WO2018045069A1 - IMMUNOTHÉRAPIES COMBINÉES À DES MODULATEURS DE SIGNALISATION DE TNFα - Google Patents

IMMUNOTHÉRAPIES COMBINÉES À DES MODULATEURS DE SIGNALISATION DE TNFα Download PDF

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WO2018045069A1
WO2018045069A1 PCT/US2017/049437 US2017049437W WO2018045069A1 WO 2018045069 A1 WO2018045069 A1 WO 2018045069A1 US 2017049437 W US2017049437 W US 2017049437W WO 2018045069 A1 WO2018045069 A1 WO 2018045069A1
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tnfa
cells
antagonist
tnfr2
mammal
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Anthony LEONARDI
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Leonardi Anthony
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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
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1793—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.

Definitions

  • Adoptive cell therapy can be an effective treatment for cancer in some patients.
  • TNF ablation has been studied as part of an adoptive cell immunotherapy regimen (Alam et al. Nat Med. Nov;21 (l l): 1337-43. (2015)), however this was done genetically rather than in a reversible way which may expose patients to unnecessary risks due to the irreversability of physiological signalling. Furthermore, the safety of genetic ablation of specific signalling proteins in T cells as part of an immunotherapy regimen is yet to be studied in humans.
  • TNF ablation has also been studied as a stand-alone immunotherapy to prevent establishment of tumor (Atretkhany, et al. Front Immunol. Apr 19;7: 147. (2016)), but has not been investigated in combination with other immunotherapies, presumably because the ablation of the TNFa signal in vivo is counterintuitive to the goal of a pro-inflammatory setting immunotherapy seeks to create.
  • An embodiment of the invention provides a method of producing T-cells for adoptive cell therapy, said method comprising:
  • a method of producing T-cells for adoptive cell therapy comprising: administering to a mammal a TNFa antagonist in combination with transferred cells in an amount effective to suppress differentiation of the T cells as part of an adoptive cell therapy regimen.
  • Another embodiment of the invention provides a method of treating or preventing cancer in a mammal comprising:
  • TNFa antagonist in an amount effective to suppress differentiation of the T cells
  • a method of treating or preventing cancer in a mammal comprising:
  • TNFa antagonist in an amount effective to suppress differentiation of the T cells following and/or during adoptive T cell immunotherapy.
  • Another embodiment of the invention provides a method of producing T-cells for adoptive cell therapy with a transduction comprising:
  • TNFa antagonist in an amount effective to suppress differentiation of the T cells during transduction of a dna/rna sequences in order to express a DNA or RNA product in the T cell for Adoptive cell transfer into a mammal.
  • Another embodiment of the invention includes a method of cancer immunotherapy including administering a TNFa antagonist and PD-1 and/or PD-L1 antagonist.
  • T cells may be desirable, in some cases, to delay T cells to exposure of TNFa until the signal becomes contextually relevant in the course of an immunotherapy regimen such as anti pd-1 or anti pd-11, or contextually relevant in an adoptive cell immunotherapy regimen.
  • Suppression of T cell endogenous signalling of TNFa may be advantageous at several points in the course of adoptive cell therapy, although this topic is not explored within the field in a physiological setting.
  • targeted nullification of a TNFa signal without the use of genetic ablation may act as an additional safety precaution if the need to reintroduce physiological activity emerges.
  • T cells may be advantageous to suppress the differentiation of T cells for a period of time, for example, while the cells are in vitro and prior to re-introducing the cells into the body, rather than simply ablating the TNF signalling ability for the lifetime of the cells, so that the cells may reacquire their physiological states following an immunotherapy regimen. Additionally, it may be advantageous to block the TNFa signal after introduction of cells to the mammal for a time.
  • the TNFa signalling of the T cells may be suppressed for a period of time, e.g., while the cells are in vitro and/or in vivo
  • the T cells may, advantageously, differentiate and acquire TNF secreting and signaling ability at a later point in time, e.g., upon re-introduction of the cells into the body or after the regression of tumor in the body or when the immunotherapy regimen is complete.
  • Adoptive cell immunotherapy refers to any treatment to prevent, treat, or cure cancer comprising of the administration or infusion of CD4+, CD8+, or any combination of T cells or white blood cells.
  • TNFa biological activity suppresses the activation and differentiation of T cells.
  • some immunotherapy regimens show differentiated T cells have decreased in vivo treatment efficacy, and/or reduced ability to be be expanded in vitro and in vivo.
  • Current studies have shown that TNF blocking antibodies alone, without regimens that include adoptive cell therapy, can prevent tumor establishment in mouse models. While the mechanism is not fully understood, it may have to do with the preservation of a lesser-differentiated pool of T cells which target tumor antigen.
  • less differentiated T cells may provide many advantages, particularly in adoptive cell therapy applications.
  • the numbers of less differentiated T cells are believed to expand to greater numbers in vitro as compared to more differentiated T cells.
  • Less differentiated T-cells may also provide increased in vivo persistence, survival, and/or anti-tumor activity as compared to more differentiated T-cells.
  • less differentiated T cells may lyse tumor cells or treat or prevent cancer more effectively as compared to more differentiated T cells. Therefore, less differentiated T-cells may be suitable for adoptive cell therapy.
  • an embodiment of the invention provides a method of producing T-cells for adoptive cell therapy comprising isolating T cells from a mammal and contacting the T- cells in vitro with a TNFa antagonist in an amount effective to suppress differentiation of the T cells.
  • the T-cells contacted with a TNFa antagonist may be less differentiated as compared to T-cells that have not been treated with a TNFa antagonist.
  • a pool of cells with a less-differentiated phenotype as enforced through TNFa signalling blockade may further buttress immunotherapies which employ T-cell activity and reactivation such as anti PD-1 and anti pd-Ll therapy.
  • an embodiment of the invention provides a method of adoptive cell immunotherapy where the tnf blocking ab is given to the t cells in vitro.
  • Another embodiment of the invention provides a method of adoptive cell
  • Another embodiment of the invention provides methods for treating or preventing cancer in a mammal comprising immunotherapy in which a TNFa blocking antibody is given to a mammal along with an anti PD-1 or anti PD-L1 immunotherapy regimen.
  • an embodiment of the invention provides a method of immunotherapy in which a TNFa blocking antibody is given to a mammal along with an anti PD-1 or anti PD- Ll immunotherapy regimen.
  • An embodiment of the invention comprises isolating T cells from a mammal.
  • the T cells may be isolated from a mammal by any suitable means known in the art.
  • the T-cells can be isolated from the mammal by a blood draw or a leukapheresis.
  • the T cell can be any T cell obtained from a mammal.
  • the T cell can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, tumors, or other tissues or fluids.
  • T cells can also be enriched for or purified, or also taken as part of a bulk lymphocyte population via leukapheresis.
  • the T cell is a human T cell. More preferably, the T cell is a T cell isolated from a human.
  • the T cell can be any type of T cell, including but not limited to, tumor infiltrating lymphocytes (TIL), CD4 + /CD8 + double positive T cells, lymphocytes containing a mixture of CD4 + and CD8 + cells, CD4 + T cells, and CD8 + T cells.
  • TIL tumor infiltrating lymphocytes
  • CD4 + /CD8 + double positive T cells lymphocytes containing a mixture of CD4 + and CD8 + cells
  • CD4 + T cells CD4 + T cells
  • CD8 + T cells CD8 + T cells.
  • the cells can be cells that are allogeneic or autologous to the mammal.
  • the cells are autologous to the mammal.
  • An embodiment of the invention comprises the administration of a TNFa-antagonist to a mammal which has received T cells as part of an adoptive cell immunotherapy regimen.
  • An embodiment of the invention comprises the administration of a TNFa-antagonist to a mammal in combination with an anti pd-1 or anti pd-11 immunotherapy regimen.
  • An embodiment of the invention comprises contacting the T-cells in vitro with a TNFa antagonist in an amount effective to suppress differentiation of the T cells.
  • contact refers to providing conditions which promote the TNFa antagonist to physically contact the T-cells.
  • Another embodiment of the invention provides a method of producing T-cells for adoptive cell therapy comprising administering to a mammal a TNFa antagonist in an amount effective to suppress differentiation of the T cells and isolating T cells from the mammal.
  • the mammal may be treated with a TNFa antagonist prior to isolating the T cells from the mammal, and the T cells may be isolated from the mammal after administering the TNFa antagonist to the mammal.
  • the T-cells isolated from the TNFa antagonist-treated mammal may be less differentiated as compared to T-cells isolated from a mammal that has not been treated with a TNFa antagonist.
  • An embodiment of the method comprises administering to a mammal a TNFa antagonist in an amount effective to suppress differentiation of the T cells.
  • the T cells may be isolated from the mammal as described herein with respect to other aspects of the invention.
  • Another embodiment of the invention provides a method of treating or preventing cancer in a mammal comprising isolating T cells from a mammal; contacting the T-cells in vitro with a TNFa antagonist in an amount effective to suppress differentiation of the T cells; and administering the TNFa antagonist-treated T cells to the mammal in an amount effective to treat or prevent cancer in the mammal.
  • the T cells may be isolated from the mammal and the T-cells may be contacted in vitro with a TNFa antagonist in an amount effective to suppress the differentiation of the T cells as described herein with respect to other aspects of the invention.
  • An embodiment of the method further comprises administering the TNFa antagonist- treated T cells to the mammal in an amount effective to treat or prevent cancer in the mammal.
  • the T cells may be isolated from the mammal prior to contacting the T-cells in vitro with a TNFa antagonist, and the T-cells may be contacted in vitro with a TNFa antagonist prior to administering the TNFa-antagonist-treated T cells to the mammal.
  • the T-cells may be contacted in vitro with a TNFa antagonist after the T-cells are isolated from the mammal, and the TNFa antagonist T cells may be administered to the mammal after contacting the isolated T-cells in vitro with a TNFa antagonist.
  • the TNFa antagonist-treated T cells that are administered to the mammal may be less differentiated as compared to T-cells that have not been contacted in vitro with TNFa antagonist.
  • An embodiment of the method may, optionally, further comprise culturing the isolated T-cells.
  • the T cells may be cultured by any suitable method known in the art such as those described, e.g., in United States Patent No. 8,034,334, United States Patent Application Publication No. 2011/0052530, and U.S. Patent Application No. 13/424,646, each of which are incorporated herein by reference in their entirety.
  • An embodiment of the method may, optionally, further comprise expanding the numbers of T-cells.
  • Expanding the numbers of T cells may comprise, for example, stimulating the T-cells in vitro with an antigen (e.g., cancer antigen) and expanding the numbers of cells using irradiated allogeneic feeder cells, OKT3 antibody, and IL-2, for example, as described in Riddell et al, Science, 257:238-241 (1992) and Dudley et al, Cancer J. Sci. Am., 6:69-77 (2000).
  • the numbers of the T cells may be expanded by any suitable method known in the art such as those described, e.g., in U.S. Patent 8,034,334, U.S. Patent Application Publication No. 2011/0052530, and U.S. Patent Application No.
  • the method may, optionally, further comprise selecting T cells capable of recognizing antigen (e.g., cancer antigen) and/or lysing cancer cells while in other embodiments, the method does not include selecting T cells capable of recognizing antigen (e.g., cancer antigen) and/or lysing cancer cells.
  • the T-cells may be selected by any suitable method known in the art such as those described, e.g., in U.S. Patent 8,034,334, U.S. Patent Application Publication No. 2011/0052530, and U.S. Patent Application No. 13/424,646, each of which are incorporated herein by reference in their entirety.
  • An embodiment of the method may, optionally, further comprise any one or more of culturing the isolated T-cells; expanding the numbers of T-cells; and selecting T cells capable of recognizing antigen (e.g., cancer antigen) and/or lysing cancer cells.
  • antigen e.g., cancer antigen
  • the method does not include selecting T cells capable of recognizing antigen (e.g., cancer antigen) and/or lysing cancer cells. Culturing the isolated T-cells; expanding the numbers of T-cells; and selecting T cells capable of recognizing antigen (e.g., cancer antigen) and/or lysing cancer cells may be carried out as described herein with respect to other aspects of the invention.
  • selecting T cells capable of recognizing antigen e.g., cancer antigen
  • lysing cancer cells may be carried out as described herein with respect to other aspects of the invention.
  • An embodiment of the invention further comprises lymphodepleting the mammal.
  • lymphodepletion include, but may not be limited to, nonmyeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, total body irradiation, etc.
  • the method may comprise administering the nonmyeloablative
  • Suppressing the differentiation of T cells may include reducing the maturation and/or proliferation of T cells. Without being bound to a particular theory or mechanism, it is believed that differentiation, activation, and proliferation are not interdependent.
  • suppressing the differentiation of T cells may include suppressing differentiation without suppressing proliferation of the cell.
  • Suppressing the differentiation of T cells can be measured by a decrease in (e.g., lack of) the production of cytokines associated with the differentiation of T cells.
  • the TNFa antagonist may decrease the T cell production of cytokines such as, for example, any or all of interferon (IFN)-y, interleukin (IL)-2, IL-4, IL-5, IL-6, IL-10, My pi alpha, Myplbeta, GM- CSF, Rantes/CCL5, CCL3, CCL4, and tumor necrosis factors (TNF) (e.g., TNFa and ⁇ ).
  • IFN interferon
  • IL-2 interleukin-2
  • IL-4 interleukin-5
  • IL-6 IL-10
  • My pi alpha Myplbeta
  • GM- CSF GM- CSF
  • Rantes/CCL5 GM- CSF
  • Rantes/CCL5 GM- CSF
  • CCL3, CCL4 tumor necrosis
  • Suppressing the differentiation of T cells can, alternatively or additionally, be measured by an increase in the expression of cell surface markers associated with a less differentiated T cell phenotype and/or a decrease in the expression of cell surface markers associated with a more differentiated phenotype.
  • Less differentiated T cell phenotypes may include, for example, a naive T-cell phenotype and/or a central memory (TCM) T cell phenotype, or cells that take up less glucose as per a 2NBDG stain.
  • More differentiated T cell phenotypes may include, for example, an exhausted T cell phenotype and/or an effector memory (TEFF) T cell phenotype, or cells that take up more glucose on a 2NBDG stain.
  • the TNFa antagonist may increase the T cell expression of cell surface markers associated with a less differentiated phenotype such as, for example, any or all of CD62L, CCR7, IL7RA, CD27, and CD28 and/or decrease the T cell expression of cell surface markers associated with a more differentiated phenotype such as, for example, any or all of KLRG-1, CD45RO, CD44, Seal, Myplalpha, Myplbeta, GM-CSF, Rantes/CCL5, CCL3, CCL4, granzymes, perforin, and CD57.
  • Assays for measuring or detecting a decrease or increase in the expression of cell surface markers are known in the art.
  • Suppressing the differentiation of T cells can, alternatively or additionally, be measured by an increase in the expression of transcription factors associated with a less differentiated T cell phenotype and/or a decrease in the expression of transcription factors associated with a more differentiated phenotype.
  • the TNFa antagonist may increase the T cell expression of transcription factors associated with a less differentiated phenotype such as, for example, any or all of Tcf7, Lefl, Klf2, Foxol, dll6ra and/or decrease the T cell expression of transcription factors associated with a more differentiated phenotype such as, for example, any or all of Tbx21, Prdml, and Gzmb.
  • Assays for measuring or detecting a decrease or increase in the expression of transcription factors are known in the art.
  • Suppressing the differentiation of T cells can, alternatively or additionally, be measured by a decrease in telomere length.
  • Less differentiated T cells may have a mean telomere length that is longer than that of more differentiated T cells.
  • T cells lose an estimated telomere length of 0.8 kb per week in culture, and that less differentiated T cells have telomeres that are about 1.4 kb longer than more differentiated T cells.
  • longer telomere lengths are associated with positive objective clinical responses in patients and persistence of the cells in vivo.
  • a "decrease” in expression (e.g., of transcription factors, proteins, cell surface markers, and/or cytokines) or telomere length includes preventing of the acquisition of the transcription factors, proteins, cells surface markers, cytokines, and/or telomere length that are markers of differentiation, and an "increase” in expression (e.g., of transcription factors, proteins, cell surface markers, cytokines) or telomere length includes maintenance (e.g., prevention of loss) of the transcription factors, proteins, cells surface markers, cytokines, and/or telomere length that are markers of low differentiation.
  • the TNFa antagonist can be any agent that inhibits the biological activity of TNFa and/or TNF Receptors TNFRl and/or TNFR2, respectively.
  • TNFa biological activity includes TNFa biological activity, TNFRl and/or TNFR2 biological activity, or both TNFa and TNFRl and/or TNFR2 biological activity.
  • the term “TNFa antagonist” collectively refers to TNFa antagonists and TNFRl and/or TNFR2 antagonists.
  • the biological activity of TNFa and/or TNFRl and/or TNFR2 may be inhibited in any manner, e.g., by inhibiting the expression of any one or more of TNFa mRNA, TNFa protein, TNFRl and/or TNFR2 mRNA, and TNFRl and/or TNFR2 protein; by inhibiting the binding of TNFa to TNFRl and/or TNFR2, and/or by inhibiting TNFa-TNFRl and/or TNFR2 signaling, as compared to that which is observed in the absence of the TNFa antagonist.
  • the biological activity may be inhibited to any degree that realizes a beneficial therapeutic effect.
  • the biological activity may be completely inhibited (i.e., prevented), while in other embodiments, the biological activity may be partially inhibited (i.e., reduced).
  • the terms "TNFa” and “TNFRl and/or TNFR2” refer to TNFa and TNFRl and/or TNFR2, respectively, in any form (e.g., mRNA or protein) and from any species (e.g., human or mouse).
  • the TNFa antagonist is an agent that inhibits TNFa-TNFRl and/or TNFR2 signaling.
  • TNFa-TNFRl and/or TNFR2 signaling can be inhibited in any manner.
  • the TNFa antagonist may inhibit the activation and/or binding of any one or more of various downstream targets of TNFa-TNFRl and/or TNFR2 signaling (e.g., nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB), mitogen-activated protein kinase (MAPK) 3/ extracellular signal-regulated kinase (ERK) 1 , MAPK8/Jun N-Terminal Protein Kinase (INK), AKT, and B-catenin).
  • NF-kappaB nuclear factor kappa-light-chain-enhancer of activated B cells
  • MAPK mitogen-activated protein kinase
  • ERK extracellular signal-regulated kinase
  • INK MAPK8/Jun N-Terminal Protein Kinase
  • AKT AKT
  • B-catenin B-catenin
  • the TNFa antagonist may be an agent that binds to TNFa or TNFRl and/or TNFR2, thereby reducing or preventing TNFa-TNFRl and/or TNFR2 signaling and inhibiting its function.
  • the TNFa antagonist that inhibits TNFa-TNFRl and/or TNFR2 signaling can be any of the antibodies or antibody fragments, antisense nucleic acids, or chemical inhibitors (e.g., small molecule or peptide (or polypeptide) inhibitor) described herein.
  • the TNFa antagonist is an agent that inhibits the binding of TNFa to TNFRl and/or TNFR2.
  • the TNFa antagonist may be any agent that binds to the TNFa protein or the TNFRl and/or TNFR2 protein, thereby reducing or preventing the binding of TNFa to TNFRl and/or TNFR2 and inhibiting its function, as well as agents that compete with the TNFa protein for the native TNFa binding site of TNFRl and/or TNFR2.
  • the agent that inhibits the binding of TNFa to TNFRl and/or TNFR2 can be any of the antibodies or antibody fragments, antisense nucleic acids, or chemical inhibitors (e.g., small molecule or peptide inhibitor) described herein.
  • the TNFa antagonist is an antibody or antibody fragment that specifically binds to TNFa or TNFRl and/or TNFR2.
  • the antibody can be any type of immunoglobulin that is known in the art.
  • the antibody can be of any isotype, e.g., IgA, IgD, IgE, IgG, IgM, etc.
  • the antibody can be monoclonal or polyclonal.
  • the antibody can be a naturally-occurring antibody, e.g., an antibody isolated and/or purified from a mammal, e.g., mouse, rabbit, goat, horse, chicken, hamster, human, etc.
  • the antibody can be a genetically-engineered antibody, e.g., a humanized antibody or a chimeric antibody.
  • the antibody can be in monomeric or polymeric form.
  • the antibody can have any level of affinity or avidity for TNFa, TNFRl and/or TNFR2, or a functional domain of TNFa or TNFRl and/or TNFR2, e.g., the TNFRl and/or TNFR2 binding portion of TNFa, or the TNFa binding portion of TNFRl and/or TNFR2.
  • Methods of testing antibodies for the ability to bind to TNFa, TNFRl and/or TNFR2, or any functional domain thereof are known in the art and include any antibody-antigen binding assay, such as, for example, radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, and competitive inhibition assays (see, e.g., Janeway et al, infra, U.S. Patent Application Publication No. 2002/0197266 Al, and U.S. Patent No. 7,338,929).
  • RIA radioimmunoassay
  • ELISA ELISA
  • Western blot Western blot
  • immunoprecipitation immunoprecipitation
  • competitive inhibition assays see, e.g., Janeway et al, infra, U.S. Patent Application Publication No. 2002/0197266 Al, and U.S. Patent No. 7,338,929.
  • Anti-TNFa and anti -TNFRl and/or TNFR2 antibodies and antibody fragments can be prepared using the TNFa and TNFRl and/or TNFR2 proteins disclosed herein and routine techniques. Suitable methods of making antibodies are known in the art. For instance, standard hybridoma methods are described in, e.g., Kohler and Milstein, Eur. J. Immunol, 5, 511-519 (1976), Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and C.A. Janeway et al. (eds.), Immunobiology, 5 th Ed., Garland Publishing, New York, NY (2001)).
  • Phage display furthermore can be used to generate an antibody.
  • phage libraries encoding antigen-binding variable (V) domains of antibodies can be generated using standard molecular biology and recombinant DNA techniques (see, e.g., Sambrook et al., supra, and Ausubel et al, supra). Phage encoding a variable region with the desired specificity are selected for specific binding to the desired antigen, and a complete or partial antibody is reconstituted comprising the selected variable domain.
  • Nucleic acid sequences encoding the reconstituted antibody are introduced into a suitable cell line, such as a myeloma cell used for hybridoma production, such that antibodies having the characteristics of monoclonal antibodies are secreted by the cell (see, e.g., Janeway et al, supra, Huse et al, supra, and U.S. Patent 6,265,150).
  • a suitable cell line such as a myeloma cell used for hybridoma production, such that antibodies having the characteristics of monoclonal antibodies are secreted by the cell (see, e.g., Janeway et al, supra, Huse et al, supra, and U.S. Patent 6,265,150).
  • Antibodies can be produced by transgenic mice that are transgenic for specific heavy and light chain immunoglobulin genes. Such methods are known in the art and described in, for example U.S. Patents 5,545,806 and 5,569,825, and Janeway et al., supra.
  • An embodiment of the invention also provides antigen binding portions of any of the antibodies described herein.
  • the antigen binding portion can be any portion that has at least one antigen binding site, such as Fab, F(ab')2, dsFv, sFv, diabodies, and triabodies.
  • a single-chain variable region fragment (sFv) antibody fragment can be generated using routine recombinant DNA technology techniques (see, e.g., Janeway et al., supra). Similarly, disulfide-stabilized variable region fragments (dsFv) can be prepared by recombinant DNA technology (see, e.g., Reiter et al, Protein Engineering, 7, 697-704 (1994)). Antibody fragments of the invention, however, are not limited to these exemplary types of antibody fragments.
  • the TNFa antagonist is a chemical inhibitor of TNFa biological activity.
  • Chemical inhibitors of TNFa and/or TNFRl and/or TNFR2 include small molecules and peptides or polypeptides that inhibit TNFa-TNFRl and/or TNFR2 signaling, bind the TNFa or TNFRl and/or TNFR2 protein or functional fragment thereof, or compete with the TNFa protein or functional fragment thereof for its native binding site of TNFRl and/or TNFR2.
  • Suitable inhibitors can include, for example, chemical compounds or a non-active fragment or mutant of TNFa.
  • the TNFa antagonist is a mutated TNFa or mutated TNFRl and/or TNFR2 transduced on the target cell population.
  • the mutation may include any insertions, deletions, and/or substitutions of one or more amino acids in any position of the TNFa protein that effectively inhibits TNFa biological activity (e.g., TNFa-TNFRl and/or TNFR2 signaling and/or binding of TNFa to TNFRl and/or TNFR2).
  • the chemical inhibitor can bind to TNFRl and/or TNFR2 and/or inhibit TNFa-TNFRl and/or TNFR2 signaling.
  • the TNFa antagonist inhibits the activation and/or binding of any one or more of NF-kappaB, MAPK3/ERK1 , MAPK8/JNK, AKT, and B-catenin, as described herein.
  • Chemical inhibitors of TNFa and/or TNFRl and/or TNFR2 can be identified using routine techniques. For example, chemical inhibitors can be tested in binding assays to identify molecules and peptides (or polypeptides) that bind to TNFa or TNFRl and/or TNFR2 with sufficient affinity to inhibit TNFa biological activity (e.g., binding of TNFa to TNFRl and/or TNFR2, and/or TNFa-TNFRl and/or TNFR2 signaling).
  • TNFa biological activity e.g., binding of TNFa to TNFRl and/or TNFR2, and/or TNFa-TNFRl and/or TNFR2 signaling.
  • competition assays can be performed to identify small-molecules and peptides (or polypeptides) that inhibit the activation and/or binding of downstream targets of TNFa-TNFRl and/or TNFR2 signaling or compete with TNFa or functional fragment thereof for binding to its native binding site of TNFRl and/or TNFR2.
  • Such techniques could be used in conjunction with mutagenesis of the TNFa protein or functional fragment thereof itself, and/or with high- throughput screens of known chemical inhibitors.
  • the functional fragment of the TNFa or TNFRl and/or TNFR2 protein can comprise any contiguous part of the TNFa or TNFRl and/or TNFR2 protein that retains a relevant biological activity of the TNFa or TNFRl and/or TNFR2 protein, e.g., binds to TNFa or TNFRl and/or TNFR2 and/or participates in TNFa-TNFRl and/or TNFR2 signaling. Any given fragment of a TNFa or TNFRl and/or TNFR2 protein can be tested for such biological activity using methods known in the art.
  • the functional fragment can comprise, consist essentially of, or consist of the TNFRl and/or TNFR2 binding portion of the TNFa protein or the TNFa binding portion of the TNFR1 and/or TNFR2 protein.
  • the functional fragment preferably comprises, for instance, about 10% or more, 25% or more, 30% or more, 50% or more, 60% or more, 80% or more, 90% or more, or even 95% or more of the parent TNFa or TNFR1 and/or TNFR2 protein, respectively.
  • the pd-1 or pd-Ll antagonist can be any agent that inhibits the biological activity of pd-1 or pd-Ll .
  • pd-1 or pd-Ll biological activity includes pd-1 or pd-Ll biological activity or both pd-1 or pd-Ll biological activity.
  • pd-1 or pd-Ll antagonist collectively refers to pd-1 or pd-Ll antagonists.
  • the biological activity of pd-1 or pd-Ll may be inhibited in any manner, e.g., by inhibiting the expression of any one or more of pd-1 or pd-Ll mRNA, pd-1 or pd-Ll protein, or by inhibiting the binding of pd-1 to pd-Ll .
  • the biological activity may be inhibited to any degree that realizes a beneficial therapeutic effect. For example, in some embodiments, the biological activity may be completely inhibited (i.e., prevented), while in other embodiments, the biological activity may be partially inhibited (i.e., reduced).
  • pd-1 or pd-Ll refer to pd-1 or pd-Ll respectively, in any form (e.g., mRNA or protein) and from any species (e.g., human or mouse).
  • the pd-1 or pd-Ll antagonist is an agent that inhibits pd-1 or pd-Ll signaling, pd-1 or pd-Ll signaling can be inhibited in any manner.
  • the pd-1 or pd-Ll antagonist may inhibit the activation and/or binding of any one or more of various downstream targets of pd-1 or pd-Ll- signaling (e.g., nuclear factor kappa- light-chain-enhancer of activated B cells (NF-kappaB), mitogen-activated protein kinase (MAPK) 3/ extracellular signal-regulated kinase (ERK) 1, MAPK8/Jun N-Terminal Protein Kinase (JNK), AKT, and B-catenin).
  • NF-kappaB nuclear factor kappa- light-chain-enhancer of activated B cells
  • MAPK mitogen-activated protein kinase
  • ERK extracellular signal-regulated kinase
  • JNK MAPK8/Jun N-Terminal Protein Kinase
  • AKT AKT
  • B-catenin B-catenin
  • the pd-1 or pd-Ll antagonist may be an agent that binds to pd-1 or pd-Ll , thereby reducing or preventing pd-1 or pd-Ll- signaling and inhibiting its function.
  • the pd-1 or pd-Ll antagonist that inhibits pd-1 or pd-Ll- signaling can be any of the antibodies or antibody fragments, antisense nucleic acids, or chemical inhibitors (e.g., small molecule or peptide (or polypeptide) inhibitor) described herein.
  • the pd-1 or pd-Ll antagonist is an agent that inhibits the binding of pd-1 to pd-Ll .
  • the pd-1 or pd-Ll antagonist may be any agent that binds to the pd-1 or pd-Ll protein, thereby reducing or preventing the binding of pd-1 to pd-Ll and inhibiting its function, as well as agents that compete with the pd-1 or pd-Ll protein for the native pd-1 or pd-Ll binding site.
  • the agent that inhibits the binding of pd-1 pd-Ll to any of its binding sites can be any of the antibodies or antibody fragments, antisense nucleic acids, or chemical inhibitors (e.g., small molecule or peptide inhibitor) described herein.
  • the pd-1 or pd-Ll antagonist is an antibody or antibody fragment that specifically binds to pd-1 or pd-Ll .
  • the antibody can be any type of immunoglobulin that is known in the art.
  • the antibody can be of any isotype, e.g., IgA, IgD, IgE, IgG, IgM, etc.
  • the antibody can be monoclonal or polyclonal.
  • the antibody can be a naturally-occurring antibody, e.g., an antibody isolated and/or purified from a mammal, e.g., mouse, rabbit, goat, horse, chicken, hamster, human, etc.
  • the antibody can be a genetically-engineered antibody, e.g., a humanized antibody or a chimeric antibody.
  • the antibody can be in monomeric or polymeric form.
  • the antibody can have any level of affinity or avidity for pd-1 or pd-Ll, or a functional domain of pd-1 or pd-Ll, e.g., the binding portion of pd-1 or pd-Ll .
  • Methods of testing antibodies for the ability to bind to pd-1 or pd-Ll , or any functional domain thereof are known in the art and include any antibody-antigen binding assay, such as, for example, radioimmunoassay (RIA), ELISA, Western blot,
  • Anti-pd-1 or pd-Ll antibodies and antibody fragments can be prepared using the pd-1 or pd-Ll proteins disclosed herein and routine techniques. Suitable methods of making antibodies are known in the art. For instance, standard hybridoma methods are described in, e.g., Kohler and Milstein, Eur. J. Immunol., 5, 511-519 (1976), Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and C.A. Janeway et al. (eds.), Immunobiology, 5 th Ed., Garland Publishing, New York, NY (2001)).
  • the TNFa antagonist is any suitable agent that inhibits the expression of any one or more of TNFa mRNA, TNFa protein, TNFR1 and/or TNFR2 mRNA, and TNFR1 and/or TNFR2 protein.
  • the TNFa antagonist can be a nucleic acid at least about 10 nucleotides in length that specifically binds to and is complementary to a target nucleic acid encoding any one or more of TNFa mRNA, TNFa protein, TNFR1 and/or TNFR2 mRNA, and TNFR1 and/or TNFR2 protein or a complement thereof.
  • the TNFa antagonist may be introduced into a host cell, wherein the cell is capable of expressing any one or more of TNFa mRNA, TNFa protein, TNFR1 and/or TNFR2 mRNA, and TNFR1 and/or TNFR2 protein, in an effective amount for a time and under conditions sufficient to interfere with expression of any one or more of TNFa mRNA, TNFa protein, TNFR1 and/or TNFR2 mRNA, and TNFR1 and/or TNFR2 protein, respectively.
  • RNA interference RNA interference
  • the TNFa antagonist may comprise an RNAi agent.
  • the RNAi agent may comprise a small interfering RNA (siRNA), a short hairpin miRNA (shMIR), a microRNA (miRNA), or an antisense nucleic acid.
  • siRNA small interfering RNA
  • shMIR short hairpin miRNA
  • miRNA microRNA
  • antisense nucleic acid e.g., siRNA, shRNA, miRNA, and/or antisense nucleic acid can comprise overhangs. That is, not all nucleotides need bind to the target sequence.
  • RNA interference nucleic acids employed can be at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, at least about 200, at least about 220, at least about 240, from about 20 to about 250, from about 40 to about 240, from about 60 to about 220, from about 80 to about 200, from about 60 to about 180, from about 80 to about 160, and/or from about 100 to about 140 nucleotides in length.
  • the RNAi agent e.g., siRNA or shRNA
  • a nucleotide sequence included in a cassette e.g., a larger nucleic acid construct such as an appropriate vector.
  • vectors include lentiviral and adenoviral vectors, as well as other vectors described herein with respect to other aspects of the invention.
  • An example of a suitable vector is described in Aagaard et al. Mol. Ther., 15(5): 938-45 (2007).
  • the resulting nucleic acid can be longer than the comprised RNAi nucleic acid, e.g., greater than about 70 nucleotides in length.
  • the RNAi agent employed cleaves the target mRNA. In other embodiments, the RNAi agent employed does not cleave the target mRNA.
  • any type of suitable siRNA, miRNA, and/or antisense nucleic acid can be employed.
  • the antisense nucleic acid comprises a nucleotide sequence
  • the siRNA may comprise, e.g., trans-acting siRNAs (tasiRNAs) and/or repeat-associated siRNAs (rasiRNAs).
  • the miRNA may comprise, e.g., a short hairpin miRNA (shMIR).
  • the TNFa antagonist may inhibit or downregulate to some degree the expression of the protein encoded by a TNFa or TNFRl and/or TNFR2 gene, e.g., at the DNA, RNA, or other level of regulation.
  • a host cell comprising a TNFa antagonist expresses none of any one or more of TNFa mRNA, TNFa protein, TNFRl and/or TNFR2 mRNA, and TNFRl and/or TNFR2 protein or lower levels of any one or more of TNFa mRNA, TNFa protein, TNFRl and/or TNFR2 mRNA, and TNFRl and/or TNFR2 protein as compared to a host cell that lacks a TNFa antagonist.
  • the TNFa antagonist such as an RNAi agent, such as a shMIR
  • the TNFa antagonist can target a nucleotide sequence of a TNFa or TNFRl and/or TNFR2 gene or mRNA encoded by the same.
  • the Pd-1 or pd-Ll antagonist is any suitable agent that inhibits the expression of any one or more of Pd-1 or pd-Ll mRNA, Pd-1 or pd-Ll protein.
  • the Pd-1 or pd-Ll antagonist can be a nucleic acid at least about 10 nucleotides in length that specifically binds to and is complementary to a target nucleic acid encoding any one or more of Pd-1 or pd-Ll mRNA, Pd-1 or pd-Ll protein, or a complement thereof.
  • the Pd-1 or pd-Ll antagonist may be introduced into a host cell, wherein the cell is capable of expressing any one or more of Pd-1 or pd-Ll mRNA, Pd-1 or pd-Ll protein in an effective amount for a time and under conditions sufficient to interfere with expression of any one or more of Pd-1 or pd-Ll mRNA, Pd-1 or pd-Ll protein, respectively.
  • RNA interference RNA interference
  • the Pd-1 or pd-Ll antagonist may comprise an RNAi agent.
  • the RNAi agent may comprise a small interfering RNA (siRNA), a short hairpin miRNA (shMIR), a microRNA (miRNA), or an antisense nucleic acid.
  • siRNA small interfering RNA
  • shMIR short hairpin miRNA
  • miRNA microRNA
  • antisense nucleic acid e.g., siRNA, shRNA, miRNA, and/or antisense nucleic acid can comprise overhangs. That is, not all nucleotides need bind to the target sequence.
  • RNA interference nucleic acids employed can be at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, at least about 200, at least about 220, at least about 240, from about 20 to about 250, from about 40 to about 240, from about 60 to about 220, from about 80 to about 200, from about 60 to about 180, from about 80 to about 160, and/or from about 100 to about 140 nucleotides in length.
  • the RNAi agent e.g., siRNA or shRNA
  • a nucleotide sequence included in a cassette e.g., a larger nucleic acid construct such as an appropriate vector.
  • vectors include lentiviral and adenoviral vectors, as well as other vectors described herein with respect to other aspects of the invention.
  • An example of a suitable vector is described in Aagaard et al. Mol. Ther., 15(5): 938-45 (2007).
  • the resulting nucleic acid can be longer than the comprised RNAi nucleic acid, e.g., greater than about 70 nucleotides in length.
  • the RNAi agent employed cleaves the target mRNA. In other embodiments, the RNAi agent employed does not cleave the target mRNA.
  • any type of suitable siRNA, miRNA, and/or antisense nucleic acid can be employed.
  • the antisense nucleic acid comprises a nucleotide sequence
  • the siRNA may comprise, e.g., trans-acting siRNAs (tasiRNAs) and/or repeat-associated siRNAs (rasiRNAs).
  • the miRNA may comprise, e.g., a short hairpin miRNA (shMIR).
  • the Pd-1 or pd-Ll antagonist may inhibit or downregulate to some degree the expression of the protein encoded by a Pd-1 or pd-Ll gene, e.g., at the DNA, RNA, or other level of regulation.
  • a host cell comprising a Pd-1 or pd-Ll antagonist expresses none of any one or more of Pd-1 or pd-Ll mRNA, Pd-1 or pd-Ll protein, or lower levels of any one or more of Pd-1 or pd-Ll mRNA, Pd-1 or pd-Ll protein as compared to a host cell that lacks a Pd-1 or pd-Ll antagonist.
  • the Pd-1 or pd-Ll antagonist such as an RNAi agent, such as a shMIR
  • RNAi agent such as a shMIR
  • the TNFa sequence is a human TNFa sequence.
  • human TNFa is assigned Gene NCBI Entrez Gene ID No. 7124, and a Mendelian Inheritance in Man (MIM) No. 191 160.
  • MIM Mendelian Inheritance in Man
  • the TNFR1 and/or TNFR2 sequences are human TNFR1 and/or TNFR2 sequences.
  • human TNFR1 and/or TNFR2 is assigned Gene NCBI Entrez Gene ID No. 7132 and 7133 respectively, and Mendelian Inheritance in Man (MIM) No. 191190 and 191191 respectively.
  • MIM Mendelian Inheritance in Man
  • the human TNFRl and/or TNFR2 genes are found on chromosome 1 at lp36.22 and 12 at 12p.13.31 , respectively.
  • the TNFa antagonist such as an RNAi agent, such as a shMIR
  • a nucleotide sequence selected from the group consisting of the 5 ' untranslated region (5 ' UTR), the 3 ' untranslated region (3 ' UTR), and the coding sequence of TNFa or TNFRl and/or TNFR2, complements thereof, and any combination thereof.
  • Any suitable TNFa or TNFRl and/or TNFR2 target sequence can be employed.
  • RNAi agents can be designed against any appropriate TNFa or TNFRl and/or TNFR2 mRNA sequence.
  • the TNFa antagonist is a TNFRl and/or TNFR2/Fc fusion protein.
  • the TNFRl and/or TNFR2/Fc fusion protein is a soluble variation of the native TNFRl and/or TNFR2 which binds TNFa protein, thereby competing with the native, cell surface TNFRl and/or TNFR2 for binding to TNFa. Accordingly, the TNFRl and/or TNFR2/Fc fusion protein may inhibit the binding of TNFa to the native TNFRl and/or TNFR2.
  • the TNFRl and/or TNFR2/Fc fusion protein may also inhibit the binding and/or activation of any one or more of various downstream targets of TNFa-TNFRl and/or TNFR2 signaling (e.g., NF-kappaB, MAPK3/ERK1, MAPK8/JNK, AKT, and B-catenin).
  • the TNFRl and/or TNFR2/Fc fusion protein may be from any mammal.
  • the TNFRl and/or TNFR2/Fc fusion protein is a human TNFRl and/or TNFR2/Fc fusion protein.
  • the cancer can be any cancer, including any of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer
  • bladder cancer e.g.,
  • TNFa antagonists described herein can be obtained by methods known in the art.
  • TNFa antagonists or s that are peptides or polypeptides can be obtained by de novo synthesis as described in references, such as Chan et al, Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2005; Peptide and Protein Drug Analysis , ed. Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwoood et al., Oxford University Press, Oxford, United Kingdom, 2000; and U. S. Patent 5,449,752.
  • TNFa antagonists or s can be recombinantly produced using standard recombinant methods.
  • the TNFa antagonist can be isolated and/or purified from a natural source, e.g., a human. Methods of isolation and purification are well-known in the art. In this respect, the TNFa antagonists may be exogenous and can be synthetic, recombinant, or of natural origin.
  • TNFa antagonists that are peptides or polypeptides can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, e.g., a disulfide bridge, or converted into an acid addition salt and/or optionally dimerized or polymerized, or conjugated.
  • Suitable pharmaceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, and sulphuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, gly colic, gluconic, succinic, and arylsulphonic acids, for example, p- toluenesulphonic acid.
  • mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, and sulphuric acids
  • organic acids such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, gly colic, gluconic, succinic, and arylsulphonic acids, for example, p- toluenesulphonic acid.
  • the methods of the invention can comprise administering two or more TNFa antagonists, any of which may be the same or different from one another or administering two or more s, any of which may be the same or different from one another.
  • the TNFa antagonist or can be provided as part of a larger polypeptide construct.
  • the TNFa antagonist or can be provided as a fusion protein comprising a TNFa antagonist or , respectively, along with other amino acid sequences or a nucleic acid encoding same.
  • the TNFa antagonist or also can be provided as part of a conjugate or nucleic acid encoding same.
  • the TNFa antagonist can be administered to the mammal by administering a nucleic acid encoding the TNFa antagonist to the mammal.
  • Nucleic acid as used herein includes “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, non- natural or altered nucleotides, and which can contain a natural, non-natural or altered intemucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified
  • Nucleic acids encoding the TNFa antagonist or (and degenerate nucleic acid sequences encoding the same amino acid sequences), can be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. See, for example, Sambrook et al, supra, and Ausubel et al., supra.
  • a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides).
  • the nucleic acids can be incorporated into a recombinant expression vector.
  • the term "recombinant expression vector” means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA or polypeptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA or polypeptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA or polypeptide expressed within the cell.
  • the vectors are not naturally- occurring as a whole. However, parts of the vectors can be naturally-occurring.
  • the recombinant expression vectors can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides.
  • the recombinant expression vectors can comprise naturally-occurring or non- naturally-occurring intemucleotide linkages, or both types of linkages.
  • the non- naturally occurring or altered nucleotides or internucleotide linkages do not hinder the transcription or replication of the vector.
  • the recombinant expression vector can be any suitable recombinant expression vector, and can be used to transform or transfect any suitable host cell. Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses.
  • the vector can be of the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla, Calif), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), or the pEX series (Clontech, Palo Alto, Calif).
  • Bacteriophage vectors such as ⁇ , GTl l, ZapII (Stratagene), EMBL4, and ⁇ 149, also can be used.
  • plant expression vectors include pBIOl, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech).
  • animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech).
  • the recombinant expression vector is a viral vector, e.g., a retroviral vector.
  • the recombinant expression vectors can be prepared using standard recombinant DNA techniques described in, for example, Sambrook et al., supra, and Ausubel et al, supra. Constructs of expression vectors, which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell. Replication systems can be derived, e.g., from ColEl, 2 ⁇ plasmid, ⁇ , SV40, bovine papilloma virus, and the like.
  • the recombinant expression vector comprises regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host cell (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA- or RNA- based.
  • regulatory sequences such as transcription and translation initiation and termination codons, which are specific to the type of host cell (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA- or RNA- based.
  • the recombinant expression vector can include one or more marker genes, which allow for selection of transformed or transfected host cells.
  • Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host cell to provide prototrophy, and the like.
  • Suitable marker genes for the expression vectors include, for instance, neomycin/G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.
  • the recombinant expression vector can comprise a native or nonnative promoter and/or stop codon operably linked to the nucleotide sequence encoding the TNFa antagonist, or to the nucleotide sequence which is complementary to the nucleotide sequence encoding the TNFa antagonist.
  • the selection of stop codons and promoters e.g., strong, weak, inducible, tissue-specific and developmental-specific, is within the ordinary skill of the artisan.
  • the combining of a nucleotide sequence with a stop codon and a promoter is also within the skill of the artisan.
  • the promoter can be a non-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter found in the long-terminal repeat of the murine stem cell virus.
  • CMV cytomegalovirus
  • the TNFa antagonist or can be administered to the mammal by administering a host cell comprising the TNFa antagonist or to the mammal.
  • a host cell refers to any type of cell that can contain the TNFa antagonist or .
  • the host cell can be a eukaryotic cell, e.g., plant, animal, fungi, or algae, or can be a prokaryotic cell, e.g., bacteria or protozoa.
  • the host cell can be a cultured cell or a primary cell, i.e., isolated directly from an organism, e.g., a human.
  • the host cell can be an adherent cell or a suspended cell, i.e., a cell that grows in suspension.
  • Suitable host cells are known in the art and include, for instance, DH5a ?. coli cells, Chinese hamster ovarian cells, monkey VERO cells, COS cells, HEK293 cells, and the like.
  • the host cell may be a prokaryotic cell, e.g., a DH5 D cell.
  • the host cell may be a mammalian cell.
  • the host cell may be a human cell.
  • the host cell can be of any cell type, can originate from any type of tissue, and can be of any developmental stage, the host cell may be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC).
  • PBL peripheral blood lymphocyte
  • PBMC peripheral blood mononuclear cell
  • the host cell may be a T cell.
  • the host cell may be any T cell described herein with respect to other aspects of the invention.
  • the TNFa antagonist or nucleic acids encoding them can be of synthetic or natural origin, and can be isolated or purified to any degree.
  • isolated and purified as used herein means having been increased in purity, wherein “purity” is a relative term, and not to be necessarily construed as absolute purity.
  • the purity can be at least about 50%, can be greater than 60%, 70% or 80%, or can be 100%.
  • the methods described herein may be used for any purpose, e.g., the treatment or prevention of disease, especially cancer.
  • the terms "treat,” and “prevent” as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect.
  • the inventive methods can provide any amount of any level of treatment or prevention of a disease in a mammal.
  • the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the disease, e.g., cancer, being treated or prevented.
  • prevention can encompass delaying the onset of the disease, or a symptom or condition thereof.
  • the disease can be any disease, including any of the diseases discussed herein.
  • the mammal referred to herein can be any mammal.
  • the term "mammal” refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Camivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order
  • Artiodactyla including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.
  • TNFa materials TNFa materials
  • TNFa materials TNFa materials
  • the dose will be determined by the efficacy of the particular TNFa material and the condition of the mammal (e.g., human), as well as the body weight of the mammal (e.g., human) to be treated.
  • the dose of the TNFa material also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular TNFa material. Typically, the attending physician will decide the dosage of the TNFa material with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, TNFa material to be administered, route of administration, and the severity of the condition being treated.
  • Administering a TNFa material to the mammal in accordance with the inventive methods may comprise administering a pharmaceutical composition comprising the TNFa material and a pharmaceutically acceptable carrier.
  • the carrier can be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of
  • the pharmaceutically acceptable carriers described herein for example, vehicles, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active agent(s) and one which has no detrimental side effects or toxicity under the conditions of use. The choice of carrier will be determined in part by the particular compounds used in the pharmaceutical composition, as well as by the particular method used to administer the TNFa material.
  • administering the TNFa material to the mammal may comprise administering the TNFa material orally, intravenously, intramuscularly, subcutaneously, or intraperitoneally.
  • the following formulations for oral, intravenous, intramuscular, subcutaneous, or intraperitoneal administration are exemplary and are in no way limiting. More than one route can be used to administer the TNFa material, and in certain instances, a particular route can provide a more immediate and more effective response than another route.
  • Oral formulations may include any suitable carrier.
  • formulations suitable for oral administration may comprise suitable carriers, such as lactose, sucrose, starch, talc magnesium stearate, crystalline cellulose, methyl cellulose, carboxymethyl cellulose, glycerin, sodium alginate or gum arabic among others.
  • Formulations suitable for parenteral administration include aqueous and nonaqueous isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and nonaqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the TNFa material can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol or hexadecyl alcohol, a glycol, such as propylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol, ketals such as 2,2-dimethy 1-1,3 -dioxolane- 4-methanol, ethers, poly(ethyleneglycol) 400, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvant
  • Oils which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.
  • Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts
  • suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl- D -aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof.
  • the parenteral formulations will typically contain, for example, from about 0.5% to about 25% by weight of the TNFa material in solution. Preservatives and buffers may be used. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having, for example, a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range, for example, from about 5% to about 15% by weight.
  • HLB hydrophile-lipophile balance
  • Suitable surfactants include polyethylene glycol sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.
  • the parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • injectable formulations are in accordance with an embodiment of the invention.
  • the requirements for effective pharmaceutical carriers for injectable compositions are well- known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), md ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)).
  • an “effective amount” or “an amount effective to treat” refers to a dose that is adequate to prevent or treat a disorder or condition; enhance T cell differentiation; suppress T cell differentiation; and/or change the expression of a protein in an individual. Amounts effective for a therapeutic or prophylactic use will depend on, for example, the stage and severity of the disorder or condition being treated, the age, weight, and general state of health of the patient, and the judgment of the prescribing physician. The size of the dose will also be determined by the TNFa material selected, method of administration, timing and frequency of administration, the existence, nature, and extent of any adverse side-effects that might accompany the administration of a particular TNFa material, and the desired physiological effect.
  • the dose of the TNFa material can be about 0.001 to about 1000 mg/kg body weight of the subject being treated/day, from about 0.01 to about 10 mg/kg body weight/day, about 0.01 mg to about 1 mg/kg body weight/day.
  • the dose may be from about 1 x 10 4 to about 1 x 10 8 cells.
  • An exemplary dose of cells may be a minimum of one million cells (1 mg cells/dose).
  • the amount or dose of the TNFa material administered should be sufficient to effect a therapeutic or prophylactic response in the subject or animal over a reasonable time frame.
  • the dose of the TNFa material should be sufficient treat a disorder or condition; enhance T cell differentiation; suppress T cell differentiation; and/or change the expression of a protein in an individual in a time period of from about 2 hours or longer, e.g., about 12 to about 24 or more hours, from the time of administration. In certain embodiments, the time period could be even longer.
  • the dose will be determined by the efficacy of the particular TNFa material and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) to be treated.
  • an assay which comprises, for example, comparing the extent to which target cells are lysed and/or IFN-g is secreted by T cells comprising a TNFa antagonist, or which have been contacted by a TNFa antagonist, upon administration of a given dose of such T cells to a mammal, among a set of mammals of which is each given a different dose of the T cells, could be used to determine a starting dose to be administered to a mammal.
  • the extent to which target cells are lysed and/or IFN-g is secreted upon administration of a certain dose can be assayed by methods known in the art.

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Abstract

L'invention concerne un procédé de production de lymphocytes T destinés à une thérapie cellulaire adoptive, comprenant les étapes consistant à : a) isoler des lymphocytes T d'un mammifère ; et mettre en contact les lymphocytes T in vitro avec un antagoniste de TNFα en quantité efficace pour supprimer la différenciation des lymphocytes T, avec ou sans la transduction d'une séquence d'ADN ou d'ARN (par exemple, récepteur de lymphocytes T, cytokine, récepteur d'antigène chimère ou micro-ARN) dans le cadre d'une immunothérapie adoptive de lymphocytes T ; ou b) administrer à un mammifère un antagoniste de TNFα en quantité efficace pour supprimer la différenciation des lymphocytes T consécutive à un régime d'immunothérapie comprenant une immunothérapie adoptive de lymphocytes T et/ou un traitement d'anticorps anti-Pd-1 et/ou anti-Pd-L1.
PCT/US2017/049437 2016-08-31 2017-08-30 IMMUNOTHÉRAPIES COMBINÉES À DES MODULATEURS DE SIGNALISATION DE TNFα WO2018045069A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110052547A1 (en) * 2001-07-02 2011-03-03 The United States of America, as represented by the Secretary, Department of Health & Human Rapamycin-resistant t cells and therapeutic uses thereof
US20110268749A1 (en) * 2007-07-31 2011-11-03 The Government of the US as Represented by the Secretary Department of Health and Human Services Treatment of cancer via targeting of il-13 receptor-alpha2

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110052547A1 (en) * 2001-07-02 2011-03-03 The United States of America, as represented by the Secretary, Department of Health & Human Rapamycin-resistant t cells and therapeutic uses thereof
US20110268749A1 (en) * 2007-07-31 2011-11-03 The Government of the US as Represented by the Secretary Department of Health and Human Services Treatment of cancer via targeting of il-13 receptor-alpha2

Non-Patent Citations (1)

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Title
BERTRAND ET AL.: "Blocking Tumor Necrosis Factor alpha Enhances CD 8 T- cell -Dependent Immunity in Experimental Melanoma", CANCER RES., vol. 75, no. 13, July 2015 (2015-07-01), pages 2619 - 28, XP055470986 *

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