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WO2019136305A1 - Thérapies à base de cellules et par inhibiteurs de points de contrôle immunitaires combinées à il-12 pour le traitement du cancer - Google Patents

Thérapies à base de cellules et par inhibiteurs de points de contrôle immunitaires combinées à il-12 pour le traitement du cancer Download PDF

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WO2019136305A1
WO2019136305A1 PCT/US2019/012423 US2019012423W WO2019136305A1 WO 2019136305 A1 WO2019136305 A1 WO 2019136305A1 US 2019012423 W US2019012423 W US 2019012423W WO 2019136305 A1 WO2019136305 A1 WO 2019136305A1
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hours
cells
cancer
cell
dose
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Lena A. Basile
Christopher E. Lawrence
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Neumedicines Inc
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Neumedicines Inc
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    • 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
    • A61K38/20—Interleukins [IL]
    • A61K38/208—IL-12
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule

Definitions

  • Cell -based therapies for cancer such as CAR T cell therapy or ACT therapy or the like, have many limitations, especially when attempting to treat solid tumors, or even
  • At least one of the first, second, third, or fourth non-treatment intervals is at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. In some embodiments, at least one of the first, second, third, or fourth non treatment intervals is no more than 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
  • each of the first dose, second dose, third dose, fourth dose, and fifth dose comprises between 5 pg and 15 pg of recombinant IL-12. In some embodiments, each of the first dose, second dose, third dose, fourth dose, and fifth dose comprises 2, 2.5, 3, 3.5, 4, 4.5, 5,
  • the IL-12 is formulated as a pharmaceutical composition comprising a buffer, a salt, and a surfactant.
  • the pharmaceutical composition comprises IL-12 at a concentration of between 1-50 pg/mL.
  • the pharmaceutical composition comprises IL-12 at a concentration of 20 pg/mL.
  • the buffer comprises sodium phosphate.
  • the IL-12 is formulated as a pharmaceutical composition comprising a buffer, a salt, and a surfactant.
  • the pharmaceutical composition comprises IL-12 at a concentration of between 1-50 pg/mL.
  • the pharmaceutical composition comprises IL-12 at a concentration of 20 pg/mL.
  • the buffer comprises sodium phosphate.
  • tumor burden in the subject at 3 months post-treatment with IL-12 is lower than that of a subject receiving the same cancer treatment but which does not receive IL-12.
  • the response rate in the subject at 3 months post-treatment with IL-12 is greater than that of a subject receiving the same cancer treatment but which does not receive IL-12.
  • the effective dose of IL-12 is selected from the group consisting of: (a) less than 1000 ng/kg; (b) less than 500 ng/kg; (c) less than 300 ng/kg; (d) less than 150 ng/kg; (e) less than 100 ng/kg; and (f) less than 50 ng/kg.
  • IL-12 is further administered one or more times following the initial IL-12 administration.
  • the methods comprise: (a) the vaccine effect comprises activation of endogenous antigen presenting cells (APC); and (b) specific APC are produced from incorporation of one or more cancer-associated antigens into the APC, which are then presented as antigens on the APC.
  • the cancer-associated APC promote the production of cytotoxic T cells (CTL).
  • the CTL comprise CD4+ T cells, CD8+ T cells, or a combination thereof.
  • the methods further comprise administering a fifth IL-12 treatment comprising a fifth dose of between 2 pg and 20 pg of recombinant IL-12 to the subject, wherein the fifth IL-12 treatment is administered after a fourth non-treatment interval of at least 8 days between the fourth IL-12 treatment and the fifth IL-12 treatment.
  • the method elicits a therapeutic response that is not diminished by tachyphylaxis.
  • the ICI therapy is administered before at least one of the first, second, third, fourth, or fifth IL-12 treatment.
  • the ICI therapy is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
  • the ICI therapy is administered after at least one of the first, second, third, fourth, or fifth IL-12 treatment. In some embodiments, the ICI therapy is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
  • At least one of the first dose, second dose, third dose, fourth dose, or fifth dose comprises between 0.5 ng and 400 ng of IL-12 per kilogram of the subject (ng/kg). In some embodiments, at least one dose comprises 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, or 400 ng/kg.
  • the effective dose of IL-12 is selected from the group consisting of: (a) less than about 1000 ng/kg; (b) less than about 500 ng/kg; (c) less than about 100 ng/kg; (d) about 100 ng/kg or less; (e) about 50 ng/kg or less; or (f) about 10 ng/kg or less.
  • each of the two components are administered at a time frame selected from the group consisting of: (a) within about 72 hours of each other; (b) within about 36 hours of each other; (c) within about 24 hours of each other; (d) within about 12 hours of each other; and (e) within about 6 hours of each other.
  • the components of the cancer vaccine are repeated one or more times.
  • the dendritic cells are activated by one or more of the ICI therapy treatments; (b) the dendritic cells are mobilized by one or more of the ICI therapy treatments to the tumor site; (c) the dendritic cells are mobilized to the tumor site by the administered of IL-12; (d) the dendritic cells are activated by the administered IL-12; or (e) any combination thereof.
  • activation of the dendritic cells involves dendritic cell maturation
  • dendropoiesis occurs at or near the tumor site. 210.
  • the dendropoiesis results in the proliferation of dendritic cells at or near the tumor site.
  • administration of IL-12 reduces the toxicity of the ICI therapy treatment.
  • the ICI therapy treatment causes necrosis of the cancer cells, apoptosis of the cancer cells, or a combination thereof.
  • the ICI therapy comprises at least one antibody, fusion protein, or compound targeted to an immune checkpoint protein selected from the list consisting of CTLA4, PD1, PDL1, LAG3, B7.1, B7-H3, B7-H4, TIM3, VISTA, CD137, OX-40, CD40, CD27, CCR4, GITR, NKG2D, and KIR. 214.
  • the ICI therapy treatment comprises at least one agent selected from the group consisting of an anti -PD- 1 antibody, an anti-CTLA4 antibody, and an anti-PD-Ll antibody.
  • cancer vaccines comprising: (a) administering one or more therapeutically effective dose(s) of an ICI therapy; and (b) administering one or more therapeutically effective dose(s) of IL-12 to the subject.
  • the ICI therapy comprises at least one antibody, fusion protein, or compound targeted to an immune checkpoint protein selected from the list consisting of CTLA4, PD1, PDL1, LAG3, B7.1, B7-H3, B7-H4, TIM3, VISTA, CD137, OX-40, CD40, CD27, CCR4, GITR, NKG2D, and KIR.
  • the methods further comprise the addition of cytokines, wherein the cytokines are selected from the group consisting of IL-12, IL-4 and IL-2.
  • CD8 and CD56 mRNA dropped compared with expression in the absence of anti-CD3, but with the exception of CD8 expression being increased with IL-2, and CD4 expression being maintained with IL-2 alone and in combination with recombinant IL-12 (HemaMax).
  • HemaMax The x-axis show the treatment groups and the y-axis show the expression levels of IFN-g, Perforin, or Granzyme B.
  • PBMCs were purified from the blood of a healthy human volunteer, and incubated for 2 days with or without anti-CD3 antibody (activation step, 4 treatment bars to the right in each graph). Cells were then studied for the expression of IFN-g, Perforin, or Granzyme B mRNA expression (quantified by qRT-PCR) after 4 days culture in the presence of IL-2, recombinant IL-12 (HemaMax), or recombinant IL-12 (HemaMax) and IL-2, or in the absence of any stimulating cytokine.
  • FIG. 30 highlights the problem and solution for cancer.
  • Cancer patients do not produce sufficient endogenous IL-12 needed for a robust immune offense. These patients are immunosuppressed and thus an exogenous recombinant IL-12 (HemaMax) is needed to replace the deficiency in IL-12 and allow restoration of a robust immune offense, supported by hematopoietic stimulation.
  • HemaMax exogenous recombinant IL-12
  • the exogenous recombinant IL-12 leads to increase patient survival.
  • the methods described herein can increase hematopoietic regeneration and immune memory.
  • the methods described herein increase the persistence of CAR-T cells. This can help overcome CAR-T cell exhaustion that can occur in typical CAR-T cell therapies.
  • the methods described herein increase the rate at which memory T-cells, including memory CAR-T cells, generate new effector T-cells.
  • the methods described herein inhibit T-cells from becoming anergic. In some embodiments, such methods can also reduce the risk of relapse.
  • the methods include administering IL-12 to decrease the numbers of or the activity of T regulatory cells (Treg) or myeloid derived suppressor (MDSC) cells found in the microenvironment of the tumor.
  • Treg or MDSC cells can counteract the efficacy of the cell based therapy.
  • administering IL-12 can increase the efficacy of an adoptive cell therapy by decreasing the ability of Treg or MDSC cells to interfere with the adoptive cell therapy.
  • the methods disclosed herein result in greater complete response (CR) and longer term remissions for adaptive T-cell therapy (ACT), including but not limited to CAR- and TCR-modified T cell cancer therapy.
  • the methods disclosed herein enable IL-12 to expand and activate the autologous or allogenic T cells, such as CAR- or TCR-modified T cells, and assist in these cells being trafficked to the tumor.
  • the systemic delivery of IL-12 localizes IL-12 to the tumor.
  • the subject to be treated by the methods disclosed herein has cancer.
  • the cancer is solid tumor type of cancer, a non-solid tumor type of cancer, a hematopoietic cancer, or a leukemia.
  • the non-solid tumor cancers treatable with the methods disclosed herein include but are not limited to leukemias.
  • examples of types of cancer treatable with the methods of the disclosure include but are not limited to, a solid tumor, carcinomas, sarcomas, lymphomas, cancers that begin in the skin, and cancers that begin in tissues that line or cover internal organs.
  • administration of a therapeutically effective dose IL-12 is less toxic that administering a therapeutically effective dose or course of IL-2.
  • IL-12 can be administered at various stages of cell-based therapy preparation or administration, e.g. transduction or culture/proliferation enhance the persistence of the genetically modified T cell or NK cells after these are infused into the cancer patient.
  • IL-12 is injected at some point before or after the infusion of the cell-based therapy provides greater efficacy over the use of the cell-based therapy alone.
  • the increased efficacy of in vivo, systemic administration of IL-12 occur whether or not IL-12 is also used in the ex vivo steps of transduction and proliferation.
  • the vaccine therapy described herein includes the addition of antigens to the culturing of the antigen presenting cells.
  • the benefits of the methods disclosed herein is a decrease in toxicity associated with IL-12 administration as, contrary to what would be expected, systemic IL-12, administered in dosing regimens of the present invention, less toxic than IL-12 delivered via gene therapy.
  • the decreased toxicity is due to more precisely controlled delivery of IL-12 via injectable delivery, as compared to gene therapy delivery. Injectable delivery of IL-12 enables both systemic and local effects of IL-12.
  • the methods further comprise removing lymphocytes from either peripheral blood, a primary or secondary tumor, or the spleen of the subject, culturing the lymphocytes following the one or more treatments, and reintroducing the cultured lymphocytes, included T cells and/or NK cells into the subject.
  • the cell extraction and culturing method is used to obtain the T cells for CAR modification or engineering of affinity enhanced TCRs.
  • the lymphocytes are cultured in the presence of one or more cytokines, such as IL-2, IL-4, IL-7, IL-15, IL-21 and IL-12.
  • administration of IL-12 with a cell-based therapy eliminates the need for these harsh conditioning regimens. Additionally, in most ACT regimens, IL-2 is administered until tolerance following injection of the T cells, but IL-2 may produce severe toxicities. In some embodiments, IL-12 eliminates the need for IL-2 and its associated toxicity.
  • time periods for IL-12 administration include but are not limited to at about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 54 hours, about 60 hours, about 66 hours, about 72 hours, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days before or after administration of the T cells, such as CAR- or TCR-modified T cells, or any combination of the foregoing time periods.
  • T cells such as CAR- or TCR-modified T cells, or any combination of the fore
  • the endogenous vaccine effect can comprise activation of endogenous cells, as delineated above, where (i) the cells are mobilized to the tumor site by the one or more T cell cancer therapy treatments, such as chimeric antigen receptor (CAR) T-cell therapy cancer treatments or affinity enhanced T cell receptor (TCR) T-cell therapy cancer treatments, (ii) the cells, as delineated above, are mobilized to the tumor site by the administration of IL-12, or (iii) a combination thereof.
  • T cell cancer therapy treatments such as chimeric antigen receptor (CAR) T-cell therapy cancer treatments or affinity enhanced T cell receptor (TCR) T-cell therapy cancer treatments
  • CAR chimeric antigen receptor
  • TCR affinity enhanced T cell receptor
  • dendropoiesis results in the proliferation of dendritic cells in the bone marrow.
  • IL-12 counterproductive to efficacy effects of IL-12. This is because the delivery of IL-12 can be more precisely controlled via injectable delivery, as compared to gene therapy delivery. Injectable delivery of IL-12 enables both systemic and local effects of IL-12.
  • the methods described herein can increase hematopoietic regeneration and immune memory.
  • the methods described herein increase the persistence of effector T-cells. This can be particularly important when using ICI, which in some cases can exhaust anti-tumor T cells.
  • the methods described herein increase the rate at which memory T-cells generate new effector T- cells.
  • the methods described herein inhibit T-cells from becoming anergic.
  • a benefit of the methods disclosed herein is that cytotoxic t-lymphocytes (CTL) produced as a result of the method can comprise one or more cell types selected from the CD8+ cells.
  • CTL cytotoxic t-lymphocytes
  • the administration of IL-12 yields newly regenerated cells due to ⁇ L-l2’s potent bone marrow expansion effects.
  • endogenous antigen presenting cells APC can be newly generated and mobilized to the tumor site following administration of IL-12.
  • APC cells are, for example, dendritic cells or macrophages.
  • IL-12 is produced mainly by dendritic cells (DC) and phagocytes (macrophages and neutrophils) once they are activated by encountering pathogenic bacteria, fungi or intracellular parasites.
  • the IL-12 receptor (IL-12R) is expressed mainly by activated T cells and NK cells.
  • NM-IL-12 is a highly potent, pleiotropic cytokine with a unique mechanism of action that augments and links innate and adaptive immunity.
  • the release of IL-12 by dendritic cells and macrophages, the natural producers of endogenous IL-12 is suppressed in cancer patients and in melanoma.
  • exogenous NM-IL-12 is needed to restore the immune system to competency.
  • IL-12 can be considered the missing link in cancer therapy.
  • the transgenic TCR comprises of the alpha and beta polypeptide chains, whereas the CAR composes one
  • Cancer typically arises in the host with a healthy immune system due, in part, to tolerance of the TCR to TAA.
  • circumventing tolerance to engender a desired immune response is achieved in several ways including: through the engineering of affinity enhanced TCR or through CARs in T cells, or through similar engineering of natural killer (NK) cells.
  • NK natural killer
  • such cell-based therapies result in an immune response that is specifically targeted at cancer cells (i.e. malignant B cells).
  • TILs extracted from cancer patient are engineered in various ways to improve efficacy.
  • T cells and NK cells, and cell lines expressing CARs are highly targeted, but additionally could offer the potential benefits of active trafficking to tumor sites, in vivo expansion and long term persistence, but these activities are still in need of optimization for CARs.
  • CAR T and CAR NK cells and cell lines large quantities of effector cells are generated that recognize and target specific antigens on the tumor cells.
  • novel TCRs are developed with enhanced binding affinity to specific TAA as compared to their native receptor.
  • novel TCRs engineered to enhance affinity or direct binding to a novel tumor associated antigen target.
  • sequences that encode the new receptor are transduced into and expressed in a patient’s T cells similar to CARs.
  • TCRs unlike CARs, TCRs generally bind both intracellular and extracellular targets, thus expanding the scope of targets that may be pursued.
  • gene transfer offers the potential to allow the introduction of countermeasures to tumor immune evasion and of safety mechanisms in both CAR and TCR systems.
  • the cell-based therapy comprises a CAR T-cell therapy delivered using a suitable gene therapy technique.
  • the cell-based therapy comprises an engineered and/or affinity enhanced TCR T-cell therapy delivered using a suitable gene therapy technique.
  • the cell-based therapy comprises a CAR NK cell therapy delivered using a suitable gene therapy technique.
  • the cell-based therapy comprises an engineered and/or affinity enhanced TCR NK cell therapy delivered using a suitable gene therapy technique.
  • the cell -based therapy comprises dendritic cell therapy or dendritic cell vaccine.
  • the methods provided herein are combined with conventional cancer treatment methods, such as chemotherapy and radiation treatments.
  • the methods provided herein incorporate a number of cell therapies, including Antigen expanded CD8+ or CD4+ T cells or NK cells (e.g. NY-ESO-l- specific cells); Engineered T cell receptor (TCR / CD3) expressed in lymphocytes; Chimeric antigen receptor (CAR) expression in lymphocytes (e.g. tumor associated antigen (TAA) + CD3/ co-stimulatory molecule); and TILs tumor infiltrating lymphocytes.
  • the cell-based therapies are based on the activation and/or expansion of dendritic cells in the presence or absence of tumor associated antigens.
  • a broad variety of tumor cells are specifically be targeted by patients’ T cells, which are redirected in an antibody-defined, major histocompatibility complex- unrestricted fashion by endowing them with a CAR or TCR.
  • CAR T cells are engineered with inducible or constitutive release of IL-12 (also known as “armored” CAR T cells).
  • dendritic cells and macrophages are usually the primary producers of IL-12.
  • “armored” CAR T cells that have a genetically engineered capability to produce and secrete IL-12, have displayed increased proliferation, enhanced persistence, up-regulation of perforin and granzyme B, as well as a central memory-like phenotype.
  • examples include the CH2CH3 portion of an immunoglobulin molecule such as IgGl.
  • an immunoglobulin molecule such as IgGl.
  • the importance of the hinge region is illustrated by studies which demonstrated that, for the same targeting construct, optimal T cell activation depends on the relative length of this spacer region and the distance of the epitope from the target cell membrane. For instance, juxtamembrane epitopes require in general longer spacer regions than those farther away from the membrane.
  • Second generation CARs generally have been engineered to include another stimulatory domain, usually derived from the intracytoplasmatic portion of costimulatory molecules, such as CD28, CD134/OX40, CD137/4-1BB, Lck, ICOS and DAP10.
  • the second generation CARs provide T cells with additional activating signals.
  • costimulatory domains are incorporated in the CAR, its activation by engagement with the respective antigen delivers both signal 1 and signal 2 to T cells, bypassing the need for costimulatory ligands and preventing potential anergy or apoptosis resulting from a solitary signal 1.
  • Many in vitro and preclinical studies comparing first and second generation CARs demonstrate improved function of T cells bearing the latter.
  • CAR constructs are effective in vitro and in animal models at mediating killing of their target cells. In some cases, few clinical studies have shown consistent anti-tumor effects. In some cases, this limitation is thought to be in part, at least for trials employing first generation CARs, due to the fact that tumor cell engagement of the CAR alone fails to sustain sufficient T-cell growth, cell numbers and activation for tumor eradication. In some cases, T cells require multiple additional, or costimulatory, signals to produce optimum activation, proliferation, persistence and survival following antigen receptor engagement. In some cases, incorporating one or more endodomains from the required costimulatory molecules into the CAR itself enhances T cell performance following chimeric receptor engagement. In some cases, even T cells expressing second generation CARs have been observed to have limited persistence and mobilization in vivo after infusion in patients, highlighting the need to find alternative methods of optimizing their expansion, persistence and mobilization of T cells after adoptive transfer.
  • NK cells can be engineered or enriched in much the same way that T cells can.
  • Natural killer (NK) cells are a subset of lymphocytes and have been shown to differentiate into NK1 and NK2 subsets, similar to the Thl and Th2 subsets of CD4+ T cells. The different NK cell subsets have distinct functions in immune responses.
  • NK cells produce many chemokines that impact dendritic cells (DCs), macrophages and neutrophils during an immune response, thus endowing NK cells with regulatory functions. More importantly, In some cases, NK cells also have“memory” properties that were previously ascribed only to T and B cells.
  • DCs dendritic cells
  • macrophages macrophages
  • neutrophils neutrophils
  • NK cells also have“memory” properties that were previously ascribed only to T and B cells.
  • gene therapy has been successfully combined with T-cell therapy to generate potent immune cells that upon administration can sustain proliferation, home to sites of malignant disease, and recycle effector functions in the tumor microenvironment.
  • gene therapy has been successfully used to enforce expression of CARs and engineer TCRs that provide T cells with ability to directly recognize tumor-associated antigens without the need for presentation by human leukocyte antigen.
  • gene transfer of CARs and TCRs are undertaken using viral-based and non-viral approaches.
  • gene therapy techniques are used to express CARs or other receptors on NK cells or increase the activity of dendritic cells.
  • nonmyeloblative preparative regimen in conjunction with total body irradiation (TBI), 2 - 12 Gy (2 Gy twice a day for 3 days).
  • TBI total body irradiation
  • iv intravenous infusion of ACT therapy.
  • ICI therapies have been used in treating various forms of cancer with several different ICI therapies.
  • the ICI therapies include antibodies specific for CTLA4 or PD-l.
  • ICI therapies resulted in serious adverse reaction, high levels of non-respondents, and even deaths.
  • Some unexpected benefits of the methods disclosed herein include (1) direct assistance by IL-12 at the tumor site in terms of the generation of anti-tumor responses; (2) proliferation and engagement of activated NK cells to assist in the generation of anti-tumor innate responses; (3) increased activation of memory T cells leading to elimination of minimal residual disease and longer remissions from the disease; (4) proliferation of CD8 + and CD4 + T cells to eradicate the tumor; and (5) bone marrow effects in terms of expansion of key lymphoid and myeloid cells to further increase the anti-tumor effects of the treatment.
  • immune checkpoints refer to a plethora of inhibitory pathways hardwired into the immune system, which, under normal physiological conditions are crucial for maintaining self-tolerance and modulating the duration and amplitude of physiological immune responses in peripheral tissues in order to minimize collateral tissue damage in response to pathogenic infection.
  • the expression of immune checkpoint proteins is often dysregulated by tumors as an important immune resistance and escape mechanism.
  • CTL4 cytotoxic T-lymphocyte-associated antigen 4
  • FDA US Food and Drug Administration
  • inhibitors of additional immune- checkpoint proteins such as programmed cell death protein 1 (PD1), provide broad and diverse opportunities to enhance antitumor immunity with the potential to produce durable clinical responses.
  • PD1 programmed cell death protein 1
  • T cell activation through blockade of immune checkpoints has been a major focus of efforts to therapeutically manipulate endogenous antitumor immunity, owing to the capacity of T cells for the selective recognition of peptides derived from proteins in all cellular compartments; their capacity to directly recognize and kill antigen-expressing cells (by CD8+ effector T cells; also known as cytotoxic T lymphocytes (CTLs)); and their ability to orchestrate diverse immune responses (by CD4+ helper T cells), which integrate adaptive and innate effector mechanisms.
  • CTLs cytotoxic T lymphocytes
  • CD4+ helper T cells CD4+ helper T cells
  • agonists of co-stimulatory receptors or antagonists of inhibitory signals are currently agents of interest in clinical testing.
  • a non-limiting list of immune checkpoint targets is shown in Table 1.
  • immune checkpoint inhibitors required to activate the immune system include inflammatory effects such as uveitis, dermatitis, colitis, hepatitis, and pancreatitis.
  • these checkpoint inhibitors have the potential to cause serious and/or life threatening side effects in numerous major organs and systems.
  • the subject to be treated by the methods disclosed herein has a cancer.
  • the cancer is solid tumor type of cancer, a non-solid tumor type of cancer, a hematopoietic cancer, or a leukemia.
  • the non-solid tumor cancers treatable with the methods disclosed herein include but are not limited to leukemias.
  • the types of cancer treatable with the methods disclosed herein include but are not limited to, a solid tumor, carcinomas, sarcomas, lymphomas, cancers that begin in the skin, and cancers that begin in tissues that line or cover internal organs.
  • types of cancer include, but are not limited to, brain cancer, including glioblastoma,
  • human IL- 12 dosages is at least 0.01 pg/dose, 0.05 pg/dose, 0.1, 0.5 pg/dose, 1 pg/dose, 2 pg/dose, 3 pg/dose, 4 pg/dose, 5 pg/dose, 6 pg/dose, 7 pg/dose, 8 pg/dose, 9 pg/dose, 10 pg/dose, 11 pg/dose, 12 pg/dose, 13 pg/dose, 14 pg/dose, 15 pg/dose, 16 pg/dose, 17 pg/dose, 18 pg/dose, 19 pg/dose, 20 pg/dose, 21 pg/dose, 22 pg/dose, 23 pg/dose, 24 pg/dose, 25 pg/dose, 26 pg/dose, 27 pg/dose, 28 pg/dose, 29 pg/dose, or 30 pg/dose.
  • IL-12 dosing schedule In some embodiments, IL-12 can be administered such that the frequency of IL-12 doses do not induce tachyphylaxis, which results in downregulation of the anti-tumor effects of IL-12.
  • a marker of tachypylaxis can be reduced induction of IFN- g or other biological markers associated with IL-12 activity. Another method to check for
  • this unexpected long exposure of IL-12 in blood is due to lymphatic absorption of the drug, which is much slower than capillary absorption of IL-12 following the exogeneous administration of IL-12 in a single low dose or infrequent multiple low dosages.
  • IL-12 dose is given no more than 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, or 20 times. In some embodiments, IL-12 dose is given 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, or 20 times.
  • the IL-12 administrations are spaced by about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days,
  • IL-12 is given as a maintenance dose following a therapeutic dosing regimen, and the maintenance dosing regimens is at a frequency of once a month, once every two months or once every three to four months, or other time periods as described herein.
  • the IL-12 maintenance dose is given at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months after the earlier IL-12 dose.
  • the IL-12 maintenance dose is given no more than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days,
  • the IL-12 maintenance dose is given about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months after the earlier IL-12 dose.
  • the IL-12 maintenance dose is given about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months after the earlier IL- 12 dose.
  • the time between treatments or doses of IL-12 can be a non-treatment interval.
  • the non-treatment interval between a first dose of IL-12 and a second dose of IL-12 can be or can be about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days.
  • the non-treatment interval is or is at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days.
  • the non-treatment interval is no more than 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. In some embodiments, the non treatment interval is or is at least 1, 2, 3, or 4 weeks. In some embodiments, the non-treatment interval is no more than 1 2, 3, or 4 weeks. In some embodiments, the non-treatment interval is or is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. In some embodiments, the non treatment interval is not more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.
  • the dosing frequency is determined by measuring the IL-12 exposure in blood as assessed by pharmacokinetic(PK) and downstream effects
  • pharmacodynamic (PD) of IL-12 in the blood of the subject such as exposure to IFN- g or other cytokines or chemokines in the target patient population, such as a patient population having a particular type of cancer.
  • the first and the second administrations of IL- 12 give similar PK and/or PD exposure in blood via PK and PD parameters such as C max , AUC, etc. Similar PK and/or PD exposure indicates that IL-12 is essentially cleared from the blood and a second dose of IL-12 result in optimal efficacyas shown by data in Example 5.
  • trafficking of major peripheral blood cells is related to the efficacy of IL- 12.
  • these trafficked cells move into sites of injury, such as a tumor, a wound, or a site of organ damage.
  • the ability of IL-12 to traffic peripheral blood cells provides a key pharmacodynamic (PD) parameter to guide effective dosing regimens.
  • PD pharmacodynamic
  • the methods disclosed herein provide several parameters to guide the dosage amount and frequency of administration of IL-12, including PK or terminal half-life of IL-12, the ability of IL-12 to traffic major peripheral blood cells out of the peripheral blood and to sites of injury or disease, IL-l2-mediated infiltration of sites of injury or disease by T cells.
  • the dosing of IL-12 is determined using various PD markers, such as cell trafficking and the presence of Th2 cytokines. These markers provide can be used to characterize a therapeutic need for a subsequent doses of IL-12.
  • Measurement of peripheral blood cell (PBC) trafficking can be performed using techniques known in the art.
  • the PBC trafficking can be characterized for different target patient populations to determine the time points of IL-12 administration for optimal results of PBC trafficking.
  • Methods of measuring an increase in PBC trafficking to site of interest include, but are not limited to, measuring an increase as compared to a base line measurement in cytotoxic T lymphocytes (CTL) directed to a tumor in peripheral blood (peripheral CTL) within one to a few days of IL-12 administration (e.g., “circulating antitumor CTL”).
  • CTL cytotoxic T lymphocytes
  • Non-limiting examples of useful measuring techniques include a high-efficiency limiting dilution analysis technique and by staining peripheral blood
  • lymphocytes with a tumor-specific antigen or antibody recognized by T cells.
  • Another method of measuring an increase in PBC trafficking to a site of interest is by evaluating infiltration of target tissue, such as neoplastic or tumor tissue, by T cells by
  • an“increase” in PBC (T cells, NK cells, monocytes, red blood cells, and/or platelets) cell trafficking to a site of interest is, for example, an about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, or an about 45% increase (e.g., as measured by an increase in CTLp, or an increase in T cells at the target site as measured by immunohi stochemi stry).
  • IL-12 traffics all major peripheral blood cells to sites of injury or disease.
  • these“markers” can be used to determine a therapeutic need for an IL-12 dosage.
  • a subsequent dose of IL-12 is not administered to a subject when an increase in serum of IL-10 or any other Th2 cytokine, such as IL-4, is observed as compared to baseline levels in the subject or patient population present prior to IL-12 administration.
  • an additional administration of a maintenance dose of IL-12 following cessation of cell-based therapy such as CAR- or TCR-modified T cell or NK cell therapy and/or ICI therapy.
  • the IL-12 maintenance dose amount is from about 0.5 ng/kg up to about 2000 ng/kg, or less than about 2000 ng/kg.
  • the IL-12 maintenance dose is administered for any therapeutically effective duration of time following cessation of therapy with T cells or NK cells (such as but not limited to CAR- or TCR-modified cells) and/or ICI therapy.
  • exemplary IL-12 maintenance dosing periods include, but are not limited to, daily (e.g., one IL-12 dose/day up to yearly (one IL-12 dose/yearly) or any time point in-between, including for example, one IL-12 dose every week, 2 weeks, 3 weeks, 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, or about 15 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months.
  • an IL- 12 maintenance dose is administered for periods of longer than 1 year, e.g., over a several year period.
  • the dose of IL-12 is less than about 1000 ng/kg, less than about 500 ng/kg, about 300 ng/kg, less than about 300 ng/kg, about 200 ng/kg, less than about 200 ng/kg, about 100 ng/kg, less than about 100 ng/kg, about 100 ng/kg or less, about 50 ng/kg or less, about 10 ng/kg or less, about 9 ng/kg or less, about 8 ng/kg or less, about 7 ng/kg or less, about 6 ng/kg or less, about 5 ng/kg or less, about 4 ng/kg or less, about 3 ng/kg or less, about 2 ng/kg or less, or about 1 ng/kg or less, with a minimum threshold amount of IL-12 administered of 0.5 ng/kg.
  • the IL-12 maintenance dose is administered for any therapeutically effective duration of time following cessation of therapy with ICIs, such as but not limited to, 1 day up to 1 year or any time point in-between, including for example 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months.
  • an IL- 12 maintenance dose is administered for periods of longer than 1 year, e.g., over a several year period.
  • the dosing protocols are consistent with the parameters detailed above, e.g., subsequent doses of IL-12 given at about twice the terminal half-life of IL- 12 for a particular patient population.
  • a second parameter relates to the ability of a subsequent dose of IL-12 to traffic peripheral blood cells to a target site, such as a site of injury or disease.
  • a third parameter is the ability of IL-12 to increase serum levels of IL-10 and other Th2 cytokines, which is undesirable. These parameters generally result in IL-12 administration at a frequency of more than every two weeks, preferably every 3-4 weeks, such as when IL-12 is given along with a cell-based therapy and/or ICI therapy. Responsive patients may receive a maintenance dose of IL-12 either monthly, or every two months, or every three to 4 months.
  • the maintenance dose of IL-12 may be at the same as the last dose of IL-12 given during the treatment regimen, or a lower dose, such as 1/3 or V 2 or V 4 of the last dose given during the therapeutic regimen.
  • cell-based therapy is administered alone or in combination with IL-12, ICIs, or combinations thereof.
  • the cell- based therapy is administered in various amounts.
  • the cells administered in the cell-based therapy comprise engineered cells.
  • the cells administered in the cell-based therapy comprise engineered cells.
  • the cell-based therapy comprises non-engineered cells.
  • the cell-based therapy is administered to the subject in an amount of at least 1 million, 2 million, 3 million, 4 million, 5 million, 6 million, 7 million, 8 million, 9 million, 10 million, 20 million, 30 million, 40 million, 50 million, 60 million, 70 million, 80 million, 90 million, 100 million, 200 million, 300 million, 400 million, 500 million, 600 million, 700 million, 800 million, 900 million, 1 billion, 2 billion, 3 billion, 4 billion, 5 billion, 6 billion, 7 billion, 8 billion, 9 billion,
  • the cell-based therapy is administered to the subject in an amount of no more than 1 million, 2 million, 3 million, 4 million, 5 million, 6 million, 7 million, 8 million, 9 million, 10 million, 20 million, 30 million, 40 million, 50 million, 60 million, 70 million, 80 million, 90 million, 100 million, 200 million, 300 million, 400 million, 500 million, 600 million, 700 million, 800 million, 900 million, 1 billion, 2 billion, 3 billion, 4 billion, 5 billion, 6 billion, 7 billion, 8 billion, 9 billion, 10 billion, 20 billion, 30 billion, 40 billion, 50 billion, 60 billion, 70 billion, 80 billion, 90 billion, or 100 billion cells.
  • the cell-based therapy is administered to the subject in an amount of about 1 million, 2 million, 3 million, 4 million, 5 million, 6 million, 7 million, 8 million, 9 million, 10 million, 20 million, 30 million, 40 million, 50 million, 60 million, 70 million, 80 million, 90 million, 100 million, 200 million, 300 million, 400 million, 500 million, 600 million, 700 million, 800 million, 900 million, 1 billion, 2 billion, 3 billion, 4 billion, 5 billion, 6 billion, 7 billion, 8 billion, 9 billion, 10 billion, 20 billion, 30 billion, 40 billion, 50 billion, 60 billion, 70 billion, 80 billion, 90 billion, or 100 billion cells.
  • the cell-based therapy is administered to the subject in an amount of at least 1 million, 2 million, 3 million, 4 million, 5 million, 6 million, 7 million, 8 million, 9 million, 10 million, 20 million, 30 million, 40 million, 50 million, 60 million, 70 million, 80 million, 90 million, 100 million, 200 million, 300 million, 400 million, 500 million, 600 million, 700 million, 800 million, 900 million, 1 billion, 2 billion, 3 billion, 4 billion, 5 billion, 6 billion, 7 billion, 8 billion, 9 billion, 10 billion, 20 billion, 30 billion, 40 billion, 50 billion, 60 billion, 70 billion, 80 billion, 90 billion, or 100 billion engineered cells.
  • the cell-based therapy is administered to the subject in an amount of about 1 million, 2 million, 3 million, 4 million, 5 million, 6 million, 7 million, 8 million, 9 million, 10 million, 20 million, 30 million, 40 million, 50 million, 60 million, 70 million, 80 million, 90 million, 100 million, 200 million, 300 million, 400 million, 500 million, 600 million, 700 million, 800 million, 900 million, 1 billion, 2 billion, 3 billion, 4 billion, 5 billion, 6 billion, 7 billion, 8 billion, 9 billion, or 10 billion per kg of weight of subject.
  • cell-based therapy is administered once. In some instances, cell- based therapy in the methods disclosed herein is administered more than once. In some embodiments, the cell-based therapy is given 1 time, 2 times, 3 times, 4 times, or 5 times.
  • the methods disclosed herein can be administered with various ICI dose amounts.
  • the dose of ICI is at least 1 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 240 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, or 1000 mg.
  • a subsequent ICI dose is given at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months after an earlier ICI dose.
  • ICI is administered once. In some instances, ICI in the methods disclosed herein is administered more than once. In some embodiments, the ICI dose is given at least 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, or 20 times. In some embodiments, the ICI dose is given no more than 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, or 20 times.
  • the IL-12 dose is given no more than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, or 4 weeks before the initiation of the treatment cycle.
  • the methods disclosed herein include a dosing schedule of IL-
  • the maintenance dose of IL-12 is given at about 1, about 2, about 3, or about 4 months following cell-based therapies, ICI therapy, radiation, or chemotherapy, In some embodiments, the maintenance dose of IL-12 is given at the following intervals following adjunctive therapy of IL-12 with cell-based therapies, ICI therapy, radiation, or chemotherapy: about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about
  • IL-12 is administered for no more than 1 day, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months. In some embodiments, IL-12 is administered for about 1 day, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months. In some embodiments, an IL-12 dose is administered for periods of longer than 1 year, e.g., over a several year period.
  • the IL-12 dose is given within a specified time frame from administration of cell-based therapy. In some embodiments, the IL-12 dose is given during the administration of cell-based therapy. In some embodiments, the IL-12 dose is given at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 30 hours, 36 hours, 42 hours, 48 hours, about 54 hours, 60 hours, 66 hours, 72 hours, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, or
  • the IL-12 dose is given no more than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 30 hours, 36 hours, 42 hours, 48 hours, about 54 hours, 60 hours, 66 hours, 72 hours, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, or 4 weeks after administration of cell-based therapy. In some embodiments, the IL-12 dose is given about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours,
  • the IL-12 dose is given within a specified time frame from administration of ICI therapy. In some embodiments, the IL-12 dose is given during the administration of ICI therapy. In some embodiments, the IL-12 dose is given at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours,
  • the IL-12 dose is given no more than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 30 hours, 36 hours, 42 hours, 48 hours, about 54 hours, 60 hours, 66 hours, 72 hours, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, or 4 weeks within administration of ICI therapy.
  • the IL-12 dose is given about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 30 hours, 36 hours, 42 hours, 48 hours, about 54 hours, 60 hours, 66 hours, 72 hours, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, or 4 weeks after administration of ICI therapy.
  • the ICI therapy is given no more than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 30 hours, 36 hours, 42 hours,
  • the ICI therapy is given no more than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours,
  • IL-12 is used for the ex vivo expansion and functional activation of TIL, and in so doing to establish next-generation TILs.
  • IL- 12 is unique in its ability to program effector cell development in human CD8+ T cells.
  • IL-12 increases the cytotoxic potency and persistence of TILs thus reducing the cell numbers required for ACT, and having the potential to reduce both costs and the duration of the expansion process.
  • IL-12 is safe and well tolerated in healthy volunteers, enhancing the utility and clinical success of such ex vivo treatment of TILs with IL- 12
  • NMA non-myeloablative chemotherapy
  • OT-l CD8+ T cells were inoculated into an irradiated syngeneic mouse and showed dramatically enhanced CD8+ T cell homeostatic proliferation when supported with an injection of IL-12.
  • homeostatic proliferation does not require antigen recognition, but does require TCR recognition of self-peptide / MHC ligands.
  • IL-12 regulates gene expression in murine CD8 T cells to favor the generation of full activated effector T (TE) cells.
  • both murine and human anti-tumor T cells can undergo ex vivo IL-12 conditioning.
  • the ex vivo IL-12 conditioning of murine splenocyte-derived CD8+ Thyl. l+ T cells led to a lO-lOO-fold increase in the persistence and anti-tumor efficacy upon adoptive transfer into lympho-depleted mice.
  • the second and the third generation CAR-modification T cells are capable of delivering enhancement of activation signal, proliferation, and production of cytokines and effector function of CAR-modified T cell in pre-clinical trials.
  • Figure 1 depicts first, second, and third generation chimeric antigen receptors.
  • the second and the third generation CAR-modified T cells are entering clinical trials now.
  • Figures 2 and 3 show a pictorial generation of a tumor targeted CAR ( Figure 2) and a pictorial of a method for generating tumor- associated antigen (TAA)-targeted T cells for treatment of cancer (Figure 3). In some cases, clinical trials have shown some promising results.
  • TAA tumor- associated antigen
  • ligand or receptor are used as targeting moiety because the corresponding receptor or ligand are often overexpressed on tumor cells, such as peptide against vascular endothelial growth factor receptor (VEGFR).
  • VEGFR vascular endothelial growth factor receptor
  • the potential immunogenicity of murine-derived scFv limits the use of CAR in clinical setting.
  • utilizing humanized or fully human antibodies to construct CAR lowers the immunogenicity and avoids immune-mediated recognition leading to elimination of the genetically modified cells.
  • CAR containing a CDg TM domain form homodimers and are incorporated into the endogenous T cell receptor (TCR) complex.
  • TCR T cell receptor
  • various transmembrane regions have been employed in CAR including those derived from CD28, CD3z, CD8, CD4, FcRy, etc.
  • IL-6 a cytokine that is secreted by T cells and macrophages in response to inflammation. So they turned to two drugs that are approved to treat inflammatory conditions like juvenile arthritis: etanercept (Enbrel®) and tocilizumab (Actemra®), the latter of which blocks IL-6 activity. The patients had“excellent responses” to the treatment. The other two teams have subsequently used tocilizumab in several patients. Both drugs could become a useful way to help manage cytokine-release syndrome because, unlike steroids, they don’t appear to affect the infused CAR T cells’ activity or proliferation.
  • the successful application of CAR cell therapy requires the identification of target antigens that are expressed only on tumor cells, thereby minimizing the risk of toxicity.
  • Table 2 is a list of tumors antigens and CARs in in vitro and in vivo trials.
  • An“ideal” tumor associated antigen (TAA) (1) has expression restricted to the tumor cell population alone; (2) has expression restricted to the tumor and otherwise non-vital tissues; (3) is expressed by all tumor cells; (4) is expressed on the tumor cell surface; and (5) is required by the tumor cell for survival.
  • TAA tumor associated antigen
  • TCRs Affinity Enhanced T Cell Receptors
  • Affinity enhanced T cell receptors are engineered receptors, which increase an immune effector cell’s ability to bind to a specific antigen beyond that of a native immune effector cell.
  • T-cell receptor (TCR) gene transfer as a strategy to create tumor-reactive T cells is an emerging approach with the potential to overcome many of the obstacles associated with conventional T-cell adoptive immunotherapy.
  • TCRs provide an alternative approach to CAR-based adaptive T-cell therapies.
  • this approach to immunotherapy also comprises the use of gene therapy to reprogram patient or donor T-cells to target and kill tumor cells.
  • the ability of a TCR to activate the T-cell and subsequent immune response depends largely on the strength of the receptor’s binding to a target antigen. In some embodiments, most naturally occurring TCRs are weak with regard to many antigens with therapeutic potential, and therefore require genetic manipulation in order to be optimized.
  • T-cells are sorted by their relative ability to recognize and react to various antigens to prepare the immune system to respond to exogenous agents while preventing autoimmunity.
  • T-cells that bind weakly to self antigens are allowed to survive and undergo further development and maturation, while T-cells that bind strongly to self-antigens, which would cause an autoimmune response under normal circumstances, are eliminated.
  • this tolerance mechanism limits the ability of naturally occurring T-cells to bind to target antigens, such as tumor antigens, with high affinity, which would allow effective elimination of tumor cells.
  • WT1 Wilms’ tumor antigen 1
  • WT1 is a transcription factor that is detected only at low levels in a subset of normal cell types, but is over-expressed 10- to 1, 000- fold in many solid tumors and leukemia cells.
  • native WTl-reactive TCR from T- cells of mice have been isolated, cloned, and mutated to produce WTl-specific TCR variants with higher binding affinity using a saturation mutagenesis screen.
  • engineered TCRs i that bind with higher affinity to WT1 in vitro and the TCR variants were transduced into T-cell lines and tested for T-cell activation against WT1 antigen ex vivo.
  • the mutant TCR with the highest binding affinity h the highest functional activity as measured by T- cell expression and release of cytokines.
  • one potential limitation of TCR-based therapy is that in their natural form, TCRs are only expressed as membrane-anchored proteins that undergo a selection process that only allows TCRs with very low affinities for natural antigens to survive.
  • TCRs are only expressed as membrane-anchored proteins that undergo a selection process that only allows TCRs with very low affinities for natural antigens to survive.
  • whether the enhanced-affmity TCR T-cells would be selected against during normal T- cell development in the thymus was studied.
  • many promising antigens for TCR-based therapies are overexpressed self-proteins, targeting these antigens, even the highest-affmity naturally occurring TCRs, may not result in adequate affinity to efficiently lyse target cells because of the elimination of self-reactive T cells by negative selection in the thymus.
  • hematopoietic stem cells were transduced with the TCR genes, and the T-cell progenitors that developed from these cells displayed negative selection with reduced function against the WT1 antigen, providing support for the hypothesis that thymic selection may be overprotective and TCRs with high binding affinity to turn or/self-anti gens could potentially be tolerated in the periphery.
  • a means of enhancing the activity or efficacy of this approach would clearly be beneficial.
  • antigens suitable for affinity enhanced TCRs of the disclosed methods include, but are not limited to, NY-ESO-l, WT1, MART, MAGE-A1, MAGE- A3, MAGE- A 10, HLA A2, ras, p53, CTAG1B, AFP, CEA, CA-125, MUC- 1, ETA, Tyrosinase, antigens related to infection with oncoviruses such as EBV and HPV, abnormal glycolipids and glycoproteins, and other target antigens known in the art.
  • NY-ESO-l is a particularly noteworthy target because this cancer antigen has shown great promise in preclinical models and in early stage clinical trials. In some cases, tumor persistence or recurrence after NY-ESO-l -specific therapy occurs, however, and the
  • the disclosed methods of co-administration with IL-12 and the resultant robust anti-tumor response improve this recurrence and overall efficacy of targeting NY-ESO-l.
  • gene transfer allows the introduction of countermeasures to tumor immune evasion and of safety mechanisms.
  • CAR- and TCR-based immunotherapy is dependent on the efficient genetic modifications of the infused T cells.
  • IL-12 is used to increase the efficiency of the desired gene transfer.
  • transposon-based system is one of the promising methods because of much higher efficiency of the transgene integration.
  • Sleeping Beauty was the first transposon system used for modification of human T lymphocytes and demonstrated stable expression of CAR with killing of targeted cancer cells in vitro as well as in animal models.
  • PiggyBac transposons mediate stable gene expression in about 20% of primary T cells before selection, and the rate was increased to 40% after selection.
  • the expression can sustain for over 9 weeks in culture through multiple logs of expansion.
  • gammaretroviruses or lentiviruses are engineered to encode the full length CAR or TCR molecule.
  • the viral genomic RNA gets retrotranscribed by the virus reverse transcriptase into DNA, which in turn gets randomly inserted into the host cell DNA through the action of a viral integrase, thus becoming part of that cell’s genome.
  • the retroviruses used are replication-defective, meaning that they are unable to complete their life cycle by proliferating and infecting other cells.
  • the Sleeping Beauty (SB) system provides DNA vectors and avoid the expense and manufacturing difficulty associated with transducing T cells with recombinant viral vectors.
  • the Sleeping Beauty (SB) system provides DNA vectors and avoid the expense and manufacturing difficulty associated with transducing T cells with recombinant viral vectors.
  • retroviral transduction is very efficient, minimizing the time to achieve clinically useful cell numbers.
  • it is an expensive method, there is a potential concern of generating replication competent retrovirus, and viral genes may be immunogenic, curtailing survival of the transduced cells.
  • earlier non-viral methods relied on electroporation of cells with a naked DNA plasmid encoding the CAR or TCR and its illegitimate recombination for stable genomic integration.
  • the cells being used for cell-based therapy are transduced with a gene or genes to express a CAR or CARs specific for particular TAA.
  • the cells being used for cell -based therapy i.e. T cells or NK cells
  • the cells being used for cell -based therapy i.e. T cells or NK cells
  • gene transfer is performed with viral vectors. In some embodiments, gene transfer is performed with non-viral vectors.
  • TILs tumor infiltrated lymphocytes
  • preconditioning of a patient prior to ACT comprises regimens with total body irradiation, lymphodepleting chemotherapy, and/or additional cytokine support. In some embodiments, preconditioning is not required in the methods disclosed herein. In some embodiments, some cancer patient populations may be unable to tolerate
  • IL-l2-activated Ua14 NKT cells there is an antitumor effect of adoptive transfer of IL-l2-activated Ua14 NKT cells.
  • adoptive transfer of IL-l2-activated Ua14 NKT cells cells of a lymphocyte lineage, prevents hepatic metastasis of B16 melanoma.
  • the injection of large amounts of IL-2, IL-4, and IFN- g which are cytokines produced by activated Val4 NKT cells, exhibited no significant inhibition of the metastasis of this melanoma.
  • TReg suppressive CD4 + CD25 + regulatory T
  • the components of the cancer vaccine are (1) one or more cell-based cancer therapies, such as but not limited to chimeric antigen receptor (CAR) T-cell therapy cancer treatments, engineered T cell receptor (TCR) T-cell therapy cancer treatments, or NK cell therapy cancer treatments, which are administered to a subject who has a cancer and (2) a therapeutically effective dose of IL-12 administered exogenously.
  • CAR chimeric antigen receptor
  • TCR engineered T cell receptor
  • NK cell therapy cancer treatments which are administered to a subject who has a cancer
  • the initially effective dose of IL-12 can be followed by maintenance dosing of IL-12 with or without repeating
  • the cell-based cancer therapy comprises obtaining blood cells, including lymphocytes, and cytotoxic T lymphocytes (CTL) or CD8+ cells, either from a donor or a patient.
  • CTL cytotoxic T lymphocytes
  • the cell-based cancer therapy comprises obtaining blood cells, including lymphocytes, and NK cells, either from a donor, a patient, or a cell line.
  • the cells are cultured to expand the cells at least two fold using current or future techniques for expansion of blood cells, preferably lymphocytes.
  • the culturing of blood cells ex vivo or in vitro comprises IL-12 in the culture media.
  • IL-12 can also be included in the transduction step.
  • these cells are administered near the time of administration of any - dose of IL-12.
  • the components when administered to a subject who has cancer produce a vaccine resulting in resistance to the cancer.
  • the cancer vaccine disclosed herein provides a treatment for the primary cancer, prevention of metastasis, and treatment of one or more metastases.
  • the combination administrations of IL-12 and blood cells from the patient comprising lymphocytes are accompanied by booster infusions of dendritic cells that have or have not been pre-exposed to antigens ex vivo.
  • the dendritic cells for the booster are cultured ex vivo with the goal of producing mature dendritic for infusion from the patient’s peripheral blood cells.
  • the dendritic culture methods include the use of GM-CSF in the culture medium.
  • the dendritic culture methods include the use of IL-12 in the culture medium.
  • the components of the cancer vaccine are (1) one or more ICI therapies, such as but not limited, antibodies, fusion proteins, or compounds that target CTLA4, PD1, PDL1, LAG3, B7.1, B7-H3, B7-H4, TIM3, VISTA, CD137, OX-40, CD40, CD27, CCR4, GITR, NKG2D, and/or KIR, which are administered to a subject who has a cancer and (2) a therapeutically effective dose of IL-12.
  • the ICI therapy comprises at least one agent capable of inhibiting an immune checkpoint protein.
  • the agents capable of inhibiting an immune checkpoint protein are administered near the time of administration of any one dose of IL-12.
  • the dose of IL-12 is less than about 1000 ng/kg, less than about 500 ng/kg, about 300 ng/kg, less than about 300 ng/kg, about 200 ng/kg, less than about 200 ng/kg, about 100 ng/kg, less than about 100 ng/kg, about 100 ng/kg or less, about 50 ng/kg or less, or about 10 ng/kg or less.
  • the IL-12 maintenance dose is administered for any therapeutically effective duration of time following cessation of therapy with T cells or NK cells, such as but not limited to, 1 day up to 1 year or any time point in-between, including for example 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months.
  • IL-12 can be administered to the subject in many ways. In some embodiments, these methods of administration include intravenous, subcutaneous, intraperitoneal, intradermal, or the like. In some embodiments, another method of administration of IL-12 is via continuous infusion. In some embodiments, the continuous infusion method has the advantage of delivery a low dose of IL-12 over longer time period, which can add to the effectiveness of present invention.
  • the components of the compositions and methods disclosed herein are administered to the subject at some time interval relative to one another that is somewhat close in time, such as within 2 weeks to 1 hour of each other (and any time point in-between these two values).
  • exemplary time points between administration of the two primary components of the method disclosed herein include, but are not limited to, within 2 weeks (e.g., 14 days), 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 108 hours, 96 hours, 84 hours, 72 hours, 60 hours, 48 hours, 36 hours, 24 hours, 12 hours, 6 hours, 3 hours and within 1 hour of each other.
  • two of the components are administered within about 24 to about 72 hours of each other in the following manner.
  • a therapeutically effective dose of IL-12 is administered about 24 to about 72 hours before the cancer treatment.
  • the cell -based therapy is administered first, followed by a therapeutically effective dose of IL-12 that is administered shortly after the administration of the cancer treatment, preferably within about 24 hours to about 72 hours after the cancer treatment, or at any time point as described above after the cancer treatment, e.g., IL-12 is administered within 2 weeks of the cancer treatment, and can be administered up to 2 weeks before the cancer treatment, up to 2 weeks after the cancer treatment, or at time point in between, or at multiple time points.
  • One of the therapeutic effects of the present disclosure is to generate immunity to the targeted cancer.
  • the targeted cancer means the cancer for which the subject is being treated.
  • the endogenous immunity that is generated has multiple effects: Some of these effects are: (1) to reduce the primary cancerous lesion, (2) to reduce the reoccurrence of the targeted cancer (reduction of micrometastases, i.e., reduction of minimal residual disease (MRD)), as well as prevention of metastasis.
  • the vaccine disclosed herein also renders the subject resistant to other forms of cancer other than the targeted cancer.
  • this effect depends on whether the tumor associated antigens (TAA) that are generated from cancer cells following the administration of the cancer treatment are tumor specific antigens or generalized antigens that are found in more than one cancer. In some embodiments, if these antigens are generalized antigens, there is an expectation that the vaccine would create resistance to other forms of cancer, other than the targeted cancer.
  • TAA tumor associated antigens
  • Tables 2 and 3 show some tumor-associated antigens, which may be specific or generalized antigens.
  • TSA tumor specific antigens
  • TSA tumor specific antigens
  • TSA arises from point mutations or other genetic alterations specific to a given tumor or group of tumors, such as fusion proteins generated by translocations, or sometimes from alterations in posttranslational modification.
  • most of the tumor antigens that are targets for the immune system are more properly defined as tumor- associated antigens (TAA) (see Tables 2 and 3).
  • this definition includes antigens that are not mutated but are differentially expressed by neoplastic and normal cells, either in time, quantity, location, or cellular context, resulting in a preferential or exclusive recognition of the tumor by the immune system.
  • carcinoembryonic antigens are normally expressed only during embryonic development, p53 and HER-2/neu are overexpressed in some cancer cells.
  • an example of tumor-associated antigens is represented by a growing family of cancer/testis antigens that are expressed only in male germ cells, and sometimes in placenta and fetal ovary.
  • tumor- associated antigens with a tissue-restricted expression are legitimate targets for immunotherapy, especially when the tumor arises from nonessential tissues, such as differentiation antigens expressed by melanoma, and prostate cancer.
  • a special class of TAA is derived from oncogenic viruses associated with some types of cancer, such as human papilloma virus E6 and E7 proteins in cervical cancer, and Epstein-Barr virus— derived antigens in lymphomas.
  • Another therapeutic effect of embodiments of the present disclosure includes the generation on anti-tumor activity within the immunological system.
  • the methods disclosed herein comprise immunostimulatory methods, activating the immune system to create a significant anti-tumor effect.
  • the anti-tumor activity is related to the generation of immunity and resistance to the targeted cancer.
  • Still other therapeutic effects of embodiments of the present disclosure include alleviation of the hematological toxicities associated with the T cell therapy cancer treatment or NK cell therapy, such as a CAR- or TCR-modified cell-based therapies.
  • the alleviation of hematological toxicities is particularly important.
  • this hematological effects stems from the hematological effects of IL-12 on bone marrow cells, and other organs related to the lymphatic system.
  • a component of the vaccine is the cancer treatment.
  • the cell-based therapy cancer treatment such as a CAR- or TCR-modified T-cell or NK cell therapy cancer treatment, are any single treatment that kills or destroys cancer cells.
  • the T-cell or NK cell therapy cancer treatment produce necrosis and/or apoptosis of the cancer cells or tissue being treated.
  • necrosis or apoptosis generated by the destruction or death of the cancer cells or tissue by a particular cancer therapy or combinations of cancer therapies generate tumor-associated antigens (TAAs), as described above, and illustrated in Tables 2 and 3.
  • TAAs tumor-associated antigens
  • therapeutic effect include alleviation of the toxicities associated with ICI therapy.
  • a reduction in ICI-related toxicity is expected for the disclosed therapies and vaccines because the additional immune response resulting from the administration of IL-12 allow for significant dose reduction of the given ICI.
  • a component of the vaccine is the ICI therapy treatment.
  • the ICI therapy treatment is any single treatment that kills or destroys cancer cells, including but not limited to, antibodies, fusion proteins, or compounds that target CTLA4, PD1, PDL1, LAG3, B7.1, B7-H3, B7-H4, TIM3, VISTA, CD137, OX-40, CD40, CD27, CCR4, GITR, NKG2D, and/or KIR.
  • the ICI therapy treatment produce necrosis and/or apoptosis of the cancer cells or tissue being treated.
  • such necrosis or apoptosis generated by the destruction or death of the cancer cells or tissue by a particular cancer therapy or combinations of cancer therapies generate tumor-associated antigens (TAAs), as described above, and illustrated in Tables 2.
  • TAAs tumor-associated antigens
  • hematopoietic tumors or cancers such as leukemia, lymphoma or myeloma or the like.
  • the cancer associated APCs are mobilized to the tumor site by the T-cell therapy cancer treatments, such as CAR- or TCR-modified cell-based therapy cancer treatments, or the ICI therapy, or IL- 12 or any other added cytokine, such as Ftl3 ligand, G-CSF or GM-CSF.
  • the cancer associated APC are generated by the vaccine of the present invention to be specific for the targeted cancer or may be more generalized and in this later case may provide immunity or resistance to more than one cancer.
  • the cancer associated APCs promote the production of cytotoxic T cells (CTL). These CTL may comprise CD4 + T cells and/or CD8 + T cells.
  • hematopoietic stem or precursor cells are mobilized to the tumor site by the components of the vaccine disclosed herein, and this mobilization of hematopoietic stem cell or precursor cells gives rise to hematopoiesis outside of the bone marrow, which in turn may give rise to the proliferation of hematopoietic cells, such as monocytes, macrophages, platelets, lymphocytes, T cells, dendritic cells and neutrophils or the like.
  • these hematopoietic stem cells or precursor cells comprises dendritic stem cell or dendritic precursor cells.
  • the mobilization of dendritic stem cell or dendritic precursor cells involves dendropoiesis at or near the tumor site, which gives rise to the proliferation of immature dendritic cells at or near the tumor site.
  • the tumor site are anywhere in the blood, bone marrow, spleen or other hematopoietic organs.
  • a vaccine is created within the body of the subject. In some embodiments, this vaccine is boostered at time points subsequent to the initial production of the vaccine within the subject.
  • cancer cells are taken from the subject prior to treatment with a cancer treatment, and are preserved in some manner, such as cyropreservation. In some embodiments, to generate the booster, the cancer cells are subject to irradiation and a dose that will cause apoptosis and/or necrosis of the cancer cells.
  • these irradiated cancer cells are then administered to the subject along with IL-12, where the irradiated cells and IL-12 are given at time points that are close in time, such as simultaneously or near simultaneously.
  • this booster generates the immunity related cells and molecules that increases resistance to the targeted cancer.
  • the methods disclosed herein comprise using the cells either autologous, i.e., from a cancer patient, or allogenic, i.e, not from the cancer patient, such as the use of cancer cell line related to the targeted cancer, as a source of tumor associated antigens for the generation of an endogenous vaccine.
  • a sufficient number of tumor cells are used.
  • the number of cancer cells preferably would comprise 1 million cells or more, however, 10,000 cells or less should be sufficient.
  • these autologous or allogenic cancer cells are then exposed to radiation in a sufficient dose to cause cellular apoptosis and/or necrosis.
  • the cancer cells are treated with a radiation dose sufficient to cause cellular lysis.
  • the cancer cells are irradiated in the presence of other agents that cause the cells to be radiosensitive, thus ensuring complete destruction of the cells.
  • these cells are injected into the patient, preferably by a subcutaneous route. In some embodiments, other injection routes are applicable. In some embodiments, administration of a therapeutically amount of IL-12 is administered before or after, or before and after, the administration of the irradiated cells to the patient. In some embodiments, administration of IL- 12 is in a single dose or repeated doses. In some embodiments, for human subjects a
  • therapeutically effective dose of IL-12 is generally less than 1000 ng/kg/day and preferably less than 500 ng/kg/day. In some embodiments, even lower doses of IL-12 are effective, such as doses of less than 100 ng/kg/day, especially when more than one dose is administered to the subject at varying time intervals.
  • the cancer cells derived from the patient are from a solid tumor or hematopoietic tumor.
  • the vaccine comprises administering a therapeutically effective dose of radiation or chemotherapy one or more in times either before or after, or before and after, the injection of the irradiated tumor cells.
  • the culture when the blood cells are lymphocytes the culture can be enriched in the population of cytotoxic lymphocytes within the population of lymphocytes.
  • the expansion of blood cells, preferably lymphocytes are performed in the presence of cytokines.
  • cytokines added to the culture are IL-12, IL-4 and IL-2. Table 4 provides an exemplary list of cytokines used to generate cytotoxic T lymphocytes. Table 4
  • tumor or disease-specific T cells are activated to produce proinflammatory cytokines and become effectors capable of killing the tumor cells.
  • recognition and killing of tumor cells by CTL are further enhanced by the up-regulation of Fas and/or major histocompatibility complex class I (MHC I) molecules on the tumor cells.
  • MHC I major histocompatibility complex class I
  • TCR T cell receptor
  • IL interleukin
  • IFN interferon.
  • engineered TCR T-cell therapy in which TCRs with enhanced affinity for specific antigens similarly promote cross-presentation and activation of T cells, and the therapeutic effect likewise are enhanced by the administration of IL-12.
  • the components of the methods disclosed herein are administered with other immune effectors, such as checkpoint inhibitors.
  • immune effectors such as checkpoint inhibitors.
  • the ultimate amplitude and quality of the response which is initiated through antigen recognition by the T cell receptor (TCR), is regulated by a balance between co-stimulatory and inhibitory signals, or “immune checkpoints.”
  • the methods disclosed herein comprise a method with at least three components: (1) a cell-based therapy such as autologous allogenic T cells, NK cell lines or primary cells, or dendritic cells which are cultured and expanded ex vivo prior to administration to a patient, disclosed herein (2) injectable administration of IL-12 disclosed herein, and (3) administering one or more immune checkpoint inhibitor (ICI) therapies, which can include but are not limited to antibodies, fusion proteins, or compounds that target CTLA4, PD1, PDL1, LAG3, B7.1, B7-H3, B7-H4, TIM3, VISTA, CD137, OX-40, CD40, CD27, CCR4, GITR, NKG2D, and/or KIR.
  • ICI immune checkpoint inhibitor
  • IL-12 is administered at any suitable time point before, during, or after the ICI, or any combination thereof.
  • Cl therapies that target other immune checkpoint proteins that are either not disclosed or have not yet been discovered are also contemplated for the purposes of the disclosed invention.
  • the cell-based therapy comprises a chimeric antigen receptor (CAR) T-cell therapy delivered using a suitable gene therapy technique.
  • the cell -based therapy comprises an engineered and/or affinity enhanced T cell receptor (TCR) T-cell therapy delivered using a suitable gene therapy technique.
  • the cell- based therapy a chimeric antigen receptor (CAR) NK cell therapy delivered using a suitable gene therapy technique.
  • the cell-based therapy comprises an engineered and/or affinity enhanced T cell receptor (TCR) NK cell therapy delivered using a suitable gene therapy technique.
  • the cell-based therapy comprises activated dendritic cell therapy.
  • the methods disclosed herein further comprise conventional cancer treatment methods, such as chemotherapy and radiation treatments.
  • conventional cancer treatment methods such as chemotherapy and radiation treatments.
  • cell-based therapies and IL- 12 delivered systemically is expected to increase the anti-tumor response by further expanding T cells and increasing their persistence, both effects being produced in vivo.
  • the methods disclosed herein result in greater complete response (CR) and/or longer term durable remissions.
  • methods disclosed herein enable IL-12 to expand the T cells, and assist in these cells being trafficked to the tumor.
  • determining “determining”,“measuring”,“evaluating”,“assessing,”“assaying,” and “analyzing” are often used interchangeably herein to refer to forms of measurement, and include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing is alternatively relative or absolute.“Detecting the presence of’ includes determining the amount of something present, as well as determining whether it is present or absent.
  • A“subject” can be a biological entity containing expressed genetic materials.
  • the biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa.
  • the subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro.
  • the subject can be a mammal.
  • the mammal can be a human.
  • the subject may be diagnosed or suspected of being at high risk for a disease.
  • the disease can be endometriosis. In some cases, the subject is not necessarily diagnosed or suspected of being at high risk for the disease.
  • in vivo is used to describe an event that takes place in a subject’s body.
  • ex vivo is used to describe an event that takes place outside of a subject’s body.
  • An“ex vivo” assay is not performed on a subject. Rather, it is performed upon a sample separate from a subject.
  • An example of an“ex vivo” assay performed on a sample is an“in vitro” assay.
  • in vitro is used to describe an event that takes places contained in a container for holding laboratory reagent such that it is separated from the living biological source organism from which the material is obtained.
  • in vitro assays can encompass cell-based assays in which cells alive or dead are employed.
  • In vitro assays can also encompass a cell-free assay in which no intact cells are employed.
  • treatment or“treating” are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient.
  • beneficial or desired results include but are not limited to a therapeutic benefit and/or a prophylactic benefit.
  • a therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject,
  • prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.
  • a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.“A treatment” is intended to target the disease state and combat it, i.e., ameliorate the disease state. The particular treatment thus will depend on the disease state to be targeted and the current or future state of medicinal therapies and therapeutic approaches.
  • a treatment may have associated toxicities.
  • Disease state refers to a condition present in a mammal whereby the health and well-being of the mammal is compromised.
  • various forms of cancer are the targeted disease states of the vaccine of the invention.
  • treatments intended to target the disease state are administered to the mammal.
  • “Chemotherapy” refers to agents useful in the treatment of cancer, including cytotoxic agents.
  • the term as used herein includes natural or synthetic agents now known or to be developed in the medical arts.
  • Examples of chemotherapy include the numerous cancer drugs that are currently available.
  • chemotherapy may include the administration of several state of the art drugs intended to treat the disease state.
  • Hematopoietic stem cells are generally the blood stem cells; there are two types: “long-term repopulating” and“short-term repopulating” hematopoietic stem cells. Short-term repopulating hematopoietic stem cells can generally produce“progenitor cells” for a short period (weeks, months or even sometimes years depending on the mammal). These are also referred to herein as hematopoietic repopulating cells.
  • Hematopoietic progenitor cells are generally the first cells to differentiate from (i.e., mature from) blood stem cells; they then differentiate (mature) into the various blood cell types and lineages.
  • human IL-12, or recombinant human IL-12 would be administered to a human, and similarly, for felines, for example, the feline IL-12, or recombinant feline IL-12, would be administered to a feline. Also disclosed herein are embodiments where the IL-12 molecule does not derive its amino acid sequence from the mammal that is the subject of the therapeutic methods disclosed herein. For the sake of illustration, human IL-12 or recombinant human IL-12 may be utilized in a feline mammal.
  • Some embodiments include IL-12 molecules where the native amino acid sequence of IL-12 is altered from the native sequence, but the IL-12 molecule functions to yield the properties of IL-12 that are disclosed herein. Alterations from the native, species-specific amino acid sequence of IL-12 include changes in the primary sequence of IL-12 and encompass deletions and additions to the primary amino acid sequence to yield variant IL-12 molecules.
  • An example of a highly derivatized IL-12 molecule is the redesigned IL-12 molecule produced by Maxygen, Inc., where the variant IL-12 molecule is produced by a DNA shuffling method.
  • Modified IL-12 molecules are also included in disclosure, such as covalent modifications to the IL-12 molecule that increase its shelf life, half-life, potency, solubility, delivery, etc., additions of polyethylene glycol groups, polypropylene glycol, etc., in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. Each of these references is incorporated herein.
  • One type of covalent modification of the IL-12 molecule is introduced into the molecule by reacting targeted amino acid residues of the IL-12 polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues of the IL-12 polypeptide.
  • Both native sequence IL-12 and amino acid sequence variants of IL-12 may be covalently modified.
  • the IL-12 molecule can be produced by various methods known in the art, including recombinant methods. Since it is often difficult to predict in advance the characteristics of a variant IL-12 polypeptide, it will be appreciated that some screening of the recovered variant will be needed to select the optimal variant.
  • a preferred method of assessing a change in the properties of variant IL-12 molecules is via the lethal irradiation rescue protocol disclosed below.
  • Other potential modifications of protein or polypeptide properties such as redox or thermal stability,
  • hydrophobicity, susceptibility to proteolytic degradation, or the tendency to aggregate with carriers or into mul timers are assayed by methods well known in the art.
  • One or more therapeutically effective dose(s) of IL-12 is any dose administered at any time intervals and for any duration that can substantially generate the vaccine effect in the subject.
  • IL-12 is an adjuvant to the chimeric antigen receptor (CAR) or engineered T cell receptor (TCR) T-cell therapy vaccine.
  • Randomtion or radiation therapy or radiation treatment refers to any therapy where any form of radiation is used to treat the disease state.
  • the instruments that produce the radiation for the radiation therapy are either those instruments currently available or to be available in the future.
  • Solid tumors generally is manifested in various cancers of body tissues, such as those solid tumors manifested in lung, breast, prostate, ovary, etc., and are cancers other than cancers of blood tissue, bone marrow or the lymphatic system.
  • Vaccine“or“vaccine effect” is a generated resistance or immunity to a targeted cancer within the body of a subject.
  • a vaccine is created within the subject by the generation of tumor-associated antigens via a disease-related treatment, such as CAR or TCR cell-based therapy, like T-cell or NK cell therapies.
  • IL-12 is acting as an adjuvant to stimulate the immune system to increase the immunological response within the subject to the antigens provided by the treatment.
  • Plasma terminal half-life (also terminal plasma half-life) is the time required to divide the plasma concentration by two after reaching pseudo-equilibrium, and not the time required to eliminate half the administered dose.
  • a method of treating a subject with cancer comprising: administering an adoptive cell therapy to the subject; and administering a first IL-12 treatment comprising a first dose of between 2 pg and 20 pg of recombinant IL-12 to the subject.
  • a first IL-12 treatment comprising a first dose of between 2 pg and 20 pg of recombinant IL-12 to the subject.
  • a second IL-12 treatment comprising a second dose of between 2 pg and 20 pg of recombinant IL-12 to the subject, wherein the second IL-12 treatment is administered after a first non-treatment interval of at least 8 days between the first IL-12 treatment and the second IL-12 treatment.
  • the method of embodiment 2 further comprising administering a third IL-12 treatment comprising a third dose of between 2 pg and 20 pg of recombinant IL-12 to the subject, wherein the third IL-12 treatment is administered after a second non-treatment interval of at least 8 days between the second IL-12 treatment and the third IL-12 treatment.
  • the method of embodiment 3 further comprising administering a fourth IL-12 treatment comprising a fourth dose of between 2 pg and 20 pg of recombinant IL- 12 to the subject, wherein the fourth IL-12 treatment is administered after a third non-treatment interval of at least 8 days between the third IL-12 treatment and the fourth IL-12 treatment. 5.
  • embodiments 1 to 10 wherein at least one of the first, second, third, or fourth non-treatment intervals is at least 1 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.
  • 13 The method of any one of embodiments 1 to 12, wherein the adoptive cell therapy is administered before at least one of the first, second, third, fourth, or fifth IL-12 treatment. 14.
  • the adoptive cell therapy is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days before at least one of the first, second, third, fourth, or fifth IL-12 treatment.
  • the adoptive cell therapy is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or at least 1, 2,
  • the IL-12 is formulated as a pharmaceutical composition comprising a buffer, a salt, and a surfactant.
  • the pharmaceutical composition comprises IL-12 at a concentration of between 1-50 pg/mL. 26.
  • the buffer comprises sodium phosphate. 28.
  • the salt comprises sodium chloride. 30.
  • the adoptive cell therapy comprises an engineered cell.
  • the engineered cell comprise a chimeric antigen receptor.
  • chimeric antigen receptor comprises a T cell receptor.
  • chimeric antigen receptor comprises an extracellular domain comprising an antigen-binding domain.
  • the antigen-binding domain is or comprises an antibody or an antibody fragment thereof.
  • the antibody fragment comprises an scFv.
  • the chimeric antigen receptor is capable of binding to a target antigen that is associated with, specific to, and/or expressed on a cancer cell.
  • the target antigen is selected from the group consisting of: wherein the target antigen i s selected from among anb6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3 , B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), a cancer-testis antigen, cancer/testis antigen IB (CTAG, also known as NY-ES0-1 and LAGE-2), carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C Motif Chemokine Ligand 1 (CCL-l ), CD 19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44, CD44
  • a cancer treatment method comprising: (a) administering one or more cell-based therapies to a subject with cancer; and (b) administering at least one therapeutically effective dose of IL-12 to the subject before, during, or after the administration of the cell-based therapy cancer treatment.
  • the one or more cell-based therapies comprises a T-cell therapy.
  • one or more cell-based therapies comprises a NK cell therapy.
  • the one or more cell -based therapies comprises a dendritic cell therapy.
  • the method of embodiment 56 wherein the method prolongs cancer remission in subjects with cancer as compared to the cell-based therapy alone.
  • the method of embodiment 63, wherein the prolongation of cancer remission results in an increase in progression free patient survival in the population treated with IL-12 and one or more cell-based therapies, as compared to the cell-based therapy alone.
  • the method of embodiment 63, wherein the prolongation of cancer remission results in an increase in overall patient survival in the population treated with IL-12 and one or more cell-based therapies, as compared to the cell-based therapy alone.
  • 67 The method of any one of embodiments 56-66, wherein the subject is a mammal.
  • 68 The method of any one of embodiments 56-67, wherein the subject is a human.
  • 69 The method of any one of embodiments 56-68, wherein the dose of IL-12 is selected from the group consisting of: (a) less than 1000 ng/kg; (b) less than 500 ng/kg; (c) less than 300 ng/kg; (d) less than 150 ng/kg; (e) less than 100 ng/kg; and (f) less than 50 ng/kg.
  • Cytotoxic T- Lymphocytes (CTL) produced as a result of the method comprise one or more cell types selected from the group consisting of CD4+ cells and CD8+ cells.
  • any one of embodiments 56-78 further comprising: (a) removing peripheral blood cells of the subject, (b) culturing the blood cells in a culture medium that activates and expands the number of cells, and (c) reintroducing the cultured blood cells into the subject.
  • a booster to the subject, wherein the booster regenerates immunity-related cells and molecules endogenously, and wherein the booster comprises cancer cells taken from the patient prior to the cancer treatment and which are irradiated prior to administration.
  • IL-12 dosing continues as a maintenance therapy after completion of the administration of at least one cell-based therapy.
  • a) IL-12 is additionally administered one or more times following the initial IL-12 administration;
  • the cell-based therapies are repeated one or more times following the initial administration; or (c) a
  • the vaccine of embodiment 92, wherein the ex -vivo culture comprises cytokines.
  • the vaccine of embodiment 93, wherein the cytokines comprise IL-12.
  • the vaccine of any one of embodiments 92-94, wherein the ex vivo culture results in a cell population comprising increased numbers of dendritic cells.
  • the vaccine of any one of embodiments 88-95, wherein the at least one cell-based therapy comprises a chimeric antigen receptor (CAR) T-cell therapy cancer treatment.
  • the vaccine of any one of embodiments 88- 96, wherein the at least one cell-based therapy is an engineered T cell receptor (TCR) T-cell therapy cancer treatment.
  • TCR engineered T cell receptor
  • a method of administering a cancer vaccine comprising: (a) administering at least one cell -based therapy to a subject who has cancer; and (b) administering a therapeutically effective dose of IL-12 to the subject, wherein the at least one cell-based therapy and IL-12 produce a vaccine effect.
  • 106 The method of any one of embodiments 98-105, wherein the vaccine effect comprises: (a) an anti-tumor response; (b) the generation of immunity to the treated cancer; (c) prevention of metastasis of the cancer; (d) treatment of metastasis of the cancer; (e) activation of endogenous antigen presenting cells (APC); or (f) any combination thereof.
  • the vaccine effect comprises: (a) an anti-tumor response; (b) the generation of immunity to the treated cancer; (c) prevention of metastasis of the cancer; (d) treatment of metastasis of the cancer; (e) activation of endogenous antigen presenting cells (APC); or (f) any combination thereof.
  • the vaccine effect comprises activation of endogenous antigen presenting cells (APC) and: (a) the antigen presenting cells are mobilized to the tumor site by the one or more chimeric antigen receptor (CAR) T-cell therapy cancer treatments or engineered T cell receptor (TCR) T-cell therapy cancer treatments; (b) the antigen presenting cells are mobilized to the tumor site by the administration of IL-12; or (c) a combination thereof.
  • the vaccine effect comprises activation of endogenous antigen presenting cells (APC) and the APC are specific for the cancer.
  • 109 The method of any one of embodiments 98-108, wherein: (a) the vaccine effect comprises activation of endogenous antigen presenting cells (APC); and (b) specific APC are produced from incorporation of one or more cancer-associated antigens into the APC, which are then presented as antigens on the APC.
  • APC endogenous antigen presenting cells
  • specific APC are produced from incorporation of one or more cancer-associated antigens into the APC, which are then presented as antigens on the APC.
  • the cancer-associated APC promote the production of cytotoxic T cells (CTL).
  • CTL cytotoxic T cells
  • the CTL comprise CD4+ T cells, CD8+ T cells, or a combination thereof.
  • a method of administering a cancer vaccine comprising: (a) isolating peripheral blood cells from a subject; (b) culturing the blood cells to expand a lymphocyte subset of cells at least 2 fold; (c) administering the cultured and/or modified lymphocytes to the subject; and (d) administering one or more therapeutically effective dose(s) of IL-12 to the subject.
  • cytokines are selected from the group consisting of IL-12, IL-4, IL-2, IL-7, IL-21, and IL-15.
  • 131. The method of any one of embodiments 127-130, wherein the lymphocytes are enriched for CTL cells.
  • the method of embodiment 133 further comprising administering a third IL-12 treatment comprising a third dose of between 2 pg and 20 pg of recombinant IL-12 to the subject, wherein the third IL-12 treatment is administered after a second non-treatment interval of at least 8 days between the second IL-12 treatment and the third IL-12 treatment. 135.
  • the method of embodiment 134 further comprising administering a fourth IL-12 treatment comprising a fourth dose of between 2 pg and 20 pg of recombinant IL-12 to the subject, wherein the fourth IL-12 treatment is administered after a third non-treatment interval of at least 8 days between the third IL-12 treatment and the fourth IL-12 treatment.
  • a fifth IL-12 treatment comprising a fifth dose of between 2 pg and 20 pg of recombinant IL-12 to the subject, wherein the fifth IL-12 treatment is administered after a fourth non -treatment interval of at least 8 days between the fourth IL-12 treatment and the fifth IL-12 treatment.
  • the effect of the treatment on the patient is monitored by various assessment methods.
  • the infused CAR-T cells are monitored for mobilization from the peripheral blood and for viability following their administration.
  • Other immune cells are also monitored for mobilization out of the peripheral blood and recovery from the bone marrow compartment.
  • Key peripheral blood cells including lymphocytes, neutrophils, platelets, red blood cells and other cells, are monitored.
  • Parameters to monitor include: overall response rate (ORR), including the partial response rate (PR) and complete response rate (CR), duration of response (DOR), progression free survival (PFS), and overall survival (OS).
  • ORR overall response rate
  • PR partial response rate
  • CR complete response rate
  • DOR duration of response
  • PFS progression free survival
  • OS overall survival
  • Example 10 Treatment of cancer patients with cell-based therapy and a pre-conditioning chemotherapy before cell-based therapy infusion
  • the patient is administered intravenously with fludarabine at 30mg/m 2 /day for 3 days on days -4, -3, -2 prior to CAR T-cell administration and with cyclophosphamide at
  • the pre-conditioning nearly ablates the bone marrow, reduces immunosuppressive cells that threaten CAR T-cell expansion, and releases endogenous intracellular inflammatory cytokines, which promote CAR T-cell activity.
  • the pre-conditioning chemotherapy results in hematological adverse effects, including persistent cytopenias, namely one or more of the following, lymphopenia, neutropenia, thrombocytopenia and anemia, with little-to-no mitigation of the adverse effects.
  • Example 11 Treatment of cancer patients with a combination of IL-12 and cell-based therapy with IL-12 as a pre-conditioning before cell-based therapy infusion
  • IL-12 provides ability to stimulate regeneration of bone marrow cells and stimulate progenitor and/or stem cells in contrast to the adverse hematological effects following a pre-conditioning chemotherapy.
  • Example 12 Treatment of cancer patients with a combination of IL-12 and cell-based therapy and pre-conditioning/chemotherapy before cell-based therapy infusion
  • the patient is IL-12 is injected subcutaneously with a dose of 10 pg of IL-12.
  • the injectable IL-12 is prepared in a formulation comprising 10 mM sodium phosphate, 150 mM sodium chloride, and 0.1% (w/v) poloxamer 188, a surfactant, pH 6.0 at a concentration of 20 pg/mL of IL-12 prior to injection.
  • the patient receives an infusion of CAR-T cell therapy intravenously.
  • the maintenance dose comprises 10 pg of IL-12 prepared in a formulation comprising 10 mM sodium phosphate, 150 mM sodium chloride, and 0.1% (w/v) poloxamer 188, a surfactant, pH 6.0 at a concentration of 20 pg/mL of IL-12 prior to injection.
  • This example describes a proof of concept experiment in which ICI therapy and IL-2 are co-administered in a murine model of glioblastoma.
  • FIG. 16 shows an exemplary Kaplan Meier plot in which mice receiving both anti-CTLA4 and IL-12 had significantly increased survival as compared to the animals in the control or mono-therapy groups.
  • the safety and efficacy of a combination therapy of a checkpoint inhibitor and IL-12 can be performed according to the following methods.
  • the present disclosure contains numerous exemplary checkpoint inhibitors that could be combined with IL-12, with one such inhibitor being OPDIVOTM (nivolumab).
  • a clinical study is performed assessing safety and efficacy presenting a novel approach of combining OPDIVOTM, which is the drug of choice for leading melanoma oncologists, with NM-IL-12 (recombinant IL-12), a key cytokine capable of augmenting the effects of checkpoint inhibition.
  • This combination of immunotherapeutics is expected to increase the number of metastatic melanoma patients who respond to checkpoint inhibition, and also increase the number of Complete Responses (CRs). Consequently, this novel combination approach can be expected to reduce the rate of morbidity and mortality associated with this deadly disease.
  • Target Profile of Combination Immunotherapy Target indication and usage: NM-IL- 12 (recombinant human IL-12) in combination with standard of care (SOC) OPDIVOTM
  • nivolumab for the treatment of unresectable or metastatic melanoma.
  • Dosage and administration A single subcutaneous (sc) injection of NM-IL-12 at the dose of 50 or 150 ng/kg following infusion of OPDIVOTM, both drugs given every two weeks until intolerability or disease progression.
  • NM-IL-12 Single use vial of 14 pg/0.80 mL (18 pg/mL), OPDIVOTM: as supplied by its manufacturer Bristol Meyers Squibb (BMS).
  • the first goal of the study is to determine the safety and preliminary efficacy of the combination of SOC OPDIVOTM (3 mg/kg every two weeks) and NM-IL-12 at two sequential dose levels, 50 ng/kg and 150 ng/kg every two weeks in an open label, phase 2a clinical trial in unresectable or metastatic melanoma patients.
  • Advanced melanoma is the most deadly form of skin cancer. When discovered early, melanoma usually can be cured with surgery alone, but once it spreads (metastasizes) throughout the body, treatment options are limited. Until recently, only two drugs were approved by the ETS Food and Drug Administration for the treatment of advanced (stage IV) metastatic melanoma. dacarbazine (DTIC), approved in 1975, remains the only chemotherapy licensed to treat the disease. Patients on dacarbazine have a one-in-eight chance of having tumors shrink.
  • DTIC dacarbazine
  • interleukin-2 an immunological (immune-boosting) therapy
  • IL-2 an immunological (immune-boosting) therapy
  • Checkpoint inhibitors are providing hope to metastatic melanoma patients after decades of research. Since 2014, three checkpoint inhibitors have received FDA approval, namely YERVOY ® (ipilimumab), KEYTRUDA ® (pembrolizumab) and OPDIVOTM
  • OPDIVOTM Despite the success of OPDIVOTM, still many patients are not responsive to the drug. OPDIVOTM when used alone has about a 40% objective response rate (ORR) in treatment naive, metastatic melanoma patients with a complete response rate (CR) of only about 6-8%. Thus, OPDIVOTM alone as a treatment leaves a gap of unmet need for the majority of metastatic melanoma patients. The same is true for KEYTRUDA ® .
  • the FDA also conditionally approved a combination of two checkpoint inhibitors for the treatment of unresectable or metastatic melanoma.
  • the approved combination therapy included an anti-CTLA and an anti -PD- 1 checkpoint inhibitor, namely YERVOY ® and OPDIVOTM respectively.
  • YERVOY ® and OPDIVOTM anti-CTLA
  • OPDIVOTM anti-PD- 1 checkpoint inhibitor
  • this combination of checkpoint inhibitors leaves much to be desired, as there is no apparent synergy in the efficacy of these two agents, as judged by response rates.
  • the toxicity of this combination was substantially increased and resulted in a greater number of patients withdrawing from treatment as they were not able to tolerate the therapy.
  • The“holy grail” of combination therapy with checkpoint inhibitors is to be able to safely and synergistically increase the number of responding patients, particularly, the increase the number of CR and increase PFS. By all accounts, the combination of YERVOY ® and OPDIVOTM missed this mark. See summary of response rates and number of adverse events in Tables 6A and 6B, excerpted from the phase 3 trial comparing OPDIVOTM and YERVOY ® alone and in combination. As shown in the tables below, the ORR was less than additive, while the number of Grade 3/4 treatment-related adverse events went from 16.3% for OPDIVOTM to 55% for the combination therapy. The number of Grade 3/4 treatment— related patient withdrawals also increased from 5.1% for OPDIVOTM to 36.4% for the combination, leaving fewer patients to benefit from the combination therapy.
  • NM-IL-12 (recombinant human IL-12) is a comprehensive immunotherapeutic with an excellent safety profile that is expected to augment the activity of OPDIVOTM in metastatic melanoma patients in a combination immunotherapy approach.
  • NM-IL-12 can be thought of as a“comprehensive immunotherapeutic” due to its diverse pleiotropic, immune effects enumerated in Figure 29. These pleiotropic effects provide for key effects on mature immune cells, as well as providing for the continuous renewal of immature immune cells from the bone marrow (hematopoietic effects). In this manner, IL-12 is a unique actor on the life cycle of immune cells, thus being able to replenish immune cells as they are consumed by their anti-tumor effector action.

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Abstract

L'invention concerne des méthodes de traitement du cancer à l'aide de l'interleukine-12 (IL-12) dans la génération de thérapies à base de cellules, comprenant des thérapies cellulaires à lymphocyte T de récepteur antigénique chimérique (CAR), de récepteur de lymphocyte T (TCR) modifié, à cellules tueuses naturelles (NK) ou à cellules dendritiques, et l'utilisation d'une combinaison d'interleukine-12 (IL-12) injectable et de telles thérapies à base de cellules, et des compositions les concernant.
PCT/US2019/012423 2018-01-04 2019-01-04 Thérapies à base de cellules et par inhibiteurs de points de contrôle immunitaires combinées à il-12 pour le traitement du cancer Ceased WO2019136305A1 (fr)

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US12371504B2 (en) 2017-10-13 2025-07-29 Harpoon Therapeutics, Inc. Trispecific proteins and methods of use
US12150960B2 (en) 2018-04-17 2024-11-26 Innovative Cellular Therapeutics Holdings, Ltd. Modified cell expansion and uses thereof
US12240915B2 (en) 2018-08-30 2025-03-04 Innovative Cellular Therapeutics Holdings, Ltd. Chimeric antigen receptor cells for treating solid tumor
US12195544B2 (en) 2018-09-21 2025-01-14 Harpoon Therapeutics, Inc. EGFR binding proteins and methods of use
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US12043654B2 (en) 2020-06-02 2024-07-23 Innovative Cellular Therapeutics Holdings, Ltd. Anti-GCC antibody and CAR thereof for treating digestive system cancer
WO2022178325A1 (fr) * 2021-02-18 2022-08-25 Engene, Inc. Polythérapie génique pour le traitement du cancer métastatique
WO2022256498A1 (fr) * 2021-06-03 2022-12-08 Harpoon Therapeutics, Inc. Protéines trispécifiques ciblant la msln et méthodes d'utilisation

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