_6alkoxy; R2 is a group having the structure:
Figure imgf000024_0002
n' is an integer having a value of 1 to 6;
Het' is a 6-membered saturated heterocycle containing one nitrogen atom wherein Het' is attached to the -(CH2)n - moiety at any carbon atom of the heterocycle;
R3 is hydrogen, Ci_8alkyl, or C3-7cycloalkylCo-6alkyl;
and salts thereof;
with the proviso that 2-butoxy-7,8-dihydro-9-[2-(piperidin-2-yl)ethyl]-8-oxoadenine is excluded.
In one embodiment, R is n-butyloxy. In another embodiment, R is (1 S)-1-methylbutoxy. In another embodiment, R is n-butylamino. In another embodiment, R is (1- methylethyl)oxy.
In one embodiment, n' is 1. In another embodiment, n' is 2. In another embodiment, n' is 3. In another embodiment, n' is 4. In another embodiment, n' is 5. In another
embodiment, n' is 6. In another embodiment, n' is 2, 3, or 4. In one embodiment, Het' is attached to the -(CH2)n- moiety at the 2-position of the heterocycle. In another embodiment, Het' is attached to the -(CH2)n- moiety at the 3- position of the heterocycle. In another embodiment, Het' is attached to the -(CH2)n - moiety at the 4-position of the heterocycle. In another embodiment, when Het' is attached to the - (CH2)n - moiety at the 3-position of the heterocycle, then the stereochemistry at the 3- position of the heterocycle is (R,S). In another embodiment, when Het' is attached to the - (CH2)n- moiety at the 3-position of the heterocycle, then the stereochemistry at the 3- position of the heterocycle is (R). In another embodiment, when Het' is attached to the - (CH2)n- moiety at the 3-position of the heterocycle, then the stereochemistry at the 3- position of the heterocycle is (S).
In one embodiment, R3 is ethyl. In another embodiment, R3 is a hydrogen atom. In another embodiment, R3 is n-propyl. In another embodiment, R3 is 1-methylethyl. In another embodiment, R3 is n-butyl. In another embodiment, R3 is 2-methylpropyl. In another embodiment, R3 is 3-methylbutyl. In another embodiment, R3 is cyclopentyl. In another embodiment, R3 is cyclopentylmethyl. In another embodiment, R3 is 2- cyclohexylethyl. In another embodiment, R3 is 1 -ethyl propyl. In another embodiment, R3 is cyclohexyl.
In other embodiments, the TLR7/8 modulators, or salts thereof, are selected from the group consisting of: 6-amino-2-butoxy-9-[(1-ethyl-3-piperidinyl)methyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butylamino)-9-[(1-ethyl-3-piperidinyl)methyl]-7,9-dihydro-8/-/-purin-8-one;
2-(butyloxy)-9-(4-piperidinylmethyl)-9/-/-purin-6-amine;
2-(butyloxy)-9-[(3S)-3-piperidinylmethyl]-9/-/-purin-6-amine;
2-(butyloxy)-9-[(3f?)-3-piperidinylmethyl]-9/-/-purin-6-amine;
2-(butyloxy)-9-[2-(4-piperidinyl)ethyl]-9/-/-purin-6-amine;
2-(butyloxy)-9-[(1-ethyl-4-piperidinyl)methyl]-9/-/-purin-6-amine;
2-(butyloxy)-9-[2-(1-ethyl-4-piperidinyl)ethyl]-9/-/-purin-6-amine;
2-(butyloxy)-9-[3-(1-ethyl-4-piperidinyl)propyl]-9/-/-purin-6-amine;
6-amino-2-(butyloxy)-9-[4-(1-ethyl-4-piperidinyl)butyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-[2-(2-piperidinyl)ethyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-[2-(1-ethyl-2-piperidinyl)ethyl]-7,9-dihydro-8/-/-purin-8-one; 6-amino-2-(buty oxy)-9-[3-(2-piperidinyl)propyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(buty oxy)-9-[3-(1-ethyl-2-piperidinyl)propyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(buty oxy)-9-[3-(1-propyl-2-piperidinyl)propyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(buty oxy)-9-{3-[1-(1-methylethyl)-2-piperidinyl]propyl}-7,9-dihydro-8/-/-purin-8- one;
6-amino-2-(buty oxy)-9-[3-(1-butyl-2-piperidinyl)propyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(buty oxy)-9-{3-[1-(2-methylpropyl)-2-piperidinyl]propyl}-7,9-dihydro-8/-/-purin-8- one;
6-amino-2-(buty oxy)-9-{3-[1-(3-methylbutyl)-2-piperidinyl]propyl}-7,9-dihydro-8/-/-purin-8- one;
6-amino-2-(buty oxy)-9-[3-(1-cyclopentyl-2-piperidinyl)propyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(buty oxy)-9-{3-[1-(cyclopentylmethyl)-2-piperidinyl]propyl}-7,9-dihydro-8/-/- purin-8-one;
6-amino-2-(buty oxy)-9-{3-[1-(2-cyclohexylethyl)-2-piperidinyl]propyl}-7,9-dihydro-8/-/-purin- 8-one;
6-amino-2-(buty oxy)-9-[(1-propyl-3-piperidinyl)methyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(buty oxy)-9-{[1-(1-methylethyl)-3-piperidinyl]methyl}-7,9-dihydro-8/-/-purin-8- one;
6-amino-2-(buty oxy)-9-[(1-butyl-3-piperidinyl)methyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(buty oxy)-9-{[1-(2-methylpropyl)-3-piperidinyl]methyl}-7,9-dihydro-8/-/-purin-8- one;
6-amino-2-(buty oxy)-9-{[1-(3-methylbutyl)-3-piperidinyl]methyl}-7,9-dihydro-8/-/-purin-8- one;
6-amino-2-(buty oxy)-9-[(1-cyclopentyl-3-piperidinyl)methyl]-7,9-dihydro-8H-purin-8-one; 6-amino-2-(buty oxy)-9-[2-(3-piperidinyl)ethyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(buty oxy)-9-[2-(1-ethyl-3-piperidinyl)ethyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(buty oxy)-9-[2-(1-propyl-3-piperidinyl)ethyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(buty oxy)-9-{2-[1-(1-methylethyl)-3-piperidinyl]ethyl}-7,9-dihydro-8/-/-purin-8- one;
6-amino-2-(buty oxy)-9-[2-(1-butyl-3-piperidinyl)ethyl]-7,9-dihydro-8/-/-purin-8-one; 6-amino-2-(butyloxy)-9-{2-[1-(2-methylpropyl)-3-piperidinyl]ethyl}-7,9-dihydro-8H-purin-8^ one;
6-amino-2-(butyloxy)-9-{2-[1-(1-ethylpropyl)-3-piperidinyl]ethyl}-7,9-dihydro-8/-/-purin-8- one;
6-amino-2-(butyloxy)-9-[2-(1-cyclopentyl-3-piperidinyl)ethyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-{2-[1-(cyclopentylmethyl)-3-piperidinyl]ethyl}-7,9-dihydro-8/-/-purin- 8-one;
6-amino-2-(butyloxy)-9-[2-(1-cyclohexyl-3-piperidinyl)ethyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-{2-[1-(2-cyclohexylethyl)-3-piperidinyl]ethyl}-7,9-dihydro-8/-/-purin- 8-one;
6-amino-2-{[(1 S)-1-methylbutyl]oxy}-9-[2-(3-piperidinyl)ethyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-9-[2-(1-ethyl-3-piperidinyl)ethyl]-2-{[(1 S)-1-methylbutyl]oxy}-7,9-dihydro-8/-/-purin- 8-one;
6-amino-2-{[(1 S)-1-methylbutyl]oxy}-9-{2-[1-(1-methylethyl)-3-piperidinyl]ethyl}-7,9-dihydro- 8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-[3-(3-piperidinyl)propyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-[3-(1-ethyl-3-piperidinyl)propyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-[3-(1-propyl-3-piperidinyl)propyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-{3-[1-(1-methylethyl)-3-piperidinyl]propyl}-7,9-dihydro-8/-/-purin-8- one;
6-amino-2-(butyloxy)-9-[3-(1-butyl-3-piperidinyl)propyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-{3-[1-(2-methylpropyl)-3-piperidinyl]propyl}-7,9-dihydro-8/-/-purin-8- one;
6-amino-2-(butyloxy)-9-{3-[1-(3-methylbutyl)-3-piperidinyl]propyl}-7,9-dihydro-8/-/-purin-8- one;
6-amino-2-(butyloxy)-9-[3-(1-cyclopentyl-3-piperidinyl)propyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-{3-[1-(cyclopentylmethyl)-3-piperidinyl]propyl}-7,9-dihydro-8/-/- purin-8-one;
6-amino-2-(butyloxy)-9-{3-[1-(2-cyclohexylethyl)-3-piperidinyl]propyl}-7,9-dihydro-8/-/-purin- 8-one;
6-amino-2-{[(1 S)-1-methylbutyl]oxy}-9-[3-(3-piperidinyl)propyl]-7,9-dihydro-8/-/-purin-8-one; 6-amino-9-[3-(1-ethyl-3-piperidinyl)pro
purin-8-one;
6-amino-2-(butyloxy)-9-[4-(3-piperidinyl)butyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-[4-(1-ethyl-3-piperidinyl)butyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-[4-(1-propyl-3-piperidinyl)butyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-{4-[1-(1-methylethyl)-3-piperidinyl]butyl}-7,9-dihydro-8/-/-purin-8- one;
6-amino-2-(butyloxy)-9-[4-(1-butyl-3-piperidinyl)butyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-{4-[1-(2-methylpropyl)-3-piperidinyl]butyl}-7,9-dihydro-8/-/-purin-8- one;
6-amino-2-(butyloxy)-9-{4-[1-(3-methylbutyl)-3-piperidinyl]butyl}-7,9-dihydro-8/-/-purin-8- one;
6-amino-2-(butyloxy)-9-[4-(1-cyclopentyl-3-piperidinyl)butyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-{4-[1-(cyclopentylmethyl)-3-piperidinyl]butyl}-7,9-dihydro-8/-/-purin- 8-one;
6-amino-2-(butyloxy)-9-{4-[1-(2-cyclohexylethyl)-3-piperidinyl]butyl}-7,9-dihydro-8/-/-purin- 8-one;
6-amino-2-{[(1 S)-1-methylbutyl]oxy}-9-[4-(3-piperidinyl)butyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-9-[4-(1-ethyl-3-piperidinyl)butyl]-2-{[(1 S)-1-methylbutyl]oxy}-7,9-dihydro-8/-/-purin- 8-one;
6-amino-2-(butyloxy)-9-[5-(3-piperidinyl)pentyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-[5-(1-ethyl-3-piperidinyl)pentyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-{5-[1-(1-methylethyl)-3-piperidinyl]pentyl}-7,9-dihydro-8/-/-purin-8- one;
6-amino-2-(butyloxy)-9-[(1-propyl-4-piperidinyl)methyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-{[1-(1-methylethyl)-4-piperidinyl]methyl}-7,9-dihydro-8/-/-purin-8- one;
6-amino-2-(butyloxy)-9-[(1-butyl-4-piperidinyl)methyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-{[1-(2-methylpropyl)-4-piperidinyl]methyl}-7,9-dihydro-8/-/-purin-8- one;
6-amino-2-(butyloxy)-9-{[1-(3-methylbutyl)-4-piperidinyl]methyl}-7,9-dihydro-8/-/-purin-8- one; 6-amino-2-(butyloxy)-9-[(1-cyclopentyl-4-piperidinyl)methyl]-7,9-dihydro-8H-purin-8-one;
6-amino-2-(butyloxy)-9-[(1-cyclohexyl-4-piperidinyl)methyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-{[(1 S)-1-methylbutyl]oxy}-9-(4-piperidinylmethyl)-7,9-dihydro-8/-/-purin-8-one;
6-amino-9-[(1-ethyl-4-piperidinyl)methyl]-2-{[(1 S)-1-methylbutyl]oxy}-7,9-dihydro-8H-purin- 8-one;
6-amino-2-{[(1 S)-1-methylbutyl]oxy}-9-{[1-(1-m^
8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-[2-(1-propyl-4-piperidinyl)ethyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-{2-[1-(1-methylethyl)-4-piperidinyl]ethyl}-7,9-dihydro-8/-/-purin-8- one;
6-amino-2-(butyloxy)-9-[2-(1-butyl-4-piperidinyl)ethyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-{2-[1-(2-methylpropyl)-4-piperidinyl]ethyl}-7,9-dihydro-8/-/-purin-8- one;
6-amino-2-(butyloxy)-9-{2-[1-(1-ethylpropyl)-4-piperidinyl]ethyl}-7,9-dihydro-8/-/-purin-8- one;
6-amino-2-(butyloxy)-9-{2-[1-(3-methylbutyl)-4-piperidinyl]ethyl}-7,9-dihydro-8/-/-purin-8- one;
6-amino-2-(butyloxy)-9-[2-(1-cyclopentyl-4-piperidinyl)ethyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-{2-[1-(cyclopentylmethyl)-4-piperidinyl]ethyl}-7,9-dihydro-8/-/-purin- 8-one;
6-amino-2-(butyloxy)-9-[2-(1-cyclohexyl-4-piperidinyl)ethyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-{2-[1-(2-cyclohexylethyl)-4-piperidinyl]ethyl}-7,9-dihydro-8/-/-purin- 8-one;
6-amino-2-{[(1 S)-1-methylbutyl]oxy}-9-[2-(4-piperidinyl)ethyl]-7,9-dihydro-8/-/-purin-8-one; 6-amino-9-[2-(1-ethyl-4-piperidinyl)ethyl]-2-{[(1 S)-1-methylbutyl]oxy}-7,9-dihydro-8/-/-purin- 8-one;
6-amino-2-{[(1 S)-1-methylbutyl]oxy}-9-{2-[1-(1-methylethyl)-4-piperidinyl]ethyl}-7,9-dihydro- 8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-[3-(4-piperidinyl)propyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-[3-(1-propyl-4-piperidinyl)propyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-{3-[1-(1-methylethyl)-4-piperidinyl]propyl}-7,9-dihydro-8/-/-purin-8- one; 6-amino-2-(butyloxy)-9-[3-(1-butyl-4-piperidinyl)propyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-{3-[1-(2-methylpropyl)-4-piperidinyl]propyl}-7,9-dihydro-8/-/-purin-8- one;
6-amino-2-(butyloxy)-9-{3-[1-(1-ethylpropyl)-4-piperidinyl]propyl}-7,9-dihydro-8/-/-purin-8- one;
6-amino-2-(butyloxy)-9-{3-[1-(3-methylbutyl)-4-piperidinyl]propyl}-7,9-dihydro-8/-/-purin-8- one;
6-amino-2-(butyloxy)-9-[3-(1-cyclopentyl-4-piperidinyl)propyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-{3-[1-(cyclopentylmethyl)-4-piperidinyl]propyl}-7,9-dihydro-8/-/- purin-8-one;
6-amino-2-(butyloxy)-9-[3-(1-cyclohexyl-4-piperidinyl)propyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-{3-[1-(2-cyclohexylethyl)-4-piperidinyl]propyl}-7,9-dihydro-8/-/-purin- 8-one;
6-amino-2-{[(1 S)-1-methylbutyl]oxy}-9-[3-(4-piperidinyl)propyl]-7,9-dihydro-8/-/-purin-8-one; 6-amino-9-[3-(1-ethyl-4-piperidinyl)propyl]-2-{[(1 S)-1-methylbutyl]oxy}-7,9-dihydro-8/-/- purin-8-one;
6-amino-2-{[(1 S)-1-methylbutyl]oxy}-9-{3-[1-(1-methylethyl)-4-piperidinyl]propyl}-7,9- dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-[4-(4-piperidinyl)butyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-[4-(1-propyl-4-piperidinyl)butyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-{4-[1-(1-methylethyl)-4-piperidinyl]butyl}-7,9-dihydro-8/-/-purin-8- one;
6-amino-2-(butyloxy)-9-[4-(1-butyl-4-piperidinyl)butyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-{4-[1-(2-methylpropyl)-4-piperidinyl]butyl}-7,9-dihydro-8/-/-purin-8- one;
6-amino-2-(butyloxy)-9-{4-[1-(1-ethylpropyl)-4-piperidinyl]butyl}-7,9-dihydro-8/-/-purin-8- one;
6-amino-2-(butyloxy)-9-{4-[1-(3-methylbutyl)-4-piperidinyl]butyl}-7,9-dihydro-8/-/-purin-8- one;
6-amino-2-(butyloxy)-9-[4-(1-cyclopentyl-4-piperidinyl)butyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-{4-[1-(cyclopentylmethyl)-4-piperidinyl]butyl}-7,9-dihydro-8/-/-purin- 8-one; 6-amino-2-(butyloxy)-9-[4-(1-cyclohexyl-4-piperidinyl)butyl]-7,9-dihydro-8H-purin-8-one;
6-amino-2-(butyloxy)-9-{4-[1-(2-cyclohexylethyl)-4-piperidinyl]butyl}-7,9-dihydro-8/-/-purin- 8-one;
6-amino-2-{[(1 S)-1-methylbutyl]oxy}-9-[4-(4-piperidinyl)butyl]-7,9-dihydro-8/-/-purin-8-one; 6-amino-9-[4-(1-ethyl-4-piperidinyl)butyl]-2-{[(1 S)-1-methylbutyl]oxy}-7,9-dihydro-8/-/-purin- 8-one;
6-amino-2-(butylamino)-9-[4-(4-pipendinyl)butyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butylamino)-9-[4-(1-ethyl-4-piperidinyl)butyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-[5-(4-piperidinyl)pentyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-[5-(1-ethyl-4-piperidinyl)pentyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-[5-(1-propyl-4-piperidinyl)pentyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-{5-[1-(1-methylethyl)-4-piperidinyl]pentyl}-7,9-dihydro-8/-/-purin-8- one;
6-amino-2-(butyloxy)-9-[5-(1-butyl-4-piperidinyl)pentyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-{5-[1-(2-methylpropyl)-4-piperidinyl]pentyl}-7,9-dihydro-8/-/-purin-8- one;
6-amino-2-(butyloxy)-9-{5-[1-(3-methylbutyl)-4-piperidinyl]pentyl}-7,9-dihydro-8/-/-purin-8- one;
6-amino-2-(butyloxy)-9-[5-(1-cyclopentyl-4-piperidinyl)pentyl]-7,9-dihydro-8/-/-purin-8-one; 6-amino-2-(butyloxy)-9-{5-[1-(cyclopentylmethyl)-4-piperidinyl]pentyl}-7,9-dihydro-8/-/-purin- 8-one;
6-amino-2-(butyloxy)-9-{5-[1-(2-cyclohexylethyl)-4-piperidinyl]pentyl}-7,9-dihydro-8/-/-purin- 8-one;
6-amino-2-(butyloxy)-9-[6-(4-piperidinyl)hexyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-[6-(1-ethyl-4-piperidinyl)hexyl]-7,9-dihydro-8/-/-purin-8-one;
6-amino-2-(butyloxy)-9-{6-[1-(1-methylethyl)-4-piperidinyl]hexyl}-7,9-dihydro-8/-/-purin-8- one, and;
6-amino-2-[(1-methylethyl)oxy]-9-[4-(4-piperidinyl)butyl]-7,9-dihydro-8/-/-purin-8-one.
In any embodiment herein, references to 'alkyl' include references to both straight-chain and branched-chain aliphatic isomers of the corresponding alkyl containing up to eight carbon atoms, for example up to six carbon atoms, or up to four carbon atoms ,or up to two carbon atoms, or one carbon atom. Such references to 'alkyl' are also applicable when an alkyl group is part of another group, for example an alkylamino or alkoxy group. Examples of such alkyl groups and groups containing alkyl groups are Ci_8 alkyl, Ci-6alkyl, Ci.6alkylamino, and Ci.6alkoxy. In any embodiment herein, references to 'cycloalkyl' refer to monocyclic alkyl groups containing between three and seven carbon atoms, for example three carbon atoms, or five carbon atoms, or six carbon atoms. Examples of such cycloalkyl groups are cyclopropyl, cyclopentyl, and cyclohexyl.
In any embodiment herein, references to 'heterocycle' or 'heterocyclyl' refer to a monocyclic saturated heterocyclic aliphatic ring containing 6 carbon atoms and one heteroatom, which heteroatom is nitrogen. Such a heterocyclic ring is piperidine or piperidinyl.
In any embodiment herein, references to 'halogen' refer to iodine, bromine, chlorine or fluorine, typically bromine, chlorine, or fluorine. References to 'halo' refer to iodo, bromo, chloro or fluoro, typically bromo, chloro, or fluoro.
In one embodiment, the TLR7/8 modulators have beneficial characteristics, e.g. inducing predominantly TLR7 activity. In other embodiments, the TLR7/8 modulators induce predominantly TLR8 activity. Beneficial cytokine profiles are described below.
In one embodiment, the TLR7/8 modulator is CRX-692, having a formula shown below. In other embodiments, the TLR7/8 modulator is CRX-672, having a formula shown below. In other embodiments, the TLR7/8 modulator is CRX-677, having a formula shown below. In other embodiments, the TLR7/8 modulator is CRX-648, having a formula shown below. In other embodiments, the TLR7/8 modulator is CRX-649, having a formula shown below. In other embodiments, the TLR7/8 modulator is CRX-706, having a formula shown below. In other embodiments, the TLR7/8 modulator is CRX-705, having a formula shown below.
Figure imgf000033_0001
It is to be understood that references herein to oxoadenine compounds of the invention mean a compound of formula (I) as the free base, or as a salt, for example a
pharmaceutically acceptable salt.
Lipidated oxoadenines Other TLR 7/8 modulators include oxoadenines that have been modified, specifically lipidated, e.g., in order to facilitate uptake into immune cells.
Lipidated oxoadenines are known in the art, for example such compounds are disclosed in PCT Application No. US20120044708 filed August 6, 2010 and in US Application No. 13/389,307. Thus in one embodiment, the TLR7/8 modulator in a combination of the invention is a compound disclosed in PCT Application No. US20120044708 filed August 6, 2010 and in US Application No. 13/389,307.
Methods of lipidating known TLR7/8 compounds are also known in the art. Thus, in another embodiment, the TLR 7/8 modulator is a lipidated version of any of the above described oxoadenines. In another embodiment, the TLR7/8 modulator is a nitrogen heterocyclyl-substituted oxoadenine molecule (e.g. any of the above described nitrogen heterocyclyl-substituted oxoadenine molecules) covalently linked to a phospho- or phosphonolipid group. In another embodiment, the TLR7/8 modulator is a lipidated oxoadenine of Formula (II):
Figure imgf000034_0001
wherein
R = C1-6alkyl, C1-6alkylamino, C1-6alkoxy, C3-6cycloalkylC1-6alkyl, C3-6cycloalkylC1- 6alkylamino, C3-6cycloalkylC1-6alkoxy, C1-6alkoxyC1-6alkyl, C1-6alkoxyC1-6alkylamino, C1-6alkoxyC1-6alkoxy; branched or unbranched and optionally terminally substituted with a hydroxyl, amino, thio, hydrazino, hydrazido, azido, acetylenyl, carboxyl, or maleimido group n = 0-6
Het is a four-, five-, or six-membered saturated heteocycle wherein X, Y = CH or N, and at least one of X and Y is a nitrogen atom Q = O, NH, or covalent bond Z = O, CH2, CF2 or covalent bond W = 0, S m = 0-6 p = 1 or 2
Figure imgf000034_0002
wherein R2 = H or straight/branched/unsaturated C4-C24 alkyl or acyl
R3 = straightybranched/unsaturated C4-C24 alkyl or acyl or a pharmaceutically acceptable salt thereof.
In one embodiment, the compounds of the subject invention are described generally by Formula III:
Figure imgf000035_0001
wherein R = C1-6alkyl, C1-6alkylamino, C1-6alkoxy, C3-6cycloalkylC1-6alkyl, C3-6cycloalkylC1- 6alkylamino, C3-6cycloalkylC1-6alkoxy, C1-6alkoxyC1-6alkyl, C1-6alkoxyC1-6alkylamino, C1-6alkoxyC1-6alkoxy; branched or unbranched. n = 0-6
X = CH or N Q = O, NH, or covalent bond
Z = O, CH2, CF2 or covalent bond
W = 0, S m = 0-6 p = 1 or 2 R2 = H or straight/branched/unsaturated C4-C24 alkyl or acyl
R3 = straight/branched/unsaturated C4-C24 alkyl or acyl In one embodiment the TLR7/8 modulator is a lipidated oxoadenine CRX-682 having the following structure:
Figure imgf000036_0001
In one embodiment the TLR7/8 modulator is a lipidated oxoadenine CRX-681 having the following structure:
Figure imgf000036_0002
Figure imgf000036_0003
Imadazoquinolines (IQs). Imadazoquinolones are known in the art as TLR7/8 modulators, e.g. agonists. In one embodiment of the invention, the TLR7/8 modulator is an IQ compound. Known IQs include imiquimod, resiquimod, and gardiquimod, shown below. These and other substituted imadazoquinolines are shown in J. Med. Chem., 2014, 57 (2), pp 339-347. In another embodiment of the invention, the TLR 7/8 modulator is a 1 H-imidazo[4,5-c]quinolines. In a further embodiment, the 1 H-imidazo[4,5-c]quinolines is one disclosed in J. Med. Chem., 2014, 57 (2), pp 339-347. In a further embodiment, the TLR7/8 modulator is selected from the group consisting of imiquimod, resiquimod, and gardiquimod. In a further embodiment the TLR7/8 is a substituted imadazoquinoline, e.g. imiquimod, resiquimod, or gardiquimod, wherein such substitution results in a cytokine profile for said substituted imadazoquinoline with increased IL-12 production.
Figure imgf000037_0001
Lipidated imadazoquinolines (IQs). In a further aspect of the invention, the
imidazoquinolines are lipidated, i.e. convalently linked or conjugated to a phospho- or phosphonolipid, e.g. in order to facilitate uptake into immune cells. Thus, in one embodiment, the TLR7/8 modulator in a combination of the invention is a lipidated imadazoquinolone. Lipidated IQs are disclosed in US Patent No. 8,624,029, and US Publication No. US201 10282061 , and PCT Application PCT/US2009/61867 filed Oct. 23, 2009. In one embodiment, the TLR7/8 modulator in a combination of the invention is a compound disclosed in US Patent No. 8,624,029, US Publication No. US20110282061 , or PCT Application PCT/US2009/61867 filed Oct. 23, 2009.
Methods of lipidating known TLR7/8 compounds are also known in the art. Thus, in another embodiment, the TLR 7/8 modulator is a lipidated version of any of the above described imadazoquinolones. In another embodiment, the TLR7/8 modulator is an imadazoquinolone covalently linked to a phospho- or phosphonolipid group. In another embodiment, the lipidated imadazoquinoline of the invention is a compound of Formula IV:
Figure imgf000038_0001
wherein
Ri = H, Ci-6 alkyl, Ci-3 alkoxyCi.3 alkyl, C3.6cycloalkylCi-3alkoxy, C3.6cycloalkylCi-3alkyl. . . n = 1-6 Y = O, NH X = O, CH2, CF2 W = O or S m = 1-2,
R2 = H or straight/branched/unsaturated C4-C24 alkyl or acyl
R3 = straight/branched/unsaturated C4-C24 alkyl or acyl (e.g. phosphatidyl,
lysophosphatidyl ether or ester when W=0, X=0, m= 1) In further embodiments, the TLR7/8 modulator is a lipidated imadazoquinoline of Formula IV with a substitution shown in Table 2. In other embodiments, the lipidated
imadazoquinoline of Formula IV is chosen from: CRX-664, CRX-650, CRX-652, CRX-651 , CRX-665, and CRX-658.
TABLE 2
Example Ref. CRX# Ri n m
No.
1 — — — — —
2 — 664 — — — 3 L1 650 H 2 1
4 L2 652 n-Bu 2 1
5 L3 651 CH2OEt 2 1
6 L4 665 CH2OEt 4 1
7 — — — — —
8 L5 658 CH2OEt 2 2
For all Examples shown: Y = W = X = O; R2 = R3 = hexadecanoyl
TLR-4 modulators
The combinations of the invention comprise TLR-4 modulators, that is molecules that modulate TLR4, for example by binding and initiating conformational changes or signaling by engaging TLR4. In certain embodiments, the combinations comprise an anti-OX40 ABP, a TLR 7/8 modulator, and a TLR4 modulator.
In one embodiment, the TLR-4 modulators are aminoalkyl glucosaminide phosphate compounds (AGPs). Toll-like receptor 4 recognizes bacterial LPS (lipopolysaccharide) and when activated initiates an innate immune response. AGPs are a monosaccharide mimetic of the lipid A protein of bacterial LPS and have been developed with ether and ester linkages on the "acyl chains" of the compound. Processes for making these compounds are known and disclosed, for example, in WO 2006/016997, U.S. Patent Nos. 7,288,640 and 6, 113,918, and WO 01/90129. Other AGPs and related processes are disclosed in U.S. Patent No. 7, 129,219, U.S. Patent No. 6,525,028 and U.S. Patent No 6,911 ,434. AGPs with ether linkages on the acyl chains employed in the composition of the invention are known and disclosed in WO 2006/016997. Of particular interest, are the aminoalkyl glucosaminide phosphate compounds set forth and described according to Formula (III) at paragraphs [0019] through [0021] in WO 2006/016997.
Aminoalkyl glucosaminide phosphate compounds employed in the present invention have the structure set forth in Formula 1 as follows:
Figure imgf000040_0001
wherein
m is 0 to 6
n is 0 to 4;
X is O or S, preferably O;
Y is O or NH;
Z is O or H;
each R1 , R2, R3 is selected independently from the group consisting of a C1-20 acyl and a C1-20 alkyl;
R4 is H or Me;
R5 is selected independently from the group consisting of -H, -OH, -(CI-C4) alkoxy, -P03R8R9, -OP03R8R9, -S03R8, -OS03R8, -NR8R9, -SR8, -CN, -N02, -CHO, - C02R8, and -CONR8R9, wherein R8 and R9 are each independently selected from H and (CI-C4) alkyl; and
each R6 and R7 is independently H or P03H2.
In Formula 1 the configuration of the 3' stereogenic centers to which the normal fatty acyl residues (that is, the secondary acyloxy or alkoxy residues, e.g., R10, R20, and R30) are attached is R or S, preferably R (as designated by Cahn-lngold-Prelog priority rules). Configuration of aglycon stereogenic centers to which R4 and R5 are attached can be R or S. All stereoisomers, both enantiomers and diastereomers, and mixtures thereof, are considered to fall within the scope of the present invention. The number of carbon atoms between heteroatom X and the aglycon nitrogen atom is determined by the variable "n", which can be an integer from 0 to 4, preferably an integer from 0 to 2.
The chain length of normal fatty acids R1 , R2, and R3 can be from about 6 to about 16 carbons, preferably from about 9 to about 14 carbons. The chain lengths can be the same or different. Some embodiments include chain lengths where R1 , R2 and R3 are 6 or 10 or 12 or 14.
Formula 1 encompasses L/D-seryl, -threonyl, -cysteinyl ether and ester lipid AGPs, both agonists and antagonists and their homologs (n=1-4), as well as various carboxylic acid bioisosteres (i.e, R5 is an acidic group capable of salt formation; the phosphate can be either on 4- or 6- position of the glucosamine unit, but preferably is in the 4-position).
In a one embodiment of the invention employing an AGP compound of Formula 1 , n is 0, R5 is C02H, R6 is P03H2, and R7 is H. This AGP compound is set forth as the structure in Formula 1a as follows:
Figure imgf000041_0001
(Formula 1a) wherein X is O or S; Y is O or NH; Z is O or H; each R1 , R2, R3 is selected independently from the group consisting of a C1-20 acyl and a C1-20 alkyl; and R4 is H or methyl.
In Formula 1a the configuration of the 3' stereogenic centers to which the normal fatty acyl residues (that is, the secondary acyloxy or alkoxy residues, e.g., R10, R20, and R30) are attached as R or S, preferably R (as designated by Cahn-lngold-Prelog priority rules). Configuration of aglycon stereogenic centers to which R4 and C02H are attached can be R or S. All stereoisomers, both enantiomers and diastereomers, and mixtures thereof, are considered to fall within the scope of the present invention. Formula 1a encompasses L/D-seryl, -threonyl, -cysteinyl ether or ester lipid AGPs, both agonists and antagonists.
In both Formula 1 and Formula 1 a, Z is O attached by a double bond or two hydrogen atoms which are each attached by a single bond. That is, the compound is ester-linked when Z=Y=0; amide-linked when Z =0 and Y=NH; and ether-linked when Z=H/H and Y=0.
Two compounds of Formula 1 are referred to as CRX-601 and CRX-527. Their structures are set forth as follows:
Figure imgf000042_0001
(CRX-601)
Figure imgf000043_0001
Additionally, another embodiment employs CRX-547 having the structure shown. CRX-547
Figure imgf000044_0001
Still other embodiments include AGPs such as CRX-602 or CRX-526 providing increased stability to AGPs having shorter secondary acyl or alkyl chains.
Figure imgf000045_0001
Figure imgf000046_0001
CRX-526
AGP Buffers
In one embodiment of the present invention, the composition comprising a TLR4 modulator, preferably an AGP, is buffered using a zwitterionoic buffer. Suitably, the zwitterionic buffer is an aminoalkanesulfonic acid or suitable salt. Examples of
amninoalkanesulfonic buffers include, but are not limlited to: HEPES, HEPPS/EPPS, MOPS, MOBS, and PIPES. In one embodiment, the buffer is a pharmaceutically acceptable buffer, suitable for use in humans, such as in for use in a commercial injection product. In another embodiment, the buffer is HEPES.
Methods of Treatment The combinations of the invention are believed to have utility in disorders wherein the engagement of OX40 and/or TLR7/8, and/or TLR4 is beneficial.
The present invention thus also provides a combination of the invention, for use in therapy, particularly in the treatment of disorders wherein the engagement of OX40 and/or TLR7/8, is beneficial, particularly cancer. The present invention thus also provides a combination of the invention, for use in therapy, particularly in the treatment of disorders wherein the engagement of OX40 and TLR7/8 and TLR4, is beneficial, such as cancer.
A further aspect of the invention provides a method of treatment of a disorder wherein engagement of OX40 and/or TLR7/8 and/or TLR4 is beneficial, comprising administering a combination of the invention.
A further aspect of the present invention provides the use of a combination of the invention in the manufacture of a medicament for the treatment of a disorder engagement of OX40 and and/or TLR7/8 and/or TLR4 is beneficial. In one embodiment, the disorder is cancer. Examples of cancers that are suitable for treatment with combination of the invention include, but are limited to, both primary and metastatic forms of head and neck, breast, lung, colon, ovary, and prostate cancers. Suitably the cancer is selected from: brain (gliomas), glioblastomas, astrocytomas, glioblastoma multiforme, Bannayan-Zonana syndrome, Cowden disease, Lhermitte-Duclos disease, breast, inflammatory breast cancer, Wilm's tumor, Ewing's sarcoma, Rhabdomyosarcoma, ependymoma,
medulloblastoma, colon, head and neck, kidney, lung, liver, melanoma, ovarian, pancreatic, prostate, sarcoma, osteosarcoma, giant cell tumor of bone, thyroid, lymphoblastic T cell leukemia, Chronic myelogenous leukemia, Chronic lymphocytic leukemia, Hairy-cell leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, AML, Chronic neutrophilic leukemia, Acute lymphoblastic T cell leukemia, plasmacytoma, Immunoblastic large cell leukemia, Mantle cell leukemia, Multiple myeloma Megakaryoblastic leukemia, multiple myeloma, acute megakaryocytic leukemia, promyelocytic leukemia, Erythroleukemia, malignant lymphoma, hodgkins lymphoma, non- hodgkins lymphoma, lymphoblastic T cell lymphoma, Burkitt's lymphoma, follicular lymphoma, neuroblastoma, bladder cancer, urothelial cancer, lung cancer, vulval cancer, cervical cancer, endometrial cancer, renal cancer, mesothelioma, esophageal cancer, salivary gland cancer, hepatocellular cancer, gastric cancer, nasopharangeal cancer, buccal cancer, cancer of the mouth, GIST (gastrointestinal stromal tumor) and testicular cancer. Additionally, examples of a cancer to be treated include Barret's adenocarcinoma; billiary tract carcinomas; breast cancer; cervical cancer; cholangiocarcinoma; central nervous system tumors including primary CNS tumors such as glioblastomas, astrocytomas (e.g. , glioblastoma multiforme) and ependymomas, and secondary CNS tumors (i.e. , metastases to the central nervous system of tumors originating outside of the central nervous system); colorectal cancer including large intestinal colon carcinoma; gastric cancer; carcinoma of the head and neck including squamous cell carcinoma of the head and neck; hematologic cancers including leukemias and lymphomas such as acute lymphoblastic leukemia, acute myelogenous leukemia (AML), myelodysplasia syndromes, chronic myelogenous leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, megakaryoblastic leukemia, multiple myeloma and erythroleukemia; hepatocellular carcinoma; lung cancer including small cell lung cancer and non-small cell lung cancer; ovarian cancer; endometrial cancer; pancreatic cancer; pituitary adenoma; prostate cancer; renal cancer; sarcoma; skin cancers including melanomas; and thyroid cancers.
The present invention relates to a method for treating or lessening the severity of a cancer selected from: brain (gliomas), glioblastomas, astrocytomas, glioblastoma multiforme, Bannayan-Zonana syndrome, Cowden disease, Lhermitte-Duclos disease, breast, colon, head and neck, kidney, lung, liver, melanoma, ovarian, pancreatic, prostate, sarcoma and thyroid.
Further, the present invention relates to a method for treating or lessening the severity of a cancer selected from ovarian, breast, pancreatic and prostate.
Moreover, the present invention relates to a method for treating or lessening the severity of pre-cancerous syndromes in a mammal, including a human, wherein the pre-cancerous syndrome is selected from: cervical intraepithelial neoplasia, monoclonal gammapathy of unknown significance (MGUS), myelodysplasia syndrome, aplastic anemia, cervical lesions, skin nevi (pre-melanoma), prostatic intraepithleial (intraductal) neoplasia (PIN), Ductal Carcinoma in situ (DCIS), colon polyps and severe hepatitis or cirrhosis.
The combination of the invention may be used alone or in combination with one or more other therapeutic agents. The invention thus provides in a further aspect a further combination comprising a combination of the invention with a further therapeutic agent or agents, compositions and medicaments comprising the combination and use of the further combination, compositions and medicaments in therapy, in particular in the treatment of diseases susceptible engagement of OX40 and/or and/or TLR7/8 and/or TLR4.
In the embodiment, the combination of the invention may be employed with other therapeutic methods of cancer treatment. In particular, in anti-neoplastic therapy, combination therapy with other chemotherapeutic, hormonal, antibody agents as well as surgical and/or radiation treatments other than those mentioned above are envisaged. Combination therapies according to the present invention thus include the administration of an anti-OX40 ABP or antibody of the invention and/or a TLR7/8 modulator and/or TLR4 modulator as well as optional use of other therapeutic agents including other anti- neoplastic agents. Such combination of agents may be administered together or separately and, when administered separately this may occur simultaneously or sequentially in any order, both close and remote in time. In one embodiment, the pharmaceutical combination includes an anti-OX40 ABP or antibody of the invention and a TLR7/8 modulator and/or a TLR4 modulator, and optionally at least one additional antineoplastic agent.
In one embodiment, the further anti-cancer therapy is surgical and/or radiotherapy.
In another embodiment, the further anti-cancer therapy is at least one additional antineoplastic agent. Any anti-neoplastic agent that has activity versus a susceptible tumor being treated may be utilized in the combination. Typical anti-neoplastic agents useful include, but are not limited to: anti-microtubule agents such as diterpenoids and vinca alkaloids; platinum coordination complexes; alkylating agents, such as nitrogen mustards,
oxazaphosphorines, alkylsulfonates, nitrosoureas, and triazenes; antibiotic agents, such as anthracyclins, actinomycins and bleomycins; topoisomerase II inhibitors, such as epipodophyllotoxins; antimetabolites, such as purine and pyrimidine analogues and anti- folate compounds; topoisomerase I inhibitors, such as camptothecins; hormones and hormonal analogues; signal transduction pathway inhibitors; non-receptor tyrosine angiogenesis inhibitors; immunotherapeutic agents; proapoptotic agents; and cell cycle signaling inhibitors.
Anti-microtubule or anti-mitotic agents are phase-specific agents that are active against the microtubules of tumor cells during M or the mitosis phase of the cell cycle. Examples of anti-microtubule agents include, but are not limited to, diterpenoids and vinca alkaloids.
Diterpenoids, which are derived from natural sources, are phase-specific anti -cancer agents that operate at the G2/M phases of the cell cycle. It is believed that the
diterpenoids stabilize the β-tubulin subunit of the microtubules, by binding with this protein. Disassembly of the protein appears then to be inhibited with mitosis being arrested and cell death following. Examples of diterpenoids include, but are not limited to, paclitaxel and its analog docetaxel. Paclitaxel, 5p,20-epoxy-1 ,2a,4,7p, 10p, 13a-hexa-hydroxytax-11-en-9-one 4,10-diacetate 2- benzoate 13-ester with (2R,3S)-N-benzoyl-3-phenylisoserine; is a natural diterpene product isolated from the Pacific yew tree Taxus brevifolia and is commercially available as an injectable solution TAXOL®. It is a member of the taxane family of terpenes. Paclitaxel has been approved for clinical use in the treatment of refractory ovarian cancer in the United States (Markman, et al., Yale Journal of Biology and Medicine, 64:583, 1991 ;
McGuire, et al., Ann. Intern, Med., 11 1 :273, 1989) and for the treatment of breast cancer (Holmes, et al., J. Nat. Cancer Inst., 83: 1797, 1991.) It is a potential candidate for treatment of neoplasms in the skin (Einzig, et. al., Proc. Am. Soc. Clin. Oncol., 20:46) and head and neck carcinomas (Forastire, et. al., Sem. Oncol., 20:56, 1990). The compound also shows potential for the treatment of polycystic kidney disease (Woo, et. al., Nature, 368:750. 1994), lung cancer and malaria. Treatment of patients with paclitaxel results in bone marrow suppression (multiple cell lineages, Ignoff, et. al, Cancer Chemotherapy Pocket Guide, 1998) related to the duration of dosing above a threshold concentration (50nM) (Kearns, et. al., Seminars in Oncology, 3(6) p.16-23, 1995).
Docetaxel, (2R,3S)- N-carboxy-3-phenylisoserine,N-te/f-butyl ester, 13-ester with 5β-20- epoxy-1 ,2a,4,7p, 10p, 13ot-hexahydroxytax-11-en-9-one 4-acetate 2-benzoate, trihydrate; is commercially available as an injectable solution as TAXOTERE®. Docetaxel is indicated for the treatment of breast cancer. Docetaxel is a semi-synthetic derivative of paclitaxel q.v., prepared using a natural precursor, 10-deacetyl-baccatin III, extracted from the needle of the European Yew tree.
Vinca alkaloids are phase-specific anti-neoplastic agents derived from the periwinkle plant. Vinca alkaloids act at the M phase (mitosis) of the cell cycle by binding specifically to tubulin. Consequently, the bound tubulin molecule is unable to polymerize into
microtubules. Mitosis is believed to be arrested in metaphase with cell death following. Examples of vinca alkaloids include, but are not limited to, vinblastine, vincristine, and vinorelbine. Vinblastine, vincaleukoblastine sulfate, is commercially available as VELBAN® as an injectable solution. Although, it has possible indication as a second line therapy of various solid tumors, it is primarily indicated in the treatment of testicular cancer and various lymphomas including Hodgkin's Disease; and lymphocytic and histiocytic lymphomas. Myelosuppression is the dose-limiting side effect of vinblastine.
Vincristine, vincaleukoblastine, 22-oxo-, sulfate, is commercially available as ONCOVIN® as an injectable solution. Vincristine is indicated for the treatment of acute leukemias and has also found use in treatment regimens for Hodgkin's and non-Hodgkin's malignant lymphomas. Alopecia and neurologic effects are the most common side effect of vincristine and to a lesser extent myelosupression and gastrointestinal mucositis effects occur.
Vinorelbine, 3',4'-didehydro -4'-deoxy-C'-norvincaleukoblastine [R-(R*,R*)-2,3- dihydroxybutanedioate (1 :2)(salt)], commercially available as an injectable solution of vinorelbine tartrate (NAVELBINE®), is a semisynthetic vinca alkaloid. Vinorelbine is indicated as a single agent or in combination with other chemotherapeutic agents, such as cisplatin, in the treatment of various solid tumors, particularly non-small cell lung, advanced breast, and hormone refractory prostate cancers. Myelosuppression is the most common dose-limiting side effect of vinorelbine.
Platinum coordination complexes: Platinum coordination complexes are non-phase- specific anti-cancer agents, which are interactive with DNA. The platinum complexes enter tumor cells, undergo, aquation and form intra- and interstrand crosslinks with DNA causing adverse biological effects to the tumor. Examples of platinum coordination complexes include, but are not limited to, oxaliplatin, cisplatin and carboplatin.
Cisplatin, cis-diamminedichloroplatinum, is commercially available as PLATINOL® as an injectable solution. Cisplatin is primarily indicated in the treatment of metastatic testicular and ovarian cancer and advanced bladder cancer.
Carboplatin, platinum, diammine [1 ,1-cyclobutane-dicarboxylate(2-)-0,0'], is commercially available as PARAPLATIN® as an injectable solution. Carboplatin is primarily indicated in the first and second line treatment of advanced ovarian carcinoma. Alkylating agents: Alkylating agents are non-phase anti-cancer specific agents and strong electrophiles. Typically, alkylating agents form covalent linkages, by alkylation, to DNA through nucleophilic moieties of the DNA molecule such as phosphate, amino, sulfhydryl, hydroxyl, carboxyl, and imidazole groups. Such alkylation disrupts nucleic acid function leading to cell death. Examples of alkylating agents include, but are not limited to, nitrogen mustards such as cyclophosphamide, melphalan, and chlorambucil; alkyl sulfonates such as busulfan; nitrosoureas such as carmustine; and triazenes, such as dacarbazine.
Cyclophosphamide, 2-[bis(2-chloroethyl)amino]tetrahydro-2H-1 ,3,2-oxazaphosphorine 2- oxide monohydrate, is commercially available as an injectable solution or tablets as CYTOXAN®. Cyclophosphamide is indicated as a single agent or in combination with other chemotherapeutic agents, in the treatment of malignant lymphomas, multiple myeloma, and leukemias.
Melphalan, 4-[bis(2-chloroethyl)amino]-L-phenylalanine, is commercially available as an injectable solution or tablets as ALKERAN®. Melphalan is indicated for the palliative treatment of multiple myeloma and non-resectable epithelial carcinoma of the ovary. Bone marrow suppression is the most common dose-limiting side effect of melphalan.
Chlorambucil, 4-[bis(2-chloroethyl)amino]benzenebutanoic acid, is commercially available as LEUKERAN® tablets. Chlorambucil is indicated for the palliative treatment of chronic lymphatic leukemia, and malignant lymphomas such as lymphosarcoma, giant follicular lymphoma, and Hodgkin's disease.
Busulfan, 1 ,4-butanediol dimethanesulfonate, is commercially available as MYLERAN® tablets. Busulfan is indicated for the palliative treatment of chronic myelogenous leukemia. Carmustine, 1 ,3-[bis(2-chloroethyl)-1-nitrosourea, is commercially available as single vials of lyophilized material as BiCNU®. Carmustine is indicated for the palliative treatment as a single agent or in combination with other agents for brain tumors, multiple myeloma, Hodgkin's disease, and non-Hodgkin's lymphomas.
Dacarbazine, 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide, is commercially available as single vials of material as DTIC-Dome®. Dacarbazine is indicated for the treatment of metastatic malignant melanoma and in combination with other agents for the second line treatment of Hodgkin's Disease.
Antibiotic anti-neoplastics: Antibiotic anti-neoplastics are non-phase-specific agents, which bind or intercalate with DNA. Typically, such action results in stable DNA complexes or strand breakage, which disrupts ordinary function of the nucleic acids leading to cell death. Examples of antibiotic anti-neoplastic agents include, but are not limited to, actinomycins such as dactinomycin, anthrocyclins, such as daunorubicin and doxorubicin; and bleomycins.
Dactinomycin, also know as Actinomycin D, is commercially available in injectable form as COSMEGEN®. Dactinomycin is indicated for the treatment of Wilm's tumor and
rhabdomyosarcoma. Daunorubicin, (8S-cis-)-8-acetyl-10-[(3-amino-2,3,6-trideoxy-a-L-lyxo-hexopyranosyl)oxy]- 7,8,9, 10-tetrahydro-6, 8,1 1-trihydroxy-1-methoxy-5, 12 naphthacenedione hydrochloride, is commercially available as a liposomal injectable form as DAUNOXOME® or as an injectable as CERUBIDINE®. Daunorubicin is indicated for remission induction in the treatment of acute nonlymphocytic leukemia and advanced HIV associated Kaposi's sarcoma.
Doxorubicin, (8S, 10S)-10-[(3-amino-2,3,6-trideoxy-a-L-lyxo-hexopyranosyl)oxy]-8- glycoloyl , 7,8,9, 10-tetrahydro-6,8, 11 -trihydroxy-1 -methoxy-5, 12 naphthacenedione hydrochloride, is commercially available as an injectable form as RUBEX® or
ADRIAMYCIN RDF®. Doxorubicin is primarily indicated for the treatment of acute lymphoblastic leukemia and acute myeloblastic leukemia, but is also a useful component in the treatment of some solid tumors and lymphomas. Bleomycin, a mixture of cytotoxic glycopeptide antibiotics isolated from a strain of
Streptomyces verticillus, is commercially available as BLENOXANE®. Bleomycin is indicated as a palliative treatment, as a single agent or in combination with other agents, of squamous cell carcinoma, lymphomas, and testicular carcinomas. Topoisomerase II inhibitors: Topoisomerase II inhibitors include, but are not limited to, epipodophyllotoxins. Epipodophyllotoxins are phase-specific anti-neoplastic agents derived from the mandrake plant. Epipodophyllotoxins typically affect cells in the S and G2 phases of the cell cycle by forming a ternary complex with topoisomerase II and DNA causing DNA strand breaks. The strand breaks accumulate and cell death follows.
Examples of epipodophyllotoxins include, but are not limited to, etoposide and teniposide.
Etoposide, 4'-demethyl-epipodophyllotoxin 9[4,6-0-(R )-ethylidene-p-D-glucopyranoside], is commercially available as an injectable solution or capsules as VePESID® and is commonly known as VP-16. Etoposide is indicated as a single agent or in combination with other chemotherapy agents in the treatment of testicular and non-small cell lung cancers.
Teniposide, 4'-demethyl-epipodophyllotoxin 9[4,6-0-(R )-thenylidene-p-D-glucopyranoside], is commercially available as an injectable solution as VUMON® and is commonly known as VM-26. Teniposide is indicated as a single agent or in combination with other chemotherapy agents in the treatment of acute leukemia in children. Anti-metabolite neoplastic agents: Anti-metabolite neoplastic agents are phase-specific anti-neoplastic agents that act at S phase (DNA synthesis) of the cell cycle by inhibiting DNA synthesis or by inhibiting purine or pyrimidine base synthesis and thereby limiting DNA synthesis. Consequently, S phase does not proceed and cell death follows.
Examples of antimetabolite anti-neoplastic agents include, but are not limited to, fluorouracil, methotrexate, cytarabine, mecaptopurine, thioguanine, and gemcitabine.
5-fluorouracil, 5-fluoro-2,4- (1 H,3H) pyrimidinedione, is commercially available as fluorouracil. Administration of 5-fluorouracil leads to inhibition of thymidylate synthesis and is also incorporated into both RNA and DNA. The result typically is cell death. 5- fluorouracil is indicated as a single agent or in combination with other chemotherapy agents in the treatment of carcinomas of the breast, colon, rectum, stomach and pancreas. Other fluoropyrimidine analogs include 5-fluoro deoxyuridine (floxuridine) and 5- fluorodeoxyuridine monophosphate.
Cytarabine, 4-amino-1-p-D-arabinofuranosyl-2 (I H)-pyrimidinone, is commercially available as CYTOSAR-U® and is commonly known as Ara-C. It is believed that cytarabine exhibits cell phase-specificity at S-phase by inhibiting DNA chain elongation by terminal incorporation of cytarabine into the growing DNA chain. Cytarabine is indicated as a single agent or in combination with other chemotherapy agents in the treatment of acute leukemia. Other cytidine analogs include, but are not limited to, 5-azacytidine and 2',2'-difluorodeoxycytidine (gemcitabine). Mercaptopurine, 1 ,7-dihydro-6H-purine-6-thione monohydrate, is commercially available as PURINETHOL®. Mercaptopurine exhibits cell phase-specificity at S-phase by inhibiting DNA synthesis by an as of yet unspecified mechanism. Mercaptopurine is indicated as a single agent or in combination with other chemotherapy agents in the treatment of acute leukemia. A useful mercaptopurine analog is azathioprine.
Thioguanine, 2-amino-1 ,7-dihydro-6H-purine-6-thione, is commercially available as TABLOID®. Thioguanine exhibits cell phase-specificity at S-phase by inhibiting DNA synthesis by an as of yet unspecified mechanism. Thioguanine is indicated as a single agent or in combination with other chemotherapy agents in the treatment of acute leukemia. Other purine analogs include pentostatin, erythrohydroxynonyladenine, fludarabine phosphate, and cladribine. Gemcitabine, 2'-deoxy-2', 2'-difluorocytidine monohydrochloride (β-isomer), is
commercially available as GEMZAR®. Gemcitabine exhibits cell phase-specificity at S- phase and by blocking progression of cells through the G1/S boundary. Gemcitabine is indicated in combination with cisplatin in the treatment of locally advanced non-small cell lung cancer and alone in the treatment of locally advanced pancreatic cancer.
Methotrexate, N-[4[[(2,4-diamino-6-pteridinyl) methyl]methylamino] benzoyl]-L-glutamic acid, is commercially available as methotrexate sodium. Methotrexate exhibits cell phase effects specifically at S-phase by inhibiting DNA synthesis, repair and/or replication through the inhibition of dyhydrofolic acid reductase which is required for synthesis of purine nucleotides and thymidylate. Methotrexate is indicated as a single agent or in combination with other chemotherapy agents in the treatment of choriocarcinoma, meningeal leukemia, non-Hodgkin's lymphoma, and carcinomas of the breast, head, neck, ovary and bladder.
Topoisomerase I inhibitors: Camptothecins, including, camptothecin and camptothecin derivatives are available or under development as Topoisomerase I inhibitors.
Camptothecins cytotoxic activity is believed to be related to its Topoisomerase I inhibitory activity. Examples of camptothecins include, but are not limited to irinotecan, topotecan, and the various optical forms of 7-(4-methylpiperazino-methylene)-10, 11-ethylenedioxy-20- camptothecin described below.
Irinotecan HCI, (4S)-4, 1 1-diethyl-4-hydroxy-9-[(4-piperidinopiperidino) carbonyloxy]-1 H- pyrano[3',4',6,7]indolizino[1 ,2-b]quinoline-3,14(4H, 12H)-dione hydrochloride, is
commercially available as the injectable solution CAMPTOSAR®. Irinotecan is a derivative of camptothecin which binds, along with its active metabolite SN-38, to the topoisomerase I - DNA complex. It is believed that cytotoxicity occurs as a result of irreparable double strand breaks caused by interaction of the topoisomerase I: DNA:
irintecan or SN-38 ternary complex with replication enzymes. Irinotecan is indicated for treatment of metastatic cancer of the colon or rectum.
Topotecan HCI, (S)-10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-1 H- pyrano[3',4',6,7]indolizino[1 ,2-b]quinoline-3, 14-(4H, 12H)-dione monohydrochloride, is commercially available as the injectable solution HYCAMTIN®. Topotecan is a derivative of camptothecin which binds to the topoisomerase I - DNA complex and prevents religation of singles strand breaks caused by Topoisomerase I in response to torsional strain of the DNA molecule. Topotecan is indicated for second line treatment of metastatic carcinoma of the ovary and small cell lung cancer.
Hormones and hormonal analogues: Hormones and hormonal analogues are useful compounds for treating cancers in which there is a relationship between the hormone(s) and growth and/or lack of growth of the cancer. Examples of hormones and hormonal analogues useful in cancer treatment include, but are not limited to, adrenocorticosteroids such as prednisone and prednisolone which are useful in the treatment of malignant lymphoma and acute leukemia in children; aminoglutethimide and other aromatase inhibitors such as anastrozole, letrazole, vorazole, and exemestane useful in the treatment of adrenocortical carcinoma and hormone dependent breast carcinoma containing estrogen receptors; progestrins such as megestrol acetate useful in the treatment of hormone dependent breast cancer and endometrial carcinoma; estrogens, androgens, and anti-androgens such as flutamide, nilutamide, bicalutamide, cyproterone acetate and 5a- reductases such as finasteride and dutasteride, useful in the treatment of prostatic carcinoma and benign prostatic hypertrophy; anti-estrogens such as tamoxifen, toremifene, raloxifene, droloxifene, iodoxyfene, as well as selective estrogen receptor modulators (SERMS) such those described in U.S. Patent Nos. 5,681 ,835, 5,877,219, and 6,207,716, useful in the treatment of hormone dependent breast carcinoma and other susceptible cancers; and gonadotropin-releasing hormone (GnRH) and analogues thereof which stimulate the release of leutinizing hormone (LH) and/or follicle stimulating hormone (FSH) for the treatment prostatic carcinoma, for instance, LHRH agonists and
antagagonists, such as goserelin acetate and luprolide.
Signal transduction pathway inhibitors: Signal transduction pathway inhibitors are inhibitors that block or inhibit a chemical process which evokes an intracellular change. As used herein, this change is cell proliferation or differentiation. Signal tranduction inhibitors useful in the present invention include inhibitors of receptor tyrosine kinases, non-receptor tyrosine kinases, SH2/SH3 domain blockers, serine/threonine kinases, phosphotidyl inositol-3 kinases, myo-inositol signaling, and Ras oncogenes.
Several protein tyrosine kinases catalyse the phosphorylation of specific tyrosyl residues in various proteins involved in the regulation of cell growth. Such protein tyrosine kinases can be broadly classified as receptor or non-receptor kinases. Receptor tyrosine kinases are transmembrane proteins having an extracellular ligand binding domain, a transmembrane domain, and a tyrosine kinase domain. Receptor tyrosine kinases are involved in the regulation of cell growth and are generally termed growth factor receptors. Inappropriate or uncontrolled activation of many of these kinases, i.e., aberrant kinase growth factor receptor activity, for example by over-expression or mutation, has been shown to result in uncontrolled cell growth. Accordingly, the aberrant activity of such kinases has been linked to malignant tissue growth. Consequently, inhibitors of such kinases could provide cancer treatment methods. Growth factor receptors include, for example, epidermal growth factor receptor (EGFr), platelet derived growth factor receptor (PDGFr), erbB2, erbB4, ret, vascular endothelial growth factor receptor (VEGFr), tyrosine kinase with immunoglobulin-like and epidermal growth factor identity domains (TIE-2), insulin growth factor -I (IGFI) receptor, macrophage colony stimulating factor (cfms), BTK, ckit, cmet, fibroblast growth factor (FGF) receptors, Trk receptors (TrkA, TrkB, and TrkC), ephrin (eph) receptors, and the RET protooncogene. Several inhibitors of growth receptors are under development and include ligand antagonists, antibodies, tyrosine kinase inhibitors and anti-sense oligonucleotides. Growth factor receptors and agents that inhibit growth factor receptor function are described, for instance, in Kath, Exp. Opin. Ther. Patents (2000) 10(6):803-818; Shawver, et al DDT, Vol 2, No. 2 February 1997; and Lofts, et al, "Growth factor receptors as targets", NEW
MOLECULAR TARGETS FOR CANCER CHEMOTHERAPY, ed. Workman, Paul and Kerr, David, CRC press 1994, London.
Tyrosine kinases, which are not growth factor receptor kinases, are termed non-receptor tyrosine kinases. Non-receptor tyrosine kinases useful in the present invention, which are targets or potential targets of anti-cancer drugs, include cSrc, Lck, Fyn, Yes, Jak, cAbl, FAK (Focal adhesion kinase), Brutons tyrosine kinase, and Bcr-Abl. Such non-receptor kinases and agents which inhibit non-receptor tyrosine kinase function are described in Sinh, et al., (1999) Journal of Hematotherapy and Stem Cell Research 8 (5): 465 - 80; and Bolen, et al., (1997) Annual review of Immunology. 15: 371-404.
SH2/SH3 domain blockers are agents that disrupt SH2 or SH3 domain binding in a variety of enzymes or adaptor proteins including, PI3-K p85 subunit, Src family kinases, adaptor molecules (She, Crk, Nek, Grb2) and Ras-GAP. SH2/SH3 domains as targets for anticancer drugs are discussed in Smithgall, et al. (1995), Journal of Pharmacological and Toxicological Methods 34(3) 125-32.
Inhibitors of Serine/Threonine Kinases including MAP kinase cascade blockers which include blockers of Raf kinases (rafk), Mitogen or Extracellular Regulated Kinase (MEKs), and Extracellular Regulated Kinases (ERKs); and Protein kinase C family member blockers including blockers of PKCs (alpha, beta, gamma, epsilon, mu, lambda, iota, zeta). IkB kinase family (IKKa, IKKb), PKB family kinases, akt kinase family members, and TGF beta receptor kinases. Such Serine/Threonine kinases and inhibitors thereof are described in Yamamoto, et al., (1999), Journal of Biochemistry 126 (5) 799-803; Brodt, et al. (2000), Biochemical Pharmacology, 60. 1 101-1107; Massague, et al. (1996) Cancer Surveys 27:41-64; Philip, P.A., and Harris (1995), Cancer Treatment and Research 78: 3- 27, Lackey, et al., Bioorganic and Medicinal Chemistry Letters, (10), 2000, 223-226; U.S. Patent No. 6,268,391 ; and Martinez-lacaci, et al, Int. J. Cancer (2000), 88(1), 44-52. Inhibitors of Phosphotidyl inositol-3 Kinase family members including blockers of PI3- kinase, ATM, DNA-PK, and Ku are also useful in the present invention. Such kinases are discussed in Abraham, R.T. (1996), Current Opinion in Immunology. 8 (3) 412-8; Canman, et al. (1998), Oncogene 17 (25) 3301-3308; Jackson, S.P. (1997), International Journal of Biochemistry and Cell Biology 29 (7):935-8; and Zhong, et al, Cancer Res, (2000) 60(6), 1541-1545.
Also useful in the present invention are Myo-inositol signaling inhibitors, such as phospholipase C blockers and Myoinositol analogues. Such signal inhibitors are described in Powis, et al., (1994) NEW MOLECULAR TARGETS FOR CANCER CHEMOTHERAPY ed., Paul Workman and David Kerr, CRC press 1994, London. Another group of signal transduction pathway inhibitors are inhibitors of Ras Oncogene. Such inhibitors include inhibitors of farnesyltransferase, geranyl-geranyl transferase, and CAAX proteases, as well as anti-sense oligonucleotides, ribozymes and immunotherapy. Such inhibitors have been shown to block ras activation in cells containing wild type mutant ras , thereby acting as antiproliferation agents. Ras oncogene inhibition is discussed in Scharovsky, et al. (2000), Journal of Biomedical Science 7(4) 292-8; Ashby, M.N. (1998), Current Opinion in Lipidology 9 (2) 99 - 102; and BioChim. Biophys. Acta, (1989) 1423(3): 19-30.
As mentioned above, antibody antagonists to receptor kinase ligand binding may also serve as signal transduction inhibitors. This group of signal transduction pathway inhibitors includes the use of humanized antibodies to the extracellular ligand binding domain of receptor tyrosine kinases. For example, Imclone C225 EGFR specific antibody (see Green, et al, Monoclonal Antibody Therapy for Solid Tumors, Cancer Treat. Rev., (2000), 26(4), 269-286); HERCEPTIN® erbB2 antibody (see Tyrosine Kinase Signalling in Breast cancer.erbB Family Receptor Tyrosine Kinases, Breast cancer Res., 2000, 2(3), 176-183); and 2CB VEGFR2 specific antibody (see Brekken, et al, "Selective Inhibition of VEGFR2 Activity by a monoclonal Anti-VEGF antibody blocks tumor growth in mice", Cancer Res. (2000) 60, 51 17-5124).
Anti-angiogenic agents: Anti-angiogenic agents, including non-receptorMEKngiogenesis inhibitors may alo be useful. Such anti-angiogenic agents such, may inhibit the effects of vascular edothelial growth factor, (for example the anti-vascular endothelial cell growth factor antibody bevacizumab [Avastin™], and may include compounds that work by other mechanisms (for example linomide, inhibitors of integrin ανβ3 function, endostatin and angiostatin);
Immunotherapeutic agents: Agents used in immunotherapeutic regimens may also be useful in combination with the compounds of formula (I). Immunotherapy approaches, including for example ex-vivo and in-vivo approaches to increase the immunogenecity of patient tumour cells, such as transfection with cytokines, such as interleukin 2, interleukin 4 or granulocyte-macrophage colony stimulating factor, approaches to decrease T-cell anergy, approaches using transfected immune cells such as cytokine-transfected dendritic cells, approaches using cytokine-transfected tumour cell lines and approaches using anti- idiotypic antibodies
Proapoptotoc agents: Agents used in proapoptotic regimens (e.g., bcl-2 antisense oligonucleotides) may also be used in the combination of the present invention.
Cell cycle signalling inhibitors: Cell cycle signalling inhibitors inhibit molecules involved in the control of the cell cycle. A family of protein kinases called cyclin-dependent kinases (CDKs) and their interaction with a family of proteins termed cyclins controls progression through the eukaryotic cell cycle. The coordinate activation and inactivation of different cyclin/CDK complexes is necessary for normal progression through the cell cycle. Several inhibitors of cell cycle signalling are under development. For instance, examples of cyclin dependent kinases, including CDK2, CDK4, and CDK6 and inhibitors for the same are described in, for instance, Rosania, et al, Exp. Opin. Ther. Patents (2000) 10(2):215-230.
In one embodiment, the combination of the present invention comprises an anti-OX40 ABP or antibody and a TLR7/8 modulator and/or a TLR4 modulator and at least one antineoplastic agent selected from: anti-microtubule agents, platinum coordination complexes, alkylating agents, antibiotic agents, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormonal analogues, signal transduction pathway inhibitors, non-receptor tyrosine MEKngiogenesis inhibitors, immunotherapeutic agents, proapoptotic agents, and cell cycle signaling inhibitors. In another embodiment, the combination of the present invention comprises an anti-OX40 ABP or antibody and a TLR7/8 modulator and/or a TLR4 modulator and at least one antineoplastic agent that is an anti-microtubule agent selected from: diterpenoids and vinca alkaloids. In a further embodiment, the at least one anti-neoplastic agent agent is a diterpenoid.
In yet a further embodiment, the at least one anti-neoplastic agent is a vinca alkaloid.
In one embodiment, the combination of the present invention comprises an anti-OX40 ABP or antibody and a TLR7/8 modulator and/or a TLR4 modulator and at least one antineoplastic agent, which is a platinum coordination complex. In a further embodiment, the at least one anti-neoplastic agent is paclitaxel, carboplatin, or vinorelbine.
In another embodiment, the at least one anti-neoplastic agent is carboplatin.
In a further embodiment, the at least one anti-neoplastic agent is vinorelbine.
In yet a further embodiment, the at least one anti-neoplastic agent is paclitaxel. In one embodiment, the combination of the present invention comprises an anti-OX40 ABP or antibody and a TLR7/8 modulator and/or a TLR4 modulator and at least one antineoplastic agent which is a signal transduction pathway inhibitor.
In a further embodiment the signal transduction pathway inhibitor is an inhibitor of a growth factor receptor kinase chosen from: VEGFR2, TIE2, PDGFR, BTK, erbB2, EGFr, IGFR-1 , TrkA, TrkB, TrkC, and c-fms.
In a further embodiment the signal transduction pathway inhibitor is an inhibitor of a serine/threonine kinase rafk, akt, or PKC-zeta.
In another embodiment the signal transduction pathway inhibitor is an inhibitor of a nonreceptor tyrosine kinase selected from the src family of kinases. In a further embodiment the signal transduction pathway inhibitor is an inhibitor of c-src.
In a further embodiment the signal transduction pathway inhibitor is an inhibitor of Ras oncogene selected from: inhibitors of farnesyl transferase and geranylgeranyl transferase.
In yet a further embodiment the signal transduction pathway inhibitor is an inhibitor of a serine/threonine kinase selected from Pi3K. In a further embodiment the signal transduction pathway inhibitor is a dual EGFr/erbB2 inhibitor, for example, N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2- (methanesulphonyl) ethyl]amino}methyl)-2-furyl]-4-quinazolinamine (structure below):
Figure imgf000061_0001
In one embodiment, the combination of the present invention comprises a compound of formula I or a salt or solvate thereof and at least one anti-neoplastic agent which is a cell cycle signaling inhibitor.
In further embodiment, the cell cycle signaling inhibitor is an inhibitor of CDK2, CDK4 or CDK6. In one embodiment, the mammal in the methods and uses of the present invention is a human.
As indicated, therapeutically effective amounts of the combinations of the invention (an anti-OX40 ABP or antibody and a TLR7/8 modulator and/or a TLR4 modulator) are administered to a human. Typically, the therapeutically effective amount of the administered agents of the present invention will depend upon a number of factors including, for example, the age and weight of the subject, the precise condition requiring treatment, the severity of the condition, the nature of the formulation, and the route of administration. Ultimately, the therapeutically effective amount will be at the discretion of the attendant physician. The following examples are intended for illustration only and are not intended to limit the scope of the invention in any way.
EXAMPLES
Example 1 : Treatment of 0X86 Monotherapy in a CT-26 syngeneic mouse model for colon cancer
The CT-26 mouse colon carcinoma (CT26.WT; ATCC #CRL-2638) cell line was obtained from ATCC. It is an N-nitroso-N-methylurethane-(NNMU) induced, undifferentiated colon carcinoma cell line known in the art. For example, it is described in: Wang M, et al. Active immunotherapy of cancer with a nonreplicating recombinant fowlpox virus encoding a model tumor-associated antigen. J. Immunol. 154: 4685-4692, 1995 (PubMed: 7722321). Rat lgG1 was obtained from Bioxcell. 0X86 (Hybridoma 134) cells were obtained from the European Cell Culture collection and manufactured by Harlan; 0X86 is the name for a tool anti-OX40 monoclonal antibody used in rodents; it is a rodent antibody that binds rodent OX40, e.g., mouse OX40 (receptor).
0X86 and rat lgG1 are diluted in diluted DPBS. For preparation of tumor, a frozen (-140°C) vial of CT-26 (mouse colon carcinoma cells), from ATCC (cat# CRL-2638, lot# 59227052) are basic RPMI (with 10% FBS) media over the following week.
CT-26 cells (passage 12) are harvested from the flask in complete medium. Cells are centrifuged and resuspended in RPMI (without FBS), this step is repeated 3 times. Cell density and viability are checked via trypan blue exclusion. Cells are then diluted to desired density (5x105 cells per ml_) and kept on ice.
Escalating doses of OX40 monoclonal antibody (mAb) 0X86 are evaluated their efficacy in reducing tumor growth. Animals are weighed and innoculated on the right hind quarter with 0.5x105 CT26 tumor cells per mouse on Day 0. A total of 130 mice are inoculated with tumor cells - assuming 30% failure rate (either too big or too small at time of start of study), the goal is to have n=10 for each group. After tumor cell innoculation, tumor growth and total body weight are measured 3 times a week for the duration of the study.
Randomization occurs on day 10 or 1 1 when the average tumor volume is approximately 100 mm3. Begining on the day of randomization, animals are dosed with 0X86 mAb or Rat lgG1 isotype i.p. biweekly, for a total of 6 doses. Mice remain on study until tumors >2000 cu mm for two consecutive measurements, they are removed from study for other reasons (i.e. weight loss >20%, ulceration on tumor, etc.) or until the end of the study. After euthanization the tumors may be removed and subject to dissociation for flow analysis and/or FFPE for IHC analysis.
Dosing treatment
Treatment Dose No. of mice
Group 1 : 0.5x105 cells per, Rat lgG1 400 ug per mouse 10-13
Group 2: 0.5x105 cells per, 0X86 400 ug per mouse 10-13 Group 3: 0.5x105 cells per, 0X86 200 ug per mouse 10-13 Group 4: 0.5x105 cells per, 0X86 100 ug per mouse 10-13 Group 5: 0.5x105 cells per, 0X86 50 ug per mouse 10-13
Day 0: sc innoculation with tumor cells
Days 1 , 4, 6, 8: Animals are weighed and checked for tumors and if present, tumors measured.
Randomization day (approx. day 10): Animals randomized and placed into cages representing appropriate groups
Dosing, biweekly through end of study: Animals are dosed ip with 0X86 or anti Rat lgG1 , where the amounts shown above are on a per mouse basis.
Measurements, triweekly through end of study: Animals are weighed and tumors will be measured
The mean tumor weights from about 10 animals are averaged. Error bars show SEM analysis. P values are calculated based on the following: P value tests the null hypothesis that the survival curves are identical in the overall populations. In other words, the null hypothesis is that the treatment did not change survival. Raw p-values adjusted for multiple comparisons via the Stepdown Bonferroni method
The above protocol used to generate the results in FIG. 1 B, and results of the individual mice can be found in Figure 4. These figures demonstrate that mice inoculated with CT-26 cells and treated with rat lgG1 developed tumors that grew unabated as expected, whereas dosing with OX40 monoclonal antibody (mAb) 0X86 led to clear inhibition of tumor growth and increased survivability when compared to the rat lgG1 control group.
EXAMPLE 2: Results of CT-26 study with treatment with TLR-4 (CRX-527)
The addition of TLR4 modulators such as CRX-527 to the above OX40 monotherapy treatment protocol can be used to study TLR4 monotherapy and the combination of anti- mOX40 immunotherapy with TLR4 modulators.
Treatment Dose No. of mice
Group 0: 0.5x105 cells per, vehicle 10-13
Group a: 0.5x105 cells per, CRX-527; 4 ug 10-13
Group b: 0.5x105 cells per, CRX-527; 20 ug 10-13
Group c: 0.5x105 cells per, CRX-527; 100 ug 10-13 Day 0: sc innoculation with tumor cells
Days 1 , 4, 6, 8: Animals were weighed and checked for tumors and measured.
Randomization day (approx. day 10): Animals randomized and placed into cages representing appropriate groups
Dosing, biweekly through end of study: Animals dosed ip with TLR compound CRX-527 at amounts shown above (per mouse), or vehicle.
Measurements, triweekly through end of study: Animals weighed and tumors measured.
The above protocol was used to generate the results in FIG. 1A and Figures 2-6 at the dosages indicated. In almost every case, Balb/c mice that were inoculated with 0.5x105 CT-26 colorectal tumor cells on the right hind quarter developed tumors that, when treated i.p. with vehicle (2% glycerol) only, and progressed as expected. TLR 4 agonists CRX- 527 (Figures 2-5) and CRX-601 (Figure 6) inhibited tumor growth in a dose-dependent manner when compared to the vehicle treated animals. Dose dependence was also seen in the survivability of the model.
EXAMPLE 3: Combination Treatment with OX40 (i.e. OX-86, an antibody raised against rodent OX40 receptor) and CRX-527
Dosing treatment 1 treatment 2 number of mice
Group 1 : 0.5x105 cells per, Rat lgG1 drug vehicle 10-13
Group 2: 0.5x105 cells per, 0X86 50 ug drug vehicle 10-13
Group 3: 0.5x105 cells per, RatlgGI CRX-527 5ug 10-13
Group 4: 0.5x105 cells per, RatlgGI CRX-527 25ug 10-13
Group 5: 0.5x105 cells per, 0X86 50 ug CRX-527 5ug 10-13
Group 6: 0.5x105 cells per, 0X86 50 ug CRX-527 25ug 10-13
Day 0: SC innoculation with tumor cells
Days 1 , 4, 6, 8: Animals checked for tumors and if present, tumors measured. Study enrollment day (approx. day 10): Animals randomized and received treatment 1.
Biweekly post enrollment: starting with day of enrollment, mice received i.p. dose biweekly for a total 6 doses.
Triweekly through end of study: Animals weighed and tumors measured When 0X86 treatment was combined with TLR4 modulator treatment (CRX-527), mice exhibited a higher reduction in tumor burden and survived longer than either treatment alone.
EXAMPLE 3: Monotherapy and Combination Treatment with anti-mOX40R antibody and TLR-4 targeting molecules in a CT-26 model
Mice are administered OX-40 antibody; a compound of Formula 1 (including a compound of Formula la, CRX-527, CRX-547, and CRX-601 (TLR-4 agonists), or a combination of both. Each treatment has significant anti-tumor activity.
There are at least two significant findings. First, in mice, anti-OX40R or combination of anti-OX40 antibody and TLR4 agonist combination each delay the growth of established CT-26 tumors relative to an untreated control group. Secondly, in mice significant antitumor effect is observed in TLR4 agonist and anti-OX40R antibody combinations as compared to monotherapy treatment.
EXAMPLE 4
n=10-13 mice/ Dosing treatment 1 treatment 2
Group
Group 1 : 0.5x105 cells per, Rat lgG1 drug vehicle
Group 2: 0.5x105 cells per, 0X86 50 ug drug vehicle
Group 3: 0.5x105 cells per, RatlgGI CRX-527 5ug
Group 4: 0.5x105 cells per, RatlgGI CRX-527 25ug
Group 5: 0.5x105 cells per, RatlgGI CRX-649 5ug
Group 6: 0.5x105 cells per, RatlgGI CRX-649 25ug
Group 7: 0.5x105 cells per, 0X86 50 ug CRX-527 5ug
Group 8: 0.5x105 cells per, 0X86 50 ug CRX-527 25ug
Group 9: 0.5x105 cells per, 0X86 50 ug CRX-649 5ug
Group 10: 0.5X105 cells per, 0X86 50 ug CRX-649 25ug Day 0: SC innoculation with tumor cells
Days 1 , 4, 6, 8: Animals were checked for tumors and if present, tumors measured.
Study enrollment day (approx. day 10): Animals were randomized and received Treatment 1
Biweekly post enrollment: starting with day of enrollment, mice received i.p. dose biweekly for a total 6 doses.
Triweekly through end of study: Animals were weighed and tumors were measured
The 25 ug dose of both CRX-527 (TLR4 agonist) and CRX-649 (TLR7/8 agonist) proved to be suboptimal in this study in terms of tumor growth inhibition when compared to the control group. There was very little observable difference in tumor growth in groups that received 5 ug of each compound when compared to control.
Combination dosing with 0X86 and either dose of CRX-649 resulted in some observable differences, but no significant difference in tumor growth at either day 22 or day 38 when compared to animals that were dosed with CRX-649/rat lgG1 , veh/OX86, or veh/rat lgG1.
Combination dosing with 0X86 and both doses of CRX-527 resulted in significant inhibition of tumor growth at both days 22 and 38 when compared to animals dosed with CRX- 527/rat lgG1 , veh/OX86, and veh/rat IgGl The reasons that the 0X86 antibody in combination with the TLR 7/8 molecule did not work well are unclear. One reason could be suboptimal dosing. Another is that the TLR7/8 machinery in mice poorly reproduces that in the human system. Thus, additional experiments using human cell tissue culture and human blood samples will be performed. EXAMPLE 5: Combination Treatment with anti-mOX40R antibody and TLR4 or TLR7/8 molecules in CT-26 model
Tumor bearing mice are administered OX-40 antibody; a compound of Formula 1
(including a compound of Formula la, CRX-527, CRX-547, and CRX-601 (TLR-4 agonists), or a TLR 7/8 compound, such as an oxoadenine, a lipidated oxoadenine, an
imadazoquinoline, and a lipidated imadazoquinoline, or a combination of all three (i.e. an OX-40 antibody, a TLR4 agonist, and a TLR7/8 compound). Using human tissue culture and human blood samples in tissue culture model for human cancer, results are obtained using an anti-hOX40 antibody as disclosed herein is tested OX-40 antibody in combination with a TLR 4 modulator, e.g. a compound of Formula 1 (including a compound of Formula la, CRX-527, CRX-547, and CRX-601 (TLR-4 agonists), and/or a TLR 7/8 modulator, such as an oxoadenine, a lipidated oxoadenine, an imadazoquinoline, and a lipidated imadazoquinoline, or a combination of all three (i.e. an OX-40 antibody, a TLR4 agonist, and a TLR7/8 compound). Anti-hOX40 antibody in combination with one or more TLR4 modulators and/or a TLR 7/8 compound has significant anti-tumor effect.
EXAMPLE 6: Combination Treatment with triple treatment combination of anti- mOX40R antibody and TLR4 and TLR7/8 in a CT-26 model.
Tumor bearing mice are administered OX-40 antibody; a compound of Formula 1
(including a compound of Formula la, CRX-527, CRX-547, and CRX-601 (TLR-4 agonists), or a TLR 7/8 compound, such as an oxoadenine, a lipidated oxoadenine, an
imadazoquinoline, and a lipidated imadazoquinoline, or a combination of all three (i.e. an OX-40 antibody, a TLR4 agonist, and a TLR7/8 compound). As with the above, human tissue culture models are used to test the triple combination and an anti-tumor effect is observed.
The OX40 Antibody Sequence Listing is below.
<140> UNKNOWN
<141> 2014-02-24
<150> PCT/US2012/024570
<151> 2012-02-09
<150> PCT/US2011/048752
<151> 2011-08-23
<160> 47
<170> Patentln version 3.
<210> 1
<211> 5
<212> PRT
<213> Mus sp.
<400> 1
Asp Tyr Ser Met His
1 5
<210> 2
<211> 17
<212> PRT
<213> Mus sp.
<400> 2
Trp lie Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys 1 5 10 15
Gly
<210> 3
<211> 13
<212> PRT
<213> Mus sp.
<400> 3
Pro Tyr Tyr Asp Tyr Val Ser Tyr Tyr Ala Met Asp Tyr
1 5 10
<210> 4
<211> 122
<212> PRT
<213> Mus sp.
<400> 4
Gin lie Gin Leu Val Gin Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15
Thr Val Lys lie Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30 Ser Met His Trp Val Lys Gin Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45
Gly Trp lie Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60
Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80
Leu Gin lie Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95
Ala Asn Pro Tyr Tyr Asp Tyr Val Ser Tyr Tyr Ala Met Asp Tyr Trp
100 105 110
Gly His Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 5
<211> 122
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
polypeptide
<400> 5
Gin Val Gin Leu Val Gin Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Met His Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Lys Trp Met 35 40 45
Gly Trp lie Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60
Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80
Leu Gin lie Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Asn Pro Tyr Tyr Asp Tyr Val Ser Tyr Tyr Ala Met Asp Tyr Trp
100 105 110 Gly Gin Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 6
<211> 458
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
polynucleotide
<400> 6
actagtacca ccatggcttg ggtgtggacc ttgctattcc tgatggcagc tgcccaaagt 60 atccaagcac aggttcagtt ggtgcagtct ggatctgagc tgaagaagcc tggagcctca 120 gtcaaggttt cctgcaaggc ttctggttat accttcacag actattcaat gcactgggtg 180 cgacaggctc caggacaagg tttaaagtgg atgggctgga taaacactga gactggtgag 240 ccaacatatg cagatgactt caagggacgg tttgtcttct ctttggacac ctctgtcagc 300 actgcctatt tgcagatcag cagcctcaaa gctgaggaca cggctgtgta ttactgtgct 360 aatccctact atgattacgt ctcttactat gctatggact actggggtca gggaaccacg 420 gtcaccgtct cctcaggtaa gaatggcctc tcaagctt 458
<210> 7
<211> 11
<212> PRT
<213> Mus sp.
<400> 7
Lys Ala Ser Gin Asp Val Ser Thr Ala Val Ala
1 5 10
<210> 8
<211> 7
<212> PRT
<213> Mus sp.
<400> 8
Ser Ala Ser Tyr Leu Tyr Thr
1 5
<210> 9
<211> 9
<212> PRT
<213> Mus sp.
<400> 9
Gin Gin His Tyr Ser Thr Pro Arg Thr
1 5 <210> 10
<211> 107
<212 > PRT
<213> Mus sp .
<400> 10
Asp He Val Met Thr Gin Ser Hi s Lys Phe Met Ser Thr Ser Val Arg 1 5 10 15
Asp Arg Val Ser He Thr Cys Lys Ala Ser Gin Asp Val Ser Thr Ala
20 25 30
Val Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu He
35 40 45
Tyr Ser Ala Ser Tyr Leu Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr He Ser Ser Val Gin Ala 65 70 75 80
Glu Asp Leu Ala Val Tyr Tyr Cys Gin Gin Hi s Tyr Ser Thr Pro Arg
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu He
100 105
<210> 11
<211> 107
<212 > PRT
<213> Arti fi cial Sequence
<220>
<223> Des cription o f Arti fi cial Sequence : Syntheti c
polypeptide
<400> 11
Asp He Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15
Asp Arg Val Thr He Thr Cys Lys Ala Ser Gin Asp Val Ser Thr Ala
20 25 30
Val Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu He
35 40 45
Tyr Ser Ala Ser Tyr Leu Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr He Ser Ser Leu Gin Pro
65 70 75 80
Glu Asp He Ala Thr Tyr Tyr Cys Gin Gin His Tyr Ser Thr Pro Arg
85 90 95
Thr Phe Gly Gin Gly Thr Lys Leu Glu He
100 105
<210> 12
<211> 416
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
polynucleotide
<400> 12
gctagcacca ccatggagtc acagattcag gtctttgtat tcgtgtttct ctggttgtct 60 ggtgttgacg gagacattca gatgacccag tctccatcct ccctgtccgc atcagtggga 120 gacagggtca ccatcacctg caaggccagt caggatgtga gtactgctgt agcctggtat 180 caacagaaac caggaaaagc ccctaaacta ctgatttact cggcatccta cctctacact 240 ggagtccctt cacgcttcag tggcagtgga tctgggacgg atttcacttt caccatcagc 300 agtctgcagc ctgaagacat tgcaacatat tactgtcagc aacattatag tactcctcgg 360 acgttcggtc agggcaccaa gctggaaatc aaacgtaagt agaatccaaa gaattc 416
<210> 13
<211> 5
<212> PRT
<213> Mus sp.
<400> 13
Ser His Asp Met
1
<210> 14
<211> 17
<212> PRT
<213> Mus sp.
<400> 14
Ala He Asn Ser Asp Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Met Glu
1 5 10 15
Arg <210> 15
<211> 11
<212> PRT
<213> Mus sp.
<400> 15
His Tyr Asp Asp Tyr Tyr Ala Trp Phe Ala Tyr
1 5 10
<210> 16
<211> 120
<212> PRT
<213> Mus sp.
<400> 16
Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Glu 1 5 10 15
Ser Leu Lys Leu Ser Cys Glu Ser Asn Glu Tyr Glu Phe Pro Ser His
20 25 30
Asp Met Ser Trp Val Arg Lys Thr Pro Glu Lys Arg Leu Glu Leu Val
35 40 45
Ala Ala lie Asn Ser Asp Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Met 50 55 60
Glu Arg Arg Phe lie lie Ser Arg Asp Asn Thr Lys Lys Thr Leu Tyr 65 70 75 80
Leu Gin Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Arg His Tyr Asp Asp Tyr Tyr Ala Trp Phe Ala Tyr Trp Gly Gin
100 105 110
Gly Thr Leu Val Thr Val Ser Ala
115 120
<210> 17
<211> 120
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
polypeptide
<400> 17
Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Tyr Glu Phe Pro Ser His
20 25 30
Asp Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Leu Val
35 40 45 Ala Ala lie Asn Ser Asp Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Met
50 55 60
Glu Arg Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg His Tyr Asp Asp Tyr Tyr Ala Trp Phe Ala Tyr Trp Gly Gin
100 105 110
Gly Thr Met Val Thr Val Ser Ser
115 120 <210> 18
<211> 451
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
polynucleotide
<400> 18
actagtacca ccatggactt cgggctcagc ttggttttcc ttgtccttat tttaaaaagt 60 gtacagtgtg aggtgcagct ggtggagtct gggggaggct tagtgcagcc tggagggtcc 120 ctgagactct cctgtgcagc ctctgaatac gagttccctt cccatgacat gtcttgggtc 180 cgccaggctc cggggaaggg gctggagttg gtcgcagcca ttaatagtga tggtggtagc 240 acctactatc cagacaccat ggagagacga ttcaccatct ccagagacaa tgccaagaac 300 tcactgtacc tgcaaatgaa cagtctgagg gccgaggaca cagccgtgta ttactgtgca 360 agacactatg atgattacta cgcctggttt gcttactggg gccaagggac tatggtcact 420 gtctcttcag gtgagtccta acttcaagct t 451
<210> 19
<211> 15
<212> PRT
<213> Mus sp. <400> 19
Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His 1 5 10 15
<210> 20
<211> 7
<212> PRT
<213> Mus
<400> 20
Leu Ala Ser Asn Leu Glu Ser
1 5
<210> 21
<211> 9
<212> PRT
<213> Mus sp.
<400> 21
Gin His Ser Arg Glu Leu Pro Leu Thr
1 5
<210> 22
<211> 111
<212> PRT
<213> Mus
<400> 22
Asp He Val Leu Thr Gin Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15
Gin Arg Ala Thr He Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser
20 25 30
Gly Tyr Ser Tyr Met His Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro
35 40 45
Lys Leu Leu He Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn He His 65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gin His Ser Arg
85 90 95
Glu Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105 110
<210> 23
<211> 111
Figure imgf000076_0001
<210> 25
<211> 5
<212> PRT
<213> Mus sp.
<400> 25
Asp Ala Trp Met
1
<210> 26
<211> 19
<212> PRT
<213> Mus sp.
<400> 26
Glu He Arg Ser Lys Ala Asn Asn His Ala Thr Tyr Tyr Ala Glu Ser
1 5 10 15
Val Asn Gly
<210> 27
<211> 8
<212> PRT
<213> Mus sp.
<400> 27
Gly Glu Val Phe Tyr Phe Asp Tyr
1 5
<210> 28
<211> 414
<212> DNA
<213> Mus sp.
<400> 28
atgtacttgg gactgaacta tgtattcata gtttttctct taaatggtgt ccagagtgaa 60 gtgaagcttg aggagtctgg aggaggcttg gtgcaacctg gaggatccat gaaactctct 120 tgtgctgcct ctggattcac ttttagtgac gcctggatgg actgggtccg ccagtctcca 180 gagaaggggc ttgagtgggt tgctgaaatt agaagcaaag ctaataatca tgcaacatac 240 tatgctgagt ctgtgaatgg gaggttcacc atctcaagag atgattccaa aagtagtgtc 300 tacctgcaaa tgaacagctt aagagctgaa gacactggca tttattactg tacgtggggg 360 gaagtgttct actttgacta ctggggccaa ggcaccactc tcacagtctc ctca 414
<210> 29
<211> 138
<212> PRT
<213> Mus sp. <400> 29
Met Tyr Leu Gly Leu Asn Tyr Val Phe He Val Phe Leu Leu Asn Gly
1 5 10 15
Val Gin Ser Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gin
20 25 30
Pro Gly Gly Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
35 40 45 Ser Asp Ala Trp Met Asp Trp Val Arg Gin Ser Pro Glu Lys Gly Leu
50 55 60
Glu Trp Val Ala Glu He Arg Ser Lys Ala Asn Asn His Ala Thr Tyr
65 70 75 80
Tyr Ala Glu Ser Val Asn Gly Arg Phe Thr He Ser Arg Asp Asp Ser
85 90 95
Lys Ser Ser Val Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr
100 105 110
Gly He Tyr Tyr Cys Thr Trp Gly Glu Val Phe Tyr Phe Asp Tyr Trp
115 120 125 Gly Gin Gly Thr Thr Leu Thr Val Ser Ser
130 135
<210> 30
<211> 448
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
polynucleotide
<400> 30
actagtacca ccatgtactt gggactgaac tatgtattca tagtttttct cttaaatggt 60 gtccagagtg aagtgaagct ggaggagtct ggaggaggct tggtgcaacc tggaggatcc 120 atgaaactct cttgtgctgc ctctggattc acttttagtg acgcctggat ggactgggtc 180 cgccagtctc cagagaaggg gcttgagtgg gttgctgaaa ttagaagcaa agctaataat 240 catgcaacat actatgctga gtctgtgaat gggaggttca ccatctcaag agatgattcc 300 aaaagtagtg tctacctgca aatgaacagc ttaagagctg aagacactgg catttattac 360 tgtacgtggg gggaagtgtt ctactttgac tactggggcc aaggcaccac tctcacagtc 420 tcctcaggtg agtccttaaa acaagctt 44
<210> 31
<211> 138
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
polypeptide
<400> 31
Met Tyr Leu Gly Leu Asn Tyr Val Phe He Val Phe Leu Leu Asn Gly
1 5 10 15
Val Gin Ser Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gin
20 25 30
Pro Gly Gly Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
35 40 45
Ser Asp Ala Trp Met Asp Trp Val Arg Gin Ser Pro Glu Lys Gly Leu
50 55 60
Glu Trp Val Ala Glu He Arg Ser Lys Ala Asn Asn His Ala Thr Tyr
65 70 75 80
Tyr Ala Glu Ser Val Asn Gly Arg Phe Thr He Ser Arg Asp Asp
85 90 95
Lys Ser Ser Val Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr
100 105 110
Gly He Tyr Tyr Cys Thr Trp Gly Glu Val Phe Tyr Phe Asp Tyr Trp
115 120 125
Gly Gin Gly Thr Thr Leu Thr Val Ser Ser
130 135
<210> 32
<211> 11
<212> PRT
<213> Mus
<400> 32
Lys Ser Ser Gin Asp He Asn Lys Tyr He Ala
1 5 10 <210> 34
<211> 8
<212> PRT
<213> Mus sp.
<400> 34
Leu Gin Tyr Asp Asn Leu Leu Thr
1 5
<210> 35
<211> 378
<212> DNA
<213> Mus sp.
<400> 35
atgagaccgt ctattcagtt cctggggctc ttgttgttct ggcttcatgg tgctcagtgt 60 gacatccaga tgacacagtc tccatcctca ctgtctgcat ctctgggagg caaagtcacc 120 atcacttgca agtcaagcca agacattaac aagtatatag cttggtacca acacaagcct 180 ggaaaaggtc ctaggctgct catacattac acatctacat tacagccagg catcccatca 240 aggttcagtg gaagtgggtc tgggagagat tattccttca gcatcagcaa cctggagcct 300 gaagatattg caacttatta ttgtctacag tatgataatc ttctcacgtt cggtgctggg 360 accaagctgg agctgaaa 378
<210> 36
<211> 126
<212> PRT
<213> Mus sp.
<400> 36
Met Arg Pro Ser He Gin Phe Leu Gly Leu Leu Leu Phe Trp Leu His
1 5 10 15
Gly Ala Gin Cys Asp He Gin Met Thr Gin Ser Pro Ser Ser Leu Ser
20 25 30
Ala Ser Leu Gly Gly Lys Val Thr He Thr Cys Lys Ser Ser Gin Asp
35 40 45
He Asn Lys Tyr He Ala Trp Tyr Gin His Lys Pro Gly Lys Gly Pro
50 55 60 Arg Leu Leu He His Tyr Thr Ser Thr Leu Gin Pro Gly He Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser He Ser
85 90 95
Asn Leu Glu Pro Glu Asp He Ala Thr Tyr Tyr Cys Leu Gin Tyr Asp
100 105 110
Asn Leu Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
115 120 125
<210> 37
<211> 413
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
polynucleotide
<400> 37
gctagcacca ccatgagacc gtctattcag ttcctggggc tcttgttgtt ctggcttcat 60 ggtgctcagt gtgacatcca gatgacacag tctccatcct cactgtctgc atctctggga 120 ggcaaagtca ccatcacttg caagtcaagc caagacatta acaagtatat agcttggtac 180 caacacaagc ctggaaaagg tcctaggctg ctcatacatt acacatctac attacagcca 240 ggcatcccat caaggttcag tggaagtggg tctgggagag attattcctt cagcatcagc 300 aacctggagc ctgaagatat tgcaacttat tattgtctac agtatgataa tcttctcacg 360 ttcggtgctg ggaccaagct ggagctgaaa cgtaagtaca cttttctgaa ttc 413
<210> 38
<211> 126
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
polypeptide
<400> 38
Met Arg Pro Ser He Gin Phe Leu Gly Leu Leu Leu Phe Trp Leu His
1 5 10 15
Gly Ala Gin Cys Asp He Gin Met Thr Gin Ser Pro Ser Ser Leu Ser
20 25 30 Ala Ser Leu Gly Gly Lys Val Thr lie Thr Cys Lys Ser Ser Gin Asp
35 40 45 lie Asn Lys Tyr lie Ala Trp Tyr Gin His Lys Pro Gly Lys Gly Pro
50 55 60
Arg Leu Leu lie His Tyr Thr Ser Thr Leu Gin Pro Gly lie Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser lie Ser
85 90 95
Asn Leu Glu Pro Glu Asp lie Ala Thr Tyr Tyr Cys Leu Gin Tyr Asp
100 105 110
Asn Leu Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
115 120 125
<210> 39
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
primer
<400> 39
cgctgttttg acctccatag
<210> 40
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
primer
<400> 40
tgaaagatga gctggaggac
<210> 41
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
primer
<400> 41
ctttcttgtc caccttggtg 20 <210> 42
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
primer
<400> 42
gctgtcctac agtcctcag
<210> 43
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
primer
<400> 43
acgtgccaag catcctcg 18
<210> 44
<211> 1407
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
polynucleotide
<400> 44
atgtacttgg gactgaacta tgtattcata gtttttctct taaatggtgt ccagagtgaa 60 gtgaagctgg aggagtctgg aggaggcttg gtgcaacctg gaggatccat gaaactctct 120 tgtgctgcct ctggattcac ttttagtgac gcctggatgg actgggtccg ccagtctcca 180 gagaaggggc ttgagtgggt tgctgaaatt agaagcaaag ctaataatca tgcaacatac 240 tatgctgagt ctgtgaatgg gaggttcacc atctcaagag atgattccaa aagtagtgtc 300 tacctgcaaa tgaacagctt aagagctgaa gacactggca tttattactg tacgtggggg 360 gaagtgttct actttgacta ctggggccaa ggcaccactc tcacagtctc ctcagcctcc 420 accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca 480 gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 540 tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 600 tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 660 tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct 720 tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 780 gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 840 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 900 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 960 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 1020 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1080 aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc 1140 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1200 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1260 tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 1320 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1380 agcctctccc tgtctccggg taaatga 1407
<210> 45
<211> 469
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
polypeptide
<400> 45
Met Tyr Leu Gly Leu Asn Tyr Val Phe He Val Phe Leu Leu Asn Gly
1 5 10 15
Val Gin Ser Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gin
20 25 30
Pro Gly Gly Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
35 40 45
Ser Asp Ala Trp Met Asp Trp Val Arg Gin Ser Pro Glu Lys Gly Leu
50 55 60
Glu Trp Val Ala Glu He Arg Ser Lys Ala Asn Asn His Ala Thr Tyr
65 70 75 80
Tyr Ala Glu Ser Val Asn Gly Arg Phe Thr He Ser Arg Asp Asp
85 90 95
Lys Ser Ser Val Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr 100 105 110
Gly lie Tyr Tyr Cys Thr Trp Gly Glu Val Phe Tyr Phe Asp Tyr Trp
115 120 125
Gly Gin Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 130 135 140
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 145 150 155 160
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
165 170 175
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
180 185 190
Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
195 200 205
Val Pro Ser Ser Ser Leu Gly Thr Gin Thr Tyr lie Thr Cys Asn Val 210 215 220
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 225 230 235 240
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
245 250 255
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
260 265 270
Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
275 280 285
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295 300
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser 305 310 315 320
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu
325 330 335
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
340 345 350 Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro
355 360 365
Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin
370 375 380
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala
385 390 395 400
Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr
405 410 415
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
420 425 430
Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser
435 440 445
Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser
450 455 460
Leu Ser Pro Gly Lys
465
<210> 46
<211> 702
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
polynucleotide
<400> 46
atgagaccgt ctattcagtt cctggggctc ttgttgttct ggcttcatgg tgctcagtgt 60 gacatccaga tgacacagtc tccatcctca ctgtctgcat ctctgggagg caaagtcacc 120 atcacttgca agtcaagcca agacattaac aagtatatag cttggtacca acacaagcct 180 ggaaaaggtc ctaggctgct catacattac acatctacat tacagccagg catcccatca 240 aggttcagtg gaagtgggtc tgggagagat tattccttca gcatcagcaa cctggagcct 300 gaagatattg caacttatta ttgtctacag tatgataatc ttctcacgtt cggtgctggg 360 accaagctgg agctgaaacg aactgtggct gcaccatctg tcttcatctt cccgccatct 420 gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc 480 agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag 540 agtgtcacag agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg 600 agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg 660 agctcgcccg tcacaaagag cttcaacagg ggagagtgtt ag 702
<210> 47
<211> 233
<212 > PRT
<213> Arti fi cial Sequence
<220>
<223> Des cription o f Arti fi cial Sequence : Syntheti c
polypeptide
<400> 47
Met Arg Pro Ser He Gin Phe Leu Gly Leu Leu Leu Phe Trp Leu Hi s
1 5 10 15
Gly Ala Gin Cys Asp He Gin Met Thr Gin Ser Pro Ser Ser Leu Ser
20 25 30
Ala Ser Leu Gly Gly Lys Val Thr He Thr Cys Lys Ser Ser Gin Asp
35 40 45
He Asn Lys Tyr He Ala Trp Tyr Gin Hi s Lys Pro Gly Lys Gly Pro
50 55 60
Arg Leu Leu He Hi s Tyr Thr Ser Thr Leu Gin Pro Gly He Pro Ser
65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser He Ser
85 90 95
Asn Leu Glu Pro Glu Asp He Ala Thr Tyr Tyr Cys Leu Gin Tyr Asp
100 105 110
Asn Leu Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr
115 120 125
Val Ala Ala Pro Ser Val Phe He Phe Pro Pro Ser Asp Glu Gin Leu
130 135 140
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
145 150 155 160
Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly
165 170 175 Asn Ser Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr 180 185 190
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
195 200 205
Lys Val Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val 210 215 220
Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230

Claims

We claim:
A combination of an antigen binding protein (ABP) that binds OX40 and at least one TLR modulator chosen from a TLR4 modulator and a TLR7/8 modulator, and wherein the ABP modulates OX40.
The combination as claimed in Claim 1 , wherein the ABP that binds OX40 is a humanized monoclonal antibody comprising: a heavy chain variable region CDR1 comprising an amino acid sequence with at least 90% identity to an amino acid sequence chosen from: SEQ ID NO: 1 and SEQ ID NO: 13; a heavy chain variable region CDR2 comprising an amino acid sequence with at least 90% identity to an amino acid sequence chosen from: SEQ ID NO: 2 and SEQ ID NO: 14; and/or a heavy chain variable region CDR3 comprising an amino acid sequence with at least 90% identity to an amino acid sequence chosen from: SEQ ID NO: 3 and SEQ ID NO: 15; a light chain variable region CDR1 comprising an amino acid sequence with at least 90% identity to an amino acid sequence chosen from: SEQ ID NO: 7 and SEQ ID NO: 19; a light chain variable region CDR2 comprising an amino acid sequence with at least 90% identity to an amino acid sequence chosen from: SEQ ID NO: 8 and SEQ ID NO: 20; and/or a light chain variable region CDR3 comprising an amino acid sequence with at least 90% identity to an amino acid sequence chosen form: SEQ ID NO: 9 and SEQ ID NO: 21.
A combination of any of the preceding claims, wherein the the ABP that binds OX40 comprises: (a) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1 ; (b) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2; (c) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO. 3; (d) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO. 7; (e) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO. 8; and (f) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO. 9.
A combination of any of the preceding claims, wherein the ABP that binds to OX40 is an antibody comprising a heavy chain variable region comprising an amino acid sequence with at least 90% identity to an amino acid sequence chosen from: SEQ ID NO: 4 and SEQ ID NO: 5, wherein the antibody further comprises a light chain variable region comprising an amino acid sequence with at least 90% identity to an amino acid sequence chosen from: SEQ ID NO: 10 and SEQ ID NO: 11.
5. A combination of any of the preceding claims, wherein the TLR modulator is a
TLR4 modulator chosen from: CRX-601 , CRX-547, CRX-602, CRX-527, and CRX- 526.
6. A combination kit comprising a combination according to any of the preceding
claims together with one or more pharmaceutically acceptable carriers.
7. A pharmaceutical composition comprising a therapeutically effective amount of an ABP or antibody of any of the preceding claims and a second pharmaceutical composition comprising a therapeutically effective amount of a at least one TLR moculator chosen from: a TLR7/8 modulator and a TLR4 modulator of any of the preceding claims.
8. A pharmaceutical composition as claimed in Claim 7, comprising an antibody
comprising an ABP or antibody comprising a CDRH1 having a sequence at least 90% identity to the amino acid sequence as set forth in SEQ ID NO: 1 , a CDRH2 having a sequence at least with a sequence at least 90% identity to the amino acid sequence as set forth in SEQ ID NO: 2, a CDRH3 having a sequence at least 90% identity to the amino acid sequence as set forth in SEQ ID NO: 3, a CDRL1 having a sequence at least with a sequence at least 90% identity to the amino acid sequence as set forth in SEQ ID NO: 7, a CDRL2 having a sequence at least with a sequence at least 90% identity to the amino acid sequence as set forth in SEQ ID NO: 8, a CDRL3 having a sequence at least with a sequence at least 90% identity to the amino acid sequence as set forth in SEQ ID NO: 9; and a TLR7/8 modulator and a TLR4 modulator, wherein the TLR4 modulator is CRX-601.
9. A pharmaceutical composition as claimed in Claim 7 or Claim 8, comprising an antibody comprising a VH region having a sequence at least with a sequence at least 90% identity to the amino acid sequence as set forth in SEQ ID NO: 4 or 5 and VL having a sequence at least with a sequence at least 90% identity to the amino acid sequence as set forth in SEQ ID NO: 10 or 11 , and a TLR7/8 modulator, wherein the TLR4 modulator is CRX-601.
10. Use of a combination or pharmaceutical composition of any of the preceding claims in the manufacture of a medicament for the treatment of cancer.
11. A method of treating cancer in an animal need thereof comprising administering a combination or pharmaceutical composition of any of the preceding claims.
12. The method of treatment as claimed in Claim 11 , wherein the animal is a human, claims. 13. The method of treatment of any of the preceding claims, wherein the ABP or
antibody and at least one TLR modulator chosen from: a TLR47/8 modulator and a TLR4 modulator are administered at the same time.
14. The method of treatment of any of the preceding claims, wherein the ABP or
antibody and the at least one TLR modulator chosen from: a TLR47/8 modulator and a TLR4 modulator are administered sequentially, in any order.
15. The method of of treatment of any of the preceding claims, wherein the ABP or antibody and at least one TLR modulator chosen from: a TLR7/8 modulator and a TLR4 modulator are administered systemically.
16. The method of treatment of any of the preceding claims, wherein the ABP or
antibody is administered systemically, and the at least one TLR modulator chosen from: a TLR7/8 modulator and a TLR4 modulator is administered intratumorally.
17. The method of treatment of any of the preceding claims, wherein the ABP or
antibody is administered intratumorally, and the at least one TLR modulator chosen from: a TLR7/8 modulator and a TLR4 modulator is administered systemically. 18. The method of treatment any of the preceding claims, wherein the ABP and the at least one TLR modulator chosen from: a TLR7/8 modulator and a TLR4 modulator are administered intratumorally.
19. The method of treatment of any of the preceding claims, wherein the cancer is a solid tumor.
20. The method of treatment of any of the preceding claims, wherein the cancer is chosen from: melanoma, lung cancer, NSCLC, kidney cancer, RCC, HCC breast cancer, metastatic breast cancer, TNBC, H&N, colon cancer, CRC, ovarian cancer, pancreatic cancer, liver cancer, HCC, prostate cancer, bladder cancer, gastric cancer, liquid tumors, hematopoetic tumors, leukemia, NHL, lymphoma, and
CLL.
PCT/IB2016/054696 2015-08-06 2016-08-03 Combined tlrs modulators with anti ox40 antibodies WO2017021912A1 (en)

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WO2019191133A1 (en) 2018-03-27 2019-10-03 Bristol-Myers Squibb Company Real-time monitoring of protein concentration using ultraviolet signal
CN111363040A (en) * 2018-12-25 2020-07-03 杭州翰思生物医药有限公司 anti-OX 40 monoclonal antibodies and uses thereof
WO2020172658A1 (en) 2019-02-24 2020-08-27 Bristol-Myers Squibb Company Methods of isolating a protein
WO2020237221A1 (en) 2019-05-23 2020-11-26 Bristol-Myers Squibb Company Methods of monitoring cell culture media
WO2020259667A1 (en) * 2019-06-28 2020-12-30 江苏恒瑞医药股份有限公司 Use of combination of tlr agonist and anti-ox40 antibody or antigen binding fragment thereof in preparation of medicament for treating tumors
CN112442121A (en) * 2020-08-20 2021-03-05 山东兴瑞生物科技有限公司 OX40 antibody, encoding gene thereof, preparation method of antibody and application of antibody in enhancing human immune function
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CN115856300A (en) * 2022-08-01 2023-03-28 南京大学 Lung cancer-related plasma exosome protein marker, antibody and application thereof
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