Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0635—B lymphocytes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/14—Blood; Artificial blood
  • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
• • 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
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/42—Cancer antigens
  • A61K40/428—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0636—T lymphocytes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0636—T lymphocytes
  • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
• • 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
• • 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
  • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
  • A61K2239/55—Lung
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/20—Cytokines; Chemokines
  • C12N2501/23—Interleukins [IL]
  • C12N2501/2302—Interleukin-2 (IL-2)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/20—Cytokines; Chemokines
  • C12N2501/23—Interleukins [IL]
  • C12N2501/2315—Interleukin-15 (IL-15)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/20—Cytokines; Chemokines
  • C12N2501/23—Interleukins [IL]
  • C12N2501/2321—Interleukin-21 (IL-21)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/50—Cell markers; Cell surface determinants
  • C12N2501/515—CD3, T-cell receptor complex
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells

Definitions

• TILs tumor infiltrating lymphocytes
• REP can result in a 1, 000-fold expansion of TILs over a l4-day period, although it requires a large excess (e.g, 200-fold) of irradiated allogeneic peripheral blood mononuclear cells (PBMCs, also known as mononuclear cells (MNCs)), often from multiple donors, as feeder cells, as well as anti-CD3 antibody (OKT3) and high doses of IL-2.
• PBMCs peripheral blood mononuclear cells
• MNCs mononuclear cells
• TILs that have undergone an REP procedure have produced successful adoptive cell therapy following host immunosuppression in patients with melanoma.
• Current infusion acceptance parameters rely on readouts of the composition of TILs (e.g ., CD28, CD8, or CD4 positivity) and on fold expansion and viability of the REP product.
• TIL manufacturing and treatment processes are limited by length, cost, sterility concerns, and other factors described herein such that the potential to treat patients which are refractory to anti -PD 1 and as such have been severely limited.
• the present invention meets this need by providing a shortened manufacturing process for use in generating TILs which can then be employed in the treatment of non-small cell lung carcinoma (NSCLC) patients whom are refractory to anti -PD- 1 treatment.
• NSCLC non-small cell lung carcinoma
• the present invention provides improved and/or shortened methods for expanding TILs and producing therapeutic populations of TILs for use in treatment of non-small cell lung carcinoma (NSCLC) patients whom are refractory to anti -PD- 1 treatment.
• NSCLC non-small cell lung carcinoma
• the present invention provides a method of treating non-small cell lung carcinoma (NSCLC) with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
• the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), and optionally irradiated allogeneic peripheral blood mononuclear cells (PBMCs); and wherein the rapid expansion is performed over a period of 14 days or less;
• NSCLC is refractory to treatment with an anti-PD-l antibody.
• “obtaining” indicates the TILs employed in the method and/or process can be derived directly from the sample (including from a surgical resection, needle biopsy, core biopsy, small biopsy, or other sample) as part of the method and/or process steps.
• ‘receiving” indicates the TILs employed in the method and/or process can be derived indirectly from the sample (including from a surgical resection, needle biopsy, core biopsy, small biopsy, or other sample) and then employed in the method and/or process, (for example, where step (a) begins will TILs that have already been derived from the sample by a separate process not included in part (a), such TILs could be refered to as “received”).
• obtaining the first population of TILs comprises a
• the refractory NSCLC has been previously treated with an anti-PD-l and/or anti -PD-L1 and/or anti-PD-L2 antibody.
• the refractory NSCLC has not been previously treated with an anti-PD-l and/or anti -PD-L1 antibody.
• the refractory NSCLC has been treated with a
• the refractory NSCLC has been previously treated with an anti-PD-l and/or anti-PD-Ll antibody and has been previously treated a chemotherapeutic agent.
• the refractory NSCLC has not been previously treated with an anti-PD-l and/or anti-PD-Ll antibody and has been previously treated a chemotherapeutic agent.
• the refractory NSCLC has been treated with a
• the refractory NSCLC has low expression of PD-L1.
• the refractory NSCLC has been previously treated with an anti-PD-l and/or anti-PD-Ll antibody and has low expression of PD-L1.
• the refractory NSCLC has not been previously treated with an anti-PD-l and/or anti-PD-Ll antibody and has low expression of PD-L1.
• the refractory NSCLC has been treated with a
• chemotherapeutic agent and has low expression of PD-L1.
• the refractory NSCLC has been treated with a
• chemotherapeutic agent but is not being currently treated with a chemotherapeutic agent and has low expression of PD-L1
• the refractory NSCLC has not been previously treated with an anti-PD-l and/or anti-PD-Ll antibody and has bulky disease at baseline.
• the refractory NSCLC has been previously treated with an anti-PD-l and/or anti-PD-Ll antibody and has bulky disease at baseline.
• the refractory NSCLC has been treated with a
• chemotherapeutic agent and has bulky disease at baseline.
• the refractory NSCLC has been treated with a
• chemotherapeutic agent but is not being currently treated with a chemotherapeutic agent and has bulky disease at baseline.
• bulky disease is indicated where the maximal tumor diameter is greater than 7 cm measured in either the transverse or coronal plane or swollen lymph nodes with a short-axis diameter of 20 mm or greater.
• the refractory NSCLC is refractory to at least two prior systemic treatment courses, not including neo-adjuvant or adjuvant therapies.
• the refractory NSCLC is refractory to an anti-PD-l antibody selected from the group consisting of nivolumab, pembrolizumab, ipilimumab, JS001, TSR- 042, pidilizumab, (BGB-A317, SHR-1210, REGN2810, MDX-1106, PDR001, anti-PD-l from clone: RMP1-14; and an anti-PD-l antibodies disclosed in U.S. Patent No. 8,008,449, durvalumab, atezolizumab, avelumab, and fragments, derivatives, variants, as well as biosimilars thereof.
• the refractory NSCLC is refractory to pembrolizumab or a biosimilar thereof.
• the refractory NSCLC is refractory to nivolumab or a biosimilar thereof.
• the refractory NSCLC is refractory to ipilimumab or a biosimilar thereof.
• the refractory NSCLC is refractory to ipilimumab or a biosimilar thereof and pembrolizumab or a biosimilar thereof.
• the refractory NSCLC is refractory to ipilimumab or a biosimilar thereof and nivolumab or a biosimilar thereof.
• the refractory NSCLC is refractory to durvalumab or a biosimilar thereof.
• the refractory NSCLC is refractory to atezolizumab or a biosimilar thereof.
• the refractory NSCLC is refractory to avelumab or a biosimilar thereof.
• the initial expansion is performed over a period of 21 days or less.
• the initial expansion is performed over a period of 14 days or less.
• the initial expansion is performed over a period of about 11 days and the rapid expansion is performed over a period of about 11 days.
• the IL-2 is present at an initial concentration of between 1000 IU/mL and 6000 IU/mL in the first cell culture medium.
• the IL-2 is present at an initial concentration of between 1000 IU/mL and 6000 IU/mL and the OKT-3 antibody is present at an initial concentration of about 30 ng/mL in the second cell culture medium.
• the initial expansion is performed using a gas permeable container.
• the rapid expansion is performed using a gas permeable container.
• the first cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.
• the second cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.
• the method further comprises the step of treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the third population of TILs to the patient.
• the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg/m 2 /day for two days followed by administration of fludarabine at a dose of 25 mg/m 2 /day for five days.
• the method further comprises the step of treating the patient with an IL-2 regimen starting on the day after administration of the third population of TILs to the patient.
• the IL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000 IU/kg of aldesleukin, or a biosimilar or variant thereof, administered as a 15-minute bolus intravenous infusion every eight hours until tolerance.
• the invention provides a method of treating non-small cell lung carcinoma (NSCLC) with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
• cancer is refractory to treatment with an anti -PD- 1 antibody.
• the refractory NSCLC has been previously treated with an anti-PD-l and/or anti -PD-L1 antibody.
• the refractory NSCLC has not been previously treated with an anti-PD-l and/or anti -PD-L1 antibody.
• the refractory NSCLC has been treated with a
• the refractory NSCLC has been previously treated with an anti-PD-l and/or anti-PD-Ll antibody and has been previously treated a chemotherapeutic agent.
• the refractory NSCLC has not been previously treated with an anti-PD-l and/or anti-PD-Ll antibody and has been previously treated a chemotherapeutic agent.
• the refractory NSCLC has been treated with a
• chemotherapeutic agent but is not being currently treated with a chemotherapeutic agent.
• the refractory NSCLC has low expression of PD-L1.
• the refractory NSCLC has been previously treated with an anti-PD-l and/or anti-PD-Ll antibody and has low expression of PD-L1.
• the refractory NSCLC has not been previously treated with an anti-PD-l and/or anti-PD-Ll antibody and has low expression of PD-L1.
• the refractory NSCLC has been treated with a chemotherapeutic agent and has low expression of PD-L1.
• the refractory NSCLC has been treated with a
• chemotherapeutic agent but is not being currently treated with a chemotherapeutic agent and has low expression of PD-L1
• the refractory NSCLC has not been previously treated with an anti-PD-l and/or anti-PD-Ll antibody and has bulky disease at baseline.
• the refractory NSCLC has been previously treated with an anti-PD-l and/or anti-PD-Ll antibody and has bulky disease at baseline.
• the refractory NSCLC has been treated with a
• chemotherapeutic agent and has bulky disease at baseline.
• the refractory NSCLC has been treated with a
• chemotherapeutic agent but is not being currently treated with a chemotherapeutic agent and has bulky disease at baseline.
• bulky disease is indicated where the maximal tumor diameter is greater than 7 cm measured in either the transverse or coronal plane or swollen lymph nodes with a short-axis diameter of 20 mm or greater.
• the refractory NSCLC is refractory to at least two prior systemic treatment courses, not including neo-adjuvant or adjuvant therapies.
• the refractory NSCLC is refractory to an anti-PD-l antibody selected from the group consisting of nivolumab, pembrolizumab, ipilimumab, JS001, TSR- 042, pidilizumab, (BGB-A317, SHR-1210, REGN2810, MDX-1106, PDR001, anti-PD-l from clone: RMP1-14; and an anti-PD-l antibodies disclosed in U.S. Patent No. 8,008,449, durvalumab, atezolizumab, avelumab, and fragments, derivatives, variants, as well as biosimilars thereof.
• an anti-PD-l antibody selected from the group consisting of nivolumab, pembrolizumab, ipilimumab, JS001, TSR- 042, pidilizumab, (BGB-A317, SHR-1210, REGN2810, MDX-1106, P
• the refractory NSCLC is refractory to pembrolizumab or a biosimilar thereof.
• the refractory NSCLC is refractory to nivolumab or a biosimilar thereof.
• the refractory NSCLC is refractory to ipilimumab or a biosimilar thereof.
• the refractory NSCLC is refractory to ipilimumab or a biosimilar thereof and pembrolizumab or a biosimilar thereof.
• the refractory NSCLC is refractory to ipilimumab or a biosimilar thereof and nivolumab or a biosimilar thereof.
• the refractory NSCLC is refractory to durvalumab or a biosimilar thereof.
• the refractory NSCLC is refractory to atezolizumab or a biosimilar thereof.
• the refractory NSCLC is refractory to avelumab or a biosimilar thereof.
• the initial expansion is performed over a period of 21 days or less.
• the initial expansion is performed over a period of 14 days or less.
• the initial expansion is performed over a period of about 11 days and the rapid expansion is performed over a period of about 11 days.
• the IL-2 is present at an initial concentration of between 1000 IU/mL and 6000 IU/mL in the first cell culture medium.
• the IL-2 is present at an initial concentration of between 1000 IU/mL and 6000 IU/mL and the OKT-3 antibody is present at an initial concentration of about 30 ng/mL in the second cell culture medium.
• the initial expansion is performed using a gas permeable container.
• the rapid expansion is performed using a gas permeable container.
• the first cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.
• the second cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.
• the method further comprises the step of treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the third population of TILs to the patient.
• the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg/m 2 /day for two days followed by administration of fludarabine at a dose of 25 mg/m 2 /day for five days.
• the method further comprises the step of treating the patient with an IL-2 regimen starting on the day after administration of the third population of TILs to the patient.
• the IL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000 IU/kg of aldesleukin, or a biosimilar or variant thereof, administered as a 15-minute bolus intravenous infusion every eight hours until tolerance.
• the invention provides a method for treating a subject with non-small cell lung carcinoma (NSCLC), wherein the cancer is refractory to treatment with an anti -PD- 1 antibody, the method comprising administering expanded tumor infiltrating lymphocytes (TILs) comprising:
• step (c) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas- permeable surface area, wherein the first expansion is performed for about 3-113- 44 days to obtain the second population of TILs, wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs, and wherein the transition from step (b) to step (c) occurs without opening the system;
• step (d) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-113-44 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs which comprises an increased subpopulation of effector T cells and/or central memory T cells relative to the second population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (c) to step (d) occurs without opening the system;
• APCs antigen presenting cells
• step (e) harvesting therapeutic population of TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system;
• step (f) transferring the harvested TIL population from step (e) to an infusion bag
• step (e) to (f) occurs without opening the system
• step (g) cryopreserving the infusion bag comprising the harvested TIL population from step (f) using a cryopreservation process
• step (h) administering a therapeutically effective dosage of the third population of TILs from the infusion bag in step (g) to the subject.
• “obtaining” indicates the TILs employed in the method and/or process can be derived directly from the sample (including from a surgical resection, needle biopsy, core biopsy, small biopsy, or other sample) as part of the method and/or process steps.
• ‘receiving” indicates the TILs employed in the method and/or process can be derived indirectly from the sample (including from a surgical resection, needle biopsy, core biopsy, small biopsy, or other sample) and then employed in the method and/or process, (for example, where step (a) begins will TILs that have already been derived from the sample by a separate process not included in part (a), such TILs could be refered to as “received”).
• the refractory NSCLC has been previously treated with an anti-PD-l and/or anti -PD-L1 antibody.
• the refractory NSCLC has not been previously treated with an anti-PD-l and/or anti -PD-L1 antibody.
• the refractory NSCLC has been treated with a
• the refractory NSCLC has been previously treated with an anti-PD-l and/or anti-PD-Ll antibody and has been previously treated a chemotherapeutic agent.
• the refractory NSCLC has not been previously treated with an anti-PD-l and/or anti-PD-Ll antibody and has been previously treated a chemotherapeutic agent.
• the refractory NSCLC has been treated with a
• chemotherapeutic agent but is not being currently treated with a chemotherapeutic agent.
• the refractory NSCLC has low expression of PD-L1.
• the refractory NSCLC has been previously treated with an anti-PD-l and/or anti-PD-Ll antibody and has low expression of PD-L1.
• the refractory NSCLC has not been previously treated with an anti-PD-l and/or anti-PD-Ll antibody and has low expression of PD-L1.
• the refractory NSCLC has been treated with a
• chemotherapeutic agent and has low expression of PD-L1.
• the refractory NSCLC has been treated with a chemotherapeutic agent but is not being currently treated with a chemotherapeutic agent and has low expression of PD-L1.
• the refractory NSCLC has not been previously treated with an anti-PD-l and/or anti-PD-Ll antibody and has bulky disease at baseline.
• the refractory NSCLC has been previously treated with an anti-PD-l and/or anti-PD-Ll antibody and has bulky disease at baseline.
• the refractory NSCLC has been treated with a chemotherapeutic agent and has bulky disease at baseline.
• the refractory NSCLC has been treated with a chemotherapeutic agent but is not being currently treated with a chemotherapeutic agent and has bulky disease at baseline.
• bulky disease is indicated where the maximal tumor diameter is greater than 7 cm measured in either the transverse or coronal plane or swollen lymph nodes with a short-axis diameter of 20 mm or greater.
• the refractory NSCLC is refractory to at least two prior systemic treatment courses, not including neo-adjuvant or adjuvant therapies.
• the refractory NSCLC is refractory to an anti-PD-l antibody selected from the group consisting of nivolumab, pembrolizumab, ipilimumab, JS001, TSR-042, pidilizumab, (BGB-A317, SHR-1210, REGN2810, MDX-1106, PDR001, anti-PD-l from clone: RMP1-14; and an anti-PD-l antibodies disclosed in U.S. Patent No. 8,008,449, durvalumab, atezolizumab, avelumab, and fragments, derivatives, variants, as well as biosimilars thereof.
• an anti-PD-l antibody selected from the group consisting of nivolumab, pembrolizumab, ipilimumab, JS001, TSR-042, pidilizumab, (BGB-A317, SHR-1210, REGN2810, MDX-1106, PDR001
• the refractory NSCLC is refractory to pembrolizumab or a biosimilar thereof.
• the refractory NSCLC is refractory to nivolumab or a biosimilar thereof.
• the refractory NSCLC is refractory to ipilimumab or a biosimilar thereof.
• the refractory NSCLC is refractory to ipilimumab or a biosimilar thereof and pembrolizumab or a biosimilar thereof.
• the refractory NSCLC is refractory to ipilimumab or a biosimilar thereof and nivolumab or a biosimilar thereof.
• the refractory NSCLC is refractory to durvalumab or a biosimilar thereof.
• the refractory NSCLC is refractory to atezolizumab or a biosimilar thereof.
• the refractory NSCLC is refractory to avelumab or a biosimilar thereof.
• the initial expansion is performed over a period of 21 days or less.
• the initial expansion is performed over a period of 14 days or less.
• the initial expansion is performed over a period of about 3-11 days and the second expansion is performed over a period of about 7-11 days. [00119] In some embodiments, the initial expansion is performed over a period of about 11 days and the rapid expansion is performed over a period of about 11 days.
• the IL-2 is present at an initial concentration of between 1000 IU/mL and 6000 IU/mL in the first cell culture medium.
• the IL-2 is present at an initial concentration of between 1000 IU/mL and 6000 IU/mL and the OKT-3 antibody is present at an initial concentration of about 30 ng/mL in the second cell culture medium.
• the initial expansion is performed using a gas permeable container.
• the rapid expansion is performed using a gas permeable container.
• the first cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.
• the second cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.
• the method further comprises the step of treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the third population of TILs to the patient.
• the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg/m 2 /day for two days followed by administration of fludarabine at a dose of 25 mg/m 2 /day for five days.
• the method further comprises the step of treating the patient with an IL-2 regimen starting on the day after administration of the third population of TILs to the patient.
• the IL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000 IU/kg of aldesleukin, or a biosimilar or variant thereof, administered as a 15-minute bolus intravenous infusion every eight hours until tolerance.
• Figure 1 Exemplary Process 2A chart providing an overview of Steps A through F.
• Figure 2 Process Flow Chart of Process 2A.
• Figure 3 Shows a diagram of an embodiment of a cryopreserved TIL exemplary manufacturing process ( ⁇ 22 days).
• Figure 4 Shows a diagram of an embodiment of process 2A, a 22-day process for TIL manufacturing.
• Figure 5 Comparison table of Steps A through F from exemplary embodiments of process 1C and process 2A.
• FIG. 7 Study Flowcharts for Combination Cohorts: Cohort 1A (MM), Cohort 2A (HNSCC), and Cohort 3A (NSCLC).
• Cy cyclophosphamide
• EOA end of assessment
• EOS end of study
• EOT end of treatment
• Flu fludarabine
• IL-2 interleukin-2
• NMA-LD nonmyeloablative lymphodepletion
• Q3W every 3 weeks
• T ⁇ L tumor infiltrating lymphocytes.
• Patients in Cohorts 1 A, 2A, and 3A will receive a single infusion of
• pembrolizumab after the completion of their tumor resection for TIL production and baseline scans before the initiation of the NMA-LD regimen.
• the next dose of pembrolizumab was not administered earlier than following the completion of IL-2 and continue Q3W ⁇ 3 days thereafter for ⁇ 2 years (24 months) or until disease progression or unacceptable toxicity, whichever occurred first.
• Figure 8 Study Flowchart for Single-agent Cohort: Cohort 3B (NSCLC).
• Cy cyclophosphamide
• EOA end of assessment
• EOS end of study
• EOT end of treatment
• Flu fludarabine
• IL-2 interleukin-2
• NMA-LD nonmyeloablative lymphodepletion
• TIL tumor infiltrating lymphocytes.
• Figure 9 Shows a diagram of an embodiment of process 2A, a 22-day process for TIL manufacturing.
• Figure 10 Provides the structures I-A and I-B, the cylinders refer to individual polypeptide binding domains.
• Structures I-A and I-B comprise three linearly-linked TNFRSF binding domains derived from e.g., 4-1BBL or an antibody that binds 4-1BB, which fold to form a trivalent protein, which is then linked to a second trivalent protein through IgGl-Fc (including CH3 and CH2 domains) is then used to link two of the trivalent proteins together through disulfide bonds (small elongated ovals), stabilizing the structure and providing an agonists capable of bringing together the intracellular signaling domains of the six receptors and signaling proteins to form a signaling complex.
• IgGl-Fc including CH3 and CH2 domains
• the TNFRSF binding domains denoted as cylinders may be scFv domains comprising, e.g., a VH and a VL chain connected by a linker that may comprise hydrophilic residues and Gly and Ser sequences for flexibility, as well as Glu and Lys for solubility.
• SEQ ID NO: 1 is the amino acid sequence of the heavy chain of muromonab.
• SEQ ID NO:2 is the amino acid sequence of the light chain of muromonab.
• SEQ ID NO:3 is the amino acid sequence of a recombinant human IL-2 protein.
• SEQ ID NO:4 is the amino acid sequence of aldesleukin.
• SEQ ID NO:5 is the amino acid sequence of a recombinant human IL-4 protein.
• SEQ ID NO:6 is the amino acid sequence of a recombinant human IL-7 protein.
• SEQ ID NO:7 is the amino acid sequence of a recombinant human IL-15 protein.
• SEQ ID NO:8 is the amino acid sequence of a recombinant human IL-21 protein.
• SEQ ID NO:9 is the amino acid sequence of human 4-1BB.
• SEQ ID NO: 10 is the amino acid sequence of murine 4-1BB.
• SEQ ID NO: 11 is the heavy chain for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).
• SEQ ID NO: 12 is the light chain for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).
• SEQ ID NO: 13 is the heavy chain variable region (VH) for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).
• SEQ ID NO: 14 is the light chain variable region (VL) for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).
• SEQ ID NO: 15 is the heavy chain CDR1 for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).
• SEQ ID NO: 16 is the heavy chain CDR2 for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).
• SEQ ID NO: 17 is the heavy chain CDR3 for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).
• SEQ ID NO: 18 is the light chain CDR1 for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).
• SEQ ID NO: 19 is the light chain CDR2 for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).
• SEQ ID NO:20 is the light chain CDR3 for the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).
• SEQ ID NO:2l is the heavy chain for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).
• SEQ ID NO:22 is the light chain for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).
• SEQ ID NO:23 is the heavy chain variable region (VH) for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).
• SEQ ID NO:24 is the light chain variable region (VL) for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).
• SEQ ID NO:25 is the heavy chain CDR1 for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).
• SEQ ID NO:26 is the heavy chain CDR2 for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).
• SEQ ID NO:27 is the heavy chain CDR3 for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).
• SEQ ID NO:28 is the light chain CDR1 for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).
• SEQ ID NO:29 is the light chain CDR2 for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).
• SEQ ID NO:30 is the light chain CDR3 for the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).
• SEQ ID NO:31 is an Fc domain for a TNFRSF agonist fusion protein.
• SEQ ID NO:32 is a linker for a TNFRSF agonist fusion protein.
• SEQ ID NO:33 is a linker for a TNFRSF agonist fusion protein.
• SEQ ID NO:34 is a linker for a TNFRSF agonist fusion protein.
• SEQ ID NO:35 is a linker for a TNFRSF agonist fusion protein.
• SEQ ID NO:36 is a linker for a TNFRSF agonist fusion protein.
• SEQ ID NO:37 is a linker for a TNFRSF agonist fusion protein.
• SEQ ID NO:38 is a linker for a TNFRSF agonist fusion protein.
• SEQ ID NO:39 is a linker for a TNFRSF agonist fusion protein.
• SEQ ID NO:40 is a linker for a TNFRSF agonist fusion protein.
• SEQ ID NO:4l is a linker for a TNFRSF agonist fusion protein.
• SEQ ID NO:42 is an Fc domain for a TNFRSF agonist fusion protein.
• SEQ ID NO:43 is a linker for a TNFRSF agonist fusion protein.
• SEQ ID NO:44 is a linker for a TNFRSF agonist fusion protein.
• SEQ ID NO:45 is a linker for a TNFRSF agonist fusion protein.
• SEQ ID NO:46 is a 4-1BB ligand (4-1BBL) amino acid sequence.
• SEQ ID NO:47 is a soluble portion of 4-1BBL polypeptide.
• SEQ ID NO:48 is a heavy chain variable region (VH) for the 4-1BB agonist antibody 4B4-1-1 version 1.
• SEQ ID NO:49 is a light chain variable region (VL) for the 4-1BB agonist antibody 4B4-1-1 version 1.
• SEQ ID NO:50 is a heavy chain variable region (VH) for the 4-1BB agonist antibody 4B4-1-1 version 2.
• SEQ ID NO:51 is a light chain variable region (VL) for the 4-1BB agonist antibody 4B4-1-1 version 2.
• SEQ ID NO:52 is a heavy chain variable region (VH) for the 4-1BB agonist antibody H39E3-2.
• SEQ ID NO:53 is a light chain variable region (VL) for the 4-1BB agonist antibody H39E3-2.
• SEQ ID NO:54 is the amino acid sequence of human 0X40.
• SEQ ID NO:55 is the amino acid sequence of murine 0X40.
• SEQ ID NO:56 is the heavy chain for the 0X40 agonist monoclonal antibody tavolixizumab (MEDI-0562).
• SEQ ID NO:57 is the light chain for the 0X40 agonist monoclonal antibody tavolixizumab (MEDI-0562).
• SEQ ID NO:58 is the heavy chain variable region (VH) for the 0X40 agonist monoclonal antibody tavolixizumab (MEDI-0562).
• SEQ ID NO:59 is the light chain variable region (VL) for the 0X40 agonist monoclonal antibody tavolixizumab (MEDI-0562).
• SEQ ID NO:60 is the heavy chain CDR1 for the 0X40 agonist monoclonal antibody tavolixizumab (MEDI-0562).
• SEQ ID NO:6l is the heavy chain CDR2 for the 0X40 agonist monoclonal antibody tavolixizumab (MEDI-0562).
• SEQ ID NO:62 is the heavy chain CDR3 for the 0X40 agonist monoclonal antibody tavolixizumab (MEDI-0562).
• SEQ ID NO:63 is the light chain CDR1 for the 0X40 agonist monoclonal antibody tavolixizumab (MEDI-0562).
• SEQ ID NO:64 is the light chain CDR2 for the 0X40 agonist monoclonal antibody tavolixizumab (MEDI-0562).
• SEQ ID NO:65 is the light chain CDR3 for the 0X40 agonist monoclonal antibody tavolixizumab (MEDI-0562).
• SEQ ID NO:66 is the heavy chain for the 0X40 agonist monoclonal antibody 11D4.
• SEQ ID NO:67 is the light chain for the 0X40 agonist monoclonal antibody 11D4.
• SEQ ID NO:68 is the heavy chain variable region (VH) for the 0X40 agonist monoclonal antibody 11D4.
• SEQ ID NO:69 is the light chain variable region (VL) for the 0X40 agonist monoclonal antibody 11D4.
• SEQ ID NO:70 is the heavy chain CDR1 for the 0X40 agonist monoclonal antibody 11D4.
• SEQ ID NO:7l is the heavy chain CDR2 for the 0X40 agonist monoclonal antibody 11D4.
• SEQ ID NO:72 is the heavy chain CDR3 for the 0X40 agonist monoclonal antibody 11D4.
• SEQ ID NO:73 is the light chain CDR1 for the 0X40 agonist monoclonal antibody 11D4.
• SEQ ID NO:74 is the light chain CDR2 for the 0X40 agonist monoclonal antibody 11D4.
• SEQ ID NO:75 is the light chain CDR3 for the 0X40 agonist monoclonal antibody 11D4.
• SEQ ID NO:76 is the heavy chain for the 0X40 agonist monoclonal antibody 18D8.
• SEQ ID NO:77 is the light chain for the 0X40 agonist monoclonal antibody 18D8.
• SEQ ID NO:78 is the heavy chain variable region (VH) for the 0X40 agonist monoclonal antibody 18D8.
• SEQ ID NO:79 is the light chain variable region (VL) for the 0X40 agonist monoclonal antibody 18D8.
• SEQ ID NO:80 is the heavy chain CDR1 for the 0X40 agonist monoclonal antibody 18D8.
• SEQ ID NO:8l is the heavy chain CDR2 for the 0X40 agonist monoclonal antibody 18D8.
• SEQ ID NO:82 is the heavy chain CDR3 for the 0X40 agonist monoclonal antibody 18D8.
• SEQ ID NO:83 is the light chain CDR1 for the 0X40 agonist monoclonal antibody 18D8.
• SEQ ID NO:84 is the light chain CDR2 for the 0X40 agonist monoclonal antibody 18D8.
• SEQ ID NO:85 is the light chain CDR3 for the 0X40 agonist monoclonal antibody 18D8.
• SEQ ID NO:86 is the heavy chain variable region (VH) for the 0X40 agonist monoclonal antibody Hul 19-122.
• SEQ ID NO:87 is the light chain variable region (VL) for the 0X40 agonist monoclonal antibody Hul 19-122.
• SEQ ID NO:88 is the heavy chain CDR1 for the 0X40 agonist monoclonal antibody Hul 19-122.
• SEQ ID NO:89 is the heavy chain CDR2 for the 0X40 agonist monoclonal antibody Hul 19-122.
• SEQ ID NO:90 is the heavy chain CDR3 for the 0X40 agonist monoclonal antibody Hul 19-122.
• SEQ ID NO:9l is the light chain CDR1 for the 0X40 agonist monoclonal antibody Hul 19-122.
• SEQ ID NO:92 is the light chain CDR2 for the 0X40 agonist monoclonal antibody Hul 19-122.
• SEQ ID NO:93 is the light chain CDR3 for the 0X40 agonist monoclonal antibody Hul 19-122.
• SEQ ID NO:94 is the heavy chain variable region (VH) for the 0X40 agonist monoclonal antibody Hul06-222.
• SEQ ID NO:95 is the light chain variable region (VL) for the 0X40 agonist monoclonal antibody Hul06-222.
• SEQ ID NO:96 is the heavy chain CDR1 for the 0X40 agonist monoclonal antibody Hu 106-222.
• SEQ ID NO:97 is the heavy chain CDR2 for the 0X40 agonist monoclonal antibody Hu 106-222.
• SEQ ID NO:98 is the heavy chain CDR3 for the 0X40 agonist monoclonal antibody Hu 106-222.
• SEQ ID NO:99 is the light chain CDR1 for the 0X40 agonist monoclonal antibody Hu 106-222.
• SEQ ID NO: 100 is the light chain CDR2 for the 0X40 agonist monoclonal antibody Hu 106-222.
• SEQ ID NO: 101 is the light chain CDR3 for the 0X40 agonist monoclonal antibody Hu 106-222.
• SEQ ID NO: 102 is an 0X40 ligand (OX40L) amino acid sequence.
• SEQ ID NO: 103 is a soluble portion of OX40L polypeptide.
• SEQ ID NO: 104 is an alternative soluble portion of OX40L polypeptide.
• SEQ ID NO: 105 is the heavy chain variable region (VH) for the 0X40 agonist monoclonal antibody 008.
• SEQ ID NO: 106 is the light chain variable region (VL) for the 0X40 agonist monoclonal antibody 008.
• SEQ ID NO: 107 is the heavy chain variable region (VH) for the 0X40 agonist monoclonal antibody 011.
• SEQ ID NO: 108 is the light chain variable region (VL) for the 0X40 agonist monoclonal antibody 011.
• SEQ ID NO: 109 is the heavy chain variable region (VH) for the 0X40 agonist monoclonal antibody 021.
• SEQ ID NO: 110 is the light chain variable region (VL) for the 0X40 agonist monoclonal antibody 021.
• SEQ ID NO: 111 is the heavy chain variable region (VH) for the 0X40 agonist monoclonal antibody 023.
• SEQ ID NO: 112 is the light chain variable region (VL) for the 0X40 agonist monoclonal antibody 023.
• SEQ ID NO: 113 is the heavy chain variable region (VH) for an 0X40 agonist monoclonal antibody.
• SEQ ID NO: 114 is the light chain variable region (VL) for an 0X40 agonist monoclonal antibody.
• SEQ ID NO: 115 is the heavy chain variable region (VH) for an 0X40 agonist monoclonal antibody.
• SEQ ID NO: 116 is the light chain variable region (VL) for an 0X40 agonist monoclonal antibody.
• SEQ ID NO: 117 is the heavy chain variable region (VH) for a humanized 0X40 agonist monoclonal antibody.
• SEQ ID NO: 118 is the heavy chain variable region (VH) for a humanized 0X40 agonist monoclonal antibody.
• SEQ ID NO: 119 is the light chain variable region (VL) for a humanized 0X40 agonist monoclonal antibody.
• SEQ ID NO: 120 is the light chain variable region (VL) for a humanized 0X40 agonist monoclonal antibody.
• SEQ ID NO: 121 is the heavy chain variable region (VH) for a humanized 0X40 agonist monoclonal antibody.
• SEQ ID NO: 122 is the heavy chain variable region (VH) for a humanized 0X40 agonist monoclonal antibody.
• SEQ ID NO: 123 is the light chain variable region (VL) for a humanized 0X40 agonist monoclonal antibody.
• SEQ ID NO: 124 is the light chain variable region (VL) for a humanized 0X40 agonist monoclonal antibody.
• SEQ ID NO: 125 is the heavy chain variable region (VH) for an 0X40 agonist monoclonal antibody.
• SEQ ID NO: 126 is the light chain variable region (VL) for an 0X40 agonist monoclonal antibody.
• SEQ ID NO: 127 is the heavy chain amino acid sequence of the PD-l inhibitor nivolumab.
• SEQ ID NO: 128 is the light chain amino acid sequence of the PD-l inhibitor nivolumab.
• SEQ ID NO: 129 is the heavy chain variable region (VH) amino acid sequence of the PD-l inhibitor nivolumab.
• SEQ ID NO: 130 is the light chain variable region (VL) amino acid sequence of the PD-l inhibitor nivolumab.
• SEQ ID NO: 131 is the heavy chain CDR1 amino acid sequence of the PD-l inhibitor nivolumab.
• SEQ ID NO: 132 is the heavy chain CDR2 amino acid sequence of the PD-l inhibitor nivolumab.
• SEQ ID NO: 133 is the heavy chain CDR3 amino acid sequence of the PD-l inhibitor nivolumab.
• SEQ ID NO: 134 is the light chain CDR1 amino acid sequence of the PD-l inhibitor nivolumab.
• SEQ ID NO: 135 is the light chain CDR2 amino acid sequence of the PD-l inhibitor nivolumab.
• SEQ ID NO: 136 is the light chain CDR3 amino acid sequence of the PD-l inhibitor nivolumab.
• SEQ ID NO: 137 is the heavy chain amino acid sequence of the PD-l inhibitor pembrolizumab.
• SEQ ID NO: 138 is the light chain amino acid sequence of the PD-l inhibitor pembrolizumab.
• SEQ ID NO: 139 is the heavy chain variable region (VH) amino acid sequence of the PD-l inhibitor pembrolizumab.
• SEQ ID NO: 140 is the light chain variable region (VL) amino acid sequence of the PD-l inhibitor pembrolizumab.
• SEQ ID NO: 141 is the heavy chain CDR1 amino acid sequence of the PD-l inhibitor pembrolizumab.
• SEQ ID NO: 142 is the heavy chain CDR2 amino acid sequence of the PD-l inhibitor pembrolizumab.
• SEQ ID NO: 143 is the heavy chain CDR3 amino acid sequence of the PD-l inhibitor pembrolizumab.
• SEQ ID NO: 144 is the light chain CDR1 amino acid sequence of the PD-l inhibitor pembrolizumab.
• SEQ ID NO: 145 is the light chain CDR2 amino acid sequence of the PD-l inhibitor pembrolizumab.
• SEQ ID NO: 146 is the light chain CDR3 amino acid sequence of the PD-l inhibitor pembrolizumab.
• SEQ ID NO: 147 is the heavy chain amino acid sequence of the PD-L1 inhibitor durvalumab.
• SEQ ID NO: 148 is the light chain amino acid sequence of the PD-L1 inhibitor durvalumab.
• SEQ ID NO: 149 is the heavy chain variable region (VH) amino acid sequence of the PD-L1 inhibitor durvalumab.
• SEQ ID NO: 150 is the light chain variable region (VL) amino acid sequence of the PD-L1 inhibitor durvalumab.
• SEQ ID NO: l5 l is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.
• SEQ ID NO: 152 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.
• SEQ ID NO: 153 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.
• SEQ ID NO: 154 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.
• SEQ ID NO: 155 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.
• SEQ ID NO: 156 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.
• SEQ ID NO: 157 is the heavy chain amino acid sequence of the PD-L1 inhibitor avelumab.
• SEQ ID NO: 158 is the light chain amino acid sequence of the PD-L1 inhibitor avelumab.
• SEQ ID NO: 159 is the heavy chain variable region (VH) amino acid sequence of the PD-L1 inhibitor avelumab.
• SEQ ID NO: 160 is the light chain variable region (VL) amino acid sequence of the PD-L1 inhibitor avelumab.
• SEQ ID NO: 161 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.
• SEQ ID NO: 162 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.
• SEQ ID NO: 163 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.
• SEQ ID NO: 164 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.
• SEQ ID NO: 165 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.
• SEQ ID NO: 166 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.
• SEQ ID NO: 167 is the heavy chain amino acid sequence of the PD-L1 inhibitor atezolizumab.
• SEQ ID NO: 168 is the light chain amino acid sequence of the PD-L1 inhibitor atezolizumab.
• SEQ ID NO: 169 is the heavy chain variable region (VH) amino acid sequence of the PD-L1 inhibitor atezolizumab.
• SEQ ID NO: 170 is the light chain variable region (VL) amino acid sequence of the PD-L1 inhibitor atezolizumab.
• SEQ ID NO: 171 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.
• SEQ ID NO: 172 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.
• SEQ ID NO: 173 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.
• SEQ ID NO: 174 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.
• SEQ ID NO: 175 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.
• SEQ ID NO: 176 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.
• T cells undergo a profound metabolic shift during the course of their maturation from naive to effector T cells (see Chang, et al., Nat. Immunol. 2016, 17, 364, hereby expressly incorporated in its entirety, and in particular for the discussion and markers of anaerobic and aerobic metabolism).
• naive T cells rely on mitochondrial respiration to produce ATP
• mature, healthy effector T cells such as TIL are highly glycolytic, relying on aerobic glycolysis to provide the bioenergetics substrates they require for proliferation, migration, activation, and anti-tumor efficacy.
• co-administration encompass administration of two or more active pharmaceutical ingredients (in a preferred embodiment of the present invention, for example, a plurality of TILs) to a subject so that both active pharmaceutical ingredients and/or their metabolites are present in the subject at the same time.
• Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present. Simultaneous administration in separate compositions and administration in a composition in which both agents are present are preferred.
• in vivo refers to an event that takes place in a subject's body.
• in vitro' refers to an event that takes places outside of a subject's body.
• In vitro assays encompass cell-based assays in which cells alive or dead are employed and may also encompass a cell-free assay in which no intact cells are employed.
• ex v/vo refers to an event which involves treating or performing a procedure on a cell, tissue and/or organ which has been removed from a subject’s body. Aptly, the cell, tissue and/or organ may be returned to the subject’s body in a method of surgery or treatment.
• rapid expansion means an increase in the number of antigen- specific TILs of at least about 3-fold (or 4-, 5-, 6-, 7-, 8-, or 9-fold) over a period of a week, more preferably at least about lO-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold) over a period of a week, or most preferably at least about lOO-fold over a period of a week.
• lO-fold or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold
• TILs tumor infiltrating lymphocytes
• TILs include, but are not limited to, CD8 + cytotoxic T cells
• TILs include both primary and secondary TILs.
• Primary TILs are those that are obtained from patient tissue samples as outlined herein (sometimes referred to as“freshly harvested”)
• secondary TILs are any TIL cell populations that have been expanded or proliferated as discussed herein, including, but not limited to bulk TILs and expanded TILs (“REP TILs” or“post-REP TILs”).
• TIL cell populations can include genetically modified TILs.
• populations generally range from 1 X 10 6 to 1 X 10 10 in number, with different TIL populations comprising different numbers.
• initial growth of primary TILs in the presence of IL-2 results in a population of bulk TILs of roughly 1 x 10 8 cells.
• REP expansion is generally done to provide populations of 1.5 x 10 9 to 1.5 x 10 10 cells for infusion.
• cryopreserved TILs herein is meant that TILs, either primary, bulk, or expanded (REP TILs), are treated and stored in the range of about -l50°C to -60°C. General methods for cryopreservation are also described elsewhere herein, including in the Examples. For clarity,“cryopreserved TILs” are distinguishable from frozen tissue samples which may be used as a source of primary TILs.
• thawed cryopreserved TILs herein is meant a population of TILs that was previously cryopreserved and then treated to return to room temperature or higher, including but not limited to cell culture temperatures or temperatures wherein TILs may be
• TILs can generally be defined either biochemically, using cell surface markers, or functionally, by their ability to infiltrate tumors and effect treatment. TILs can be generally categorized by expressing one or more of the following biomarkers: CD4, CD8, TCR ab, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-l, and CD25. Additionally and
• TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into a patient.
• cryopreservation media or“cryopreservation medium” refers to any medium that can be used for cryopreservation of cells. Such media can include media comprising 7% to 10% DMSO. Exemplary media include CryoStor CS10, Hyperthermasol, as well as combinations thereof.
• the term“CS10” refers to a cryopreservation medium which is obtained from Stemcell Technologies or from Biolife Solutions. The CS10 medium may be referred to by the trade name“CryoStor® CS10”.
• the CS10 medium is a serum-free, animal component-free medium which comprises DMSO.
• central memory T cell refers to a subset of T cells that in the human are CD45R0+ and constitutively express CCR7 (CCR7 hl ) and CD62L (CD62 hl )
• the surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R.
• Transcription factors for central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1.
• Central memory T cells primarily secret IL-2 and CD40L as effector molecules after TCR triggering.
• Central memory T cells are predominant in the CD4 compartment in blood, and in the human are proportionally enriched in lymph nodes and tonsils.
• effector memory T cell refers to a subset of human or mammalian T cells that, like central memory T cells, are CD45R0+, but have lost the constitutive expression of CCR7 (CCR7 10 ) and are heterogeneous or low for CD62L expression
• the surface phenotype of central memory T cells also includes TCR, CD3,
• CD127 IL-7R
• IL-15R Transcription factors for central memory T cells include BLIMP 1. Effector memory T cells rapidly secret high levels of inflammatory cytokines following antigenic stimulation, including interferon-g, IL-4, and IL-5. Effector memory T cells are predominant in the CD8 compartment in blood, and in the human are proportionally enriched in the lung, liver, and gut. CD8+ effector memory T cells carry large amounts of perforin.
• closed system refers to a system that is closed to the outside
• Closed systems include, for example, but are not limited to closed G-containers. Once a tumor segment is added to the closed system, the system is no opened to the outside environment until the TILs are ready to be administered to the patient.
• fragmenting includes mechanical fragmentation methods such as crushing, slicing, dividing, and morcellating tumor tissue as well as any other method for disrupting the physical structure of tumor tissue.
• peripheral blood mononuclear cells refers to a peripheral blood cell having a round nucleus, including lymphocytes (T cells, B cells, NK cells) and monocytes.
• lymphocytes T cells, B cells, NK cells
• monocytes preferably, the peripheral blood mononuclear cells are irradiated allogeneic peripheral blood mononuclear cells.
• PBMCs are a type of antigen-presenting cell.
• anti-CD3 antibody refers to an antibody or variant thereof, e.g, a monoclonal antibody and including human, humanized, chimeric or murine antibodies which are directed against the CD3 receptor in the T cell antigen receptor of mature T cells.
• Anti- CD3 antibodies include OKT-3, also known as muromonab.
• Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3e.
• Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.
• the term“OKT-3” refers to a monoclonal antibody or biosimilar or variant thereof, including human, humanized, chimeric, or murine antibodies, directed against the CD3 receptor in the T cell antigen receptor of mature T cells, and includes commercially-available forms such as OKT-3 (30 ng/mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab or variants, conservative amino acid substitutions, glycoforms, or biosimilars thereof.
• the amino acid sequences of the heavy and light chains of muromonab are given in Table 1 (SEQ ID NO: 1 and SEQ ID NO:2).
• a hybridoma capable of producing OKT-3 is deposited with the American Type Culture Collection and assigned the ATCC accession number CRL 8001.
• a hybridoma capable of producing OKT-3 is also deposited with European Collection of Authenticated Cell Cultures (ECACC) and assigned Catalogue No. 86022706.
• IL-2 refers to the T cell growth factor known as interleukin-2, and includes all forms of IL-2 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof.
• IL-2 is described, e.g., in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated by reference herein.
• IL-2 encompasses human, recombinant forms of IL-2 such as aldesleukin (PROLEUKIN, available commercially from multiple suppliers in 22 million IU per single use vials), as well as the form of recombinant IL-2 commercially supplied by CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-209-b) and other commercial equivalents from other vendors.
• aldesleukin PROLEUKIN, available commercially from multiple suppliers in 22 million IU per single use vials
• CELLGRO GMP CellGenix, Inc.
• ProSpec-Tany TechnoGene Ltd. East Brunswick, NJ, USA
• Aldesleukin (des-alanyl-l, serine-l25 human IL- 2) is a nonglycosylated human recombinant form of IL-2 with a molecular weight of approximately 15 kDa.
• the amino acid sequence of aldesleukin suitable for use in the invention is given in Table 2 (SEQ ID NO:4).
• the term IL-2 also encompasses pegylated forms of IL-2, as described herein, including the pegylated IL2 prodrug NKTR-214, available from Nektar Therapeutics, South San Francisco, CA, USA. NKTR-214 and pegylated IL-2 suitable for use in the invention is described in U.S. Patent Application Publication No.
• IL-4 refers to the cytokine known as interleukin 4, which is produced by Th2 T cells and by eosinophils, basophils, and mast cells.
• IL-4 regulates the differentiation of naive helper T cells (ThO cells) to Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70.
• Th2 T cells Upon activation by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop.
• IL-4 also stimulates B cell proliferation and class II MHC expression, and induces class switching to IgE and IgGi expression from B cells.
• Recombinant human IL-4 suitable for use in the invention is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-211) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, Cat. No. Gibco CTP0043).
• the amino acid sequence of recombinant human IL-4 suitable for use in the invention is given in Table 2 (SEQ ID NO:5).
• IL-7 refers to a glycosylated tissue- derived cytokine known as interleukin 7, which may be obtained from stromal and epithelial cells, as well as from dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate the development of T cells. IL-7 binds to the IL-7 receptor, a heterodimer consisting of IL-7 receptor alpha and common gamma chain receptor, which in a series of signals important for T cell development within the thymus and survival within the periphery.
• Recombinant human IL-7 suitable for use in the invention is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, Cat. No. Gibco PHC0071).
• the amino acid sequence of recombinant human IL-7 suitable for use in the invention is given in Table 2 (SEQ ID NO:6).
• IL-15 refers to the T cell growth factor known as interleukin- 15, and includes all forms of IL-2 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof.
• IL-15 is described, e.g ., in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated by reference herein.
• IL-15 shares b and g signaling receptor subunits with IL-2.
• Recombinant human IL-15 is a single, non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular mass of 12.8 kDa.
• Recombinant human IL-15 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, Cat. No. 34-8159-82).
• the amino acid sequence of recombinant human IL-15 suitable for use in the invention is given in Table 2 (SEQ ID NO:7).
• IL-21 refers to the pleiotropic cytokine protein known as interleukin-21, and includes all forms of IL-21 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-21 is described, e.g. , in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95, the disclosure of which is incorporated by reference herein. IL-21 is primarily produced by natural killer T cells and activated human CD4 + T cells.
• Recombinant human IL- 21 is a single, non-glycosylated polypeptide chain containing 132 amino acids with a molecular mass of 15.4 kDa.
• Recombinant human IL-21 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-21 recombinant protein, Cat. No. 14-8219-80).
• the amino acid sequence of recombinant human IL-21 suitable for use in the invention is given in Table 2 (SEQ ID NO:8).
• compositions of the present invention can be administered by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the tumor infiltrating lymphocytes (e.g.
• secondary TILs or genetically modified cytotoxic lymphocytes described herein may be administered at a dosage of 10 4 to 10 11 cells/kg body weight (e.g., 10 5 to 10 6 , 10 5 to 10 10 , 10 5 to 10 11 , l0 6 to 10 10 , l0 6 to l0 u , l0 7 to 10 11 , 10 7 to 10 10 , 10 8 to 10 11 , 10 8 to 10 10 , 10 9 to 10 11 , or 10 9 to 10 10 cells/kg body weight), including all integer values within those ranges.
• Tumor infiltrating lymphocytes including in some cases, genetically modified cytotoxic lymphocytes
• compositions may also be administered multiple times at these dosages.
• the tumor infiltrating lymphocytes can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et ah, New Eng. J. of Med. 319: 1676, 1988).
• the optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.
• microenvironment may refer to the solid or
• the tumor microenvironment refers to a complex mixture of“cells, soluble factors, signaling molecules, extracellular matrices, and mechanical cues that promote neoplastic transformation, support tumor growth and invasion, protect the tumor from host immunity, foster therapeutic resistance, and provide niches for dominant metastases to thrive,” as described in Swartz, et al, Cancer Res., 2012, 72, 2473.
• tumors express antigens that should be recognized by T cells, tumor clearance by the immune system is rare because of immune suppression by the microenvironment.
• the invention includes a method of treating a cancer with a population of TILs, wherein a patient is pre-treated with non-myeloablative chemotherapy prior to an infusion of TILs according to the invention.
• the population of TILs may be provided wherein a patient is pre-treated with nonmyeloablative
• the non-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/d for 2 days (days 27 and 26 prior to TIL infusion) and fludarabine 25 mg/m2/d for 5 days (days 27 to 23 prior to TIL infusion).
• the patient receives an intravenous infusion of IL-2 intravenously at 720,000 IU/kg every 8 hours to physiologic tolerance.
• some embodiments of the invention utilize a lymphodepletion step (sometimes also referred to as“immunosuppressive conditioning”) on the patient prior to the introduction of the rTILs of the invention.
• a lymphodepletion step sometimes also referred to as“immunosuppressive conditioning”
• an effective amount or“therapeutically effective amount” refers to that amount of a compound or combination of compounds as described herein that is sufficient to effect the intended application including, but not limited to, disease treatment.
• therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated (e.g., the weight, age and gender of the subject), the severity of the disease condition, or the manner of administration.
• the term also applies to a dose that will induce a particular response in target cells (e.g., the reduction of platelet adhesion and/or cell migration).
• the specific dose will vary depending on the particular compounds chosen, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which the compound is carried.
• treatment refers to obtaining a desired pharmacologic and/or physiologic effect.
• the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
• “Treatment”, as used herein covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it;
• Treatment encompasses delivery of a composition that can elicit an immune response or confer immunity in the absence of a disease condition, e.g., in the case of a vaccine.
• nucleic acid or protein when used with reference to portions of a nucleic acid or protein indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship to each other in nature.
• the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source, or coding regions from different sources.
• a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
• sequence identity refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity.
• the percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. Suitable programs to determine percent sequence identity include for example the BLAST suite of programs available from the U.S. Government’s National Center for Biotechnology Information BLAST web site.
• Comparisons between two sequences can be carried using either the BLASTN or BLASTP algorithm.
• BLASTN is used to compare nucleic acid sequences
• BLASTP is used to compare amino acid sequences.
• ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or MegAlign, available from DNASTAR, are additional publicly available software programs that can be used to align sequences.
• One skilled in the art can determine appropriate parameters for maximal alignment by particular alignment software. In certain embodiments, the default parameters of the alignment software are used.
• the term“variant” encompasses but is not limited to antibodies or fusion proteins which comprise an amino acid sequence which differs from the amino acid sequence of a reference antibody by way of one or more substitutions, deletions and/or additions at certain positions within or adjacent to the amino acid sequence of the reference antibody.
• the variant may comprise one or more conservative substitutions in its amino acid sequence as compared to the amino acid sequence of a reference antibody. Conservative substitutions may involve, e.g., the substitution of similarly charged or uncharged amino acids.
• the variant retains the ability to specifically bind to the antigen of the reference antibody.
• the term variant also includes pegylated antibodies or proteins.
• TILs tumor infiltrating lymphocytes
• TILs include, but are not limited to, CD8 + cytotoxic T cells
• TILs include both primary and secondary TILs.“Primary TILs” are those that are obtained from patient tissue samples as outlined herein (sometimes referred to as“freshly harvested”), and“secondary TILs” are any TIL cell populations that have been expanded or proliferated as discussed herein, including, but not limited to bulk TILs, expanded TILs (“REP TILs”) as well as“reREP TILs” as discussed herein.
• reREP TILs can include for example second expansion TILs or second additional expansion TILs (such as, for example, those described in Step D of Figure 8, including TILs referred to as reREP TILs).
• TILs can generally be defined either biochemically, using cell surface markers, or functionally, by their ability to infiltrate tumors and effect treatment. TILs can be generally categorized by expressing one or more of the following biomarkers: CD4, CD8, TCR ab, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-l, and CD25. Additionally, and
• TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into a patient.
• TILS may further be characterized by potency - for example, TILS may be considered potent if, for example, interferon (IFNy) release is greater than about 50 pg/mL, greater than about 100 pg/mL, greater than about 150 pg/mL, or greater than about 200 pg/mL, greater than about 300 pg/mL, greater than about 400 pg/mL, greater than about 500 pg/mL, greater than about 600 pg/mL, greater than about 700 pg/mL, greater than about 800 pg/mL, greater than about 900 pg/mL, greater than about 1000 pg/mL.
• IFNy interferon
• deoxy ribonucleotide encompasses natural and synthetic, unmodified and modified deoxyribonucleotides. Modifications include changes to the sugar moiety, to the base moiety and/or to the linkages between deoxyribonucleotide in the oligonucleotide.
• RNA defines a molecule comprising at least one ribonucleotide residue.
• ribonucleotide defines a nucleotide with a hydroxyl group at the 2' position of a b-D-ribofuranose moiety.
• RNA includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides.
• Nucleotides of the RNA molecules described herein may also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.
• pharmaceutically acceptable carrier or“pharmaceutically acceptable excipient” are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art.
• any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in therapeutic compositions of the invention is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the described compositions and methods.
• the terms“about” and“approximately” mean within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, more preferably still within 10%, and even more preferably within 5% of a given value or range.
• the allowable variation encompassed by the terms “about” or“approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art.
• the terms“about” and“approximately” mean that dimensions, sizes, formulations, parameters, shapes and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art.
• a dimension, size, formulation, parameter, shape or other quantity or characteristic is “about” or“approximate” whether or not expressly stated to be such. It is noted that embodiments of very different sizes, shapes and dimensions may employ the described arrangements.
• transitional terms“comprising,”“consisting essentially of,” and“consisting of,” when used in the appended claims, in original and amended form, define the claim scope with respect to what unrecited additional claim elements or steps, if any, are excluded from the scope of the claim(s).
• the term“comprising” is intended to be inclusive or open-ended and does not exclude any additional, unrecited element, method, step or material.
• compositions, methods, and kits described herein that embody the present invention can, in alternate embodiments, be more specifically defined by any of the transitional terms“comprising,”“consisting essentially of,” and“consisting of.”
• the terms“antibody” and its plural form“antibodies” refer to whole immunoglobulins and any antigen-binding fragment (“antigen-binding portion”) or single chains thereof.
• An“antibody” further refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding portion thereof.
• Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
• the heavy chain constant region is comprised of three domains, CH1, CH2 and CH3.
• Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
• the light chain constant region is comprised of one domain, CL.
• the VH and VL regions of an antibody may be further subdivided into regions of hypervariability, which are referred to as complementarity determining regions (CDR) or hypervariable regions (HVR), and which can be interspersed with regions that are more conserved, termed framework regions (FR).
• CDR complementarity determining regions
• HVR hypervariable regions
• FR framework regions
• Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
• the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen epitope or epitopes.
• the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g ., effector cells) and the first component (Cl
• an antigen refers to a substance that induces an immune response.
• an antigen is a molecule capable of being bound by an antibody or a TCR if presented by major histocompatibility complex (MHC) molecules.
• MHC major histocompatibility complex
• the term “antigen”, as used herein, also encompasses T cell epitopes.
• An antigen is additionally capable of being recognized by the immune system.
• an antigen is capable of inducing a humoral immune response or a cellular immune response leading to the activation of B lymphocytes and/or T lymphocytes. In some cases, this may require that the antigen contains or is linked to a Th cell epitope.
• An antigen can also have one or more epitopes (e.g., B- and T-epitopes).
• an antigen will preferably react, typically in a highly specific and selective manner, with its corresponding antibody or TCR and not with the multitude of other antibodies or TCRs which may be induced by other antigens.
• the terms“monoclonal antibody,”“mAb,”“monoclonal antibody composition,” or their plural forms refer to a preparation of antibody molecules of single molecular composition.
• a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
• Monoclonal antibodies specific to certain receptors can be made using knowledge and skill in the art of injecting test subjects with suitable antigen and then isolating hybridomas expressing antibodies having the desired sequence or functional characteristics.
• DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g ., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies).
• the hybridoma cells serve as a preferred source of such DNA.
• the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Recombinant production of antibodies will be described in more detail below.
• host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein
• antigen-binding portion or“antigen-binding fragment” of an antibody (or simply“antibody portion” or“fragment”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
• binding fragments encompassed within the term“antigen binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a domain antibody (dAb) fragment (Ward, et al, Nature, 1989, 341, 544-546), which may consist of a VH or a VL domain; and (vi) an isolated complementarity determining region (CDR).
• a Fab fragment a monovalent fragment consisting of the VL, VH, CL and CH1 domains
• a F(ab')2 fragment a bivalent fragment comprising two Fab
• the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules known as single chain Fv (scFv); see, e.g., Bird, et al., Science 1988, 242, 423-426; and Huston, et al., Proc. Natl. Acad. Sci. USA 1988, 85, 5879-5883).
• scFv antibodies are also intended to be encompassed within the terms“antigen-binding portion” or“antigen-binding fragment” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
• human antibody is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences.
• the human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences ( e.g ., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
• human antibody is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
• human monoclonal antibody refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences.
• the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.
• recombinant human antibody includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (such as a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies isolated from a host cell transformed to express the human antibody, e.g, from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences.
• Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences.
• such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
• isotype refers to the antibody class (e.g., IgM or IgGl) that is encoded by the heavy chain constant region genes.
• phrases“an antibody recognizing an antigen” and“an antibody specific for an antigen” are used interchangeably herein with the term“an antibody which binds specifically to an antigen.”
• the term“human antibody derivatives” refers to any modified form of the human antibody, including a conjugate of the antibody and another active pharmaceutical ingredient or antibody.
• the terms“conjugate,”“antibody-drug conjugate”,“ADC,” or “immunoconjugate” refers to an antibody, or a fragment thereof, conjugated to another therapeutic moiety, which can be conjugated to antibodies described herein using methods available in the art.
• humanized antibody “humanized antibodies,” and“humanized” are intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.
• Humanized forms of non-human (for example, murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human
• humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a 15 hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
• donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
• humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
• the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
• the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
• Fc immunoglobulin constant region
• the antibodies described herein may also be modified to employ any Fc variant which is known to impart an improvement (e.g, reduction) in effector function and/or FcR binding.
• the Fc variants may include, for example, any one of the amino acid substitutions disclosed in International Patent Application Publication Nos.
• chimeric antibody is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.
• A“diabody” is a small antibody fragment with two antigen-binding sites.
• the fragments comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH).
• VH heavy chain variable domain
• VL light chain variable domain
• VH-VL or VL-VH linker that is too short to allow pairing between the two domains on the same chain
• the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
• Diabodies are described more fully in, e.g ., European Patent No. EP 404,097, International Patent Publication No. WO 93/11161; and Bolliger, et al. , Proc. Natl. Acad. Sci. USA 1993, 90, 6444-6448.
• glycosylation refers to a modified derivative of an antibody.
• An aglycoslated antibody lacks glycosylation.
• Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen.
• Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
• Aglycosylation may increase the affinity of the antibody for antigen, as described in U.S. Patent Nos. 5,714,350 and 6,350,861.
• an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures.
• altered glycosylation patterns have been demonstrated to increase the ability of antibodies.
• carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered
• the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (alpha (1,6) fucosyltransferase), such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates.
• the Ms704, Ms705, and Ms709 FUT8-/- cell lines were created by the targeted disruption of the FUT8 gene in CHO/DG44 cells using two replacement vectors (see e.g. U.S.
• European Patent No. EP 1,176,195 describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit
• hypofucosylation by reducing or eliminating the alpha 1,6 bond-related enzyme, and also describes cell lines which have a low enzyme activity for adding fucose to the N- acetyl glucosamine that binds to the Fc region of the antibody or does not have the enzyme activity, for example the rat myeloma cell line YB2/0 (ATCC CRL 1662).
• International Patent Publication WO 03/035835 describes a variant CHO cell line, Lee 13 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, et al, ./. Biol. Chem. 2002, 277, 26733-26740.
• WO 99/54342 describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., beta(l,4)- N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana, et al. , Nat. Biotech. 1999, 77, 176-180).
• the fucose residues of the antibody may be cleaved off using a fucosidase enzyme.
• the fucosidase alpha-L-fucosidase removes fucosyl residues from antibodies as described in Tarentino, et al, Biochem. 1975, 14, 5516-5523.
• PEG polyethylene glycol
• Pegylation refers to a modified antibody, or a fragment thereof, that typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment.
• PEG polyethylene glycol
• Pegylation may, for example, increase the biological (e.g., serum) half-life of the antibody.
• the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer).
• polyethylene glycol is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (Ci-Cio)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide.
• the antibody to be pegylated may be an aglycosylated antibody. Methods for pegylation are known in the art and can be applied to the antibodies of the invention, as described for example in European Patent Nos. EP 0154316 and EP 0401384 and ET.S. Patent No.
• biosimilar means a biological product, including a monoclonal antibody or protein, that is highly similar to a ET.S. licensed reference biological product notwithstanding minor differences in clinically inactive components, and for which there are no clinically meaningful differences between the biological product and the reference product in terms of the safety, purity, and potency of the product.
• a similar biological or “biosimilar” medicine is a biological medicine that is similar to another biological medicine that has already been authorized for use by the European Medicines Agency.
• biosimilar is also used synonymously by other national and regional regulatory agencies.
• Biological products or biological medicines are medicines that are made by or derived from a biological source, such as a bacterium or yeast. They can consist of relatively small molecules such as human insulin or erythropoietin, or complex molecules such as
• IL-2 protein is aldesleukin
• PROLEEIKIN a protein approved by drug regulatory authorities with reference to aldesleukin is a“biosimilar to” aldesleukin or is a“biosimilar thereof’ of aldesleukin.
• a similar biological or“biosimilar” medicine is a biological medicine that is similar to another biological medicine that has already been authorized for use by the European Medicines Agency (EMA).
• EMA European Medicines Agency
• the relevant legal basis for similar biological applications in Europe is Article 6 of Regulation (EC) No 726/2004 and Article 10(4) of Directive 2001/83/EC, as amended and therefore in Europe, the biosimilar may be authorized, approved for authorization or subject of an application for authorization under Article 6 of Regulation (EC) No 726/2004 and Article 10(4) of Directive 2001/83/EC.
• the already authorized original biological medicinal product may be referred to as a“reference medicinal product” in Europe.
• Some of the requirements for a product to be considered a biosimilar are outlined in the CHMP Guideline on Similar Biological Medicinal Products.
• product specific guidelines including guidelines relating to monoclonal antibody biosimilars, are provided on a product-by-product basis by the EMA and published on its website.
• a biosimilar as described herein may be similar to the reference medicinal product by way of quality characteristics, biological activity, mechanism of action, safety profiles and/or efficacy.
• the biosimilar may be used or be intended for use to treat the same conditions as the reference medicinal product.
• a biosimilar as described herein may be deemed to have similar or highly similar quality characteristics to a reference medicinal product.
• a biosimilar as described herein may be deemed to have similar or highly similar biological activity to a reference medicinal product.
• a biosimilar as described herein may be deemed to have a similar or highly similar safety profile to a reference medicinal product.
• a biosimilar as described herein may be deemed to have similar or highly similar efficacy to a reference medicinal product.
• a biosimilar in Europe is compared to a reference medicinal product which has been authorized by the EMA.
• the biosimilar may be compared to a biological medicinal product which has been authorized outside the European Economic Area (a non-EEA authorized“comparator”) in certain studies. Such studies include for example certain clinical and in vivo non-clinical studies.
• a biological medicinal product which has been authorized outside the European Economic Area (a non-EEA authorized“comparator”) in certain studies. Such studies include for example certain clinical and in vivo non-clinical studies.
• the term“biosimilar” also relates to a biological medicinal product which has been or may be compared to a non-EEA authorized comparator.
• Certain biosimilars are proteins such as antibodies, antibody fragments (for example, antigen binding portions) and fusion proteins.
• a protein biosimilar may have an amino acid sequence that has minor modifications in the amino acid structure (including for example deletions, additions, and/or substitutions of amino acids) which do not significantly affect the function of the polypeptide.
• the biosimilar may comprise an amino acid sequence having a sequence identity of 97% or greater to the amino acid sequence of its reference medicinal product, e.g., 97%, 98%, 99% or 100%.
• the biosimilar may comprise one or more post-translational modifications, for example, although not limited to, glycosylation, oxidation, deamidation, and/or truncation which is/are different to the post-translational modifications of the reference medicinal product, provided that the differences do not result in a change in safety and/or efficacy of the medicinal product.
• the biosimilar may have an identical or different glycosylation pattern to the reference medicinal product.
• the biosimilar may have a different glycosylation pattern if the differences address or are intended to address safety concerns associated with the reference medicinal product. Additionally, the biosimilar may deviate from the reference medicinal product in for example its strength, pharmaceutical form, formulation, excipients and/or presentation, providing safety and efficacy of the medicinal product is not compromised.
• the biosimilar may comprise differences in for example pharmacokinetic (PK) and/or
• biosimilar exhibits different binding characteristics as compared to the reference medicinal product, wherein the different binding characteristics are considered by a Regulatory Authority such as the EMA not to be a barrier for authorization as a similar biological product.
• Regulatory Authority such as the EMA not to be a barrier for authorization as a similar biological product.
• biosimilar is also used synonymously by other national and regional regulatory agencies.
• process 2A An exemplary TIL process known as process 2A containing some of these features is depicted in Figure 2, and some of the advantages of this embodiment of the present invention over process 1C are described in Figures F and G. An embodiment of process 2A is shown Figure 1.
• the present invention can include a step relating to the restimulation of cryopreserved TILs to increase their metabolic activity and thus relative health prior to transplant into a patient, and methods of testing said metabolic health.
• TILs are generally taken from a patient sample and manipulated to expand their number prior to transplant into a patient.
• the TILs may be optionally genetically manipulated as discussed below.
• the TILs may be cryopreserved. Once thawed, they may also be restimulated to increase their metabolism prior to infusion into a patient.
• the first expansion (including processes referred to as the preREP as well as processes shown in Figure 1 as Step A) is shortened to 3 to 14 days and the second expansion (including processes referred to as the REP as well as processes shown in Figure 1 as Step B) is shorted to 7 to 14 days, as discussed in detail below as well as in the examples and figures.
• the first expansion (for example, an expansion described as Step B in Figure 1) is shortened to 11 days and the second expansion (for example, an expansion as described in Step D in Figure 1) is shortened to 11 days.
• the combination of the first expansion and second expansion (for example, expansions described as Step B and Step D in Figure 1) is shortened to 22 days, as discussed in detail below and in the examples and figures.
• TILs are initially obtained from a patient tumor sample (“primary TILs”) and then expanded into a larger population for further manipulation as described herein, optionally cryopreserved, restimulated as outlined herein and optionally evaluated for phenotype and metabolic parameters as an indication of TIL health.
• a patient tumor sample may be obtained using methods known in the art, generally via surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells.
• multilesional sampling is used.
• surgical resection, needle biopsy, core biopsy, small biopsy, or other means for obtaining a sample that contains a mixture of tumor and TIL cells includes multilesional sampling (z.e., obtaining samples from one or more tumor cites and/or locations in the patient, as well as one or more tumors in the same location or in close proximity).
• the tumor sample may be from any solid tumor, including primary tumors, invasive tumors or metastatic tumors.
• the tumor sample may also be a liquid tumor, such as a tumor obtained from a hematological malignancy.
• the solid tumor may be of lung tissue.
• useful TILs are obtained from non-small cell lung carcinoma (NSCLC).
• the tumor sample is generally fragmented using sharp dissection into small pieces of between 1 to about 8 mm 3 , with from about 2-3 mm 3 being particularly useful.
• the TILs are cultured from these fragments using enzymatic tumor digests.
• Such tumor digests may be produced by incubation in enzymatic media (e.g ., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg/mL gentamicine, 30 units/mL of DNase and 1.0 mg/mL of collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator).
• enzymatic media e.g ., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg/mL gentamicine, 30 units/mL of DNase and 1.0 mg/mL of collagenase
• Tumor digests may be produced by placing the tumor in enzymatic media and mechanically dissociating the tumor for approximately 1 minute, followed by incubation for 30 minutes at 37 °C in 5% CO2, followed by repeated cycles of mechanical dissociation and incubation under the foregoing conditions until only small tissue pieces are present.
• a density gradient separation using FICOLL branched hydrophilic polysaccharide may be performed to remove these cells.
• Alternative methods known in the art may be used, such as those described in U.S. Patent Application Publication No. 2012/0244133 Al, the disclosure of which is incorporated by reference herein. Any of the foregoing methods may be used in any of the embodiments described herein for methods of expanding TILs or methods treating a cancer.
• the harvested cell suspension is called a“primary cell population” or a “freshly harvested” cell population.
• fragmentation includes physical fragmentation, including for example, dissection as well as digestion.
• the fragmentation is physical fragmentation.
• the fragmentation is dissection.
• the fragmentation is by digestion.
• TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from patients.
• TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from patients.
• the tumor undergoes physical fragmentation after the tumor sample is obtained in, for example, Step A (as provided in Figure 1). In some embodiments, the fragmentation occurs before
• cryopreservation occurs after cryopreservation. In some embodiments, the fragmentation occurs after obtaining the tumor and in the absence of any cryopreservation. In some embodiments, the tumor is fragmented and 10, 20, 30, 40 or more fragments or pieces are placed in each container for the first expansion. In some embodiments, the tumor is fragmented and 30 or 40 fragments or pieces are placed in each container for the first expansion. In some embodiments, the tumor is fragmented and 40 fragments or pieces are placed in each container for the first expansion. In some embodiments, the multiple fragments comprise about 4 to about 50 fragments, wherein each fragment has a volume of about 27 mm 3 .
• the multiple fragments comprise about 30 to about 60 fragments with a total volume of about 1300 mm 3 to about 1500 mm 3 . In some embodiments, the multiple fragments comprise about 50 fragments with a total volume of about 1350 mm 3 . In some embodiments, the multiple fragments comprise about 50 fragments with a total mass of about 1 gram to about 1.5 grams. In some
• the multiple fragments comprise about 4 fragments.
• the TILs are obtained from tumor fragments.
• the tumor fragment is obtained by sharp dissection.
• the tumor fragment is between about 1 mm 3 and 10 mm 3 .
• the tumor fragment is between about 1 mm 3 and 8 mm 3 .
• the tumor fragment is about 1 mm 3 .
• the tumor fragment is about 2 mm 3 .
• the tumor fragment is about 3 mm 3 . In some embodiments, the tumor fragment is about 4 mm 3 . In some embodiments, the tumor fragment is about 5 mm 3 . In some embodiments, the tumor fragment is about 6 mm 3 . In some embodiments, the tumor fragment is about 7 mm 3 . In some embodiments, the tumor fragment is about 8 mm 3 . In some embodiments, the tumor fragment is about 9 mm 3 . In some embodiments, the tumor fragment is about 10 mm 3 . In some embodiments, the tumors are 1-4 mmx 1-4 mm 1-4 mm. In some embodiments, the tumors are 1 mmx 1 mm x 1 mm.
• the tumors are 2 mm x 2 mm x 2 mm. In some embodiments, the tumors are 3 mm x 3 mm x 3 mm. In some embodiments, the tumors are 4 mmx 4 mm x 4 mm.
• the tumors are resected in order to minimize the amount of hemorrhagic, necrotic, and/or fatty tissues on each piece. In some embodiments, the tumors are resected in order to minimize the amount of hemorrhagic tissue on each piece. In some embodiments, the tumors are resected in order to minimize the amount of necrotic tissue on each piece. In some embodiments, the tumors are resected in order to minimize the amount of fatty tissue on each piece.
• the tumor fragmentation is performed in order to maintain the tumor internal structure. In some embodiments, the tumor fragmentation is performed without preforming a sawing motion with a scalpel.
• the TILs are obtained from tumor digests. In some embodiments, tumor digests were generated by incubation in enzyme media, for example but not limited to RPMI 1640, 2 mM GlutaMAX, 10 mg/mL gentamicin, 30 U/mL DNase, and 1.0 mg/mL collagenase, followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in enzyme media, the tumor can be mechanically dissociated for approximately 1 minute.
• enzyme media for example but not limited to RPMI 1640, 2 mM GlutaMAX, 10 mg/mL gentamicin, 30 U/mL DNase, and 1.0 mg/mL collagenase, followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor
• the solution can then be incubated for 30 minutes at 37 °C in 5% CO2 and it then mechanically disrupted again for approximately 1 minute. After being incubated again for 30 minutes at 37 °C in 5% CO2, the tumor can be mechanically disrupted a third time for approximately 1 minute.
• 1 or 2 additional mechanical dissociations were applied to the sample, with or without 30 additional minutes of incubation at 37 °C in 5% CO2.
• a density gradient separation using Ficoll can be performed to remove these cells.
• the harvested cell suspension prior to the first expansion step is called a“primary cell population” or a“freshly harvested” cell population.
• cells can be optionally frozen after sample harvest and stored frozen prior to entry into the expansion described in Step B, which is described in further detail below, as well as exemplified in Figure 1.
• the present methods provide for obtaining young TILs, which are capable of increased replication cycles upon administration to a subject/patient and as such may provide additional therapeutic benefits over older TILs (i.e., TILs which have further undergone more rounds of replication prior to administration to a subject/patient).
• the diverse antigen receptors of T and B lymphocytes are produced by somatic recombination of a limited, but large number of gene segments. These gene segments: V (variable), D (diversity), J (joining), and C (constant), determine the binding specificity and downstream applications of immunoglobulins and T-cell receptors (TCRs).
• the present invention provides a method for generating TILs which exhibit and increase the T-cell repertoire diversity. In some embodiments, the TILs obtained by the present method exhibit an increase in the T-cell repertoire diversity.
• the TILs obtained by the present method exhibit an increase in the T-cell repertoire diversity as compared to freshly harvested TILs and/or TILs prepared using other methods than those provide herein including for example, methods other than those embodied in Figure 1.
• the TILs obtained by the present method exhibit an increase in the T-cell repertoire diversity as compared to freshly harvested TILs and/or TILs prepared using methods referred to as process 1C, as exemplified in Figure 5 and/or Figure 6.
• the TILs obtained in the first expansion exhibit an increase in the T-cell repertoire diversity.
• the increase in diversity is an increase in the immunoglobulin diversity and/or the T-cell receptor diversity.
• the diversity is in the immunoglobulin is in the immunoglobulin heavy chain.
• the diversity is in the immunoglobulin is in the immunoglobulin light chain.
• the diversity is in the T-cell receptor.
• the diversity is in one of the T-cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors.
• TCR T-cell receptor
• TCR T-cell receptor
• beta T-cell receptor
• TCRab TCRab
• the resulting cells are cultured in serum containing IL-2 under conditions that favor the growth of TILs over tumor and other cells.
• the tumor digests are incubated in 2 mL wells in media comprising inactivated human AB serum with 6000 IU/mL of IL-2.
• This primary cell population is cultured for a period of days, generally from 3 to 14 days, resulting in a bulk TIL population, generally about 1 x 10 8 bulk TIL cells.
• this primary cell population is cultured for a period of 7 to 14 days, resulting in a bulk TIL population, generally about 1 c 10 8 bulk TIL cells.
• this primary cell population is cultured for a period of 10 to 14 days, resulting in a bulk TIL population, generally about 1 c 10 8 bulk TIL cells. In some embodiments, this primary cell population is cultured for a period of about 11 days, resulting in a bulk TIL population, generally about 1 c 10 8 bulk TIL cells.
• expansion of TILs may be performed using an initial bulk TIL expansion step (for example such as those described in Step B of Figure 1, which can include processes referred to as pre-REP) as described below and herein, followed by a second expansion (Step D, including processes referred to as rapid expansion protocol (REP) steps) as described below under Step D and herein, followed by optional cryopreservation, and followed by a second Step D (including processes referred to as restimulation REP steps) as described below and herein.
• the TILs obtained from this process may be optionally characterized for phenotypic characteristics and metabolic parameters as described herein.
• each well can be seeded with 1 c 10 6 tumor digest cells or one tumor fragment in 2 mL of complete medium (CM) with IL-2 (6000 IU/mL; Chiron Corp., Emeryville, CA).
• CM complete medium
• IL-2 6000 IU/mL
• the tumor fragment is between about 1 mm 3 and 10 mm 3 .
• the first expansion culture medium is referred to as“CM”, an abbreviation for culture media.
• CM for Step B consists of RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg/mL gentamicin.
• gas-permeable flasks with a 40 mL capacity and a 10 cm 2 gas-permeable silicon bottom (for example, G-RexlO; Wilson Wolf Manufacturing, New Brighton, MN) (Fig. 1)
• each flask was loaded with 10-40 x 10 6 viable tumor digest cells or 5-30 tumor fragments in 10-40 mL of CM with IL-2.
• Both the G- RexlO and 24-well plates were incubated in a humidified incubator at 37°C in 5% CO2 and 5 days after culture initiation, half the media was removed and replaced with fresh CM and IL- 2 and after day 5, half the media was changed every 2-3 days.
• the resulting cells are cultured in serum containing IL-2 under conditions that favor the growth of TILs over tumor and other cells.
• the tumor digests are incubated in 2 mL wells in media comprising inactivated human AB serum (or, in some cases, as outlined herein, in the presence of aAPC cell population) with 6000 IU/mL of IL-2.
• This primary cell population is cultured for a period of days, generally from 10 to 14 days, resulting in a bulk TIL
• the growth media during the first expansion comprises IL-2 or a variant thereof.
• the IL is recombinant human IL-2 (rhIL-2).
• the IL-2 stock solution has a specific activity of 20-30x 10 6 IU/mg for a 1 mg vial.
• the IL-2 stock solution has a specific activity of 20 x 10 6 IU/mg for a 1 mg vial.
• the IL-2 stock solution has a specific activity of 25 x 10 6 IU/mg for a 1 mg vial.
• the IL-2 stock solution has a specific activity of 30x 10 6 IU/mg for a 1 mg vial. In some embodiments, the IL- 2 stock solution has a final concentration of 4-8 x lO 6 IU/mg of IL-2. In some embodiments, the IL- 2 stock solution has a final concentration of 5-7x l0 6 IU/mg of IL-2. In some embodiments, the IL- 2 stock solution has a final concentration of 6 c 10 6 IU/mg of IL-2. In some embodiments, the IL-2 stock solution is prepare as described in Example 5.
• the first expansion culture media comprises about 10,000 IU/mL of IL-2, about 9,000 IU/mL of IL-2, about 8,000 IU/mL of IL-2, about 7,000 IU/mL of IL-2, about 6000 IU/mL of IL-2 or about 5,000 IU/mL of IL-2.
• the first expansion culture media comprises about 9,000 IU/mL of IL-2 to about 5,000 IU/mL of IL-2. In some embodiments, the first expansion culture media comprises about 8,000 IU/mL of IL-2 to about 6,000 IU/mL of IL-2. In some embodiments, the first expansion culture media comprises about 7,000 IU/mL of IL-2 to about 6,000 IU/mL of IL-2. In some embodiments, the first expansion culture media comprises about 6,000 IU/mL of IL-2. In an embodiment, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU/mL of IL-2. In an embodiment, the cell culture medium further comprises IL-2.
• the cell culture medium comprises about 3000 IU/mL of IL-2.
• the cell culture medium comprises about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL of IL-2.
• the cell culture medium comprises between 1000 and 2000 IU/mL, between 2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000 IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between 7000 and 8000 IU/mL, or about 8000 IU/mL of IL-2.
• first expansion culture media comprises about 500 IU/mL of IL-15, about 400 IU/mL of IL-15, about 300 IU/mL of IL-15, about 200 IU/mL of IL-15, about 180 IU/mL of IL-15, about 160 IU/mL of IL-15, about 140 IU/mL of IL-15, about 120 IU/mL of IL-15, or about 100 IU/mL of IL-15.
• the first expansion culture media comprises about 500 IU/mL of IL-15 to about 100 IU/mL of IL-15.
• the first expansion culture media comprises about 400 IU/mL of IL-15 to about 100 IU/mL of IL-15. In some embodiments, the first expansion culture media comprises about 300 IU/mL of IL-15 to about 100 IU/mL of IL-15. In some embodiments, the first expansion culture media comprises about 200 IU/mL of IL-15. In some embodiments, the cell culture medium comprises about 180 IU/mL of IL-15. In an embodiment, the cell culture medium further comprises IL-15. In a preferred embodiment, the cell culture medium comprises about 180 IU/mL of IL-15.
• first expansion culture media comprises about 20 IU/mL of IL-21, about 15 IU/mL of IL-21, about 12 IU/mL of IL-21, about 10 IU/mL of IL-21, about 5 IU/mL of IL-21, about 4 IU/mL of IL-21, about 3 IU/mL of IL-21, about 2 IU/mL of IL-21, about 1 IU/mL of IL-21, or about 0.5 IU/mL of IL-21.
• the first expansion culture media comprises about 20 IU/mL of IL-21 to about 0.5 IU/mL of IL-21.
• the first expansion culture media comprises about 15 IU/mL of IL-21 to about 0.5 IU/mL of IL-21. In some embodiments, the first expansion culture media comprises about 12 IU/mL of IL-21 to about 0.5 IU/mL of IL-21. In some embodiments, the first expansion culture media comprises about 10 IU/mL of IL-21 to about 0.5 IU/mL of IL-21. In some embodiments, the first expansion culture media comprises about 5 IU/mL of IL-21 to about 1 IU/mL of IL-21. In some embodiments, the first expansion culture media comprises about 2 IU/mL of IL-21. In some embodiments, the cell culture medium comprises about 1 IU/mL of IL-21. In some embodiments, the cell culture medium comprises about 0.5 IU/mL of IL-21. In an embodiment, the cell culture medium further comprises IL-21. In a preferred embodiment, the cell culture medium comprises about 1 IU/mL of IL-21.
• the cell culture medium comprises OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng/mL of OKT-3 antibody. In an embodiment, the cell culture medium comprises about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1 pg/mL of OKT-3 antibody.
• the cell culture medium comprises between 0.1 ng/mL and 1 ng/mL, between 1 ng/mL and 5 ng/mL, between 5 ng/mL and 10 ng/mL, between 10 ng/mL and 20 ng/mL, between 20 ng/mL and 30 ng/mL, between 30 ng/mL and 40 ng/mL, between 40 ng/mL and 50 ng/mL, and between 50 ng/mL and 100 ng/mL of OKT-3 antibody.
• the cell culture medium does not comprise OKT-3 antibody.
• the OKT- 3 antibody is muromonab.
• the cell culture medium comprises one or more TNFRSF agonists in a cell culture medium.
• the TNFRSF agonist comprises a 4- 1BB agonist.
• the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of urelumab, utomilumab, EU-101, a fusion protein, and fragments, derivatives, variants, biosimilars, and combinations thereof.
• the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of between 0.1 pg/mL and 100 pg/mL.
• the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of between 20 pg/mL and 40 pg/mL.
• the cell culture medium further comprises IL-2 at an initial concentration of about 3000 IU/mL and OKT-3 antibody at an initial concentration of about 30 ng/mL, and wherein the one or more TNFRSF agonists comprises a 4-1BB agonist.
• the first expansion culture medium is referred to as“CM”, an abbreviation for culture media. In some embodiments, it is referred to as CM1 (culture medium 1). In some embodiments, CM consists of RPMI 1640 with GlutaMAX,
• each flask was loaded with 10-40c10 6 viable tumor digest cells or 5-30 tumor fragments in l0-40mL of CM with IL-2.
• the CM is the CM1 described in the Examples, see , Example 1.
• the first expansion occurs in an initial cell culture medium or a first cell culture medium.
• the initial cell culture medium or the first cell culture medium comprises IL-2.
• the first expansion (including processes such as for example those described in Step B of Figure 1, which can include those sometimes referred to as the pre-REP) process is shortened to 3-14 days, as discussed in the examples and figures.
• the first expansion (including processes such as for example those described in Step B of Figure 1, which can include those sometimes referred to as the pre- REP) is shortened to 7 to 14 days, as discussed in the Examples and shown in Figures 4 and 5, as well as including for example, an expansion as described in Step B of Figure 1.
• the first expansion of Step B is shortened to 10-14 days.
• the first expansion is shortened to 11 days, as discussed in, for example, an expansion as described in Step B of Figure 1.
• the first TIL expansion can proceed for 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, or 14 days. In some embodiments, the first TIL expansion can proceed for 1 day to 14 days. In some embodiments, the first TIL expansion can proceed for 2 days to 14 days. In some
• the first TIL expansion can proceed for 3 days to 14 days. In some embodiments, the first TIL expansion can proceed for 3 days to 14 days. In some embodiments, the first TIL expansion can proceed for 3 days to 14 days. In some embodiments, the first TIL expansion can proceed for 3 days to 14 days.
• the first TIL expansion can proceed for 4 days to 14 days. In some embodiments, the first TIL expansion can proceed for 4 days to 14 days. In some embodiments, the first TIL expansion can proceed for 4 days to 14 days.
• the first TIL expansion can proceed for 5 days to 14 days. In some embodiments, the first TIL expansion can proceed for 5 days to 14 days. In some embodiments, the first TIL expansion can proceed for 5 days to 14 days. In some embodiments, the first TIL expansion can proceed for 5 days to 14 days. In some embodiments, the first TIL expansion can proceed for 5 days to 14 days. In some embodiments, the first TIL expansion can proceed for 5 days to 14 days. In some embodiments, the first TIL expansion can proceed for 5 days to 14 days.
• the first TIL expansion can proceed for 6 days to 14 days. In some embodiments, the first TIL expansion can proceed for 6 days to 14 days. In some embodiments, the first TIL expansion can proceed for 6 days to 14 days. In some embodiments, the first TIL expansion can proceed for 6 days to 14 days. In some embodiments, the first TIL expansion can proceed for 6 days to 14 days. In some embodiments, the first TIL expansion can proceed for 6 days to 14 days. In some embodiments, the first TIL expansion can proceed for 6 days to 14 days.
• the first TIL expansion can proceed for 7 days to 14 days. In some embodiments, the first TIL expansion can proceed for 7 days to 14 days.
• the first TIL expansion can proceed for 8 days to 14 days. In some embodiments, the first TIL expansion can proceed for 8 days to 14 days. In some embodiments, the first TIL expansion can proceed for 8 days to 14 days. In some embodiments, the first TIL expansion can proceed for 8 days to 14 days. In some embodiments, the first TIL expansion can proceed for 8 days to 14 days. In some embodiments, the first TIL expansion can proceed for 8 days to 14 days. In some embodiments, the first TIL expansion can proceed for 8 days to 14 days.
• the first TIL expansion can proceed for 9 days to 14 days. In some embodiments, the first TIL expansion can proceed for 9 days to 14 days. In some embodiments, the first TIL expansion can proceed for 9 days to 14 days.
• the first TIL expansion can proceed for 10 days to 14 days. In some embodiments, the first TIL expansion can proceed for 10 days to 14 days. In some embodiments, the first TIL expansion can proceed for 10 days to 14 days. In some embodiments, the first TIL expansion can proceed for 10 days to 14 days. In some embodiments, the first TIL expansion can proceed for 10 days to 14 days. In some embodiments, the first TIL expansion can proceed for 10 days to 14 days. In some embodiments, the first TIL expansion can proceed for 10 days to 14 days.
• the first TIL expansion can proceed for 11 days to 14 days. In some embodiments, the first TIL expansion can proceed for 11 days to 14 days. In some embodiments, the first TIL expansion can proceed for 11 days to 14 days. In some embodiments, the first TIL expansion can proceed for 11 days to 14 days.
• the first TIL expansion can proceed for 12 days to 14 days. In some embodiments, the first TIL expansion can proceed for 12 days to 14 days. In some embodiments, the first TIL expansion can proceed for 12 days to 14 days.
• the first TIL expansion can proceed for 13 days to 14 days. In some embodiments, the first TIL expansion can proceed for 13 days to 14 days. In some embodiments, the first TIL expansion can proceed for 13 days to 14 days.
• the first TIL expansion can proceed for 14 days. In some embodiments, the first TIL expansion can proceed for 1 day to 11 days. In some embodiments, the first TIL expansion can proceed for 2 days to 11 days. In some embodiments, the first TIL expansion can proceed for 3 days to 11 days. In some embodiments, the first TIL expansion can proceed for 4 days to 11 days. In some embodiments, the first TIL expansion can proceed for 5 days to 11 days. In some embodiments, the first TIL expansion can proceed for 6 days to 11 days. In some embodiments, the first TIL expansion can proceed for 7 days to 11 days. In some embodiments, the first TIL expansion can proceed for 8 days to 11 days. In some embodiments, the first TIL expansion can proceed for 14 days. In some embodiments, the first TIL expansion can proceed for 1 day to 11 days. In some embodiments, the first TIL expansion can proceed for 2 days to 11 days. In some embodiments, the first TIL expansion can proceed for 3 days to 11 days. In some embodiments, the first TIL expansion can proceed for 4 days to 11 days
• the first TIL expansion can proceed for 9 days to 11 days. In some embodiments, the first TIL expansion can proceed for 9 days to 11 days.
• the first TIL expansion can proceed for 10 days to 11 days. In some embodiments, the first TIL expansion can proceed for 11 days.
• a combination of IL-2, IL-7, IL-15, and/or IL-21 are employed as a combination during the first expansion.
• IL-2, IL-7, IL- 15, and/or IL-21 as well as any combinations thereof can be included during the first expansion, including for example during a Step B processes according to Figure 1, as well as described herein.
• a combination of IL-2, IL-15, and IL-21 are employed as a combination during the first expansion.
• IL-2, IL-15, and IL-21 as well as any combinations thereof can be included during Step B processes according to Figure 1 and as described herein.
• the first expansion (including processes referred to as the pre-REP; for example, Step B according to Figure 1) process is shortened to 3 to 14 days, as discussed in the examples and figures. In some embodiments, the first expansion of Step B is shortened to 7 to 14 days. In some embodiments, the first expansion of Step B is shortened to 10 to 14 days. In some embodiments, the first expansion is shortened to 11 days.
• the first expansion is performed in a closed system bioreactor.
• a closed system is employed for the TIL expansion, as described herein.
• a single bioreactor is employed.
• the single bioreactor employed is for example a G-REX -10 or a G-REX -100.
• the closed system bioreactor is a single bioreactor.
• the bulk TIL population obtained from the first expansion can be cryopreserved immediately, using the protocols discussed herein below.
• the TIL population obtained from the first expansion referred to as the second TIL population
• a second expansion which can include expansions sometimes referred to as REP
• the first TIL population (sometimes referred to as the bulk TIL population) or the second TIL population (which can in some embodiments include populations referred to as the REP TIL populations) can be subjected to genetic modifications for suitable treatments prior to expansion or after the first expansion and prior to the second expansion.
• the TILs obtained from the first expansion are stored until phenotyped for selection.
• the TILs obtained from the first expansion are not stored and proceed directly to the second expansion.
• the TILs obtained from the first expansion are not cryopreserved after the first expansion and prior to the second expansion.
• the transition from the first expansion to the second expansion occurs at about 3 days, 4, days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days from when fragmentation occurs.
• the transition from the first expansion to the second expansion occurs at about 3 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs at about 4 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs at about 4 days to 10 days from when
• the transition from the first expansion to the second expansion occurs at about 7 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs at about 14 days from when fragmentation occurs.
• the transition from the first expansion to the second expansion occurs at 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, or 14 days from when fragmentation occurs.
• the transition from the first expansion to the second expansion occurs 1 day to 14 days from when fragmentation occurs.
• the first TIL expansion can proceed for 2 days to 14 days.
• the transition from the first expansion to the second expansion occurs 3 days to 14 days from when fragmentation occurs.
• the transition from the first expansion to the second expansion occurs 4 days to 14 days from when fragmentation occurs.
• the transition from the first expansion to the second expansion occurs 5 days to 14 days from when fragmentation occurs.
• the transition from the first expansion to the second expansion occurs
• the transition from the first expansion to the second expansion occurs 7 days to 14 days from when
• the transition from the first expansion to the second expansion occurs 8 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 9 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 10 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 11 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 12 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 13 days to 14 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 14 days from when fragmentation occurs.
• the transition from the first expansion to the second expansion occurs 1 day to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 2 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 3 days to 11 days from when
• the transition from the first expansion to the second expansion occurs 4 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 5 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 6 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs
• the transition from the first expansion to the second expansion occurs 8 days to 11 days from when
• the transition from the first expansion to the second expansion occurs 9 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 10 days to 11 days from when fragmentation occurs. In some embodiments, the transition from the first expansion to the second expansion occurs 11 days from when fragmentation occurs.
• the TILs are not stored after the first expansion and prior to the second expansion, and the TILs proceed directly to the second expansion (for example, in some embodiments, there is no storage during the transition from Step B to Step D as shown in Figure 1).
• the transition occurs in closed system, as described herein.
• the TILs from the first expansion, the second population of TILs proceeds directly into the second expansion with no transition period.
• the transition from the first expansion to the second expansion is performed in a closed system bioreactor.
• a closed system is employed for the TIL expansion, as described herein.
• a single bioreactor is employed.
• the single bioreactor employed is for example a G-REX -10 or a G-REX -100.
• the closed system bioreactor is a single bioreactor.
• the expansion methods described herein generally use culture media with high doses of a cytokine, in particular IL-2, as is known in the art.
• cytokines for the rapid expansion and or second expansion of TILS is additionally possible, with combinations of two or more of IL-2, IL-15 and IL-21 as is generally outlined in International Publication No. WO 2015/189356 and W International Publication No. WO 2015/189357, hereby expressly incorporated by reference in their entirety.
• possible combinations include IL-2 and IL-15, IL-2 and IL- 21, IL-15 and IL-21 and IL-2, IL-15 and IL-21, with the latter finding particular use in many embodiments.
• the use of combinations of cytokines specifically favors the generation of lymphocytes, and in particular T-cells as described therein.
• the TIL cell population is expanded in number after harvest and initial bulk processing for example, after Step A and Step B, and the transition referred to as Step C, as indicated in Figure 1).
• This further expansion is referred to herein as the second expansion, which can include expansion processes generally referred to in the art as a rapid expansion process (REP; as well as processes as indicated in Step D of Figure 1).
• the second expansion is generally accomplished using a culture media comprising a number of components, including feeder cells, a cytokine source, and an anti-CD3 antibody, in a gas- permeable container.
• the second expansion or second TIL expansion (which can include expansions sometimes referred to as REP; as well as processes as indicated in Step D of Figure 1) of TIL can be performed using any TIL flasks or containers known by those of skill in the art.
• the second TIL expansion can proceed for 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.
• the second TIL expansion can proceed for about 7 days to about 14 days.
• the second TIL expansion can proceed for about 8 days to about 14 days.
• the second TIL expansion can proceed for about 9 days to about 14 days.
• the second TIL expansion can proceed for about 10 days to about 14 days.
• the second TIL expansion can proceed for about 11 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 12 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 13 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 14 days. [00419] In an embodiment, the second expansion can be performed in a gas permeable container using the methods of the present disclosure (including for example, expansions referred to as REP; as well as processes as indicated in Step D of Figure 1). For example,
• TILs can be rapidly expanded using non-specific T-cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin- 15 (IL-15).
• the non-specific T-cell receptor stimulus can include, for example, an anti-CD3 antibody, such as about 30 ng/ml of OKT3, a mouse monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA) or UHCT-l (commercially available from BioLegend, San Diego, CA, USA).
• TILs can be expanded to induce further stimulation of the TILs in vitro by including one or more antigens during the second expansion, including antigenic portions thereof, such as epitope(s), of the cancer, which can be optionally expressed from a vector, such as a human leukocyte antigen A2 (HLA-A2) binding peptide, e.g. , 0.3 mM MART-l :26- 35 (27 L) or gpl 00:209-217 (210M), optionally in the presence of a T-cell growth factor, such as 300 IU/mL IL-2 or IL-15.
• HLA-A2 human leukocyte antigen A2
• TIL may also be rapidly expanded by re-stimulation with the same antigen(s) of the cancer pulsed onto HLA-A2-expressing antigen-presenting cells.
• the TILs can be further re-stimulated with, e.g, example, irradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.
• the re-stimulation occurs as part of the second expansion.
• the second expansion occurs in the presence of irradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.
• the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU/mL of IL-2. In an
• the cell culture medium comprises about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL of IL-2.
• the cell culture medium comprises between 1000 and 2000 IU/mL, between 2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000 IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between 7000 and 8000 IU/mL, or between 8000 IU/mL of IL-2.
• the cell culture medium comprises OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng/mL of OKT-3 antibody.
• the cell culture medium comprises about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1 pg/mL of OKT-3 antibody.
• the cell culture medium comprises between 0.1 ng/mL and 1 ng/mL, between 1 ng/mL and 5 ng/mL, between 5 ng/mL and 10 ng/mL, between 10 ng/mL and 20 ng/mL, between 20 ng/mL and 30 ng/mL, between 30 ng/mL and 40 ng/mL, between 40 ng/mL and 50 ng/mL, and between 50 ng/mL and 100 ng/mL of OKT-3 antibody.
• the cell culture medium does not comprise OKT-3 antibody.
• the OKT- 3 antibody is muromonab.
• the cell culture medium comprises one or more TNFRSF agonists in a cell culture medium.
• the TNFRSF agonist comprises a 4- 1BB agonist.
• the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of urelumab, utomilumab, EU-101, a fusion protein, and fragments, derivatives, variants, biosimilars, and combinations thereof.
• the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of between 0.1 pg/mL and 100 pg/mL.
• the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of between 20 pg/mL and 40 pg/mL.
• the cell culture medium further comprises IL-2 at an initial concentration of about 3000 IU/mL and OKT-3 antibody at an initial concentration of about 30 ng/mL, and wherein the one or more TNFRSF agonists comprises a 4-1BB agonist.
• a combination of IL-2, IL-7, IL-15, and/or IL-21 are employed as a combination during the second expansion.
• IL-2, IL-7, IL-15, and/or IL-21 as well as any combinations thereof can be included during the second expansion, including for example during a Step D processes according to Figure 1, as well as described herein.
• a combination of IL-2, IL-15, and IL-21 are employed as a combination during the second expansion.
• IL-2, IL-15, and IL-21 as well as any combinations thereof can be included during Step D processes according to Figure 1 and as described herein.
• the second expansion can be conducted in a supplemented cell culture medium comprising IL-2, OKT-3, antigen-presenting feeder cells, and optionally a TNFRSF agonist.
• the second expansion occurs in a supplemented cell culture medium.
• the supplemented cell culture medium comprises IL-2, OKT-3, and antigen-presenting feeder cells.
• the second cell culture medium comprises IL-2, OKT-3, and antigen-presenting cells (APCs; also referred to as antigen-presenting feeder cells).
• the second expansion occurs in a cell culture medium comprising IL-2, OKT-3, and antigen-presenting feeder cells (i.e., antigen presenting cells).
• the second expansion culture media comprises about 500 IU/mL of IL-15, about 400 IU/mL of IL-15, about 300 IU/mL of IL-15, about 200 IU/mL of IL-15, about 180 IU/mL of IL-15, about 160 IU/mL of IL-15, about 140 IU/mL of IL-15, about 120 IU/mL of IL-15, or about 100 IU/mL of IL-15.
• the second expansion culture media comprises about 500 IU/mL of IL-15 to about 100 IU/mL of IL-15.
• the second expansion culture media comprises about 400 IU/mL of IL-15 to about 100 IU/mL of IL-15. In some embodiments, the second expansion culture media comprises about 300 IU/mL of IL-15 to about 100 IU/mL of IL-15. In some embodiments, the second expansion culture media comprises about 200 IU/mL of IL-15. In some embodiments, the cell culture medium comprises about 180 IU/mL of IL-15. In an embodiment, the cell culture medium further comprises IL-15. In a preferred embodiment, the cell culture medium comprises about 180 IU/mL of IL-15.
• the second expansion culture media comprises about 20 IU/mL of IL-21, about 15 IU/mL of IL-21, about 12 IU/mL of IL-21, about 10 IU/mL of IL- 21, about 5 IU/mL of IL-21, about 4 IU/mL of IL-21, about 3 IU/mL of IL-21, about 2 IU/mL of IL-21, about 1 IU/mL of IL-21, or about 0.5 IU/mL of IL-21.
• the second expansion culture media comprises about 20 IU/mL of IL-21 to about 0.5 IU/mL of IL-21.
• the second expansion culture media comprises about 15 IU/mL of IL-21 to about 0.5 IU/mL of IL-21. In some embodiments, the second expansion culture media comprises about 12 IU/mL of IL-21 to about 0.5 IU/mL of IL-21. In some
• the second expansion culture media comprises about 10 IU/mL of IL-21 to about 0.5 IU/mL of IL-21. In some embodiments, the second expansion culture media comprises about 5 IU/mL of IL-21 to about 1 IU/mL of IL-21. In some embodiments, the second expansion culture media comprises about 2 IU/mL of IL-21. In some embodiments, the cell culture medium comprises about 1 IU/mL of IL-21. In some embodiments, the cell culture medium comprises about 0.5 IU/mL of IL-21. In an embodiment, the cell culture medium further comprises IL-21. In a preferred embodiment, the cell culture medium comprises about 1 IU/mL of IL-21.
• the antigen-presenting feeder cells are PBMCs.
• the ratio of TILs to PBMCs and/or antigen-presenting cells in the rapid expansion and/or the second expansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500.
• the ratio of TILs to PBMCs in the rapid expansion and/or the second expansion is between 1 to 50 and 1 to 300.
• the ratio of TILs to PBMCs in the rapid expansion and/or the second expansion is between 1 to 100 and 1 to 200.
• REP and/or the second expansion is performed in flasks with the bulk TILs being mixed with a 100- or 200-fold excess of inactivated feeder cells, 30 mg/mL OKT3 anti-CD3 antibody and 3000 IU/mL IL-2 in 150 ml media.
• Media replacement is done (generally 2/3 media replacement via respiration with fresh media) until the cells are transferred to an alternative growth chamber.
• Alternative growth chambers include G-REX flasks and gas permeable containers as more fully discussed below.
• the second expansion (which can include processes referred to as the REP process) is shortened to 7-14 days, as discussed in the examples and figures. In some embodiments, the second expansion is shortened to 11 days.
• REP and/or the second expansion may be performed using T- 175 flasks and gas permeable bags as previously described (Tran, et al ., J. Immunother. 2008, 37, 742-51; Dudley, et al, J. Immunother. 2003, 26, 332-42) or gas permeable cultureware (G-Rex flasks).
• the second expansion (including expansions referred to as rapid expansions) is performed in T-175 flasks, and about 1 x 10 6 TILs suspended in 150 mL of media may be added to each T-175 flask.
• the TILs may be cultured in a 1 to 1 mixture of CM and AIM-V medium, supplemented with 3000 IU per mL of IL-2 and 30 ng per ml of anti-CD3.
• the T-175 flasks may be incubated at 37° C in 5% CO2. Half the media may be exchanged on day 5 using 50/50 medium with 3000 IU per mL of IL-2.
• cells from two T-175 flasks may be combined in a 3 L bag and 300 mL of AIM V with 5% human AB serum and 3000 IU per mL of IL-2 was added to the 300 ml of TIL suspension. The number of cells in each bag was counted every day or two and fresh media was added to keep the cell count between 0.5 and 2.0 x 10 6 cells/mL.
• the second expansion (which can include expansions referred to as REP, as well as those referred to in Step D of Figure 1) may be performed in 500 mL capacity gas permeable flasks with 100 cm gas-permeable silicon bottoms (G-Rex 100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA), 5 x 10 6 or 10 x 10 6 TIL may be cultured with PBMCs in 400 mL of 50/50 medium, supplemented with 5% human AB serum, 3000 IU per mL of IL-2 and 30 ng per ml of anti- CD3 (OKT3).
• the G-Rex 100 flasks may be incubated at 37°C in 5% CO2.
• TIL may be removed and placed into centrifuge bottles and centrifuged at 1500 rpm (491 x g) for 10 minutes.
• the TIL pellets may be re-suspended with 150 mL of fresh medium with 5% human AB serum, 3000 IU per mL of IL-2, and added back to the original G-Rex 100 flasks.
• the TIL in each G-Rex 100 may be suspended in the 300 mL of media present in each flask and the cell suspension may be divided into 3 100 mL aliquots that may be used to seed 3 G-Rex 100 flasks.
• AIM-V with 5% human AB serum and 3000 IU per mL of IL-2 may be added to each flask.
• the G-Rex 100 flasks may be incubated at 37° C in 5% CO2 and after 4 days 150 mL of AIM-V with 3000 IU per mL of IL-2 may be added to each G-REX 100 flask.
• the cells may be harvested on day 14 of culture.
• the second expansion (including expansions referred to as REP) is performed in flasks with the bulk TILs being mixed with a 100- or 200-fold excess of inactivated feeder cells, 30 mg/mL OKT3 anti-CD3 antibody and 3000 IU/mL IL-2 in 150 ml media.
• media replacement is done until the cells are transferred to an alternative growth chamber.
• 2/3 of the media is replaced by respiration with fresh media.
• alternative growth chambers include G- REX flasks and gas permeable containers as more fully discussed below.
• the second expansion (including expansions referred to as REP) is performed and further comprises a step wherein TILs are selected for superior tumor reactivity.
• Any selection method known in the art may be used.
• the methods described in U.S. Patent Application Publication No. 2016/0010058 Al, the disclosures of which are incorporated herein by reference, may be used for selection of TILs for superior tumor reactivity.
• a cell viability assay can be performed after the second expansion (including expansions referred to as the REP expansion), using standard assays known in the art.
• a trypan blue exclusion assay can be done on a sample of the bulk TILs, which selectively labels dead cells and allows a viability assessment.
• TIL samples can be counted and viability determined using a Cellometer K2 automated cell counter (Nexcelom Bioscience, Lawrence, MA).
• viability is determined according to the standard Cellometer K2 Image Cytometer Automatic Cell Counter protocol.
• the second expansion (including expansions referred to as REP) of TIL can be performed using T-175 flasks and gas-permeable bags as previously described (Tran KQ, Zhou J, Durflinger KH, et ak, 2008, ./ Immwiother. , 31 :742-751, and Dudley ME, Wunderlich JR, Shelton TE, et al. 2003, J Immunother. , 26:332-342) or gas-per meable G-Rex flasks.
• the second expansion is performed using flasks.
• the second expansion is performed using gas-permeable G-Rex flasks.
• the second expansion is performed in T-175 flasks, and about 1 x 10 6 TIL are suspended in about 150 mL of media and this is added to each T-175 flask.
• the TIL are cultured with irradiated (50 Gy) allogeneic PBMC as“feeder” cells at a ratio of 1 to 100 and the cells were cultured in a 1 to 1 mixture of CM and AIM-V medium (50/50 medium), supplemented with 3000 IU/mL of IL-2 and 30 ng/mL of anti-CD3.
• the T-175 flasks are incubated at 37°C in 5% CO2.
• half the media is changed on day 5 using 50/50 medium with 3000 IU/mL of IL-2.
• cells from 2 T-175 flasks are combined in a 3 L bag and 300 mL of AIM-V with 5% human AB serum and 3000 IU/mL of IL-2 is added to the 300 mL of TIL suspension.
• the number of cells in each bag can be counted every day or two and fresh media can be added to keep the cell count between about 0.5 and about 2.0 x 10 6 cells/mL.
• the second expansion (including expansions referred to as REP) are performed in 500 mL capacity flasks with 100 cm 2 gas-permeable silicon bottoms (G-Rex 100, Wilson Wolf) (Fig. 1), about 5xl0 6 or lOxlO 6 TIL are cultured with irradiated allogeneic PBMC at a ratio of 1 to 100 in 400 mL of 50/50 medium, supplemented with 3000 IU/mL of IL-2 and 30 ng / mL of anti-CD3.
• the G-Rex 100 flasks are incubated at 37°C in 5% CO2.
• TILs are expanded serially in G-Rex 100 flasks
• the TIL in each G-Rex 100 are suspended in the 300 mL of media present in each flask and the cell suspension was divided into three 100 mL aliquots that are used to seed 3 G-Rex 100 flasks.
• AIM-V with 5% human AB serum and 3000 IU/mL of IL-2 is added to each flask.
• the G-Rex 100 flasks are incubated at 37°C in 5% CO2 and after 4 days 150 mL of AIM-V with 3000 IU/mL of IL-2 is added to each G-Rex 100 flask.
• the cells are harvested on day 14 of culture.
• the diverse antigen receptors of T and B lymphocytes are produced by somatic recombination of a limited, but large number of gene segments. These gene segments: V (variable), D (diversity), J (joining), and C (constant), determine the binding specificity and downstream applications of immunoglobulins and T-cell receptors (TCRs).
• the present invention provides a method for generating TILs which exhibit and increase the T-cell repertoire diversity.
• the TILs obtained by the present method exhibit an increase in the T-cell repertoire diversity.
• the TILs obtained in the second expansion exhibit an increase in the T-cell repertoire diversity.
• the increase in diversity is an increase in the immunoglobulin diversity and/or the T-cell receptor diversity.
• the diversity is in the immunoglobulin is in the immunoglobulin heavy chain.
• the diversity is in the immunoglobulin is in the immunoglobulin light chain.
• the diversity is in the T-cell receptor.
• the diversity is in one of the T-cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors.
• TCR T-cell receptor
• TCR TCR beta
• TCRab i.e., TCRa/b.
• the second expansion culture medium (e.g ., sometimes referred to as CM2 or the second cell culture medium), comprises IL-2, OKT-3, as well as the antigen-presenting feeder cells (APCs), as discussed in more detail below.
• the second expansion is performed in a closed system bioreactor.
• a closed system is employed for the TIL expansion, as described herein.
• a single bioreactor is employed.
• the single bioreactor employed is for example a G-REX -10 or a G-REX -100.
• the closed system bioreactor is a single bioreactor.
• the second expansion procedures described herein require an excess of feeder cells during REP TIL expansion and/or during the second expansion.
• the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units from healthy blood donors.
• PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation.
• the allogenic PBMCs are inactivated, either via irradiation or heat treatment, and used in the REP procedures, as described in the examples, which provides an exemplary protocol for evaluating the replication incompetence of irradiate allogeneic PBMCs.
• PBMCs are considered replication incompetent and accepted for use in the TIL expansion procedures described herein if the total number of viable cells on day 14 is less than the initial viable cell number put into culture on day 0 of the REP and/or day 0 of the second expansion (z.e., the start day of the second expansion).
• PBMCs are considered replication incompetent and accepted for use in the TIL expansion procedures described herein if the total number of viable cells, cultured in the presence of OKT3 and IL-2, on day 7 and day 14 has not increased from the initial viable cell number put into culture on day 0 of the REP and/or day 0 of the second expansion (z.e., the start day of the second expansion).
• the PBMCs are cultured in the presence of 30 ng/ml OKT3 antibody and 3000 IU/ml IL-2.
• PBMCs are considered replication incompetent and accepted for use in the TIL expansion procedures described herein if the total number of viable cells, cultured in the presence of OKT3 and IL-2, on day 7 and day 14 has not increased from the initial viable cell number put into culture on day 0 of the REP and/or day 0 of the second expansion (z.e., the start day of the second expansion).
• the PBMCs are cultured in the presence of 5-60 ng/ml OKT3 antibody and 1000-6000 IU/ml IL-2.
• the PBMCs are cultured in the presence of 10-50 ng/ml OKT3 antibody and 2000-5000 IU/ml IL-2.
• the PBMCs are cultured in the presence of 20- 40 ng/ml OKT3 antibody and 2000-4000 IU/ml IL-2. In some embodiments, the PBMCs are cultured in the presence of 25-35 ng/ml OKT3 antibody and 2500-3500 IU/ml IL-2.
• the antigen-presenting feeder cells are PBMCs. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In an embodiment, the ratio of TILs to antigen-presenting feeder cells in the second expansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500. In an embodiment, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1 to 50 and 1 to 300. In an embodiment, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1 to 100 and 1 to 200.
• the second expansion procedures described herein require a ratio of about 2.5xl0 9 feeder cells to about lOOxlO 6 TILs. In another embodiment, the second expansion procedures described herein require a ratio of about 2.5xl0 9 feeder cells to about 50xl0 6 TILs. In yet another embodiment, the second expansion procedures described herein require about 2.5xl0 9 feeder cells to about 25xl0 6 TILs.
• the second expansion procedures described herein require an excess of feeder cells during the second expansion.
• the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units from healthy blood donors.
• PBMCs peripheral blood mononuclear cells
• the PBMCs are obtained using standard methods such as Ficoll- Paque gradient separation.
• aAPC artificial antigen-presenting cells are used in place of PBMCs.
• the allogenic PBMCs are inactivated, either via irradiation or heat treatment, and used in the TIL expansion procedures described herein, including the exemplary procedures described in the figures and examples.
• artificial antigen presenting cells are used in the second expansion as a replacement for, or in combination with, PBMCs.
• the expansion methods described herein generally use culture media with high doses of a cytokine, in particular IL-2, as is known in the art.
• a cytokine in particular IL-2
• IL-2 cytokine
• IL-15 IL-15 and IL-21
• possible combinations include IL-2 and IL-15, IL-2 and IL- 21, IL-15 and IL-21 and IL-2, IL-15 and IL-21, with the latter finding particular use in many embodiments.
• the use of combinations of cytokines specifically favors the generation of lymphocytes, and in particular T-cells as described therein.
• cells can be harvested.
• the TILs are harvested after one, two, three, four or more expansion steps, for example as provided in Figure 1. In some embodiments the TILs are harvested after two expansion steps, for example as provided in Figure 1.
• TILs can be harvested in any appropriate and sterile manner, including for example by centrifugation. Methods for TIL harvesting are well known in the art and any such know methods can be employed with the present process. In some embodiments, TILS are harvest using an automated system.
• Cell harvesters and/or cell processing systems are commercially available from a variety of sources, including, for example, Fresenius Kabi, Tomtec Life Science, Perkin Elmer, and Inotech Biosystems International, Inc. Any cell based harvester can be employed with the present methods.
• the cell harvester and/or cell processing systems is a membrane-based cell harvester.
• cell harvesting is via a cell processing system, such as the LOVO system (manufactured by Fresenius Kabi).
• LOVO cell processing system also refers to any instrument or device manufactured by any vendor that can pump a solution comprising cells through a membrane or filter such as a spinning membrane or spinning filter in a sterile and/or closed system environment, allowing for continuous flow and cell processing to remove supernatant or cell culture media without pelletization.
• the cell harvester and/or cell processing system can perform cell separation, washing, fluid-exchange, concentration, and/or other cell processing steps in a closed, sterile system.
• the harvest for example, Step E according to Figure 1, is performed from a closed system bioreactor.
• a closed system is employed for the TIL expansion, as described herein.
• a single bioreactor is employed.
• the single bioreactor employed is for example a G-REX -10 or a G-REX -100.
• the closed system bioreactor is a single bioreactor.
• Step E according to Figure 1 is performed according to the processes described in Example G.
• the closed system is accessed via syringes under sterile conditions in order to maintain the sterility and closed nature of the system.
• a closed system as described in Example G is employed.
• TILs are harvested according to the methods described in Example G.
• TILs between days 1 and 11 are harvested using the methods as described in Section 8.5 (referred to as the Day 11 TIL harvest in Example G).
• TILs between days 12 and 22 are harvested using the methods as described in Section 8.12 (referred to as the Day 22 TIL harvest in Example G).
• Steps A through E as provided in an exemplary order in Figure 1 and as outlined in detailed above and herein are complete, cells are transferred to a container for use in administration to a patient.
• cells are transferred to a container for use in administration to a patient.
• a therapeutically sufficient number of TILs are obtained using the expansion methods described above, they are transferred to a container for use in administration to a patient.
• TILs expanded using APCs of the present disclosure are administered to a patient as a pharmaceutical composition.
• the pharmaceutical composition In an embodiment, the
• TILs expanded using PBMCs of the present disclosure may be administered by any suitable route as known in the art.
• the T-cells are administered as a single intra-arterial or intravenous infusion, which preferably lasts approximately 30 to 60 minutes.
• Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic.
• the culture media used in expansion methods described herein also includes an anti- CD3 antibody.
• An anti-CD3 antibody in combination with IL-2 induces T cell activation and cell division in the TIL population. This effect can be seen with full length antibodies as well as Fab and F(ab’)2 fragments, with the former being generally preferred; see, e.g., Tsoukas et al , J Immunol 1985, 135 , 1719, hereby incorporated by reference in its entirety.
• anti-human CD3 antibodies that find use in the invention, including anti-human CD3 polyclonal and monoclonal antibodies from various mammals, including, but not limited to, murine, human, primate, rat, and canine antibodies.
• the OKT3 anti-CD3 antibody is used (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA).
• the TNFRSF agonist is a 4-1BB (CD137) agonist.
• the 4-1BB agonist may be any 4-1BB binding molecule known in the art.
• the 4-1BB binding molecule may be a monoclonal antibody or fusion protein capable of binding to human or mammalian 4-1BB.
• the 4-1BB agonists or 4-1BB binding molecules may comprise an immunoglobulin heavy chain of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g, IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
• the 4-1BB agonist or 4-1BB binding molecule may have both a heavy and a light chain.
• the term binding molecule also includes antibodies (including full length antibodies), monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g, bispecific antibodies), human, humanized or chimeric antibodies, and antibody fragments, e.g, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, epitope-binding fragments of any of the above, and engineered forms of antibodies, e.g, scFv molecules, that bind to 4-1BB.
• the 4-1BB agonist is an antigen binding protein that is a fully human antibody.
• the 4-1BB agonist is an antigen binding protein that is a humanized antibody.
• 4- 1BB agonists for use in the presently disclosed methods and compositions include anti-4-lBB antibodies, human anti-4- 1BB antibodies, mouse anti-4- 1BB antibodies, mammalian anti -4- 1BB antibodies, monoclonal anti-4-lBB antibodies, polyclonal anti-4- 1BB antibodies, chimeric anti-4-lBB antibodies, anti-4-lBB adnectins, anti-4-lBB domain antibodies, single chain anti-4-lBB fragments, heavy chain anti-4-lBB fragments, light chain anti-4-lBB fragments, anti-4-lBB fusion proteins, and fragments, derivatives, conjugates, variants, or biosimilars thereof.
• the 4-1BB agonist is an agonistic, anti-4-lBB humanized or fully human monoclonal antibody (i.e., an antibody derived from a single cell line).
• the 4-1BB agonist is EU-101 (Eutilex Co. Ltd.), utomilumab, or urelumab, or a fragment, derivative, conjugate, variant, or biosimilar thereof.
• the 4-1BB agonist is utomilumab or urelumab, or a fragment, derivative, conjugate, variant, or biosimilar thereof.
• the 4-1BB agonist or 4-1BB binding molecule may also be a fusion protein.
• a multimeric 4-1BB agonist such as a trimeric or hexameric 4-1BB agonist (with three or six ligand binding domains) may induce superior receptor (4-1BBL) clustering and internal cellular signaling complex formation compared to an agonistic monoclonal antibody, which typically possesses two ligand binding domains.
• Trimeric (trivalent) or hexameric (or hexavalent) or greater fusion proteins comprising three TNFRSF binding domains and IgGl-Fc and optionally further linking two or more of these fusion proteins are described, e.g., in Gieffers, el al. , Mol. Cancer
• the 4-1BB agonist is a monoclonal antibody or fusion protein that binds specifically to 4-1BB antigen in a manner sufficient to reduce toxicity.
• the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that abrogates antibody-dependent cellular toxicity (ADCC), for example NK cell cytotoxicity.
• the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that abrogates antibody-dependent cell phagocytosis (ADCP).
• the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that abrogates complement-dependent cytotoxicity (CDC). In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein which abrogates Fc region functionality.
• the 4-1BB agonists are characterized by binding to human 4- 1BB (SEQ ID NO:9) with high affinity and agonistic activity.
• the 4-1BB agonist is a binding molecule that binds to human 4-1BB (SEQ ID NO:9).
• the 4-1BB agonist is a binding molecule that binds to murine 4-1BB (SEQ ID NO: 10).
• Table 6 The amino acid sequences of 4-1BB antigen to which a 4-1BB agonist or binding molecule binds are summarized in Table 6.
• compositions, processes and methods described include a
• 4-1BB agonist that binds human or murine 4-1BB with a KD of about 100 pM or lower, binds human or murine 4-1BB with a KD of about 90 pM or lower, binds human or murine 4-1BB with a KD of about 80 pM or lower, binds human or murine 4-1BB with a KD of about 70 pM or lower, binds human or murine 4-1BB with a KD of about 60 pM or lower, binds human or murine 4-1BB with a KD of about 50 pM or lower, binds human or murine 4-1BB with a KD of about 40 pM or lower, or binds human or murine 4-1BB with a KD of about 30 pM or lower.
• compositions, processes and methods described include a 4- IBB agonist that binds to human or murine 4- IBB with a kassoc of about 7.5 c 10 5 l/M s or faster, binds to human or murine 4-1BB with a kassoc of about 7.5 x 10 5 l/M s or faster, binds to human or murine 4-1BB with a kassoc of about 8 x 10 5 l/M s or faster, binds to human or murine 4-1BB with a kassoc of about 8.5 x 10 5 l/M s or faster, binds to human or murine 4- 1BB with a kassoc of about 9 c 10 ⁇ 1/M s or faster, binds to human or murine 4-1BB with a kassoc of about 9.5 c 10 ⁇ 1/M s or faster, or binds to human or murine 4
• compositions, processes and methods described include a 4- 1BB agonist that binds to human or murine 4- 1BB with a kdissoc of about 2 x 10 '5 l/s or slower, binds to human or murine 4- 1BB with a kdissoc of about 2.
• compositions, processes and methods described include a 4-1BB agonist that binds to human or murine 4-1BB with an ICso of about 10 nM or lower, binds to human or murine 4-1BB with an ICso of about 9 nM or lower, binds to human or murine 4-1BB with an ICso of about 8 nM or lower, binds to human or murine 4-1BB with an IC50 of about 7 nM or lower, binds to human or murine 4-1BB with an IC50 of about 6 nM or lower, binds to human or murine 4-1BB with an IC50 of about 5 nM or lower, binds to human or murine 4-1BB with an IC50 of about 4 nM or lower, binds to human or murine 4-1BB with an IC50 of about 3 nM or lower, binds to human or murine 4-1BB with an IC50 of about 2 nM or lower, or
• the 4-1BB agonist is utomilumab, also known as PF- 05082566 or MOR-7480, or a fragment, derivative, variant, or biosimilar thereof.
• Utomilumab is available from Pfizer, Inc.
• Utomilumab is an immunoglobulin G2-lambda, anti - ⁇ Homo sapiens TNFRSF9 (tumor necrosis factor receptor (TNFR) superfamily member 9, 4-1BB, T cell antigen ILA, CD137)], Homo sapiens (fully human) monoclonal antibody.
• TNFRSF9 tumor necrosis factor receptor (TNFR) superfamily member 9
• 4-1BB T cell antigen ILA, CD137
• Table EE The amino acid sequences of utomilumab are set forth in Table EE.
• Utomilumab comprises glycosylation sites at Asn59 and Asn292; heavy chain intrachain disulfide bridges at positions 22-96 (VH-VL), 143-199 (CH UCL), 256-316 (CH2) and 362-420 (CH3); light chain intrachain disulfide bridges at positions 22’-87’ (VH-VL) and l36’-l95’ (CH UCL); interchain heavy chain-heavy chain disulfide bridges at IgG2A isoform positions 218-218, 219-219, 222-222, and 225-225, at IgG2A/B isoform positions 218-130, 219-219, 222-222, and 225- 225, and at IgG2B isoform positions 219-130 (2), 222-222, and 225-225; and interchain heavy chain-light chain disulfide bridges at IgG2A isoform positions 130-213’ (2), IgG2A/B isoform positions 218-21
• a 4-1BB agonist comprises a heavy chain given by SEQ ID NO: 11 and a light chain given by SEQ ID NO: 12.
• a 4-1BB agonist comprises heavy and light chains having the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively, or antigen binding fragments, Fab fragments, single-chain variable fragments (scFv), variants, or conjugates thereof.
• a 4-1BB agonist comprises heavy and light chains that are each at least 99% identical to the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively.
• a 4-1BB agonist comprises heavy and light chains that are each at least 98% identical to the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. In an embodiment, a 4-1BB agonist comprises heavy and light chains that are each at least 97% identical to the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. In an embodiment, a 4-1BB agonist comprises heavy and light chains that are each at least 96% identical to the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. In an embodiment, a 4-1BB agonist comprises heavy and light chains that are each at least 95% identical to the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively.
• the 4-1BB agonist comprises the heavy and light chain CDRs or variable regions (VRs) of utomilumab.
• the 4-1BB agonist heavy chain variable region (VH) comprises the sequence shown in SEQ ID NO: 13
• the 4-1BB agonist light chain variable region (VL) comprises the sequence shown in SEQ ID NO: 14, and conservative amino acid substitutions thereof.
• a 4-1BB agonist comprises VH and VL regions that are each at least 99% identical to the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively.
• a 4-1BB agonist comprises VH and VL regions that are each at least 98% identical to the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. In an embodiment, a 4-1BB agonist comprises VH and VL regions that are each at least 97% identical to the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. In an embodiment, a 4-1BB agonist comprises VH and VL regions that are each at least 96% identical to the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. In an embodiment, a 4-1BB agonist comprises VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. In an embodiment, a 4-1BB agonist comprises an scFv antibody comprising VH and VL regions that are each at least 99% identical to the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14.
• a 4-1BB agonist comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively, and conservative amino acid substitutions thereof, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO:20, respectively, and conservative amino acid substitutions thereof.
• the 4-1BB agonist is a 4-1BB agonist biosimilar monoclonal antibody approved by drug regulatory authorities with reference to utomilumab.
• the biosimilar monoclonal antibody comprises an 4-1BB antibody comprising an amino acid sequence which has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of a reference medicinal product or reference biological product and which comprises one or more post-translational
• the biosimilar is a 4-1BB agonist antibody authorized or submitted for authorization, wherein the 4-1BB agonist antibody is provided in a formulation which differs from the formulations of a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is utomilumab.
• the 4-1BB agonist antibody may be authorized by a drug regulatory authority such as the U.S. FDA and/or the European Union’s EMA.
• the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is utomilumab.
• the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is utomilumab.
• the 4-1BB agonist is the monoclonal antibody urelumab, also known as BMS-663513 and 20H4.9.h4a, or a fragment, derivative, variant, or biosimilar thereof.
• Urelumab is available from Bristol-Myers Squibb, Inc., and Creative Biolabs, Inc.
• Urelumab is an immunoglobulin G4-kappa, anti - ⁇ Homo sapiens TNFRSF9 (tumor necrosis factor receptor superfamily member 9, 4-1BB, T cell antigen ILA, CD137)], Homo sapiens (fully human) monoclonal antibody.
• the amino acid sequences of urelumab are set forth in Table EE.
• Urelumab comprises N-glycosylation sites at positions 298 (and 298”); heavy chain intrachain disulfide bridges at positions 22-95 (VH-VL), 148-204 (CHUCL), 262-322 (CH2) and 368-426 (CH3) (and at positions 22”-95”, l48”-204”, 262”-322”, and 368”- 426”); light chain intrachain disulfide bridges at positions 23’-88’ (VH-VL) and l36’-l96’ (CH!
• urelumab (and at positions 23”’-88”’ and l36”’-l96’”); interchain heavy chain-heavy chain disulfide bridges at positions 227-227” and 230-230”; and interchain heavy chain-light chain disulfide bridges at 135-216’ and l35”-2l6”’.
• the preparation and properties of urelumab and its variants and fragments are described in U.S. Patent Nos. 7,288,638 and 8,962,804, the disclosures of which are incorporated by reference herein.
• the preclinical and clinical characteristics of urelumab are described in Segal, et al, Clin. Cancer Res. 2016, available at http :/dx. doi.org/ 10.1158/1078-0432.
• a 4-1BB agonist comprises a heavy chain given by SEQ ID NO:2l and a light chain given by SEQ ID NO:22.
• a 4-1BB agonist comprises heavy and light chains having the sequences shown in SEQ ID NO:2l and SEQ ID NO:22, respectively, or antigen binding fragments, Fab fragments, single-chain variable fragments (scFv), variants, or conjugates thereof.
• a 4-1BB agonist comprises heavy and light chains that are each at least 99% identical to the sequences shown in SEQ ID NO:2l and SEQ ID NO:22, respectively.
• a 4-1BB agonist comprises heavy and light chains that are each at least 98% identical to the sequences shown in SEQ ID NO:2l and SEQ ID NO:22, respectively. In an embodiment, a 4-1BB agonist comprises heavy and light chains that are each at least 97% identical to the sequences shown in SEQ ID NO:2l and SEQ ID NO:22, respectively. In an embodiment, a 4-1BB agonist comprises heavy and light chains that are each at least 96% identical to the sequences shown in SEQ ID NO:2l and SEQ ID NO:22, respectively. In an embodiment, a 4-1BB agonist comprises heavy and light chains that are each at least 95% identical to the sequences shown in SEQ ID NO:2l and SEQ ID NO:22, respectively.
• the 4-1BB agonist comprises the heavy and light chain CDRs or variable regions (VRs) of urelumab.
• the 4-1BB agonist heavy chain variable region (VH) comprises the sequence shown in SEQ ID NO:23
• the 4-1BB agonist light chain variable region (VL) comprises the sequence shown in SEQ ID NO:24, and conservative amino acid substitutions thereof.
• a 4-1BB agonist comprises VH and VL regions that are each at least 99% identical to the sequences shown in SEQ ID NO:23 and SEQ ID NO:24, respectively.
• a 4-1BB agonist comprises VH and VL regions that are each at least 98% identical to the sequences shown in SEQ ID NO:23 and SEQ ID NO:24, respectively. In an embodiment, a 4-1BB agonist comprises VH and VL regions that are each at least 97% identical to the sequences shown in SEQ ID NO:23 and SEQ ID NO:24, respectively. In an embodiment, a 4-1BB agonist comprises VH and VL regions that are each at least 96% identical to the sequences shown in SEQ ID NO:23 and SEQ ID NO:24, respectively. In an embodiment, a 4-1BB agonist comprises VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO:23 and SEQ ID NO:24, respectively. In an embodiment, a 4-1BB agonist comprises an scFv antibody comprising VH and VL regions that are each at least 99% identical to the sequences shown in SEQ ID NO:23 and SEQ ID NO:24.
• a 4-1BB agonist comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively, and conservative amino acid substitutions thereof, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively, and conservative amino acid substitutions thereof.
• the 4-1BB agonist is a 4-1BB agonist biosimilar monoclonal antibody approved by drug regulatory authorities with reference to urelumab.
• the biosimilar monoclonal antibody comprises an 4-1BB antibody comprising an amino acid sequence which has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of a reference medicinal product or reference biological product and which comprises one or more post-translational
• the biosimilar is a 4-1BB agonist antibody authorized or submitted for authorization, wherein the 4-1BB agonist antibody is provided in a formulation which differs from the formulations of a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is urelumab.
• the 4-1BB agonist antibody may be authorized by a drug regulatory authority such as the U.S. FDA and/or the European Union’s EMA.
• the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is urelumab.
• the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is urelumab.
• the 4-1BB agonist is selected from the group consisting of 1D8, 3 El or, 4B4 (BioLegend 309809), H4-1BB-M127 (BD Pharmingen 552532), BBK2 (Thermo Fisher MS621PABX), 145501 (Leinco Technologies B591), the antibody produced by cell line deposited as ATCC No. HB-11248 and disclosed in LT.S. Patent No. 6,974,863, 5F4 (BioLegend 31 1503), C65-485 (BD Pharmingen 559446), antibodies disclosed in U.S.
• Patent Application Publication No. US 2005/0095244 antibodies disclosed in U.S. Patent No. 7,288,638 (such as 20H4.9-IgGl (BMS-663031)), antibodies disclosed in U.S. Patent No. 6,887,673 (such as 4E9 or BMS-554271), antibodies disclosed in U.S. Patent No. 7,214,493, antibodies disclosed in U.S. Patent No. 6,303,121, antibodies disclosed in U.S. Patent No. 6,569,997, antibodies disclosed in U.S. Patent No. 6,905,685 (such as 4E9 or BMS-554271), antibodies disclosed in U.S. Patent No.
• 6,362,325 (such as 1D8 or BMS-469492; 3H3 or BMS-469497; or 3E1), antibodies disclosed in U.S. Patent No. 6,974,863 (such as 53A2); antibodies disclosed in U.S. Patent No. 6,210,669 (such as 1D8, 3B8, or 3E1), antibodies described in U.S. Patent No. 5,928,893, antibodies disclosed in U.S. Patent No. 6,303,121, antibodies disclosed in U.S. Patent No. 6,569,997, antibodies disclosed in International Patent Application Publication Nos.
• the 4-1BB agonist is a 4-1BB agonistic fusion protein described in International Patent Application Publication Nos. WO 2008/025516 Al, WO 2009/007120 Al, WO 2010/003766 Al, WO 2010/010051 Al, and WO 2010/078966 Al; U.S. Patent Application Publication Nos. US 2011/0027218 Al, US 2015/0126709 Al, US
• the 4-1BB agonist is a 4-1BB agonistic fusion protein as depicted in Structure I-A (C-terminal Fc-antibody fragment fusion protein) or Structure I-B (N-terminal Fc-antibody fragment fusion protein), or a fragment, derivative, conjugate, variant, or biosimilar thereof, as provided in Figure 10.
• the cylinders refer to individual polypeptide binding domains.
• Structures I-A and I-B comprise three linearly -linked TNFRSF binding domains derived from e.g ., 4-1BBL or an antibody that binds 4-1BB, which fold to form a trivalent protein, which is then linked to a second triavelent protein through IgGl-Fc (including CH3 and CH2 domains) is then used to link two of the trivalent proteins together through disulfide bonds (small elongated ovals), stabilizing the structure and providing an agonists capable of bringing together the intracellular signaling domains of the six receptors and signaling proteins to form a signaling complex.
• the TNFRSF binding domains denoted as cylinders may be scFv domains comprising, e.g. , a VH and a VL chain connected by a linker that may comprise hydrophilic residues and Gly and Ser sequences for flexibility, as well as Glu and Lys for solubility.
• Any scFv domain design may be used, such as those described in de Marco, Microbial Cell Factories , 2011, 10, 44; Ahmad, et al, Clin. & Dev. Immunol. 2012, 980250; Monnier, et al. , Antibodies, 2013, 2, 193-208; or in references incorporated elsewhere herein. Fusion protein structures of this form are described in U.S. Patent Nos. 9,359,420, 9,340,599, 8,921,519, and 8,450,460, the disclosures of which are incorporated by reference herein.
• the Fc domain preferably comprises a complete constant domain (amino acids 17-230 of SEQ ID NO:31) the complete hinge domain (amino acids 1-16 of SEQ ID NO:3 l) or a portion of the hinge domain ( e.g ., amino acids 4-16 of SEQ ID NO: 31).
• Preferred linkers for connecting a C-terminal Fc-antibody may be selected from the embodiments given in SEQ ID NO:32 to SEQ ID NO:4l, including linkers suitable for fusion of additional polypeptides.
• Amino acid sequences for the other polypeptide domains of structure I-B are given in Table HH. If an Fc antibody fragment is fused to the N-terminus of an TNRFSF fusion protein as in structure I-B, the sequence of the Fc module is preferably that shown in SEQ ID NO:42, and the linker sequences are preferably selected from those embodiments set forth in SED ID NO:43 to SEQ ID NO:45.
• a 4-1BB agonist fusion protein according to structures I-A or I-B comprises one or more 4-1BB binding domains selected from the group consisting of a variable heavy chain and variable light chain of utomilumab, a variable heavy chain and variable light chain of urelumab, a variable heavy chain and variable light chain of utomilumab, a variable heavy chain and variable light chain selected from the variable heavy chains and variable light chains described in Table GG, any combination of a variable heavy chain and variable light chain of the foregoing, and fragments, derivatives, conjugates, variants, and biosimilars thereof.
• a 4-1BB agonist fusion protein according to structures I-A or I-B comprises one or more 4-1BB binding domains comprising a 4-1BBL sequence. In an embodiment, a 4-1BB agonist fusion protein according to structures I-A or I-B comprises one or more 4-1BB binding domains comprising a sequence according to SEQ ID NO:46. In an embodiment, a 4-1BB agonist fusion protein according to structures I-A or I-B comprises one or more 4-1BB binding domains comprising a soluble 4-1BBL sequence. In an embodiment, a 4-1BB agonist fusion protein according to structures I-A or I-B comprises one or more 4- 1BB binding domains comprising a sequence according to SEQ ID NO:47.
• a 4-1BB agonist fusion protein according to structures I-A or I-B comprises one or more 4-1BB binding domains that is a scFv domain comprising VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively, wherein the VH and VL domains are connected by a linker.
• a 4-1BB agonist fusion protein according to structures I-A or I-B comprises one or more 4-1BB binding domains that is a scFv domain comprising VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO: 23 and SEQ ID NO:24, respectively, wherein the VH and VL domains are connected by a linker.
• a 4-1BB agonist fusion protein according to structures I-A or I-B comprises one or more 4-1BB binding domains that is a scFv domain comprising VH and VL regions that are each at least 95% identical to the VH and VL sequences given in Table 11, wherein the VH and VL domains are connected by a linker.
• the 4-1BB agonist is a 4-1BB agonistic single-chain fusion polypeptide comprising (i) a first soluble 4-1BB binding domain, (ii) a first peptide linker,
• a second soluble 4-1BB binding domain (iii) a second soluble 4-1BB binding domain, (iv) a second peptide linker, and (v) a third soluble 4-1BB binding domain, further comprising an additional domain at the N-terminal and/or C-terminal end, and wherein the additional domain is a Fab or Fc fragment domain.
• the 4-1BB agonist is a 4-1BB agonistic single-chain fusion polypeptide comprising (i) a first soluble 4-1BB binding domain, (ii) a first peptide linker, (iii) a second soluble 4-1BB binding domain, (iv) a second peptide linker, and (v) a third soluble 4-1BB binding domain, further comprising an additional domain at the N-terminal and/or C-terminal end, wherein the additional domain is a Fab or Fc fragment domain, wherein each of the soluble 4-1BB domains lacks a stalk region (which contributes to trimerisation and provides a certain distance to the cell membrane, but is not part of the 4-1BB binding domain) and the first and the second peptide linkers independently have a length of 3-8 amino acids.
• the 4-1BB agonist is a 4-1BB agonistic single-chain fusion polypeptide comprising (i) a first soluble tumor necrosis factor (TNF) superfamily cytokine domain, (ii) a first peptide linker, (iii) a second soluble TNF superfamily cytokine domain, (iv) a second peptide linker, and (v) a third soluble TNF superfamily cytokine domain, wherein each of the soluble TNF superfamily cytokine domains lacks a stalk region and the first and the second peptide linkers independently have a length of 3-8 amino acids, and wherein each TNF superfamily cytokine domain is a 4-1BB binding domain.
• TNF tumor necrosis factor
• the 4-1BB agonist is a 4-1BB agonistic scFv antibody comprising any of the foregoing VH domains linked to any of the foregoing VL domains.
• the 4-1BB agonist is BPS Bioscience 4-1BB agonist antibody catalog no. 79097-2, commercially available from BPS Bioscience, San Diego, CA, USA.
• the 4-1BB agonist is Creative Biolabs 4-1BB agonist antibody catalog no. MOM-18179, commercially available from Creative Biolabs, Shirley, NY, USA.
• the TNFRSF agonist is an 0X40 (CD 134) agonist.
• the 0X40 agonist may be any 0X40 binding molecule known in the art.
• the 0X40 binding molecule may be a monoclonal antibody or fusion protein capable of binding to human or mammalian 0X40.
• the 0X40 agonists or 0X40 binding molecules may comprise an immunoglobulin heavy chain of any isotype (e.g ., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g, IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
• the 0X40 agonist or 0X40 binding molecule may have both a heavy and a light chain.
• the term binding molecule also includes antibodies (including full length antibodies), monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g, bispecific antibodies), human, humanized or chimeric antibodies, and antibody fragments, e.g, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, epitope-binding fragments of any of the above, and engineered forms of antibodies, e.g, scFv molecules, that bind to 0X40.
• the 0X40 agonist is an antigen binding protein that is a fully human antibody.
• the 0X40 agonist is an antigen binding protein that is a humanized antibody.
• 0X40 agonists for use in the presently disclosed methods and compositions include anti- 0X40 antibodies, human anti-OX40 antibodies, mouse anti-OX40 antibodies, mammalian anti-OX40 antibodies, monoclonal anti -0X40 antibodies, polyclonal anti-OX40 antibodies, chimeric anti -0X40 antibodies, anti -0X40 adnectins, anti-OX40 domain antibodies, single chain anti-OX40 fragments, heavy chain anti-OX40 fragments, light chain anti-OX40 fragments, anti-OX40 fusion proteins, and fragments, derivatives, conjugates, variants, or biosimilars thereof.
• the 0X40 agonist is an agonistic, anti-OX40 humanized or fully human monoclonal antibody (i.e., an antibody derived from a single cell line).
• the 0X40 agonist or 0X40 binding molecule may also be a fusion protein.
• 0X40 fusion proteins comprising an Fc domain fused to OX40L are described, for example, in Sadun, et al. , J. Immunother. 2009, 182, 1481-89.
• a multimeric 0X40 agonist such as a trimeric or hexameric 0X40 agonist (with three or six ligand binding domains), may induce superior receptor (OX40L) clustering and internal cellular signaling complex formation compared to an agonistic monoclonal antibody, which typically possesses two ligand binding domains.
• Trimeric (trivalent) or hexameric (or hexavalent) or greater fusion proteins comprising three TNFRSF binding domains and IgGl-Fc and optionally further linking two or more of these fusion proteins are described, e.g., in Gieffers, el al. , Mol. Cancer Therapeutics 2013, 12, 2735-47.
• the 0X40 agonist is a monoclonal antibody or fusion protein that binds specifically to 0X40 antigen in a manner sufficient to reduce toxicity.
• the 0X40 agonist is an agonistic 0X40 monoclonal antibody or fusion protein that abrogates antibody-dependent cellular toxicity (ADCC), for example NK cell cytotoxicity.
• ADCC antibody-dependent cellular toxicity
• the 0X40 agonist is an agonistic 0X40 monoclonal antibody or fusion protein that abrogates antibody- dependent cell phagocytosis (ADCP). In some embodiments, the 0X40 agonist is an agonistic 0X40 monoclonal antibody or fusion protein that abrogates complement-dependent cytotoxicity (CDC). In some embodiments, the 0X40 agonist is an agonistic 0X40 monoclonal antibody or fusion protein which abrogates Fc region functionality.
• ADCP antibody- dependent cell phagocytosis
• CDC complement-dependent cytotoxicity
• the 0X40 agonist is an agonistic 0X40 monoclonal antibody or fusion protein which abrogates Fc region functionality.
• the 0X40 agonists are characterized by binding to human 0X40 (SEQ ID NO:54) with high affinity and agonistic activity.
• the 0X40 agonist is a binding molecule that binds to human 0X40 (SEQ ID NO:54).
• the 0X40 agonist is a binding molecule that binds to murine 0X40 (SEQ ID NO:55).
• Table 12 The amino acid sequences of 0X40 antigen to which an 0X40 agonist or binding molecule binds are summarized in Table 12. TABLE 12: Amino acid sequences of 0X40 antigens.
• compositions, processes and methods described include a 0X40 agonist that binds human or murine 0X40 with a KD of about 100 pM or lower, binds human or murine 0X40 with a KD of about 90 pM or lower, binds human or murine 0X40 with a KD of about 80 pM or lower, binds human or murine 0X40 with a KD of about 70 pM or lower, binds human or murine 0X40 with a KD of about 60 pM or lower, binds human or murine 0X40 with a KD of about 50 pM or lower, binds human or murine 0X40 with a KD of about 40 pM or lower, or binds human or murine 0X40 with a KD of about 30 pM or lower.
• compositions, processes and methods described include a 0X40 agonist that binds to human or murine 0X40 with a kassoc of about 7.5 x 10 5 l/M s or faster, binds to human or murine 0X40 with a kassoc of about 7.5 x 10 5 l/M s or faster, binds to human or murine 0X40 with a kassoc of about 8 x 10 5 l/M s or faster, binds to human or murine 0X40 with a kassoc of about 8.5 x 10 5 l/M s or faster, binds to human or murine 0X40 with a kassoc of about 9 x 10 5 l/M s or faster, binds to human or murine 0X40 with a kassoc of about 9.5 x 10 5 l/M s or faster, or
• compositions, processes and methods described include a 0X40 agonist that binds to human or murine 0X40 with a kdissoc of about 2 x 10 5 l/s or slower, binds to human or murine 0X40 with a kdissoc of about 2.1 x 10 5 l/s or slower , binds to human or murine 0X40 with a kdissoc of about 2.2 x 10 5 l/s or slower, binds to human or murine 0X40 with a kdissoc of about 2.3 x 10 5 l/s or slower, binds to human or murine 0X40 with a kdissoc of about 2.4 x 10 5 l/s or slower, binds to human or murine 0X40 with a kdissoc of about 2.5 x 10 5 l/s or slower, binds to human or murine 0X40 with a
• compositions, processes and methods described include 0X40 agonist that binds to human or murine 0X40 with an ICso of about 10 nM or lower, binds to human or murine 0X40 with an ICso of about 9 nM or lower, binds to human or murine 0X40 with an ICso of about 8 nM or lower, binds to human or murine 0X40 with an IC50 of about 7 nM or lower, binds to human or murine 0X40 with an IC50 of about 6 nM or lower, binds to human or murine 0X40 with an IC50 of about 5 nM or lower, binds to human or murine 0X40 with an IC50 of about 4 nM or lower, binds to human or murine 0X40 with an IC50 of about 3 nM or lower, binds to human or murine 0X40 with an IC50 of about 10 nM or lower, bind
• the 0X40 agonist is tavolixizumab, also known as
• Tavolixizumab is available from the Medlmmune subsidiary of AstraZeneca, Inc. Tavolixizumab is immunoglobulin Gl-kappa, anti -[Homo sapiens
• TNFRSF4 tumor necrosis factor receptor (TNFR) superfamily member 4, 0X40, CD 134)
• TNFRSF4 tumor necrosis factor receptor (TNFR) superfamily member 4, 0X40, CD 1344
• the amino acid sequences of tavolixizumab are set forth in Table KK.
• Tavolixizumab comprises N-glycosylation sites at positions 301 and 301”, with fucosylated complex bi-antennary CHO-type glycans; heavy chain intrachain disulfide bridges at positions 22-95 (VH-VL), 148-204 (CHECL), 265-325 (CH2) and 371-429 (CH3) (and at positions 22”-95”, l48”-204”, 265”-325”, and 37l”-429”); light chain intrachain disulfide bridges at positions 23’-88’ (VH-VL) and l34’-l94’ (CHECL) (and at positions 23”’-88’” and l34”’-l94”’); interchain heavy chain-heavy chain disulfide bridges at positions 230-230” and 233-233”; and interchain heavy chain-light chain disulfide bridges at 224-214’ and 224”-2l4”’.
• a 0X40 agonist comprises a heavy chain given by SEQ ID NO:56 and a light chain given by SEQ ID NO:57.
• a 0X40 agonist comprises heavy and light chains having the sequences shown in SEQ ID NO:56 and SEQ ID NO:57, respectively, or antigen binding fragments, Fab fragments, single-chain variable fragments (scFv), variants, or conjugates thereof.
• a 0X40 agonist comprises heavy and light chains that are each at least 99% identical to the sequences shown in SEQ ID NO:56 and SEQ ID NO:57, respectively.
• a 0X40 agonist comprises heavy and light chains that are each at least 98% identical to the sequences shown in SEQ ID NO:56 and SEQ ID NO:57, respectively. In an embodiment, a 0X40 agonist comprises heavy and light chains that are each at least 97% identical to the sequences shown in SEQ ID NO:56 and SEQ ID NO:57, respectively. In an embodiment, a 0X40 agonist comprises heavy and light chains that are each at least 96% identical to the sequences shown in SEQ ID NO:56 and SEQ ID NO:57, respectively. In an embodiment, a 0X40 agonist comprises heavy and light chains that are each at least 95% identical to the sequences shown in SEQ ID NO:56 and SEQ ID NO:57, respectively.
• the 0X40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of tavolixizumab.
• the 0X40 agonist heavy chain variable region (VH) comprises the sequence shown in SEQ ID NO:58
• the 0X40 agonist light chain variable region (VL) comprises the sequence shown in SEQ ID NO:59, and conservative amino acid substitutions thereof.
• a 0X40 agonist comprises VH and VL regions that are each at least 99% identical to the sequences shown in SEQ ID NO:58 and SEQ ID NO:59, respectively.
• a 0X40 agonist comprises VH and VL regions that are each at least 98% identical to the sequences shown in SEQ ID NO:58 and SEQ ID NO:59, respectively. In an embodiment, a 0X40 agonist comprises VH and VL regions that are each at least 97% identical to the sequences shown in SEQ ID NO: 58 and SEQ ID NO:59, respectively. In an embodiment, a 0X40 agonist comprises VH and VL regions that are each at least 96% identical to the sequences shown in SEQ ID NO: 58 and SEQ ID NO:59, respectively.
• a 0X40 agonist comprises VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO: 58 and SEQ ID NO: 59, respectively.
• an 0X40 agonist comprises an scFv antibody comprising VH and VL regions that are each at least 99% identical to the sequences shown in SEQ ID NO:58 and SEQ ID NO:59.
• a 0X40 agonist comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 60, SEQ ID NO:6l, and SEQ ID NO:62, respectively, and conservative amino acid substitutions thereof, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 63, SEQ ID NO:64, and SEQ ID NO:65, respectively, and conservative amino acid substitutions thereof.
• the 0X40 agonist is a 0X40 agonist biosimilar monoclonal antibody approved by drug regulatory authorities with reference to tavolixizumab.
• the biosimilar monoclonal antibody comprises an 0X40 antibody comprising an amino acid sequence which has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of a reference medicinal product or reference biological product and which comprises one or more post-translational modifications as compared to the reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is tavolixizumab.
• the one or more post-translational modifications are selected from one or more of: glycosylation, oxidation, deamidation, and truncation.
• the biosimilar is a 0X40 agonist antibody authorized or submitted for authorization, wherein the 0X40 agonist antibody is provided in a formulation which differs from the formulations of a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is tavolixizumab.
• the 0X40 agonist antibody may be authorized by a drug regulatory authority such as the U.S. FDA and/or the European Union’s EMA.
• the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is tavolixizumab.
• the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is tavolixizumab.
• the 0X40 agonist is 11D4, which is a fully human antibody available from Pfizer, Inc.
• the preparation and properties of 11D4 are described in U.S. Patent Nos. 7,960,515; 8,236,930; and 9,028,824, the disclosures of which are incorporated by reference herein.
• the amino acid sequences of 11D4 are set forth in Table LL.
• a 0X40 agonist comprises a heavy chain given by SEQ ID NO:66 and a light chain given by SEQ ID NO:67.
• a 0X40 agonist comprises heavy and light chains having the sequences shown in SEQ ID NO:66 and SEQ ID NO: 67, respectively, or antigen binding fragments, Fab fragments, single-chain variable fragments (scFv), variants, or conjugates thereof.
• a 0X40 agonist comprises heavy and light chains that are each at least 99% identical to the sequences shown in SEQ ID NO:66 and SEQ ID NO:67, respectively.
• a 0X40 agonist comprises heavy and light chains that are each at least 98% identical to the sequences shown in SEQ ID NO:66 and SEQ ID NO:67, respectively. In an embodiment, a 0X40 agonist comprises heavy and light chains that are each at least 97% identical to the sequences shown in SEQ ID NO:66 and SEQ ID NO:67, respectively. In an embodiment, a 0X40 agonist comprises heavy and light chains that are each at least 96% identical to the sequences shown in SEQ ID NO:66 and SEQ ID NO:67, respectively. In an embodiment, a 0X40 agonist comprises heavy and light chains that are each at least 95% identical to the sequences shown in SEQ ID NO:66 and SEQ ID NO:67, respectively.
• the 0X40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of 11D4.
• the 0X40 agonist heavy chain variable region (VH) comprises the sequence shown in SEQ ID NO:68
• the 0X40 agonist light chain variable region (VL) comprises the sequence shown in SEQ ID NO:69, and conservative amino acid substitutions thereof.
• a 0X40 agonist comprises VH and VL regions that are each at least 99% identical to the sequences shown in SEQ ID NO:68 and SEQ ID NO:69, respectively.
• a 0X40 agonist comprises VH and VL regions that are each at least 98% identical to the sequences shown in SEQ ID NO:68 and SEQ ID NO:69, respectively. In an embodiment, a 0X40 agonist comprises VH and VL regions that are each at least 97% identical to the sequences shown in SEQ ID NO: 68 and SEQ ID NO:69, respectively. In an embodiment, a 0X40 agonist comprises VH and VL regions that are each at least 96% identical to the sequences shown in SEQ ID NO: 68 and SEQ ID NO:69, respectively. In an embodiment, a 0X40 agonist comprises VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO: 68 and SEQ ID NO:69, respectively.
• a 0X40 agonist comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO:70, SEQ ID NO:7l, and SEQ ID NO:72, respectively, and conservative amino acid substitutions thereof, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 73, SEQ ID NO:74, and SEQ ID NO:75, respectively, and conservative amino acid substitutions thereof.
• the 0X40 agonist is a 0X40 agonist biosimilar monoclonal antibody approved by drug regulatory authorities with reference to 11D4.
• the biosimilar monoclonal antibody comprises an 0X40 antibody comprising an amino acid sequence which has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of a reference medicinal product or reference biological product and which comprises one or more post-translational modifications as compared to the reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is 11D4.
• the one or more post- translational modifications are selected from one or more of: glycosylation, oxidation, deamidation, and truncation.
• the biosimilar is a 0X40 agonist antibody authorized or submitted for authorization, wherein the 0X40 agonist antibody is provided in a formulation which differs from the formulations of a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is 11D4.
• the 0X40 agonist antibody may be authorized by a drug regulatory authority such as the U.S. FDA and/or the European Union’s EMA.
• the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is 11D4.
• the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is 11D4.
• the 0X40 agonist is 18D8, which is a fully human antibody available from Pfizer, Inc.
• the preparation and properties of 18D8 are described in U.S. Patent Nos. 7,960,515; 8,236,930; and 9,028,824, the disclosures of which are incorporated by reference herein.
• the amino acid sequences of 18D8 are set forth in Table MM.
• a 0X40 agonist comprises a heavy chain given by SEQ ID NO:76 and a light chain given by SEQ ID NO:77.
• a 0X40 agonist comprises heavy and light chains having the sequences shown in SEQ ID NO:76 and SEQ ID NO:77, respectively, or antigen binding fragments, Fab fragments, single-chain variable fragments (scFv), variants, or conjugates thereof.
• a 0X40 agonist comprises heavy and light chains that are each at least 99% identical to the sequences shown in SEQ ID NO:76 and SEQ ID NO:77, respectively.
• a 0X40 agonist comprises heavy and light chains that are each at least 98% identical to the sequences shown in SEQ ID NO:76 and SEQ ID NO:77, respectively. In an embodiment, a 0X40 agonist comprises heavy and light chains that are each at least 97% identical to the sequences shown in SEQ ID NO:76 and SEQ ID NO:77, respectively. In an embodiment, a 0X40 agonist comprises heavy and light chains that are each at least 96% identical to the sequences shown in SEQ ID NO:76 and SEQ ID NO:77, respectively. In an embodiment, a 0X40 agonist comprises heavy and light chains that are each at least 95% identical to the sequences shown in SEQ ID NO:76 and SEQ ID NO:77, respectively.
• the 0X40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of 18D8.
• the 0X40 agonist heavy chain variable region (VH) comprises the sequence shown in SEQ ID NO:78
• the 0X40 agonist light chain variable region (VL) comprises the sequence shown in SEQ ID NO:79, and
• a 0X40 agonist comprises VH and VL regions that are each at least 99% identical to the sequences shown in SEQ ID NO:78 and SEQ ID NO:79, respectively. In an embodiment, a 0X40 agonist comprises VH and VL regions that are each at least 98% identical to the sequences shown in SEQ ID NO:78 and SEQ ID NO:79, respectively. In an embodiment, a 0X40 agonist comprises VH and VL regions that are each at least 97% identical to the sequences shown in SEQ ID NO:78 and SEQ ID NO:79, respectively.
• a 0X40 agonist comprises VH and VL regions that are each at least 96% identical to the sequences shown in SEQ ID NO:78 and SEQ ID NO:79, respectively. In an embodiment, a 0X40 agonist comprises VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO:78 and SEQ ID NO:79, respectively.
• a 0X40 agonist comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO:80, SEQ ID NO:8l, and SEQ ID NO:82, respectively, and conservative amino acid substitutions thereof, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO:83, SEQ ID NO:84, and SEQ ID NO:85, respectively, and conservative amino acid substitutions thereof.
• the 0X40 agonist is a 0X40 agonist biosimilar monoclonal antibody approved by drug regulatory authorities with reference to 18D8.
• the biosimilar monoclonal antibody comprises an 0X40 antibody comprising an amino acid sequence which has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of a reference medicinal product or reference biological product and which comprises one or more post-translational modifications as compared to the reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is 18D8.
• the one or more post- translational modifications are selected from one or more of: glycosylation, oxidation, deamidation, and truncation.
• the biosimilar is a 0X40 agonist antibody authorized or submitted for authorization, wherein the 0X40 agonist antibody is provided in a formulation which differs from the formulations of a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is 18D8.
• the 0X40 agonist antibody may be authorized by a drug regulatory authority such as the U.S. FDA and/or the European Union’s EMA.
• the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is 18D8.
• the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is 18D8.
• the 0X40 agonist is Hul 19-122, which is a humanized antibody available from GlaxoSmithKline plc.
• Hul 19-122 is a humanized antibody available from GlaxoSmithKline plc.
• the preparation and properties of Hul 19-122 are described in U.S. Patent Nos. 9,006,399 and 9,163,085, and in International Patent Publication No. WO 2012/027328, the disclosures of which are incorporated by reference herein.
• the amino acid sequences of Hul 19-122 are set forth in Table NN.
• the 0X40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of Hul 19-122.
• the 0X40 agonist heavy chain variable region (VH) comprises the sequence shown in SEQ ID NO:86
• the 0X40 agonist light chain variable region (VL) comprises the sequence shown in SEQ ID NO:87, and conservative amino acid substitutions thereof.
• a 0X40 agonist comprises VH and VL regions that are each at least 99% identical to the sequences shown in SEQ ID NO:86 and SEQ ID NO:87, respectively.
• a 0X40 agonist comprises VH and VL regions that are each at least 98% identical to the sequences shown in SEQ ID NO:86 and SEQ ID NO:87, respectively. In an embodiment, a 0X40 agonist comprises VH and VL regions that are each at least 97% identical to the sequences shown in SEQ ID NO: 86 and SEQ ID NO:87, respectively. In an embodiment, a 0X40 agonist comprises VH and VL regions that are each at least 96% identical to the sequences shown in SEQ ID NO: 86 and SEQ ID NO:87, respectively. In an embodiment, a 0X40 agonist comprises VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO: 86 and SEQ ID NO:87, respectively.
• a 0X40 agonist comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:90, respectively, and conservative amino acid substitutions thereof, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO:9l, SEQ ID NO:92, and SEQ ID NO:93, respectively, and conservative amino acid substitutions thereof.
• the 0X40 agonist is a 0X40 agonist biosimilar monoclonal antibody approved by drug regulatory authorities with reference to Hul 19-122.
• the biosimilar monoclonal antibody comprises an 0X40 antibody comprising an amino acid sequence which has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of a reference medicinal product or reference biological product and which comprises one or more post-translational
• the biosimilar is a 0X40 agonist antibody authorized or submitted for authorization, wherein the 0X40 agonist antibody is provided in a formulation which differs from the formulations of a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is Hul 19-122.
• the 0X40 agonist antibody may be authorized by a drug regulatory authority such as the U.S. FDA and/or the European Union’s EMA.
• the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is Hul 19- 122.
• the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is Hul 19- 122
• the 0X40 agonist is Hul06-222, which is a humanized antibody available from GlaxoSmithKline plc.
• Hul06-222 is a humanized antibody available from GlaxoSmithKline plc.
• the preparation and properties of Hu 106-222 are described in U.S. Patent Nos. 9,006,399 and 9,163,085, and in International Patent Publication No. WO 2012/027328, the disclosures of which are incorporated by reference herein.
• the amino acid sequences of Hul06-222 are set forth in Table OO.
• the 0X40 agonist comprises the heavy and light chain CDRs or variable regions (VRs) of Hul06-222.
• the 0X40 agonist heavy chain variable region (VH) comprises the sequence shown in SEQ ID NO:94
• the 0X40 agonist light chain variable region (VL) comprises the sequence shown in SEQ ID NO:95, and conservative amino acid substitutions thereof.
• a 0X40 agonist comprises VH and VL regions that are each at least 99% identical to the sequences shown in SEQ ID NO:94 and SEQ ID NO:95, respectively.
• a 0X40 agonist comprises VH and VL regions that are each at least 98% identical to the sequences shown in SEQ ID NO:94 and SEQ ID NO:95, respectively. In an embodiment, a 0X40 agonist comprises VH and VL regions that are each at least 97% identical to the sequences shown in SEQ ID NO: 94 and SEQ ID NO:95, respectively. In an embodiment, a 0X40 agonist comprises VH and VL regions that are each at least 96% identical to the sequences shown in SEQ ID NO: 94 and SEQ ID NO:95, respectively. In an embodiment, a 0X40 agonist comprises VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO: 94 and SEQ ID NO:95, respectively.
• a 0X40 agonist comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 96, SEQ ID NO: 97, and SEQ ID NO:98, respectively, and conservative amino acid substitutions thereof, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 99, SEQ ID NO: 100, and SEQ ID NO: 101, respectively, and conservative amino acid substitutions thereof.
• the 0X40 agonist is a 0X40 agonist biosimilar monoclonal antibody approved by drug regulatory authorities with reference to Hul06-222.
• the biosimilar monoclonal antibody comprises an 0X40 antibody comprising an amino acid sequence which has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of a reference medicinal product or reference biological product and which comprises one or more post-translational
• the biosimilar is a 0X40 agonist antibody authorized or submitted for authorization, wherein the 0X40 agonist antibody is provided in a formulation which differs from the formulations of a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is Hul06-222.
• the 0X40 agonist antibody may be authorized by a drug regulatory authority such as the U.S. FDA and/or the European Union’s EMA.
• the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is Hul06- 222.
• the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is Hul06- 222
• the 0X40 agonist antibody is MEDI6469 (also referred to as 9B12).
• MEDI6469 is a murine monoclonal antibody. Weinberg, et al. , J. Immunother. 2006, 29, 575-585.
• the 0X40 agonist is an antibody produced by the 9B12 hybridoma, deposited with Biovest Inc. (Malvern, MA, USA), as described in Weinberg, et al, J. Immunother. 2006, 29, 575-585, the disclosure of which is hereby incorporated by reference in its entirety.
• the antibody comprises the CDR sequences of MEDI6469.
• the antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence of MEDI6469.
• the 0X40 agonist is L 106 BD (Pharmingen Product #340420).
• the 0X40 agonist comprises the CDRs of antibody L106 (BD
• the 0X40 agonist comprises a heavy chain variable region sequence and/or a light chain variable region sequence of antibody L106 (BD Pharmingen Product #340420).
• the 0X40 agonist is ACT35 (Santa Cruz Biotechnology, Catalog #20073).
• the 0X40 agonist comprises the CDRs of antibody ACT35 (Santa Cruz Biotechnology, Catalog #20073).
• the 0X40 agonist comprises a heavy chain variable region sequence and/or a light chain variable region sequence of antibody ACT35 (Santa Cruz Biotechnology, Catalog #20073).
• the 0X40 agonist is the murine monoclonal antibody anti-mCDl34/mOX40 (clone 0X86), commercially available from InVivoMAb, BioXcell Inc, West Riverside, NH.
• the 0X40 agonist is selected from the 0X40 agonists described in International Patent Application Publication Nos. WO 95/12673, WO 95/21925, WO 2006/121810, WO 2012/027328, WO 2013/028231, WO 2013/038191, and WO
• the 0X40 agonist is an 0X40 agonistic fusion protein as depicted in Structure I-A (C-terminal Fc-antibody fragment fusion protein) or Structure I-B (N-terminal Fc-antibody fragment fusion protein), or a fragment, derivative, conjugate, variant, or biosimilar thereof.
• Structure I-A and I-B are described above and in U.S. Patent Nos. 9,359,420, 9,340,599, 8,921,519, and 8,450,460, the disclosures of which are incorporated by reference herein. Amino acid sequences for the polypeptide domains of structure I-A are given in Table GG.
• the Fc domain preferably comprises a complete constant domain (amino acids 17-230 of SEQ ID NO:3 l) the complete hinge domain (amino acids 1-16 of SEQ ID NO: 31) or a portion of the hinge domain ( e.g ., amino acids 4-16 of SEQ ID NO:3 l).
• Preferred linkers for connecting a C-terminal Fc-antibody may be selected from the embodiments given in SEQ ID NO:32 to SEQ ID NO:4l, including linkers suitable for fusion of additional polypeptides.
• amino acid sequences for the polypeptide domains of structure I-B are given in Table HH.
• the sequence of the Fc module is preferably that shown in SEQ ID NO:42, and the linker sequences are preferably selected from those embodiments set forth in SED ID NO:43 to SEQ ID NO:45.
• an 0X40 agonist fusion protein according to structures I-A or I- B comprises one or more 0X40 binding domains selected from the group consisting of a variable heavy chain and variable light chain of tavolixizumab, a variable heavy chain and variable light chain of 11D4, a variable heavy chain and variable light chain of 18D8, a variable heavy chain and variable light chain of Hu 119-122, a variable heavy chain and variable light chain of Hu 106-222, a variable heavy chain and variable light chain selected from the variable heavy chains and variable light chains described in Table OO, any combination of a variable heavy chain and variable light chain of the foregoing, and fragments, derivatives, conjugates, variants, and biosimilars thereof.
• an 0X40 agonist fusion protein according to structures I-A or I- B comprises one or more 0X40 binding domains comprising an OX40L sequence.
• an 0X40 agonist fusion protein according to structures I-A or I-B comprises one or more 0X40 binding domains comprising a sequence according to SEQ ID NO: 102.
• an 0X40 agonist fusion protein according to structures I-A or I-B comprises one or more 0X40 binding domains comprising a soluble OX40L sequence.
• a 0X40 agonist fusion protein according to structures I-A or I-B comprises one or more 0X40 binding domains comprising a sequence according to SEQ ID NO: 103. In an embodiment, a 0X40 agonist fusion protein according to structures I-A or I-B comprises one or more 0X40 binding domains comprising a sequence according to SEQ ID NO: 104.
• an 0X40 agonist fusion protein according to structures I-A or I- B comprises one or more 0X40 binding domains that is a scFv domain comprising VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO: 58 and SEQ ID NO: 59, respectively, wherein the VH and VL domains are connected by a linker.
• an 0X40 agonist fusion protein according to structures I-A or I-B comprises one or more 0X40 binding domains that is a scFv domain comprising VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO: 68 and SEQ ID NO: 69, respectively, wherein the VH and VL domains are connected by a linker.
• an 0X40 agonist fusion protein according to structures I-A or I-B comprises one or more 0X40 binding domains that is a scFv domain comprising VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO:78 and SEQ ID NO:79, respectively, wherein the VH and VL domains are connected by a linker.
• an 0X40 agonist fusion protein according to structures I-A or I-B comprises one or more 0X40 binding domains that is a scFv domain comprising VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO: 86 and SEQ ID NO: 87, respectively, wherein the VH and VL domains are connected by a linker.
• an 0X40 agonist fusion protein according to structures I-A or I-B comprises one or more 0X40 binding domains that is a scFv domain comprising VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO:94 and SEQ ID NO:95, respectively, wherein the VH and VL domains are connected by a linker.
• an 0X40 agonist fusion protein according to structures I-A or I-B comprises one or more 0X40 binding domains that is a scFv domain comprising VH and VL regions that are each at least 95% identical to the VH and VL sequences given in Table 18, wherein the VH and VL domains are connected by a linker.
• the 0X40 agonist is a 0X40 agonistic single-chain fusion polypeptide comprising (i) a first soluble 0X40 binding domain, (ii) a first peptide linker,
• a second soluble 0X40 binding domain (iii) a second soluble 0X40 binding domain, (iv) a second peptide linker, and (v) a third soluble 0X40 binding domain, further comprising an additional domain at the N-terminal and/or C-terminal end, and wherein the additional domain is a Fab or Fc fragment domain.
• the 0X40 agonist is a 0X40 agonistic single-chain fusion polypeptide comprising (i) a first soluble 0X40 binding domain, (ii) a first peptide linker, (iii) a second soluble 0X40 binding domain, (iv) a second peptide linker, and (v) a third soluble 0X40 binding domain, further comprising an additional domain at the N-terminal and/or C-terminal end, wherein the additional domain is a Fab or Fc fragment domain wherein each of the soluble 0X40 binding domains lacks a stalk region (which contributes to trimerisation and provides a certain distance to the cell membrane, but is not part of the 0X40 binding domain) and the first and the second peptide linkers independently have a length of 3-8 amino acids.
• the 0X40 agonist is an 0X40 agonistic single-chain fusion polypeptide comprising (i) a first soluble tumor necrosis factor (TNF) superfamily cytokine domain, (ii) a first peptide linker, (iii) a second soluble TNF superfamily cytokine domain,
• TNF tumor necrosis factor
• each of the soluble TNF superfamily cytokine domains lacks a stalk region and the first and the second peptide linkers independently have a length of 3-8 amino acids, and wherein the TNF superfamily cytokine domain is an 0X40 binding domain.
• the 0X40 agonist is MEDI6383.
• MEDI6383 is an 0X40 agonistic fusion protein and can be prepared as described in U.S. Patent No. 6,312,700, the disclosure of which is incorporated by reference herein.
• the 0X40 agonist is an 0X40 agonistic scFv antibody comprising any of the foregoing VH domains linked to any of the foregoing VL domains.
• the 0X40 agonist is Creative Biolabs 0X40 agonist monoclonal antibody MOM-18455, commercially available from Creative Biolabs, Inc., Shirley, NY, USA.
• the 0X40 agonist is 0X40 agonistic antibody clone Ber-ACT35 commercially available from BioLegend, Inc., San Diego, CA, USA.
• a cell viability assay can be performed after the first expansion
• a trypan blue exclusion assay can be done on a sample of the bulk TILs, which selectively labels dead cells and allows a viability assessment.
• Other assays for use in testing viability can include but are not limited to the Alamar blue assay; and the MTT assay.
• cell counts and/or viability are measured.
• the expression of markers such as but not limited CD3, CD4, CD8, and CD56, as well as any other disclosed or described herein, can be measured by flow cytometry with antibodies, for example but not limited to those commercially available from BD Bio-sciences (BD Biosciences, San Jose, CA) using a FACSCantoTM flow cytometer (BD Biosciences).
• the cells can be counted manually using a disposable c-chip hemocytometer (VWR, Batavia, IL) and viability can be assessed using any method known in the art, including but not limited to trypan blue staining.
• the cell viability can also be assayed based on USSN 15/863,634, incorporated by reference herein in its entirety.
• the bulk TIL population can be cryopreserved immediately, using the protocols discussed below.
• the bulk TIL population can be subjected to REP and then cryopreserved as discussed below.
• the bulk or REP TIL populations can be subjected to genetic modifications for suitable treatments.
• a method for assaying TILs for viability and/or further use in administration to a subject comprises:
• step (viii) determining based on the ratio in step (vii) whether the thawed population of TILs is suitable for administration to a patient;
• step (ix) administering a therapeutically effective dosage of the thawed third population of TILs to the patient when the ratio of the number of TILs in the fourth population of TILs to the number of TILs in the third population of TILs is determined to be greater than 5: 1 in step (viii).
• the TILs are assayed for viability after step (vii).
• the present disclosure also provides further methods for assaying TILs.
• the disclosure provides a method for assaying TILs comprising:
• step (iv) determining based on the ratio in step (iii) whether the first population of TILs is suitable for use in therapeutic administration to a patient;
• step (v) determining the first population of TILs is suitable for use in therapeutic administration when the ratio of the number of TILs in the second population of TILs to the number of TILs in the first population of TILs is determined to be greater than 5: 1 in step (iv).
• the ratio of the number of TILs in the second population of TILs to the number of TILs in the portion of the first population of TILs is greater than 50: 1.
• the method further comprises performing expansion of the entire first population of cryopreserved TILs from step (i) according to the methods as described in any of the embodiments provided herein.
• the method further comprises administering the entire first population of cryopreserved TILs from step (i) to the patient.
• a method for expanding TILs may include using about 5,000 mL to about 25,000 mL of cell medium, about 5,000 mL to about 10,000 mL of cell medium, or about 5,800 mL to about 8,700 mL of cell medium.
• the media is a serum free medium.
• the media in the first expansion is serum free.
• the media in the second expansion is serum free. .
• the media in the first expansion and the second are both serum free.
• expanding the number of TILs uses no more than one type of cell culture medium.
• any suitable cell culture medium may be used, e.g ., AIM-V cell medium (L-glutamine, 50 mM streptomycin sulfate, and 10 pM gentamicin sulfate) cell culture medium (Invitrogen, Carlsbad CA).
• AIM-V cell medium L-glutamine, 50 mM streptomycin sulfate, and 10 pM gentamicin sulfate cell culture medium (Invitrogen, Carlsbad CA).
• the inventive methods advantageously reduce the amount of medium and the number of types of medium required to expand the number of TIL.
• expanding the number of TIL may comprise feeding the cells no more frequently than every third or fourth day.
• Expanding the number of cells in a gas permeable container simplifies the procedures necessary to expand the number of cells by reducing the feeding frequency necessary to expand the cells.
• the cell medium in the first and/or second gas permeable container is unfiltered.
• the use of unfiltered cell medium may simplify the procedures necessary to expand the number of cells.
• the cell medium in the first and/or second gas permeable container lacks beta-mercaptoethanol (BME).
• the duration of the method comprising obtaining a tumor tissue sample from the mammal; culturing the tumor tissue sample in a first gas permeable container containing cell medium therein; obtaining TILs from the tumor tissue sample; expanding the number of TILs in a second gas permeable container containing cell medium for a duration of about 7 to 14 days, e.g ., about 11 days.
• pre-REP is about 7 to 14 days, e.g. , about 11 days.
• REP is about 7 to 14 days, e.g. , about 11 days.
• TILs are expanded in gas-permeable containers.
• Gas-permeable containers have been used to expand TILs using PBMCs using methods, compositions, and devices known in the art, including those described in U.S. Patent Application Publication No. 2005/0106717 Al, the disclosures of which are incorporated herein by reference.
• TILs are expanded in gas-permeable bags.
• TILs are expanded using a cell expansion system that expands TILs in gas permeable bags, such as the Xuri Cell Expansion System W25 (GE Healthcare).
• TILs are expanded using a cell expansion system that expands TILs in gas permeable bags, such as the WAVE Bioreactor System, also known as the Xuri Cell Expansion System W5 (GE Healthcare).
• the cell expansion system includes a gas permeable cell bag with a volume selected from the group consisting of about 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, and about 10 L.
• TILs can be expanded in G-Rex flasks (commercially available from Wilson Wolf Manufacturing). Such embodiments allow for cell populations to expand from about 5xl0 5 cells/cm 2 to between lOxlO 6 and 30xl0 6 cells/cm 2 . In an embodiment this is without feeding. In an embodiment, this is without feeding so long as medium resides at a height of about 10 cm in the G-Rex flask. In an embodiment this is without feeding but with the addition of one or more cytokines. In an embodiment, the cytokine can be added as a bolus without any need to mix the cytokine with the medium.
• the TILs are optionally genetically engineered to include additional functionalities, including, but not limited to, a high-affinity T cell receptor (TCR), e.g ., a TCR targeted at a tumor-associated antigen such as MAGE-l, HER2, or NY-ESO-l, or a chimeric antigen receptor (CAR) which binds to a tumor-associated cell surface molecule (e.g, mesothelin) or lineage-restricted cell surface molecule (e.g, CD 19).
• TCR high-affinity T cell receptor
• CAR chimeric antigen receptor
• cryopreservation can occur at numerous points throughout the TIL expansion process.
• the expanded population of TILs after the second expansion (as provided for example, according to Step D of Figure 1) can be cryopreserved.
• Cryopreservation can be generally accomplished by placing the TIL population into a freezing solution, e.g, 85% complement inactivated AB serum and 15% dimethyl sulfoxide (DMSO). The cells in solution are placed into cryogenic vials and stored for 24 hours at -80 °C, with optional transfer to gaseous nitrogen freezers for cryopreservation. See, Sadeghi, et al, Acta
• the TILs are cryopreserved in 5% DMSO. In some embodiments, the TILs are cryopreserved in cell culture media plus 5% DMSO. In some embodiments, the TILs are cryopreserved according to the methods provided in Examples F and G.
• the cells are removed from the freezer and thawed in a 37 °C water bath until approximately 4/5 of the solution is thawed.
• the cells are generally resuspended in complete media and optionally washed one or more times.
• the thawed TILs can be counted and assessed for viability as is known in the art.
• the present invention provides for the use of closed systems during the TIL culturing process.
• closed systems allow for preventing and/or reducing microbial contamination, allow for the use of fewer flasks, and allow for cost reductions.
• the closed system uses two containers.
• Sterile connecting devices produce sterile welds between two pieces of compatible tubing. This procedure permits sterile connection of a variety of containers and tube diameters.
• the closed systems include luer lock and heat sealed systems as described in for example, Example G.
• the closed system is accessed via syringes under sterile conditions in order to maintain the sterility and closed nature of the system.
• a closed system as described in Example G is employed.
• the TILs are formulated into a final product formulation container according to the method described in Example G, section“Final Formulation and Fill”.
• the closed system uses one container from the time the tumor fragments are obtained until the TILs are ready for administration to the patient or cryopreserving.
• the first container is a closed G-container and the population of TILs is centrifuged and transferred to an infusion bag without opening the first closed G-container.
• the infusion bag is a HypoThermosol-containing infusion bag.
• a closed system or closed TIL cell culture system is characterized in that once the tumor sample and/or tumor fragments have been added, the system is tightly sealed from the outside to form a closed environment free from the invasion of bacteria, fungi, and/or any other microbial
• the reduction in microbial contamination is between about 5% and about 100%. In some embodiments, the reduction in microbial contamination is between about 5% and about 95%. In some embodiments, the reduction in microbial contamination is between about 5% and about 90%. In some embodiments, the reduction in microbial contamination is between about 10% and about 90%. In some embodiments, the reduction in microbial contamination is between about 15% and about 85%.
• the reduction in microbial contamination is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or about 100%.
• the closed system allows for TIL growth in the absence and/or with a significant reduction in microbial contamination.
• pH, carbon dioxide partial pressure and oxygen partial pressure of the TIL cell culture environment each vary as the cells are cultured. Consequently, even though a medium appropriate for cell culture is circulated, the closed environment still needs to be constantly maintained as an optimal environment for TIL proliferation. To this end, it is desirable that the physical factors of pH, carbon dioxide partial pressure and oxygen partial pressure within the culture liquid of the closed environment be monitored by means of a sensor, the signal whereof is used to control a gas exchanger installed at the inlet of the culture environment, and the that gas partial pressure of the closed environment be adjusted in real time according to changes in the culture liquid so as to optimize the cell culture environment.
• the present invention provides a closed cell culture system which incorporates at the inlet to the closed environment a gas exchanger equipped with a monitoring device which measures the pH, carbon dioxide partial pressure and oxygen partial pressure of the closed environment, and optimizes the cell culture environment by automatically adjusting gas concentrations based on signals from the monitoring device.
• the pressure within the closed environment is continuously or intermittently controlled. That is, the pressure in the closed environment can be varied by means of a pressure maintenance device for example, thus ensuring that the space is suitable for growth of TILs in a positive pressure state, or promoting exudation of fluid in a negative pressure state and thus promoting cell proliferation.
• optimal culture components for proliferation of the TILs can be substituted or added, and including factors such as IL-2 and/or OKT3, as well as combination, can be added.
• Either the bulk TIL population or the expanded population of TILs can be optionally cryopreserved.
• cryopreservation occurs on therapeutic TIL population.
• cryopreservation occurs on the TILs harvested after the second expansion.
• cryopreservation occurs on the TILs in exemplary Step F of Figure 1.
• the TILs are cryopreserved in the infusion bag.
• the TILs are cryopreserved prior to placement in an infusion bag.
• the TILs are cryopreserved and not placed in an infusion bag.
• cryopreservation is performed using a cryopreservation medium.
• the cryopreservation media contains dimethylsulfoxide (DMSO). This is generally accomplished by putting the TIL population into a freezing solution, e.g. 85% complement inactivated AB serum and 15% dimethyl sulfoxide (DMSO). The cells in solution are placed into cryogenic vials and stored for 24 hours at -80 °C, with optional transfer to gaseous nitrogen freezers for cryopreservation. See, Sadeghi, et al, Acta
• the cells are removed from the freezer and thawed in a 37 °C water bath until approximately 4/5 of the solution is thawed.
• the cells are generally resuspended in complete media and optionally washed one or more times.
• the thawed TILs can be counted and assessed for viability as is known in the art.
• a population of TILs is cryopreserved using CS10 cryopreservation media (CryoStor 10, BioLife Solutions).
• a population of TILs is cryopreserved using a cryopreservation media containing
• DMSO dimethylsulfoxide
• a population of TILs is cryopreserved using about a 1 : 1 (voLvol) ratio of CS10 and cell culture media, further comprising additional IL-2.
• cryopreservation can occur at numerous points throughout the TIL expansion process.
• the bulk TIL population after the first expansion according to Step B or the expanded population of TILs after the one or more second expansions according to Step D can be cryopreserved.
• Cryopreservation can be generally accomplished by placing the TIL population into a freezing solution, e.g ., 85% complement inactivated AB serum and 15% dimethyl sulfoxide (DMSO). The cells in solution are placed into cryogenic vials and stored for 24 hours at -80 °C, with optional transfer to gaseous nitrogen freezers for cryopreservation. See Sadeghi, et al, Acta
• the cells are removed from the freezer and thawed in a 37 °C water bath until approximately 4/5 of the solution is thawed.
• the cells are generally resuspended in complete media and optionally washed one or more times.
• the thawed TILs can be counted and assessed for viability as is known in the art.
• the Step B TIL population can be cryopreserved immediately, using the protocols discussed below.
• the bulk TIL population can be subjected to Step C and Step D and then cryopreserved after Step D.
• the Step B or Step D TIL populations can be subjected to genetic modifications for suitable treatments.
• TILs expanded using the methods of the present disclosure are administered to a patient as a pharmaceutical composition.
• the pharmaceutical composition is a suspension of TILs in a sterile buffer.
• TILs expanded using PBMCs of the present disclosure may be administered by any suitable route as known in the art.
• the T-cells are administered as a single intra-arterial or intravenous infusion, which preferably lasts approximately 30 to 60 minutes.
• Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic
• any suitable dose of TILs can be administered.
• from about 2.3> ⁇ l0 10 to about 137 10 10 TILs are administered, with an average of around 78 10 10 TILs, particularly if the cancer is NSCLC.
• about 1.2* 10 10 to about 4.3 x 10 10 of TILs are administered.
• about 3xl0 10 to about l2xl0 10 TILs are administered.
• about 4xl0 10 to about l0xl0 10 TILs are administered.
• about 5xl0 10 to about 8xl0 10 TILs are administered.
• about 6xl0 10 to about 8xl0 10 TILs are administered.
• about 7xl0 10 to about 8xl0 10 TILs are administered.
• therapeutically effective dosage is about 2.3xl0 10 to about 13.7 c 10 10 .
• therapeutically effective dosage is about 7.8x 10 10 TILs, particularly of the cancer is NSCLC.
• therapeutically effective dosage is about L2xl0 10 to about 4.3 xlO 10 of TILs.
• therapeutically effective dosage is about 3xl0 10 to about l2xl0 10 TILs.
• therapeutically effective dosage is about 4xl0 10 to about IO c IO 10
• therapeutically effective dosage is about 5xl0 10 to about 8xl0 10
• therapeutically effective dosage is about 6xl0 10 to about 8xl0 10
• therapeutically effective dosage is about 7xl0 10 to about 8xl0 10
• therapeutically effective dosage is about lxl0 9 to about l50xl0 9 TILs. In some embodiments, therapeutically effective dosage is about lxl0 9 to about l40xl0 9 TILs. In some embodiments, therapeutically effective dosage is about lxl0 9 to about l30xl0 9 TILs. In some embodiments, therapeutically effective dosage is about lxl0 9 to about l20xl0 9 TILs. In some embodiments, therapeutically effective dosage is about lxl0 9 to about H0xl0 9 TILs.
• therapeutically effective dosage is about lxl0 9 to about l00xl0 9 TILs. In some embodiments, therapeutically effective dosage is about lxl0 9 to about 90xl0 9 TILs. In some embodiments, therapeutically effective dosage is about lxl0 9 to about 80xl0 9 TILs. In some embodiments, therapeutically effective dosage is about lxl0 9 to about 70xl0 9 TILs. In some embodiments, therapeutically effective dosage is about lxl0 9 to about 60xl0 9 TILs. In some embodiments, therapeutically effective dosage is about lxl0 9 to about 50xl0 9 TILs.
• therapeutically effective dosage is about lxl0 9 to about 40xl0 9 TILs. In some embodiments, therapeutically effective dosage is about lxl0 9 to about 30xl0 9 TILs. In some embodiments, therapeutically effective dosage is about lxl0 9 to about 20xl0 9 TILs. In some embodiments, therapeutically effective dosage is about lxl0 9 to about l0xl0 9 TILs. In some embodiments, therapeutically effective dosage is about lxl0 9 to about 5xl0 9 TILs.
• the number of the TILs provided in the pharmaceutical compositions of the invention is about lxlO 6 , 2xl0 6 , 3xl0 6 , 4xl0 6 , 5xl0 6 , 6xl0 6 , 7xl0 6 , 8 c 10 6 , 9 c 10 6 , I c IO 7 , 2 c 10 7 , 3 c 10 7 , 4 c 10 7 , 5 c 10 7 , 6 c 10 7 , 7 c 10 7 , 8 c 10 7 , 9 c 10 7 , I c IO 8 , 2xl0 8 , 3 c 10 8 , 4 c 10 8 , 5 c 10 8 , 6 c 10 8 , 7 c 10 8 , 8 c 10 8 , 9 c 10 8 , I c IO 9 , 2 c 10 9 , 3 c 10 9 , 4 c 10 9 , 5 c 10 9 , 2 c 10 9 , 4
• the number of the TILs provided in the pharmaceutical compositions of the invention is in the range of 1 x 10 6 to 5 c 10 6 , 5 c 10 6 to lxlO 7 , lxlO 7 to 5xl0 7 , 5xl0 7 to lxlO 8 , lxlO 8 to 5xl0 8 , 5xl0 8 to lxlO 9 , lxlO 9 to 5 c 10 9 , 5 c 10 9 to lxlO 10 , lxlO 10 to 5xl0 10 , 5xl0 10 to lxlO 11 , 5xl0 u to lxlO 12 , lxlO 12 to 5 c 10 12 , and 5xl0 12 to lxlO 13 .
• the concentration of the TILs provided in the pharmaceutical compositions of the invention is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v of the pharmaceutical composition.
• the concentration of the TILs provided in the pharmaceutical compositions of the invention is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25% 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%, 13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%, 10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%, 5.25% 5%,
• the concentration of the TILs provided in the pharmaceutical compositions of the invention is in the range from about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about 0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12% or about 1% to about 10% w/w, w/v or v/v of the pharmaceutical composition.
• the concentration of the TILs provided in the pharmaceutical compositions of the invention is in the range from about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w/w, w/v or v/v of the pharmaceutical composition.
• the amount of the TILs provided in the pharmaceutical compositions of the invention is equal to or less than 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01
• the amount of the TILs provided in the pharmaceutical compositions of the invention is more than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004 g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065
• the TILs provided in the pharmaceutical compositions of the invention are effective over a wide dosage range.
• the exact dosage will depend upon the route of administration, the form in which the compound is administered, the gender and age of the subject to be treated, the body weight of the subject to be treated, and the preference and experience of the attending physician.
• the clinically-established dosages of the TILs may also be used if appropriate.
• the amounts of the pharmaceutical compositions administered using the methods herein, such as the dosages of TILs, will be dependent on the human or mammal being treated, the severity of the disorder or condition, the rate of administration, the disposition of the active pharmaceutical ingredients and the discretion of the prescribing physician.
• TILs may be administered in a single dose.
• TILs may be administered in multiple doses. Dosing may be once, twice, three times, four times, five times, six times, or more than six times per year. Dosing may be once a month, once every two weeks, once a week, or once every other day. Administration of TILs may continue as long as necessary.
• an effective dosage of TILs is about 1 c 10 6 , 2x 10 6 , 3 c 10 6 , 4 c 10 6 , 5 c 10 6 , 6 c 10 6 , 7 c 10 6 , 8 c 10 6 , 9 c 10 6 , I c IO 7 , 2 c 10 7 , 3 c 10 7 , 4 c 10 7 , 5 c 10 7 , 6 c 10 7 , 7xl0 7 , 8 c 10 7 , 9 c 10 7 , I c IO 8 , 2 c 10 8 , 3 c 10 8 , 4 c 10 8 , 5 c 10 8 , 6 c 10 8 , 7 c 10 8 , 8 c 10 8 , 9 c 10 8 , I c IO 9 , 2xl0 9 , 3 c 10 9 , 4 c 10 9 , 5 c 10 8 , 6 c 10 8 , 7 c 10 8 , 8 c 10 8
• an effective dosage of TILs is in the range of 1 c 10 6 to 5 c 10 6 , 5xl0 6 to lxlO 7 , lxlO 7 to 5 c 10 7 , 5 c 10 7 to lxlO 8 , lxlO 8 to 5 c 10 8 , 5xl0 8 to lxlO 9 , lxlO 9 to 5 c 10 9 , 5xl0 9 to lxlO 10 , lxlO 10 to 5 c 10 10 , 5xl0 10 to lxlO 11 , 5xl0 u to lxlO 12 , lxlO 12 to 5xl0 12 , and5xl0 12 to lxlO 13 .
• an effective dosage of TILs is in the range of about 0.01 mg/kg to about 4.3 mg/kg, about 0.15 mg/kg to about 3.6 mg/kg, about 0.3 mg/kg to about 3.2 mg/kg, about 0.35 mg/kg to about 2.85 mg/kg, about 0.15 mg/kg to about 2.85 mg/kg, about 0.3 mg to about 2.15 mg/kg, about 0.45 mg/kg to about 1.7 mg/kg, about 0.15 mg/kg to about 1.3 mg/kg, about 0.3 mg/kg to about 1.15 mg/kg, about 0.45 mg/kg to about 1 mg/kg, about 0.55 mg/kg to about 0.85 mg/kg, about 0.65 mg/kg to about 0.8 mg/kg, about 0.7 mg/kg to about 0.75 mg/kg, about 0.7 mg/kg to about 2.15 mg/kg, about 0.85 mg/kg to about 2 mg/kg, about 1 mg/kg to about 1.85 mg/kg, about 1.15 mg/kg to about 1.7 mg/kg,
• an effective dosage of TILs is in the range of about 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25 mg to about 200 mg, about 1 mg to about 50 mg, about 5 mg to about 45 mg, about 10 mg to about 40 mg, about 15 mg to about 35 mg, about 20 mg to about 30 mg, about 23 mg to about 28 mg, about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg to about 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg, or about 95 mg to about 105 mg, about 98 mg to about 102 mg, about 150 mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about 230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg, about 195 mg to about 205 mg, or about 198 to about 207 mg.
• An effective amount of the TILs may be administered in either single or multiple doses by any of the accepted modes of administration of agents having similar utilities, including intranasal and transdermal routes, by intra-arterial injection, intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, topically, by transplantation, or by inhalation.
• Methods of treatment begin with the initial TIL collection and culture of TILs. Such methods have been both described in the art by, for example, Jin et al, ./. Immunotherapy , 2012, 35(3):283-292, incorporated by reference herein in its entirety. Embodiments of methods of treatment are described throughout the sections below, including the Examples.
• the expanded TILs produced according the methods described herein, including for example as described in Steps A through F above or according to Steps A through F above (also as shown, for example, in Figure 1) find particular use in the treatment of patients with cancer (for example, as described in Goff, et al., J. Clinical Oncology , 2016, 34(20):2389- 239, as well as the supplemental content; incorporated by reference herein in its entirety.
• TIL were grown from resected deposits of metastatic melanoma as previously described (see, Dudley, et al., J Immunother ., 2003, 26:332-342; incorporated by reference herein in its entirety). Fresh tumor can be dissected under sterile conditions.
• a representative sample can be collected for formal pathologic analysis. Single fragments of 2 mm 3 to 3 mm 3 may be used. In some embodiments, 5, 10, 15, 20, 25 or 30 samples per patient are obtained. In some embodiments, 20, 25, or 30 samples per patient are obtained. In some embodiments, 20, 22, 24, 26, or 28 samples per patient are obtained. In some embodiments, 24 samples per patient are obtained. Samples can be placed in individual wells of a 24-well plate, maintained in growth media with high-dose IL-2 (6,000 IU/mL), and monitored for destruction of tumor and/or proliferation of TIL. Any tumor with viable cells remaining after processing can be enzymatically digested into a single cell suspension and cryopreserved, as described herein.
• successfully grown TIL can be sampled for phenotype analysis (CD3, CD4, CD8, and CD56) and tested against autologous tumor when available. TIL can be considered reactive if overnight coculture yielded interferon-gamma (IFN-g) levels > 200 pg/mL and twice background. (Goff, et al., J Immunother., 2010, 33:840-847; incorporated by reference herein in its entirety).
• cultures with evidence of autologous reactivity or sufficient growth patterns can be selected for a second expansion (for example, a second expansion as provided in according to Step D of Figure 1), including second expansions that are sometimes referred to as rapid expansion (REP).
• a second expansion for example, a second expansion as provided in according to Step D of Figure 1
• expanded TILs with high autologous reactivity are selected for an additional second expansion.
• TILs with high autologous reactivity for example, high proliferation during second expansion as provided in Step D of Figure 1
• TILs with high autologous reactivity are selected for an additional second expansion according to Step D of Figure 1.
• TILs Cell phenotypes of cryopreserved samples of infusion bag TIL can be analyzed by flow cytometry (e.g. , FlowJo) for surface markers CD3, CD4, CD8, CCR7, and CD45RA (BD BioSciences), as well as by any of the methods described herein. Serum cytokines were measured by using standard enzyme-linked immunosorbent assay techniques. A rise in serum IFN-g was defined as >100 pg/mL and greater than 4 3 baseline levels. [00590] In some embodiments, the TILs produced by the methods provided herein, for example those exemplified in Figure 1, provide for a surprising improvement in clinical efficacy of the TILs.
• flow cytometry e.g. , FlowJo
• Serum cytokines were measured by using standard enzyme-linked immunosorbent assay techniques. A rise in serum IFN-g was defined as >100 pg/mL and greater than 4 3 baseline levels.
• the TILs produced by the methods provided herein exhibit increased clinical efficacy as compared to TILs produced by methods other than those described herein, including for example, methods other than those exemplified in Figure 1.
• the methods other than those described herein include methods referred to as process 1C and/or Generation 1 (Gen 1).
• the increased efficacy is measured by DCR, ORR, and/or other clinical responses.
• the TILS produced by the methods provided herein exhibit a similar time to response and safety profile compared to TILs produced by methods other than those described herein, including for example, methods other than those exemplified in Figure 1, for example the Gen 1 process.
• IFN-gamma is indicative of treatment efficacy and/or increased clinical efficacy.
• IFN-g in the blood of subjects treated with TILs is indicative of active TILs.
• a potency assay for IFN-g production is employed.
• IFN-g production is another measure of cytotoxic potential. IFN-g production can be measured by determining the levels of the cytokine IFN-g in the blood, serum, or TILs ex vivo of a subject treated with TILs prepared by the methods of the present invention, including those as described for example in Figure 1. In some
• an increase in IFN-g is indicative of treatment efficacy in a patient treated with the TILs produced by the methods of the present invention.
• IFN-g is increased one-fold, two-fold, three-fold, four-fold, or five-fold or more as compared to an untreated patient and/or as compared to a patient treated with TILs prepared using other methods than those provide herein including for example, methods other than those embodied in Figure 1.
• IFN-g secretion is increased one-fold as compared to an untreated patient and/or as compared to a patient treated with TILs prepared using other methods than those provide herein including for example, methods other than those embodied in Figure 1.
• IFN-g secretion is increased two-fold as compared to an untreated patient and/or as compared to a patient treated with TILs prepared using other methods than those provide herein including for example, methods other than those embodied in Figure 1. In some embodiments, IFN-g secretion is increased three-fold as compared to an untreated patient and/or as compared to a patient treated with TILs prepared using other methods than those provide herein including for example, methods other than those embodied in Figure 1. In some embodiments, IFN-g secretion is increased four-fold as compared to an untreated patient and/or as compared to a patient treated with TILs prepared using other methods than those provide herein including for example, methods other than those embodied in Figure 1.
• IFN-g secretion is increased five-fold as compared to an untreated patient and/or as compared to a patient treated with TILs prepared using other methods than those provide herein including for example, methods other than those embodied in Figure 1.
• IFN-g is measured using a Quantikine ELISA kit.
• IFN-g is measured in TILs ex vivo of a subject treated with TILs prepared by the methods of the present invention, including those as described for example in Figure 1.
• IFN-g is measured in blood of a subject treated with TILs prepared by the methods of the present invention, including those as described for example in Figure 1.
• IFN-g is measured in TILs serum of a subject treated with TILs prepared by the methods of the present invention, including those as described for example in Figure 1.
• the TILs prepared by the methods of the present invention exhibit increased polyclonality as compared to TILs produced by other methods, including those not exemplified in Figure 1, such as for example, methods referred to as process 1C methods.
• significantly improved polyclonality and/or increased polyclonality is indicative of treatment efficacy and/or increased clinical efficacy.
• polyclonality refers to the T-cell repertoire diversity.
• an increase in polyclonality can be indicative of treatment efficacy with regard to administration of the TILs produced by the methods of the present invention.
• polyclonality is increased one-fold, two-fold, ten-fold, lOO-fold, 500-fold, or 1000-fold as compared to TILs prepared using methods than those provide herein including for example, methods other than those embodied in Figure 1.
• polyclonality is increased one-fold as compared to an untreated patient and/or as compared to a patient treated with TILs prepared using other methods than those provide herein including for example, methods other than those embodied in Figure 1.
• polyclonality is increased two-fold as compared to an untreated patient and/or as compared to a patient treated with TILs prepared using other methods than those provide herein including for example, methods other than those embodied in Figure 1.
• polyclonality is increased ten-fold as compared to an untreated patient and/or as compared to a patient treated with TILs prepared using other methods than those provide herein including for example, methods other than those embodied in Figure 1.
• polyclonality is increased lOO-fold as compared to an untreated patient and/or as compared to a patient treated with TILs prepared using other methods than those provide herein including for example, methods other than those embodied in Figure 1.
• polyclonality is increased 500-fold as compared to an untreated patient and/or as compared to a patient treated with TILs prepared using other methods than those provide herein including for example, methods other than those embodied in Figure 1.
• polyclonality is increased lOOO-fold as compared to an untreated patient and/or as compared to a patient treated with TILs prepared using other methods than those provide herein including for example, methods other than those embodied in Figure 1.
• Measures of efficacy can include the disease control rate (DCR) as well as overall response rate (ORR), as known in the art as well as described herein.
• DCR disease control rate
• ORR overall response rate
• compositions and methods described herein can be used in a method for treating non-small-cell lung cancer (NSCLC), wherein the NSCLC is refractory to treatment with an anti-PD-l antibody.
• the anti-PD-l antibody includes, e.g ., but is not limited to nivolumab (BMS-936558, Bristol-Myers Squibb; Opdivo®), pembrolizumab (lambrolizumab, MK03475 or MK-3475, Merck; Keytruda®), ipilimumab (Yervoy®), humanized anti-PD-l antibody JS001 (ShangHai JunShi), monoclonal anti-PD-l antibody TSR-042 (Tesaro, Inc.), Pidilizumab (anti-PD-l mAh CT-011, Medivation), anti-PD-l monoclonal Antibody BGB-A317 (BeiGene), and/or anti-PD-l antibody.
• the PD-l antibody is from clone:
• RMP1-14 (rat IgG) - BioXcell cat# BP0146.
• Other suitable antibodies suitable for use in co administration methods with TILs produced according to Steps A through F as described herein are anti-PD-l antibodies disclosed in U.S. Patent No. 8,008,449, herein incorporated by reference.
• the antibody or antigen-binding portion thereof binds specifically to PD-L1 and inhibits its interaction with PD-l, thereby increasing immune activity.
• Any antibodies known in the art which bind to PD-L1 and disrupt the interaction between the PD-l and PD-L1, and stimulates an anti-tumor immune response, are include.
• antibodies that target PD-L1 and are in clinical trials include BMS-936559 (Bristol-Myers Squibb) and MPDL3280A (Genentech).
• BMS-936559 Bristol-Myers Squibb
• MPDL3280A Genetech
• Other suitable antibodies that target PD-L1 are disclosed in U.S. Patent No. 7,943,743, herein incorporated by reference. It will be understood by one of ordinary skill that any antibody which binds to PD-l or PD-L1, disrupts the PD-1/PD-L1 interaction, and stimulates an anti-tumor immune response, are included.
• the NSCLC has been treated with an anti -PD-l antibody. In some embodiments, the NSCLC has been treated with an anti-PD-Ll antibody.
• the NSCLC subject is treatment naive. In some embodiments, the NSCLC has not been treated with an anti-PD-l antibody. In some embodiments, the NSCLC has not been treated with an anti-PD-Ll antibody. In some embodiments, the NSCLC has been previously treated with a chemotherapeutic agent. In some embodiments, the NSCLC has been previously treated with a chemotherapeutic agent but is no longer being treated with the chemotherapeutic agent. In some embodiments, the NSCLC patient is anti-PD-l/PD-Ll naive. In some embodiments, the NSCLC subject has low expression of PD-L1.
• the NSCLC subject has treatment naive NSCLC or is post-chemotherapeutic treatment but anti-PD-l/PD-Ll naive. In some embodiments, the NSCLC subject is treatment naive NSCLC or post-chemotherapuetic treatment but anti-PD-l/PD-Ll naive and has low expression of PD-L1. In some embodiments, the NSCLC subject has bulky disease at baseline. In some embodiments, the subject has bulky disease at baseline and has low expression of PD-L1.
• the NSCLC subject has treatment naive NSCLC or post chemotherapy but anti-PD-l/PD-Ll naive who have low expression of PD- Ll and/or have bulky disease at baseline.
• bulky disease is indicated where the maximal tumor diameter is greater than 7 cm measured in either the transverse or coronal plane.
• bulky disease is indicated when there are swollen lymph nodes with a short-axis diameter of 20 mm or greater.
• the chemotherapeutic includes a standard of care therapeutic for NSCLC.
• the TILs prepared by the methods of the present invention exhibit increased polyclonality as compared to TILs produced by other methods, including those not exemplified in Figure 1, such as for example, methods referred to as process 1C methods.
• significantly improved polyclonality and/or increased polyclonality is indicative of treatment efficacy and/or increased clinical efficacy for cancer treatment.
• polyclonality refers to the T-cell repertoire diversity.
• an increase in polyclonality can be indicative of treatment efficacy with regard to administration of the TILs produced by the methods of the present invention.
• polyclonality is increased one-fold, two-fold, ten-fold, lOO-fold, 500-fold, or 1000-fold as compared to TILs prepared using methods than those provide herein including for example, methods other than those embodied in Figure 1.
• polyclonality is increased one-fold as compared to an untreated patient and/or as compared to a patient treated with TILs prepared using other methods than those provide herein including for example, methods other than those embodied in Figure 1.
• polyclonality is increased two-fold as compared to an untreated patient and/or as compared to a patient treated with TILs prepared using other methods than those provide herein including for example, methods other than those embodied in Figure 1.
• polyclonality is increased ten-fold as compared to an untreated patient and/or as compared to a patient treated with TILs prepared using other methods than those provide herein including for example, methods other than those embodied in Figure 1.
• polyclonality is increased lOO-fold as compared to an untreated patient and/or as compared to a patient treated with TILs prepared using other methods than those provide herein including for example, methods other than those embodied in Figure 1.
• polyclonality is increased 500-fold as compared to an untreated patient and/or as compared to a patient treated with TILs prepared using other methods than those provide herein including for example, methods other than those embodied in Figure 1.
• polyclonality is increased lOOO-fold as compared to an untreated patient and/or as compared to a patient treated with TILs prepared using other methods than those provide herein including for example, methods other than those embodied in Figure 1.
• the subject to be treated has Stage III or Stage IV NSCLC (squamous, adenocarcinoma, large cell carcinoma). In some embodiments, the subject to be treated has Stage III NSCLC (squamous, adenocarcinoma, large cell
• the subject to be treated has Stage IV NSCLC (squamous, adenocarcinoma, large cell carcinoma).
• the subject to be treated has an oncogene-driven tumor and had been treated with at least one effective targeted therapy directed toward the oncogene.
• the subject to be treated has Stage III or Stage IV NSCLC (squamous, adenocarcinoma, large cell carcinoma) and were previously treated with for example, a PD-l inhibitor or PD-L1 inhibitor, such as for example, for example, anti-PD- 1 and/or anti-PD-Ll.
• the subject to be treated has Stage III NSCLC (squamous, adenocarcinoma, large cell carcinoma) and were previously treated with systemic therapy including for example, anti-PD-l and/or anti-PD-Ll.
• the subject to be treated has Stage IV NSCLC (squamous, adenocarcinoma, large cell carcinoma) and were previously treated with systemic therapy including for example, anti-PD-l and/or anti-PD-Ll.
• the subject to be treated has Stage III NSCLC (squamous, adenocarcinoma, large cell carcinoma) and were previously treated with systemic therapy including anti-PD-l.
• the subject to be treated has Stage IV NSCLC (squamous, adenocarcinoma, large cell carcinoma) and were previously treated with systemic therapy including anti-PD-l.
• the subject to be treated has Stage III NSCLC (squamous, adenocarcinoma, large cell carcinoma) and were previously treated with systemic therapy including anti-PD-Ll.
• the subject to be treated has Stage IV NSCLC (squamous, adenocarcinoma, large cell carcinoma) and were previously treated with systemic therapy including anti-PD-Ll.
• the subject to be treated has an oncogene-driven tumor and had been treated with at least one effective targeted therapy directed toward the oncogene.
• the subject to be treated has a histologically or pathologically confirmed diagnosis of Stage III or Stage IV NSCLC (squamous, nonsquamous,
• adenocarcinoma large cell carcinoma
• the subject to be treated are immunotherapy naive.
• the subject to be treated has may have received up to 3 prior systemic anticancer therapies, including for example, systemic therapy in the adjuvant or neoadjuvant setting, or as part of definitive chemoradiotherapy.
• the subject to be treated has oncogene mutations, including for example mutations in EGFR, ALK, and/or ROS.
• the subject to be treated had previously received systemic therapy with for example, a PD-l inhibitor or PD-L1 inhibitor, such as for example, anti-PD- 1 and/or anti-PD-Ll, as part of ⁇ 3 prior lines of systemic therapy.
• the subject to be treated has oncogene mutations, including for example mutations in EGFR, ALK, and/or ROS.
• the subject to be treated has at least 1 resectable lesion (or aggregate lesions) of at least about 1.5 cm in diameter post-resection for use in TIL preparation.
• the subject to be treated had a washout period from one or more prior anticancer therapies of a minimum duration, prior to the first study treatment (i.e., start of nonmyeloablative lymphodepletion (NMA-LD) or pembrolizumab)
• the subject to be treated had prior targeted therapy with an epidermal growth factor receptor (EGFR), MEK, BRAF, ALK, ROS1 and/or other-targeted agents (including, for example, erlotinib, afatinib, dacomitinib, osimertinib, crizotinib, ceritinib, and/or lorlatinib) and a minimum washout of prior treatment of at least 14 days prior to the start of TIL treatment.
• EGFR epidermal growth factor receptor
• the subject to be treated had adjuvant, neoadjuvant or definitive chemotherapy and/or chemoradiation and a minimum washout of prior treatment of at least 21 days prior to the start of treatment.
• the subject to be treated had prior checkpoint-targeted therapy with for example, a PD-l inhibitor or PD-L1 inhibitor, such as for example, an anti- PD-l, and anti-PD-Ll, other mAbs, and/or vaccines and a minimum washout period of greater than or equal to 21 days before the start of nonmyeloablative lymphodepletion (NMA-LD).
• a PD-l inhibitor or PD-L1 inhibitor such as for example, an anti- PD-l, and anti-PD-Ll, other mAbs, and/or vaccines and a minimum washout period of greater than or equal to 21 days before the start of nonmyeloablative lymphodepletion (NMA-LD).
• PD-l which is also known as CD279, belongs to the CD28 family, and in humans is encoded by the Pdcdl gene on chromosome 2.
• PD-l consists of one immunoglobulin (Ig) superfamily domain, a transmembrane region, and an intracellular domain containing an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM).
• Ig immunoglobulin
• ITIM immunoreceptor tyrosine-based inhibitory motif
• ITSM immunoreceptor tyrosine-based switch motif
• PD-l and its ligands are known to play a key role in immune tolerance, as described in Keir, et al ., Annu. Rev. Immunol. 2008, 26, 677-704.
• PD-l provides inhibitory signals that negatively regulate T cell immune responses.
• PD-L1 also known as B7-H1 or CD274
• PD-L2 also known as B7-DC or CD273
• PD-L1 is a 290 amino acid transmembrane protein encoded by the Cd274 gene on human chromosome 9.
• Blocking the interaction between PD-l and its ligands PD-L1 and PD-L2 by use of a PD-l inhibitor, a PD-L1 inhibitor, and/or a PD-L2 inhibitor can overcome immune resistance, as demonstrated in recent clinical studies, such as that described in Topalian, et al. , N Eng. J. Med. 2012, 366, 2443-54.
• PD-L1 is expressed on many tumor cell lines, while PD-L2 is expressed is expressed mostly on dendritic cells and a few tumor lines.
• T cells which inducibly express PD-l after activation
• PD-l is also expressed on B cells, natural killer cells, macrophages, activated monocytes, and dendritic cells.
• the PD-l inhibitor may be any PD-l inhibitor or PD-l blocker known in the art. In particular, it is one of the PD-l inhibitors or blockers described in more detail in the following paragraphs.
• the terms“inhibitor,”“antagonist,” and“blocker” are used interchangeably herein in reference to PD-l inhibitors.
• references herein to a PD-l inhibitor that is an antibody may refer to a compound or antigen binding fragments, variants, conjugates, or biosimilars thereof.
• references herein to a PD-l inhibitor may also refer to a small molecule compound or a pharmaceutically acceptable salt, ester, solvate, hydrate, cocrystal, or prodrug thereof.
• the PD-l inhibitor is an antibody (i.e., an anti-PD-l antibody), a fragment thereof, including Fab fragments, or a single-chain variable fragment (scFv) thereof.
• the PD-l inhibitor is a polyclonal antibody.
• the PD-l inhibitor is a monoclonal antibody.
• the PD-l inhibitor competes for binding with PD-l, and/or binds to an epitope on PD-l.
• the antibody competes for binding with PD-l, and/or binds to an epitope on PD-l.
• the PD-l inhibitor is one that binds human PD-l with a KD of about 100 pM or lower, binds human PD-l with a KD of about 90 pM or lower, binds human PD-l with a KD of about 80 pM or lower, binds human PD-l with a KD of about 70 pM or lower, binds human PD-l with a KD of about 60 pM or lower, binds human PD-l with a KD of about 50 pM or lower, binds human PD-l with a KD of about 40 pM or lower, binds human PD-l with a KD of about 30 pM or lower, binds human PD-l with a KD of about 20 pM or lower, binds human PD-l with a KD of about 10 pM or lower, or binds human PD-l with a KD of about 1
• the PD-l inhibitor is one that binds to human PD-l with a kassoc of about 7.5 x 10 5 l/M s or faster, binds to human PD-l with a kassoc of about 7.5 x 10 5 l/M-s or faster, binds to human PD-l with a kassoc of about 8 x 10 5 l/M s or faster, binds to human PD-l with a kassoc of about 8.5 x 10 5 l/M s or faster, binds to human PD-l with a kassoc of about 9 x 10 5 l/M s or faster, binds to human PD-l with a kassoc of about 9.5 c 10 5 l/M s or faster, or binds to human PD-l with a kassoc of about 1 c 10 6 l/
• the PD-l inhibitor is one that binds to human PD-l with a kdissoc of about 2 x 10 5 l/s or slower, binds to human PD-l with a kdissoc of about 2.1 x 10 5 l/s or slower , binds to human PD-l with a kdissoc of about 2.2 x 10 5 l/s or slower, binds to human PD-l with a kdissoc of about 2.3 x 10 5 l/s or slower, binds to human PD-l with a kdissoc of about 2.4 x 10 5 l/s or slower, binds to human PD-l with a kdissoc of about 2.5 x 10 5 l/s or slower, binds to human PD-l with a kdissoc of about 2.6 x 10 5 l/s or slower or
• the PD-l inhibitor is one that blocks or inhibits binding of human PD-L1 or human PD-L2 to human PD-l with an ICso of about 10 nM or lower, blocks or inhibits binding of human PD-L1 or human PD-L2 to human PD-l with an ICso of about 9 nM or lower, blocks or inhibits binding of human PD-L1 or human PD-L2 to human PD-l with an ICso of about 8 nM or lower, blocks or inhibits binding of human PD-L1 or human PD-L2 to human PD-l with an ICso of about 7 nM or lower, blocks or inhibits binding of human PD-L1 or human PD-L2 to human PD-l with an ICso of about 6 nM or lower, blocks or inhibits binding of human PD-L1 or human PD-L2 to human PD-l with an ICso of about 5 nM
• the PD-l inhibitor is nivolumab (commercially available as OPDIVO from Bristol-Myers Squibb Co.), or biosimilars, antigen-binding fragments, conjugates, or variants thereof.
• Nivolumab is a fully human IgG4 antibody blocking the PD- 1 receptor.
• the anti -PD-l antibody is an immunoglobulin G4 kappa, anti- (human CD274) antibody.
• Nivolumab is assigned Chemical Abstracts Service (CAS) registry number 946414-94-4 and is also known as 5C4, BMS-936558, MDX-l 106, and ONO-4538. The preparation and properties of nivolumab are described in U.S.
• Patent No. 8,008,449 and International Patent Publication No. WO 2006/121168 the disclosures of which are incorporated by reference herein.
• the clinical safety and efficacy of nivolumab in various forms of cancer has been described in Wang, et al, Cancer Immunol Res. 2014, 2, 846-56; Page, et al., Ann. Rev. Med., 2014, 65, 185-202; and Weber, et al, J. Clin. Oncology, 2013, 37, 4311-4318, the disclosures of which are incorporated by reference herein.
• the amino acid sequences of nivolumab are set forth in Table 48.
• Nivolumab has intra-heavy chain disulfide linkages at 22-96,140-196, 254-314, 360-418, 22"-96", 140"-196", 254"-314", and 360"-4l8"; intra-light chain disulfide linkages at 23'-88', 134'- 194', 23"'-88"', and 134'"- 194'"; inter-heavy-light chain disulfide linkages at 127-214', 127"-214'", inter-heavy-heavy chain disulfide linkages at 219-219" and 222-222"; and N-glycosylation sites (H CFh 84.4) at 290, 290".
• a PD-l inhibitor comprises a heavy chain given by SEQ ID NO:463 and a light chain given by SEQ ID NO: 128.
• a PD-l inhibitor comprises heavy and light chains having the sequences shown in SEQ ID NO:463 and SEQ ID NO: 128, respectively, or antigen binding fragments, Fab fragments, single-chain variable fragments (scFv), variants, or conjugates thereof.
• a PD-l inhibitor comprises heavy and light chains that are each at least 99% identical to the sequences shown in SEQ ID NO:463 and SEQ ID NO: 128, respectively.
• a PD-l inhibitor comprises heavy and light chains that are each at least 98% identical to the sequences shown in SEQ ID NO:463 and SEQ ID NO: 128, respectively. In an embodiment, a PD-l inhibitor comprises heavy and light chains that are each at least 97% identical to the sequences shown in SEQ ID NO:463 and SEQ ID NO: 128, respectively. In an embodiment, a PD-l inhibitor comprises heavy and light chains that are each at least 96% identical to the sequences shown in SEQ ID NO:463 and SEQ ID NO: 128, respectively. In an embodiment, a PD-l inhibitor comprises heavy and light chains that are each at least 95% identical to the sequences shown in SEQ ID NO:463 and SEQ ID NO: 128, respectively.
• the PD-l inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of nivolumab.
• the PD-l inhibitor heavy chain variable region (VH) comprises the sequence shown in SEQ ID NO: 129
• the PD-l inhibitor light chain variable region (VL) comprises the sequence shown in SEQ ID NO: 130, and conservative amino acid substitutions thereof.
• a PD-l inhibitor comprises VH and VL regions that are each at least 99% identical to the sequences shown in SEQ ID NO: 129 and SEQ ID NO: 130, respectively.
• a PD-l inhibitor comprises VH and VL regions that are each at least 98% identical to the sequences shown in SEQ ID NO: 129 and SEQ ID NO: 130, respectively. In an embodiment, a PD-l inhibitor comprises VH and VL regions that are each at least 97% identical to the sequences shown in SEQ ID NO: 129 and SEQ ID NO: 130, respectively. In an embodiment, a PD-l inhibitor comprises VH and VL regions that are each at least 96% identical to the sequences shown in SEQ ID NO: 129 and SEQ ID NO: 130, respectively. In an embodiment, a PD-l inhibitor comprises VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO: 129 and SEQ ID NO: 130, respectively.
• a PD-l inhibitor comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 131, SEQ ID NO: 132, and SEQ ID NO: 133, respectively, and conservative amino acid substitutions thereof, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 134, SEQ ID NO: 135, and SEQ ID NO: 136, respectively, and conservative amino acid
• the antibody competes for binding with, and/or binds to the same epitope on PD-l as any of the aforementioned antibodies.
• the PD-l inhibitor is an anti -PD-l biosimilar monoclonal antibody approved by drug regulatory authorities with reference to nivolumab.
• the biosimilar comprises an anti -PD-l antibody comprising an amino acid sequence which has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of a reference medicinal product or reference biological product and which comprises one or more post-translational modifications as compared to the reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is nivolumab.
• the one or more post-translational modifications are selected from one or more of: glycosylation, oxidation, deamidation, and truncation.
• the biosimilar is an anti -PD-l antibody authorized or submitted for authorization, wherein the anti-PD-l antibody is provided in a formulation which differs from the formulations of a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is nivolumab.
• the anti-PD-l antibody may be authorized by a drug regulatory authority such as the ET.S. FDA and/or the European Union’s EMA.
• the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is nivolumab.
• the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is nivolumab.
• the PD-l inhibitor comprises pembrolizumab
• Pembrolizumab is assigned CAS registry number 1374853-91-4 and is also known as lambrolizumab, MK-3475, and SCH- 900475. Pembrolizumab has an immunoglobulin G4, anti-(human protein PDCD1
• pembrolizumab may also be described as immunoglobulin G4, anti-(human programmed cell death 1); humanized mouse monoclonal [228-L-proline(HlO-S>P)]Y4 heavy chain (134- 2l8')-disulfide with humanized mouse monoclonal k light chain dimer (226-226" :229-229”)- bisdisulfide.
• the properties, uses, and preparation of pembrolizumab are described in International Patent Publication No. WO 2008/156712 Al, ET.S. Patent No. 8,354,509 and U.S. Patent Application Publication Nos.
• pembrolizumab in various forms of cancer is described in Fuerst, Oncology Times, 2014, 36, 35-36; Robert, et al., Lancet, 2014, 384, 1109-17; and Thomas, et al, Exp. Opin. Biol. Ther., 2014, 14, 1061-1064.
• the amino acid sequences of pembrolizumab are set forth in Table 49.
• Pembrolizumab includes the following disulfide bridges: 22-96, 22"-96", 23'-92', 23"'-92"', 134-218', 134"-218"', 138'-198', 138"'-198"', 147- 203, 147"-203", 226-226", 229-229", 261-321, 261"-321", 367-425, and 367"-425", and the following glycosylation sites (N): Asn-297 and Asn-297".
• Pembrolizumab is an IgG4/kappa isotype with a stabilizing S228P mutation in the Fc region; insertion of this mutation in the IgG4 hinge region prevents the formation of half molecules typically observed for IgG4 antibodies.
• Pembrolizumab is heterogeneously glycosylated at Asn297 within the Fc domain of each heavy chain, yielding a molecular weight of approximately 149 kDa for the intact antibody.
• the dominant glycoform of pembrolizumab is the fucosylated agalacto
• diantennary glycan form (GOF).
• a PD-l inhibitor comprises a heavy chain given by SEQ ID NO: 137 and a light chain given by SEQ ID NO: 138.
• a PD-l inhibitor comprises heavy and light chains having the sequences shown in SEQ ID NO: 137 and SEQ ID NO: 138, respectively, or antigen binding fragments, Fab fragments, single-chain variable fragments (scFv), variants, or conjugates thereof.
• a PD-l inhibitor comprises heavy and light chains that are each at least 99% identical to the sequences shown in SEQ ID NO: 137 and SEQ ID NO: 138, respectively.
• a PD-l inhibitor comprises heavy and light chains that are each at least 98% identical to the sequences shown in SEQ ID NO: 137 and SEQ ID NO: 138, respectively. In an embodiment, a PD-l inhibitor comprises heavy and light chains that are each at least 97% identical to the sequences shown in SEQ ID NO: 137 and SEQ ID NO: 138, respectively. In an embodiment, a PD-l inhibitor comprises heavy and light chains that are each at least 96% identical to the sequences shown in SEQ ID NO: 137 and SEQ ID NO: 138, respectively. In an embodiment, a PD-l inhibitor comprises heavy and light chains that are each at least 95% identical to the sequences shown in SEQ ID NO: 137 and SEQ ID NO: 138, respectively.
• the PD-l inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of pembrolizumab.
• the PD-l inhibitor heavy chain variable region (VH) comprises the sequence shown in SEQ ID NO: 139
• the PD-l inhibitor light chain variable region (VL) comprises the sequence shown in SEQ ID NO: 140, and conservative amino acid substitutions thereof.
• a PD-l inhibitor comprises VH and VL regions that are each at least 99% identical to the sequences shown in SEQ ID NO: 139 and SEQ ID NO: 140, respectively.
• a PD-l inhibitor comprises VH and VL regions that are each at least 98% identical to the sequences shown in SEQ ID NO: 139 and SEQ ID NO: 140, respectively. In an embodiment, a PD-l inhibitor comprises VH and VL regions that are each at least 97% identical to the sequences shown in SEQ ID NO: 139 and SEQ ID NO: 140, respectively. In an embodiment, a PD-l inhibitor comprises VH and VL regions that are each at least 96% identical to the sequences shown in SEQ ID NO: 139 and SEQ ID NO: 140, respectively. In an embodiment, a PD-l inhibitor comprises VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO: 139 and SEQ ID NO: 140, respectively.
• a PD-l inhibitor comprises the heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 141, SEQ ID NO: 142, and SEQ ID NO: 143, respectively, and conservative amino acid substitutions thereof, and light chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 144, SEQ ID NO: 145, and SEQ ID NO: 146, respectively, and conservative amino acid substitutions thereof.
• the antibody competes for binding with, and/or binds to the same epitope on PD-l as any of the aforementioned antibodies.
• the PD-l inhibitor is an anti -PD-l biosimilar monoclonal antibody approved by drug regulatory authorities with reference to pembrolizumab.
• the biosimilar comprises an anti -PD-l antibody comprising an amino acid sequence which has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of a reference medicinal product or reference biological product and which comprises one or more post-translational modifications as compared to the reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is pembrolizumab.
• the one or more post-translational modifications are selected from one or more of: glycosylation, oxidation, deamidation, and truncation.
• the biosimilar is an anti -PD-l antibody authorized or submitted for authorization, wherein the anti -PD-l antibody is provided in a formulation which differs from the formulations of a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is pembrolizumab.
• the anti-PD-l antibody may be authorized by a drug regulatory authority such as the U.S. FDA and/or the European Pinion’s EMA.
• the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is pembrolizumab.
• the biosimilar is provided as a composition which further comprises one or more excipients, wherein the one or more excipients are the same or different to the excipients comprised in a reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is pembrolizumab.
• the PD-l inhibitor is a commercially-available anti-PD-l monoclonal antibody, such as anti-m-PD-l clones J43 (Cat # BE0033-2) and RMP1-14 (Cat # BE0146) (Bio X Cell, Inc., West Riverside, NH, ETSA).
• a number of commercially- available anti-PD-l antibodies are known to one of ordinary skill in the art.
• the PD-l inhibitor is an antibody disclosed in ET.S. Patent No. 8,354,509 or ET.S. Patent Application Publication Nos. 2010/0266617 Al, 2013/0108651 Al, 2013/0109843 A2, the disclosures of which are incorporated by reference herein.
• the PD-l inhibitor is an anti-PD-l antibody described in U.S. Patent Nos. 8,287,856, 8,580,247, and 8,168,757 and ET.S. Patent Application Publication Nos.
• the PD-l inhibitor is an anti-PD-l antibody disclosed in ET.S. Patent No. 8,735,553 Bl, the disclosure of which is incorporated herein by reference.
• the PD-l inhibitor is pidilizumab, also known as CT-011, which is described in ET.S. Patent No. 8,686,119, the disclosure of which is incorporated by reference herein.
• the PD-l inhibitor may be a small molecule or a peptide, or a peptide derivative, such as those described in ET.S. Patent Nos. 8,907,053; 9,096,642; and 9,044,442 and ET.S. Patent Application Publication No. ETS 2015/0087581; l,2,4-oxadiazole compounds and derivatives such as those described in ET.S. Patent Application Publication No. 2015/0073024; cyclic peptidomimetic compounds and derivatives such as those described in ET.S. Patent Application Publication No. ETS 2015/0073042; cyclic compounds and derivatives such as those described in ET.S. Patent Application Publication No. ETS 2015/0125491; l,3,4-oxadiazole and l,3,4-thiadiazole compounds and derivatives such as those described in International Patent Application Publication No. WO 2015/033301;
• the PD-L1 or PD-L2 inhibitor may be any PD-L1 or PD-L2 inhibitor, antagonist, or blocker known in the art. In particular, it is one of the PD-L1 or PD- L2 inhibitors, antagonist, or blockers described in more detail in the following paragraphs.
• the terms“inhibitor,”“antagonist,” and“blocker” are used interchangeably herein in reference to PD-L1 and PD-L2 inhibitors.
• references herein to a PD- Ll or PD-L2 inhibitor that is an antibody may refer to a compound or antigen-binding fragments, variants, conjugates, or biosimilars thereof.
• references herein to a PD-L1 or PD-L2 inhibitor may refer to a compound or a pharmaceutically acceptable salt, ester, solvate, hydrate, cocrystal, or prodrug thereof.
• the compositions, processes and methods described herein include a PD-L1 or PD-L2 inhibitor.
• the PD-L1 or PD-L2 inhibitor is a small molecule.
• the PD-L1 or PD-L2 inhibitor is an antibody (i.e., an anti -PD- 1 antibody), a fragment thereof, including Fab fragments, or a single-chain variable fragment (scFv) thereof.
• the PD-L1 or PD-L2 inhibitor is a polyclonal antibody.
• the PD-L1 or PD-L2 inhibitor is a monoclonal antibody.
• the PD-L1 or PD-L2 inhibitor competes for binding with PD-L1 or PD-L2, and/or binds to an epitope on PD-L1 or PD-L2.
• the antibody competes for binding with PD-L1 or PD-L2, and/or binds to an epitope on PD-L1 or PD-L2.
• the PD-L1 inhibitors provided herein are selective for PD- Ll, in that the compounds bind or interact with PD-L1 at substantially lower concentrations than they bind or interact with other receptors, including the PD-L2 receptor.
• the compounds bind to the PD-L1 receptor at a binding constant that is at least about a 2-fold higher concentration, about a 3-fold higher concentration, about a 5-fold higher concentration, about a lO-fold higher concentration, about a 20-fold higher concentration, about a 30-fold higher concentration, about a 50-fold higher concentration, about a lOO-fold higher concentration, about a 200-fold higher concentration, about a 300- fold higher concentration, or about a 500-fold higher concentration than to the PD-L2 receptor.
• the PD-L2 inhibitors provided herein are selective for PD- L2, in that the compounds bind or interact with PD-L2 at substantially lower concentrations than they bind or interact with other receptors, including the PD-L1 receptor.
• the compounds bind to the PD-L2 receptor at a binding constant that is at least about a 2-fold higher concentration, about a 3-fold higher concentration, about a 5-fold higher concentration, about a lO-fold higher concentration, about a 20-fold higher concentration, about a 30-fold higher concentration, about a 50-fold higher concentration, about a lOO-fold higher concentration, about a 200-fold higher concentration, about a 300- fold higher concentration, or about a 500-fold higher concentration than to the PD-L1 receptor.
• tumor cells express PD-L1, and that T cells express PD-l.
• PD-L1 expression by tumor cells is not required for efficacy of PD-l or PD-L1 inhibitors or blockers.
• the tumor cells express PD-L1.
• the tumor cells do not express PD-L1.
• the methods can include a combination of a PD-l and a PD-L1 antibody, such as those described herein, in combination with a TIL.
• a combination of a PD-l and a PD-L1 antibody and a TIL may be simultaneous or sequential.
• the PD-L1 and/or PD-L2 inhibitor is one that binds human PD-L1 and/or PD-L2 with a KD of about 100 pM or lower, binds human PD-L1 and/or PD-L2 with a KD of about 90 pM or lower, binds human PD-L1 and/or PD-L2 with a KD of about 80 pM or lower, binds human PD-L1 and/or PD-L2 with a KD of about 70 pM or lower, binds human PD-L1 and/or PD-L2 with a KD of about 60 pM or lower, a KD of about 50 pM or lower, binds human PD-L1 and/or PD-L2 with a KD of about 40 pM or lower, or binds human PD-L1 and/or PD-L2 with a KD of about 30 pM
• the PD-L1 and/or PD-L2 inhibitor is one that binds to human PD-L1 and/or PD-L2 with a kassoc of about 7.5 x 10 5 l/M s or faster, binds to human PD-L1 and/or PD-L2 with a kassoc of about 8 x 10 5 l/M s or faster, binds to human PD-L1 and/ or PD-L2 with a kassoc of about 8.5 x 10 5 l/M s or faster, binds to human PD-L1 and/or PD-L2 with a kassoc of about 9 x 10 5 l/M s or faster, binds to human PD-L1 and/or PD-L2 with a kassoc of about 9.5 x 10 5 l/M s and/or faster, or binds to human PD-L1 and/or PD
• the PD-L1 and/or PD-L2 inhibitor is one that binds to human PD-L1 or PD-L2 with a kdissoc of about 2 x 10 5 l/s or slower, binds to human PD-l with a kdissoc of about 2.1 x 10 5 l/s or slower , binds to human PD-l with a kdissoc of about 2.2 x 10 5 l/s or slower, binds to human PD-l with a kdissoc of about 2.3 x 10 5 l/s or slower, binds to human PD-l with a kdissoc of about 2.4 x 10 5 l/s or slower, binds to human PD-l with a kdissoc of about 2.5 x 10 5 l/s or slower, binds to human PD-l with a kdissoc of about a kd
• the PD-L1 and/or PD-L2 inhibitor is one that blocks or inhibits binding of human PD-L1 or human PD-L2 to human PD-l with an ICso of about 10 nM or lower; blocks or inhibits binding of human PD-L1 or human PD-L2 to human PD-l with an ICso of about 9 nM or lower; blocks or inhibits binding of human PD-L1 or human PD-L2 to human PD-l with an ICso of about 8 nM or lower; blocks or inhibits binding of human PD-L1 or human PD-L2 to human PD-l with an ICso of about 7 nM or lower; blocks or inhibits binding of human PD-L1 or human PD-L2 to human PD-l with an ICso of about 6 nM or lower; blocks or inhibits binding of human PD-L1 or human PD-L2 to human PD-l with an ICso of about 6 n
• the PD-L1 inhibitor is durvalumab, also known as MEDI4736 (which is commercially available from Medimmune, LLC, Gaithersburg, Maryland, a subsidiary of AstraZeneca pic.), or antigen-binding fragments, conjugates, or variants thereof.
• the PD-L1 inhibitor is an antibody disclosed in U.S. Patent No. 8,779,108 or U.S. Patent Application Publication No. 2013/0034559, the disclosures of which are incorporated by reference herein.
• the clinical efficacy of durvalumab has been described in Page, et aI., Ahh. Rev. Med., 2014, 65, 185-202; Brahmer, el al.
• the amino acid sequences of durvalumab are set forth in Table 50.
• the durvalumab monoclonal antibody includes disulfide linkages at 22-96, 22"-96", 23'-89', 23"'-89"', 135'-195', 135"'-195"', 148- 204, 148"-204", 215'-224, 215"'-224", 230-230", 233-233", 265-325, 265"-325", 371-429, and 371"-429'; and N-glycosylation sites at Asn-30l and Asn-30l".
• a PD-L1 inhibitor comprises a heavy chain given by SEQ ID NO: 147 and a light chain given by SEQ ID NO: 148.
• a PD-L1 inhibitor comprises heavy and light chains having the sequences shown in SEQ ID NO: 147 and SEQ ID NO: 148, respectively, or antigen binding fragments, Fab fragments, single-chain variable fragments (scFv), variants, or conjugates thereof.
• a PD-L1 inhibitor comprises heavy and light chains that are each at least 99% identical to the sequences shown in SEQ ID NO: 147 and SEQ ID NO: 148, respectively.
• a PD-L1 inhibitor comprises heavy and light chains that are each at least 98% identical to the sequences shown in SEQ ID NO: 147 and SEQ ID NO: 148, respectively. In an embodiment, a PD-L1 inhibitor comprises heavy and light chains that are each at least 97% identical to the sequences shown in SEQ ID NO: 147 and SEQ ID NO: 148, respectively. In an embodiment, a PD-L1 inhibitor comprises heavy and light chains that are each at least 96% identical to the sequences shown in SEQ ID NO: 147 and SEQ ID NO: 148, respectively. In an embodiment, a PD-L1 inhibitor comprises heavy and light chains that are each at least 95% identical to the sequences shown in SEQ ID NO: 147 and SEQ ID NO: 148, respectively.
• the PD-L1 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of durvalumab.
• the PD-L1 inhibitor heavy chain variable region (VH) comprises the sequence shown in SEQ ID NO: 149
• the PD-L1 inhibitor light chain variable region (VL) comprises the sequence shown in SEQ ID NO: 150, and conservative amino acid substitutions thereof.
• a PD-L1 inhibitor comprises VH and VL regions that are each at least 99% identical to the sequences shown in SEQ ID NO: 149 and SEQ ID NO: 150, respectively.
• a PD-L1 inhibitor comprises VH and VL regions that are each at least 98% identical to the sequences shown in SEQ ID NO: 149 and SEQ ID NO: 150, respectively. In an embodiment, a PD-L1 inhibitor comprises VH and VL regions that are each at least 97% identical to the sequences shown in SEQ ID NO: 149 and SEQ ID NO: 150, respectively. In an embodiment, a PD-L1 inhibitor comprises VH and VL regions that are each at least 96% identical to the sequences shown in SEQ ID NO: 149 and SEQ ID NO: 150, respectively. In an embodiment, a PD-L1 inhibitor comprises VH and VL regions that are each at least 95% identical to the sequences shown in SEQ ID NO: 149 and SEQ ID NO: 150, respectively.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Biomedical Technology (AREA)
• Immunology (AREA)
• Chemical & Material Sciences (AREA)
• Biotechnology (AREA)
• Zoology (AREA)
• Organic Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Wood Science & Technology (AREA)
• Genetics & Genomics (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Cell Biology (AREA)
• Hematology (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• Animal Behavior & Ethology (AREA)
• General Engineering & Computer Science (AREA)
• Microbiology (AREA)
• Biochemistry (AREA)
• Epidemiology (AREA)
• Medicinal Chemistry (AREA)
• Pharmacology & Pharmacy (AREA)
• Developmental Biology & Embryology (AREA)
• Virology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Priority Applications (19)

Applications Claiming Priority (4)

Related Child Applications (2)

Publications (2)

Family

ID=69701469

Family Applications (1)

Country Status (19)

Cited By (8)

Citations (112)