Cytokine encoding lentiviral vectors and uses thereof for making tumor infiltrating lymphocytes

Abstract

Provided herein are cytokine-encoding nucleic acid molecules, such as recombinant expression vectors, and uses thereof to make modified tumor infiltrating lymphocytes (TILs), wherein the modified TILs include one or more immunomodulatory agents (e.g, cytokines) associated with their cell surface. The immunomodulatory agents associated with the TILs provide a localized immunostimulatory effect that can advantageously enhance TIL survival, proliferation and / or anti-tumor activity in a patient recipient. As such, the compositions and methods disclosed herein provide effective cancer therapies.

Description

Attorney Docket No.116983-5126-WO / AR / TW CYTOKINE ENCODING LENTIVIRAL VECTORS AND USES THEREOF FOR MAKING TUMOR INFILTRATING LYMPHOCYTES CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 513,550, filed on July 13, 2023, U.S. Provisional Patent Application No.63 / 622,008, filed on January 17, 2024, U.S. Provisional Patent Application No.63 / 562,210, filed on March 6, 2024, and U.S. Provisional Patent Application No.63 / 649,302, filed on May 17, 2024, which are hereby incorporated by reference in their entireties. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing, which has been submitted electronically in XML ST.26 format and is hereby incorporated by reference in its entirety. Said XML copy, created on July 10, 2024, is named 116983-5126- WO_Sequence_Listing and is 209,365 bytes in size. BACKGROUND

[0003] TIL cell therapy has shown clinical benefit for patients with solid tumors (Chesney et al., Journal for ImmunoTherapy of Cancer 2022;10:e005755; 2. Schoenfeld et al., SITC Annual Meeting 2021). However, an immunosuppressive tumor microenvironment (TME) may abrogate the full potential of TIL cell therapy (Granhøj et al., Expert Opin Biol Th. 2022;1–15). The proinflammatory cytokine IL-12, known for its capability to increase IFN-γ production and promote type 1 immune responses, reshapes the TME and has a potential to augment antitumor activity.

[0004] The current TIL expansion process results in both tumor specific TILs (TS-TILs) and bystander TILs in the expanded TIL product. The frequency of neoantigen-specific clones in TIL infusion products, and their ability to mobilize upon in vitro expansion correlate with TIL efficacy in melanoma (Kristensen N.P., et al. J Clin. Invest.2022;132:e150535; Chiffelle, J., et al., bioRxiv 2023; the contents of which are hereby incorporated by reference in their entireties). Bystander T cells can have a Treg-like effect of stealing IL-2 and homostatic cytokines from effector TS-TILs. Bystander TILs also outcompete TS-TIL for nutrients and oxygen during the REP period, reducing their total numbers because: a) bystander TILs tend to be younger and fitter (less-differentiated) T cells and thus have more DB1 / 149057740.1 1Attorney Docket No.116983-5126-WO / AR / TW proliferative potential; and b) TIL growth is dependent on metabolic state and phenotypic state of TILs, i.e. expression of costimulatory receptors. Bystander TILs take up valuable tumor real-estate after adoptive transfer and create a “traffic jam” getting to tumor. Allen, et al., Science, 2022, 378, eaba1624.

[0005] Thus, there remains a need for improved TIL therapies for the treatment of cancers. Disclosed herein are gene-edited TILs with an inducible and membrane-bound IL-12, which showed superior cytotoxic function. The present invention further provides an improved expansion process for preferentially producing TS-TILs over bystander TILs. BRIEF SUMMARY

[0006] Some embodiments of the present disclosure provide a nucleic acid molecule comprising a nucleotide sequence encoding a tethered IL-12 (TeIL-12), wherein the TeIL-12 is under the control of a nuclear factor of activated T-cells (NFAT)-responsive promoter.

[0007] Some embodiments of the present disclosure provide a nucleic acid molecule comprising a nucleotide sequence encoding a tethered IL-12 (TeIL-12), wherein the TeIL-12 is under the control of a nuclear factor of activated T-cells (NFAT)-responsive promoter, and wherein the TeIL-12 comprises an amino acid sequence set forth in SEQ ID NO:62.

[0008] Some embodiments of the present disclosure provide a nucleic acid molecule comprising a nucleotide sequence encoding a tethered IL-12 (TeIL-12), wherein the TeIL-12 is under the control of a nuclear factor of activated T-cells (NFAT)-responsive promoter, and a nucleotide sequence encoding a tethered IL-15 (TeIL-15), wherein the TeIL-15 is under the control of an EF1α promoter.

[0009] Some embodiments of the present disclosure provide a nucleic acid molecule comprising a nucleotide sequence encoding a tethered IL-12 (TeIL-12), wherein the TeIL-12 is under the control of a nuclear factor of activated T-cells (NFAT)-responsive promoter, and a nucleotide sequence encoding a tethered IL-15 (TeIL-15), wherein the TeIL-15 is under the control of an EF1α promoter, and wherein the TeIL-15 has the amino acid sequence of SEQ ID NO:73.

[0010] In some embodiments, the TeIL-12 comprises a membrane anchor, and a human IL- 12 p40 subunit fused to a human IL-12 p35 subunit. In some embodiments, the human IL-12 p35 subunit has the amino acid sequence of SEQ ID NO:60 and the human IL-12 p40 subunit has the amino acid sequence of SEQ ID NO:61. In some embodiments, the TeIL-12 DB1 / 149057740.1 2Attorney Docket No.116983-5126-WO / AR / TW comprises an amino acid sequence set forth in SEQ ID NO:62. In some embodiments, the nucleotide sequence encoding the TeIL-12 is set forth in SEQ ID NO:162. In some embodiments, the nucleic acid molecule further comprises a nucleotide sequence encoding a cytokine selected from the group consisting of IL-2, IL-6, IL-7, IL-9, IL-15, IL-18, IL-21, IL- 23, IL-27, IL-33, IFN gamma, TNFa, IFN alpha, IFN beta, GM-CSF, GCSF, or a variant thereof. In some embodiments, the cytokine is under the control of a constitutive promoter. In some embodiments, the constitutive promoter is selected from the group consisting of an EF1α promotor, a CMV promotor, a CAG promotor, an MND promotor, and an SSFV promoter. In some embodiments, the cytokine is under the control of a constitutive promoter. In some embodiments, the constitutive promoter is an EF1α promoter. In some embodiments, the cytokine is IL-15 or a variant thereof. In some embodiments, the IL-15 is a human IL-15. In some embodiments, the human IL-15 has the amino acid sequence of SEQ ID NO:64. In some embodiments, the IL-15 is a tethered IL-15 (TeIL-15). In some embodiments, the TeIL- 15 comprises a membrane anchor, and a human IL-15. In some embodiments, the TeIL-15 has the amino acid sequence of SEQ ID NO:73. In some embodiments, the cytokine is IL-2 or a variant thereof. In some embodiments, the IL-2 is a human IL-2. In some embodiments, the human IL-2 has the amino acid sequence of SEQ ID NO:138. In some embodiments, the IL-2 is a tethered IL-2 (TeIL-2). In some embodiments, the TeIL-2 has the amino acid sequence of SEQ ID NO:139. In some embodiments, the nucleic acid molecule further comprises a nucleotide sequence encoding a truncated CD19 (tCD19). In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence set forth in SEQ ID NO:148- 150. In some embodiments, the nucleic acid molecule further comprises a nucleotide sequence encoding an shRNA. In some embodiments, the shRNA inhibits the expression of an immune checkpoint gene. In some embodiments, the immune checkpoint gene is selected from the group consisting of PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11, and BCOR. In some embodiments, the shRNA is a PD-1 shRNA. In some embodiments, the PD-1 shRNA comprises a nucleic acid sequence set forth in SEQ ID NO:141-147. In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence set forth in SEQ ID NO:151. DB1 / 149057740.1 3Attorney Docket No.116983-5126-WO / AR / TW

[0011] Some embodiments of the present disclosure provide a recombinant expression vector comprising the nucleic acid molecule disclosed herein.

[0012] Some embodiments of the present disclosure provide a recombinant expression vector comprising a nucleotide sequence encoding a tethered IL-12 (TeIL-12), wherein the TeIL-12 is under the control of a nuclear factor of activated T-cells (NFAT)-responsive promoter.

[0013] Some embodiments of the present disclosure provide a recombinant expression vector comprising a nucleotide sequence encoding a tethered IL-12 (TeIL-12), wherein the TeIL-12 is under the control of a nuclear factor of activated T-cells (NFAT)-responsive promoter, and wherein the TeIL-12 comprises an amino acid sequence set forth in SEQ ID NO:62.

[0014] Some embodiments of the present disclosure provide a recombinant expression vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding a tethered IL-12 (TeIL-12), wherein the TeIL-12 is under the control of a nuclear factor of activated T-cells (NFAT)-responsive promoter, and a nucleotide sequence encoding a tethered IL-15 (TeIL-15), wherein the TeIL-15 is under the control of an EF1α promoter.

[0015] Some embodiments of the present disclosure provide a recombinant expression vector comprising a nucleic acid molecule comprising a nucleotide sequence encoding a tethered IL-12 (TeIL-12), wherein the TeIL-12 is under the control of a nuclear factor of activated T-cells (NFAT)-responsive promoter, and a nucleotide sequence encoding a tethered IL-15 (TeIL-15), wherein the TeIL-15 is under the control of an EF1α promoter, and wherein the TeIL-15 has the amino acid sequence of SEQ ID NO:73.

[0016] Some embodiments of the present disclosure provide a recombinant expression vector comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:166.

[0017] In some embodiments, the vector is a transfer vector. In some embodiments, the transfer vector is derived from the human immunodeficiency virus-1 (HIV-1) and is replication deficient. In some embodiments, the transfer vector is a pLenti-IRES-EGFP vector.

[0018] Some embodiments of the present disclosure provide a lentiviral expression system comprising the recombinant expression vector disclosed herein and one or more helper plasmids encoding an Env protein, a Gag protein, a Pol protein, and a Rev protein. In some DB1 / 149057740.1 4Attorney Docket No.116983-5126-WO / AR / TW embodiments, the lentiviral expression system comprises a helper plasmid encoding the Env protein, the Gag protein, the Pol protein, and the Rev protein. In some embodiments, the lentiviral expression system comprises a helper plasmid encoding the Env protein, and a helper plasmid encoding the Gag protein, the Pol protein, and the Rev protein. In some embodiments, the lentiviral expression system comprises a helper plasmid encoding the Env protein, the Gag protein, and the Pol protein, and a helper plasmid encoding the Rev protein. In some embodiments, the lentiviral expression system comprises a helper plasmid encoding the Env protein and the Rev protein, and a helper plasmid encoding the Gag protein and the Pol protein. In some embodiments, the Env protein is selected from the group consisting of Ba-EVTR, VSV-G, and RD114. In some embodiments, the Env protein comprises a Ba- EVTR protein.

[0019] Some embodiments of the present disclosure provide a method of making a recombinant lentiviral particle, comprising: a) culturing a population of packaging cells in a cell culture medium; b) contacting the population of packaging cells with a lentiviral expression system disclosed herein; and c) harvesting the supernatant of the cell culture medium, wherein the supernatant comprises the recombinant lentiviral particle.

[0020] Some embodiments of the present disclosure provide a recombinant lentiviral RNA molecule comprising a nucleic acid molecule disclosed herein.

[0021] Some embodiments of the present disclosure provide a recombinant lentiviral proviral DNA molecule comprising a nucleic acid molecule disclosed herein.

[0022] Some embodiments of the present disclosure provide a recombinant lentiviral particle comprising a recombinant lentiviral RNA molecule disclosed herein. In some embodiments, the recombinant lentiviral particle is produced by a packaging cell line disclosed herein. In some embodiments, the recombinant lentiviral particle comprises an Env protein selected from the group consisting of Ba-EVTR, VSV-G, and RD114. In some embodiments, the recombinant lentiviral particle comprises an Env protein that is Ba-EVTR. In some embodiments, the recombinant lentiviral particle further comprises a reverse transcriptase. In some embodiments, the recombinant lentiviral particle further comprises a capsid enclosing the recombinant lentiviral RNA molecule.

[0023] Some embodiments of the present disclosure provide a gene-edited tumor infiltrating lymphocyte (TIL) comprising a recombinant lentiviral proviral DNA disclosed DB1 / 149057740.1 5Attorney Docket No.116983-5126-WO / AR / TW herein. In some embodiments, the recombinant lentiviral proviral DNA is integrated into the genome of the gene-edited TIL.

[0024] Some embodiments of the present disclosure provide a gene-edited tumor infiltrating lymphocyte (TIL) expressing an exogenous tethered IL-12 (TeIL-12).

[0025] Some embodiments of the present disclosure provide a gene-edited tumor infiltrating lymphocyte (TIL) expressing an exogenous tethered IL-12 (TeIL-12), wherein the TeIL-12 comprises an amino acid sequence set forth in SEQ ID NO:62.

[0026] Some embodiments of the present disclosure provide a gene-edited tumor infiltrating lymphocyte (TIL) expressing an exogenous tethered IL-12 (TeIL-12), and an exogenous tethered IL-15 (TeIL-15).

[0027] Some embodiments of the present disclosure provide a gene-edited tumor infiltrating lymphocyte (TIL) expressing an exogenous tethered IL-12 (TeIL-12), and an exogenous tethered IL-15 (TeIL-15), wherein the TeIL-15 has the amino acid sequence of SEQ ID NO:73.

[0028] Some embodiments of the present disclosure provide a gene-edited tumor infiltrating lymphocyte (TIL) expressing an exogenous tethered IL-12 (TeIL-12), and an exogenous tethered IL-15 (TeIL-15), wherein the TeIL-12 comprises an amino acid sequence set forth in SEQ ID NO:62, and wherein the TeIL-15 has the amino acid sequence of SEQ ID NO:73.

[0029] In some embodiments, the TeIL-12 comprises a membrane anchor, and a human IL- 12 p40 subunit fused to a human IL-12 p35 subunit. In some embodiments, the human IL-12 p35 subunit has the amino acid sequence of SEQ ID NO:60 and the human IL-12 p40 subunit has the amino acid sequence of SEQ ID NO:61. In some embodiments, the TeIL-12 comprises an amino acid sequence set forth in SEQ ID NO:62. In some embodiments, the gene-edited TIL further expresses a cytokine selected from the group consisting of IL-2, IL-6, IL-7, IL-9, IL-15, IL-18, IL-21, IL-23, IL-27, IL-33, IFN gamma, TNFa, IFN alpha, IFN beta, GM-CSF, GCSF, and a variant thereof. In some embodiments, the cytokine is IL-15 or a variant thereof. In some embodiments, the IL-15 is a human IL-15. In some embodiments, the human IL-15 has the amino acid sequence of SEQ ID NO:64. In some embodiments, the IL- 15 is a tethered IL-15 (TeIL-15). In some embodiments, the TeIL-15 comprises a membrane anchor, and a human IL-15. In some embodiments, the TeIL-15 has the amino acid sequence of SEQ ID NO:73. DB1 / 149057740.1 6Attorney Docket No.116983-5126-WO / AR / TW

[0030] In some embodiments, the gene-edited TIL comprises a nucleic acid molecule disclosed herein. In some embodiments, the gene-edited TIL comprises a recombinant lentiviral particle disclosed herein. In some embodiments, the gene-edited TIL comprises a recombinant lentiviral RNA molecule disclosed herein. In some embodiments, the gene- edited TIL comprises a recombinant lentiviral proviral DNA disclosed herein. In some embodiments, the recombinant lentiviral proviral DNA is integrated into the genome of the gene-edited TIL.

[0031] Some embodiments of the present disclosure provide a method of making a population of tumor-infiltrating lymphocytes (TILs), comprising culturing the population of TILs in a cell culture medium, wherein the cell culture medium comprises IL-15, IL-21, and L-Arginine.

[0032] Some embodiments of the present disclosure provide a method of making a population of tumor-infiltrating lymphocytes (TILs), comprising culturing the population of TILs in a cell culture medium, wherein the cell culture medium comprises IL-15, IL-21, L- Arginine, and NAD+.

[0033] Some embodiments of the present disclosure provide a method of making a population of tumor-infiltrating lymphocytes (TILs), comprising: a) performing a first expansion by culturing a population of TILs in a first cell culture medium for about 3-14 days; b) performing a second expansion by culturing the population of TILs in a second cell culture medium for about 7-14 days, wherein the second cell culture medium comprises IL- 15, IL-21, and L-Arginine.

[0034] Some embodiments of the present disclosure provide a method of making a population of tumor-infiltrating lymphocytes (TILs), comprising: a) performing a first expansion by culturing a population of TILs in a first cell culture medium for about 3-14 days; b) performing a second expansion by culturing the population of TILs in a second cell culture medium for about 7-14 days, wherein the second cell culture medium comprises IL- 15, IL-21, L-Arginine, and NAD+.

[0035] Some embodiments of the present disclosure provide a method of making a population of gene-edited tumor-infiltrating lymphocytes (TILs), comprising: a) performing a first expansion by culturing a population of TILs in a first cell culture medium; b) performing a second expansion by culturing the population of TILs in a second cell culture medium; and DB1 / 149057740.1 7Attorney Docket No.116983-5126-WO / AR / TW c) at any time, transducing the population of TILs with the recombinant lentiviral particle disclosed herein to produce the population of gene-edited TILs.

[0036] Some embodiments of the present disclosure provide a method of making a population of gene-edited tumor-infiltrating lymphocytes (TILs), comprising: a) performing a first expansion by culturing a population of TILs in a first cell culture medium; b) performing a second expansion by culturing the population of TILs in a second cell culture medium, wherein the second cell culture medium comprises IL-15 and IL-21; and c) at any time, transducing the population of TILs with the recombinant lentiviral particle disclosed herein to produce the population of gene-edited TILs.

[0037] Some embodiments of the present disclosure provide a method of making a population of gene-edited tumor-infiltrating lymphocytes (TILs), comprising: a) performing a first expansion by culturing a population of TILs in a first cell culture medium; b) performing a second expansion by culturing the population of TILs in a second cell culture medium, wherein the second cell culture medium comprises IL-15, IL-21, and L-Arginine; and c) at any time, transducing the population of TILs with the recombinant lentiviral particle disclosed herein to produce the population of gene-edited TILs.

[0038] Some embodiments of the present disclosure provide a method of making a population of gene-edited tumor-infiltrating lymphocytes (TILs), comprising: a) performing a first expansion by culturing a population of TILs in a first cell culture medium; b) performing a second expansion by culturing the population of TILs in a second cell culture medium for about 7-14 days, wherein the second cell culture medium comprises IL-15, IL-21, L- Arginine, and NAD+; and c) at any time, transducing the population of TILs with the recombinant lentiviral particle disclosed herein to produce the population of gene-edited TILs.

[0039] Some embodiments of the present disclosure provide a method of making a population of gene-edited tumor-infiltrating lymphocytes (TILs), comprising: a) performing a first expansion by culturing a population of TILs in a first cell culture medium; b) transducing the population of TILs with the recombinant lentiviral particle disclosed herein; and c) performing a second expansion by culturing the population of TILs in a second cell culture medium to produce the population of gene-edited TILs.

[0040] Some embodiments of the present disclosure provide a method of making a population of gene-edited tumor-infiltrating lymphocytes (TILs), comprising: a) performing a DB1 / 149057740.1 8Attorney Docket No.116983-5126-WO / AR / TW first expansion by culturing a population of TILs in a first cell culture medium; b) transducing the population of TILs with a recombinant lentiviral particle comprising a recombinant lentiviral RNA molecule comprising a nucleotide encoding a TeIL-12; and c) performing a second expansion by culturing the population of TILs in a second cell culture medium to produce the population of gene-edited TILs.

[0041] Some embodiments of the present disclosure provide a method of making a population of gene-edited tumor-infiltrating lymphocytes (TILs), comprising: a) performing a first expansion by culturing a population of TILs in a first cell culture medium; b) transducing the population of TILs with a recombinant lentiviral particle comprising a recombinant lentiviral RNA molecule comprising a nucleotide encoding a TeIL-12 and a nucleotide encoding a TeIL-15; and c) performing a second expansion by culturing the population of TILs in a second cell culture medium to produce the population of gene-edited TILs.

[0042] Some embodiments of the present disclosure provide a method of making a population of gene-edited tumor-infiltrating lymphocytes (TILs), comprising: a) performing a first expansion by culturing a population of TILs in a first cell culture medium; b) performing a second expansion by culturing the population of TILs in a second cell culture medium; and c) at any time, transducing the population of TILs with the recombinant lentiviral particle disclosed herein to produce the population of gene-edited TILs, wherein the population of gene-edited TILs expresses a TeIL-12.

[0043] Some embodiments of the present disclosure provide a method of making a population of gene-edited tumor-infiltrating lymphocytes (TILs), comprising: a) performing a first expansion by culturing a population of TILs in a first cell culture medium; b) performing a second expansion by culturing the population of TILs in a second cell culture medium; and c) at any time, transducing the population of TILs with the recombinant lentiviral particle disclosed herein to produce the population of gene-edited TILs, wherein the population of gene-edited TILs expresses a TeIL-12, and wherein the TeIL-12 comprises an amino acid sequence set forth in SEQ ID NO:62.

[0044] Some embodiments of the present disclosure provide a method of making a population of gene-edited tumor-infiltrating lymphocytes (TILs), comprising: a) performing a first expansion by culturing a population of TILs in a first cell culture medium; b) performing a second expansion by culturing the population of TILs in a second cell culture medium; and DB1 / 149057740.1 9Attorney Docket No.116983-5126-WO / AR / TW c) at any time, transducing the population of TILs with the recombinant lentiviral particle disclosed herein to produce the population of gene-edited TILs, wherein the population of gene-edited TILs expresses a TeIL-12 and a TeIL-15, wherein the TeIL-12 comprises an amino acid sequence set forth in SEQ ID NO:62, and wherein the TeIL-15 has the amino acid sequence of SEQ ID NO:73.

[0045] In some embodiments, the method further comprises activating the population of TILs for 1 day or 2 days before transducing the population of TILs with the recombinant lentiviral particle. In some embodiments, the activating step comprises contacting the population of TILs with a cytokine selected from the group consisting of IL-2, IL-15, IL-21, IL-7, and a combination thereof. In some embodiments, the activating step comprises contacting the population of TILs with 20ng / mL IL-15. In some embodiments, the activating step comprises contacting the population of TILs with 10 ng / mL IL-7. In some embodiments, the activating step comprises contacting the population of TILs with TransAct. In some embodiments, the activating step comprises contacting the population of TILs with TransAct at a ratio of 1:100. In some embodiments, the transducing step is conducted in the presence of RetroNectin or Vectofusin-1. In some embodiments, the transducing step comprises centrifugation. In some embodiments, the transducing step is conducted in the presence of Lentibooster. In some embodiments, the transducing step is conducted after the first expansion step and before the second expansion step. In some embodiments, the activating step is conducted after the first expansion step and before the second expansion step. In some embodiments, the method further comprises resting the population of TILs for 2 day or 3 days after the transducing step.

[0046] In some embodiments, the population of TILs is obtained from a tumor digest. In some embodiments, the method further comprises a priming step. In some embodiments, the priming step is performed before the first expansion step. In some embodiments, the priming step is performed in the presence of IFNγ. In some embodiments, IFNγ is present at a concentration of 200 ng / mL during the priming step. In some embodiments, the priming step is performed in the presence of IL-2, IL-15, and / or IL-21. In some embodiments, the IL-2 is at a concentration of 3000 IU / mL or lower during the priming step. In some embodiments, the IL-15 and / or IL-21 is present at a concentration of about 1 ng / mL to about 100 ng / mL during the priming step. In some embodiments, the IL-15 and / or IL-21 is present at a concentration of about 10 ng / mL during the priming step. In some embodiments, the priming step lasts for 24-48 hours. In some embodiments, the priming step lasts for about 30 hours. In DB1 / 149057740.1 10Attorney Docket No.116983-5126-WO / AR / TW some embodiments, the method further comprises an enrichment step. In some embodiments, the enrichment step is performed after the priming step and before the first expansion step. In some embodiments, the population of TILs is enriched for CD137+ T cells. In some embodiments, the population of TILs is enriched for CD137+ and CD39+T cells. In some embodiments, the population of TILs is enriched for CD137+ and CD200+ T cells. In some embodiments, the population of TILs is enriched for CD137+ and OX40+ T cells. In some embodiments, the population of TILs is enriched for CD137+, CD200+ and CD39+T cells. In some embodiments, the population of TILs is enriched for CD137+, CD200+ and OX40+ T cells. In some embodiments, the population of TILs is enriched for CD137+, CD39+ and OX40+ T cells. In some embodiments, the population of TILs is enriched for CD137+, CD200+, CD39+ and OX40+ T cells. In some embodiments, the first expansion is conducted in the presence of feeder cells. In some embodiments, the feeder cells are T cell-depleted PBMCs. In some embodiments, the first expansion is performed in the presence of IL-2, IL- 15, and / or IL-21. In some embodiments, the IL-2 is at a concentration of 3000 IU / mL or lower during the first expansion. In some embodiments, the IL-15 and / or IL-21 is present at a concentration of about 1 ng / mL to about 100 ng / mL during the first expansion. In some embodiments, the IL-15 and / or IL-21 is present at a concentration of about 10 ng / mL during the first expansion. In some embodiments, the first expansion is performed over a period of about 3-11 days. In some embodiments, the first expansion is performed over a period of about 9 days. In some embodiments, the second cell culture medium comprises IL-15 and IL- 21. In some embodiments, the second cell culture medium comprises IL-15 at a concentration of about 10 ng / mL and IL-21 at a concentration of about 10 ng / mL. In some embodiments, the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), and L- arginine. In some embodiments, the L-arginine is present at a concentration of about 1 mM to about 10 mM. In some embodiments, the L-arginine is present at a concentration of about 5 mM. In some embodiments, the second cell culture medium comprises an NAD+ booster. In some embodiments, the NAD+ booster is selected from the group consisting of L-Trp, NR, NMN, NAD+, NAM and P7C3 activator. In some embodiments, the second cell culture medium comprises an inhibitor of GSK-3α / β. In some embodiments, the inhibitor of GSK- 3α / β is selected from the group consisting of SB415286, SB216763, CHIR99021, AR- AO14418, TZD8, TWS119, Lithium Chloride hydrate, BIO, and 3F8. In some embodiments, the second expansion is performed over a period of about 7-11 days. In some embodiments, the second expansion is performed over a period of about 10 days. DB1 / 149057740.1 11Attorney Docket No.116983-5126-WO / AR / TW

[0047] In some embodiments, the first cell culture medium or the second cell culture medium comprises IL-2. In some embodiments, the IL-2 is at a concentration of 3000 IU / mL or lower. In some embodiments, the first cell culture medium or the second cell culture medium contains no added IL-2. In some embodiments, the first cell culture medium or the second cell culture medium comprises IL-15 and / or IL-21 at a concentration of about 1 ng / mL to about 100 ng / mL. In some embodiments, the first cell culture medium or the second cell culture medium comprises IL-15 and / or IL-21 at a concentration of about 10 ng / mL. In some embodiments, the first cell culture medium comprises IL-2 and IL-21. In some embodiments, the first cell culture medium comprises IL-2 at 3000 IU / mL and IL-21 at a concentration of about 10 ng / mL. In some embodiments, the second cell culture medium comprises IL-15 and IL-21. In some embodiments, the second cell culture medium comprises IL-15 at a concentration of about 10 ng / mL and IL-21 at a concentration of about 10 ng / mL. In some embodiments, the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), and a protein kinase B (AKT) inhibitor. In some embodiments, the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosine, Oridonin, Herbacetin, Tehranolide, Isoliquiritigenin, Scutellarin, and Honokiol. In some embodiments, the AKT inhibitor is AZD5363. In some embodiments, the AKT inhibitor is at a concentration of about 0.1 µM to about 10 µM. In some embodiments, the AKT inhibitor is at a concentration of about 1 µM. In some embodiments, the first expansion is performed over a period of about 11 days. In some embodiments, the second expansion is performed over a period of about 11 days.

[0048] Some embodiments of the present disclosure provide a method of making a population of gene-edited tumor-infiltrating lymphocytes (TILs), comprising: a) priming a tumor digest comprising a population of TILs in the presence of IFNγ; b) enriching the population of TILs for CD137+ T cells; c) performing a first expansion by culturing the population of TILs enriched for CD137+ T cells in a first cell culture medium; d) transducing the population of TILs enriched for CD137+ T cells with a recombinant lentiviral particle comprising a nucleic acid sequence encoding TeIL-12 to produce the population of gene- edited TILs; and e) performing a second expansion by culturing the population of gene-edited TILs in a second cell culture medium.

[0049] In some embodiments, the TeIL-12 is under the control of a nuclear factor of activated T-cells (NFAT)-responsive promoter. In some embodiments, the TeIL-12 DB1 / 149057740.1 12Attorney Docket No.116983-5126-WO / AR / TW comprises a membrane anchor, and a human IL-12 p40 subunit fused to a human IL-12 p35 subunit. In some embodiments, the human IL-12 p35 subunit has the amino acid sequence of SEQ ID NO:60 and the human IL-12 p40 subunit has the amino acid sequence of SEQ ID NO:61. In some embodiments, the TeIL-12 comprises an amino acid sequence set forth in SEQ ID NO:62. In some embodiments, the nucleotide sequence encoding the TeIL-12 is set forth in SEQ ID NO:162. In some embodiments, the method further comprises activating the population of TILs for 1 day or 2 days before transducing the population of TILs with the recombinant lentiviral particle. In some embodiments, the activating step comprises contacting the population of TILs with TransAct. In some embodiments, the activating step comprises contacting the population of TILs with TransAct at a ratio of 1:100. In some embodiments, the transducing step is conducted at a TIL concentration of 105cells / mL. In some embodiments, the transducing step is conducted at a multiplicity of infection (MOI) of about 10 to about 40. In some embodiments, the transducing step is conducted in the presence of RetroNectin or Vectofusin-1. In some embodiments, the transducing step comprises centrifugation. In some embodiments, the transducing step is conducted in the presence of Lentibooster. In some embodiments, the method further comprises resting the population of TILs for 2 day or 3 days after the transducing step. In some embodiments, IFNγ is present at a concentration of 200 ng / mL during the priming step. In some embodiments, the priming step is performed in the presence of IL-2, IL-15, and / or IL-21. In some embodiments, the IL- 2 is at a concentration of 3000 IU / mL or lower during the priming step. In some embodiments, the IL-15 and / or IL-21 is present at a concentration of about 1 ng / mL to about 100 ng / mL during the priming step. In some embodiments, the IL-15 and / or IL-21 is present at a concentration of about 10 ng / mL during the priming step. In some embodiments, the priming step lasts for 24-48 hours. In some embodiments, the priming step lasts for about 30 hours.

[0050] In some embodiments, the population of TILs is obtained from a tumor digest. In some embodiments, the method further comprises a priming step. In some embodiments, the priming step is performed before the first expansion step. In some embodiments, the priming step is performed in the presence of IFNγ. In some embodiments, IFNγ is present at a concentration of 200 ng / mL during the priming step. In some embodiments, the priming step is performed in the presence of IL-2, IL-15, and / or IL-21. In some embodiments, the IL-2 is at a concentration of 3000 IU / mL or lower during the priming step. In some embodiments, the IL-15 and / or IL-21 is present at a concentration of about 1 ng / mL to about 100 ng / mL DB1 / 149057740.1 13Attorney Docket No.116983-5126-WO / AR / TW during the priming step. In some embodiments, the IL-15 and / or IL-21 is present at a concentration of about 10 ng / mL during the priming step. In some embodiments, the priming step lasts for 24-48 hours. In some embodiments, the priming step lasts for about 30 hours. In some embodiments, the first expansion is conducted in the presence of feeder cells. In some embodiments, the feeder cells are T cell-depleted PBMCs. In some embodiments, the first expansion is performed in the presence of IL-2, IL-15, and / or IL-21. In some embodiments, the IL-2 is at a concentration of 3000 IU / mL or lower during the first expansion. In some embodiments, the IL-15 and / or IL-21 is present at a concentration of about 1 ng / mL to about 100 ng / mL during the first expansion. In some embodiments, the IL-15 and / or IL-21 is present at a concentration of about 10 ng / mL during the first expansion. In some embodiments, the first expansion is performed over a period of about 3-11 days. In some embodiments, the first expansion is performed over a period of about 9 days. In some embodiments, the second cell culture medium comprises IL-15 and IL-21. In some embodiments, the second cell culture medium comprises IL-15 at a concentration of about 10 ng / mL and IL-21 at a concentration of about 10 ng / mL. In some embodiments, the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), and L-arginine. In some embodiments, the L-arginine is present at a concentration of about 1 mM to about 10 mM. In some embodiments, the L-arginine is present at a concentration of about 5 mM. In some embodiments, the second cell culture medium comprises an NAD+ booster. In some embodiments, the NAD+ booster is selected from the group consisting of L-Trp, NR, NMN, NAD+, NAM and P7C3 activator. In some embodiments, the second cell culture medium comprises an inhibitor of GSK-3α / β. In some embodiments, the inhibitor of GSK-3α / β is selected from the group consisting of SB415286, SB216763, CHIR99021, AR-AO14418, TZD8, TWS119, Lithium Chloride hydrate, BIO, and 3F8. In some embodiments, the second expansion is performed over a period of about 7-11 days. In some embodiments, the second expansion is performed over a period of about 10 days.

[0051] Some embodiments of the present disclosure provide a method of making a population of gene-edited tumor-infiltrating lymphocytes (TILs), comprising: a) priming a tumor digest comprising a population of TILs in the presence of IFNγ; b) enriching the population of TILs for CD137+ T cells; c) performing a first expansion by culturing the population of TILs enriched for CD137+ T cells in a first cell culture medium; d) transducing the population of TILs enriched from CD137+ T cells with a recombinant lentiviral particle comprising a nucleic acid sequence encoding TeIL-12 and TeIL-15 to produce the population DB1 / 149057740.1 14Attorney Docket No.116983-5126-WO / AR / TW of gene-edited TILs; and e) performing a second expansion by culturing the population of gene-edited TILs in a second cell culture medium.

[0052] In some embodiments, the TeIL-12 is under the control of a nuclear factor of activated T-cells (NFAT)-responsive promoter. In some embodiments, the TeIL-15 is under the control of a constitutive promoter. In some embodiments, the constitutive promoter is an EF1α promotor. In some embodiments, the NFAT promoter and the constitutive promoter are introduced in the same nucleic acid of the coding sequence. In some embodiments, the TeIL- 12 comprises a membrane anchor, and a human IL-12 p40 subunit fused to a human IL-12 p35 subunit. In some embodiments, the human IL-12 p35 subunit has the amino acid sequence of SEQ ID NO:60 and the human IL-12 p40 subunit has the amino acid sequence of SEQ ID NO:61. In some embodiments, the TeIL-12 comprises an amino acid sequence set forth in SEQ ID NO:62. In some embodiments, the nucleotide sequence encoding the TeIL- 12 is set forth in SEQ ID NO:162. In some embodiments, the TeIL-15 comprises an amino acid sequence set forth in SEQ ID NO:73. In some embodiments, the method further comprises activating the population of TILs for 1 day or 2 days before transducing the population of TILs with the recombinant lentiviral particle. In some embodiments, the activating step comprises contacting the population of TILs with TransAct. In some embodiments, the activating step comprises contacting the population of TILs with TransAct at a ratio of 1:100. In some embodiments, the method further comprises resting the population of TILs for 2 day or 3 days after the transducing step. In some embodiments, IFNγ is present at a concentration of 200 ng / mL during the priming step. In some embodiments, the priming step is performed in the presence of IL-2, IL-15, and / or IL-21. In some embodiments, the IL-2 is at a concentration of 3000 IU / mL or lower during the priming step. In some embodiments, the IL-15 and / or IL-21 is present at a concentration of about 1 ng / mL to about 100 ng / mL during the priming step. In some embodiments, the IL-15 and / or IL-21 is present at a concentration of about 10 ng / mL during the priming step. In some embodiments, the priming step lasts for 24-48 hours. In some embodiments, the priming step lasts for about 30 hours.

[0053] In some embodiments, the population of TILs is obtained from a tumor digest. In some embodiments, the method further comprises a priming step. In some embodiments, the priming step is performed before the first expansion step. In some embodiments, the priming step is performed in the presence of IFNγ. In some embodiments, IFNγ is present at a concentration of 200 ng / mL during the priming step. In some embodiments, the priming step DB1 / 149057740.1 15Attorney Docket No.116983-5126-WO / AR / TW is performed in the presence of IL-2, IL-15, and / or IL-21. In some embodiments, the IL-2 is at a concentration of 3000 IU / mL or lower during the priming step. In some embodiments, the IL-15 and / or IL-21 is present at a concentration of about 1 ng / mL to about 100 ng / mL during the priming step. In some embodiments, the IL-15 and / or IL-21 is present at a concentration of about 10 ng / mL during the priming step. In some embodiments, the priming step lasts for 24-48 hours. In some embodiments, the priming step lasts for about 30 hours. In some embodiments, the first expansion is conducted in the presence of feeder cells. In some embodiments, the feeder cells are T cell-depleted PBMCs. In some embodiments, the first expansion is performed in the presence of IL-2, IL-15, and / or IL-21. In some embodiments, the IL-2 is at a concentration of 3000 IU / mL or lower during the first expansion. In some embodiments, the IL-15 and / or IL-21 is present at a concentration of about 1 ng / mL to about 100 ng / mL during the first expansion. In some embodiments, the IL-15 and / or IL-21 is present at a concentration of about 10 ng / mL during the first expansion. In some embodiments, the first expansion is performed over a period of about 3-11 days. In some embodiments, the first expansion is performed over a period of about 9 days. In some embodiments, the second cell culture medium comprises IL-15 and IL-21. In some embodiments, the second cell culture medium comprises IL-15 at a concentration of about 10 ng / mL and IL-21 at a concentration of about 10 ng / mL. In some embodiments, the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), and L-arginine. In some embodiments, the L-arginine is present at a concentration of about 1 mM to about 10 mM. In some embodiments, the L-arginine is present at a concentration of about 5 mM. In some embodiments, the second cell culture medium comprises an NAD+ booster. In some embodiments, the NAD+ booster is selected from the group consisting of L-Trp, NR, NMN, NAD+, NAM and P7C3 activator. In some embodiments, the second cell culture medium comprises an inhibitor of GSK-3α / β. In some embodiments, the inhibitor of GSK-3α / β is selected from the group consisting of SB415286, SB216763, CHIR99021, AR-AO14418, TZD8, TWS119, Lithium Chloride hydrate, BIO, and 3F8. In some embodiments, the second expansion is performed over a period of about 7-11 days. In some embodiments, the second expansion is performed over a period of about 10 days.

[0054] Some embodiments of the present disclosure provide a therapeutic population of TILs produced by a method of making a population of gene-edited TILs disclosed herein. In some embodiments, about 15-60% of the population of TILs expresses the TeIL-12. In some embodiments, more than 30% of the population of TILs expresses the TeIL-12. In some DB1 / 149057740.1 16Attorney Docket No.116983-5126-WO / AR / TW embodiments, more than 50% of the population of TILs expresses the TeIL-12. In some embodiments, about 15-60% of the population of TILs expresses the TeIL-15. In some embodiments, more than 30% of the population of TILs expresses the TeIL-15. In some embodiments, more than 50% of the population of TILs expresses the TeIL-15. In some embodiments, about 15-60% of the population of TILs expresses the TeIL-2. In some embodiments, more than 30% of the population of TILs expresses the TeIL-2. In some embodiments, more than 50% of the population of TILs expresses the TeIL-2. In some embodiments, more than 30% of the population of TILs has reduced expression of PD-1. In some embodiments, more than 40% of the population of TILs has reduced expression of PD- 1. In some embodiments, more than 60% of the population of TILs has reduced expression of PD-1.

[0055] Some embodiments of the present disclosure provide a pharmaceutical composition comprising a therapeutic population of TILs disclosed herein.

[0056] Some embodiments of the present disclosure provide a method of treating a cancer in a patient in need thereof comprising: a) resecting a tumor sample from the patient, wherein the tumor sample comprises a population of TILs; b) performing a first expansion by culturing the population of TILs in a first cell culture medium; c) performing a second expansion by culturing the population of TILs in a second cell culture medium; d) at any time, transducing the population of TILs with a recombinant lentiviral particle disclosed herein to produce a therapeutic population of gene-edited TILs; and e) administering the therapeutic population of gene-edited TILs to the patient. In some embodiments, the method further comprises activating the population of TILs for 1 day or 2 days before transducing the population of TILs with the recombinant lentiviral particle. In some embodiments, the activating step comprises contacting the population of TILs with a cytokine selected from the group consisting of IL-2, IL-15, IL-21, IL-7, and a combination thereof. In some embodiments, the activating step comprises contacting the population of TILs with 20ng / mL IL-15. In some embodiments, the activating step comprises contacting the population of TILs with 10 ng / mL IL-7. In some embodiments, the activating step comprises contacting the population of TILs with TransAct. In some embodiments, the activating step comprises contacting the population of TILs with TransAct at a ratio of 1:100. In some embodiments, the transducing step is conducted at a TIL concentration of 105cells / mL. In some embodiments, the transducing step is conducted at a multiplicity of infection (MOI) of about 10 to about 40. In some embodiments, the transducing step is conducted in the presence of DB1 / 149057740.1 17Attorney Docket No.116983-5126-WO / AR / TW RetroNectin or Vectofusin-1. In some embodiments, the transducing step comprises centrifugation. In some embodiments, the transducing step is conducted in the presence of Lentibooster. In some embodiments, the transducing step is conducted after the first expansion step and before the second expansion step. In some embodiments, the activating step is conducted after the first expansion step and before the second expansion step. In some embodiments, the method further comprises resting the population of TILs for 2 day or 3 days after the transducing step.

[0057] In some embodiments, the population of TILs is obtained from a tumor digest. In some embodiments, the method further comprises a priming step. In some embodiments, the priming step is performed before the first expansion step. In some embodiments, the priming step is performed in the presence of IFNγ. In some embodiments, IFNγ is present at a concentration of 200 ng / mL during the priming step. In some embodiments, the priming step is performed in the presence of IL-2, IL-15, and / or IL-21. In some embodiments, the IL-2 is at a concentration of 3000 IU / mL or lower during the priming step. In some embodiments, the IL-15 and / or IL-21 is present at a concentration of about 1 ng / mL to about 100 ng / mL during the priming step. In some embodiments, the IL-15 and / or IL-21 is present at a concentration of about 10 ng / mL during the priming step. In some embodiments, the priming step lasts for 24-48 hours. In some embodiments, the priming step lasts for about 30 hours. In some embodiments, the method further comprises an enrichment step. In some embodiments, the enrichment step is performed after the priming step and before the first expansion step. In some embodiments, the population of TILs is enriched for CD137+ T cells. In some embodiments, the population of TILs is enriched for CD137+ and CD39+ T cells. In some embodiments, the population of TILs is enriched for CD137+ and CD200+ T cells. In some embodiments, the population of TILs is enriched for CD137+ and OX40+ T cells. In some embodiments, the population of TILs is enriched for CD137+, CD200+ and CD39+ T cells. In some embodiments, the population of TILs is enriched for CD137+, CD200+ and OX40+ T cells. In some embodiments, the population of TILs is enriched for CD137+, CD39+ and OX40+ T cells. In some embodiments, the population of TILs is enriched for CD137+, CD200+, CD39+ and OX40+ T cells. In some embodiments, the first expansion is conducted in the presence of feeder cells. In some embodiments, the feeder cells are T cell-depleted PBMCs. In some embodiments, the first expansion is performed in the presence of IL-2, IL- 15, and / or IL-21. In some embodiments, the IL-2 is at a concentration of 3000 IU / mL or lower during the first expansion. In some embodiments, the IL-15 and / or IL-21 is present at a DB1 / 149057740.1 18Attorney Docket No.116983-5126-WO / AR / TW concentration of about 1 ng / mL to about 100 ng / mL during the first expansion. In some embodiments, the IL-15 and / or IL-21 is present at a concentration of about 10 ng / mL during the first expansion. In some embodiments, the first expansion is performed over a period of about 3-11 days. In some embodiments, the first expansion is performed over a period of about 9 days. In some embodiments, the second cell culture medium comprises IL-15 and IL- 21. In some embodiments, the second cell culture medium comprises IL-15 at a concentration of about 10 ng / mL and IL-21 at a concentration of about 10 ng / mL. In some embodiments, the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), and L- arginine. In some embodiments, the L-arginine is present at a concentration of about 1 mM to about 10 mM. In some embodiments, the L-arginine is present at a concentration of about 5 mM. In some embodiments, the second cell culture medium comprises an NAD+ booster. In some embodiments, the NAD+ booster is selected from the group consisting of L-Trp, NR, NMN, NAD+, NAM and P7C3 activator. In some embodiments, the second cell culture medium comprises an inhibitor of GSK-3α / β. In some embodiments, the inhibitor of GSK- 3α / β is selected from the group consisting of SB415286, SB216763, CHIR99021, AR- AO14418, TZD8, TWS119, Lithium Chloride hydrate, BIO, and 3F8. In some embodiments, the second expansion is performed over a period of about 7-11 days. In some embodiments, the second expansion is performed over a period of about 10 days.

[0058] In some embodiments, the first cell culture medium or the second cell culture medium comprises IL-2. In some embodiments, the IL-2 is at a concentration of 3000 IU / mL or lower. In some embodiments, the first cell culture medium or the second cell culture medium contains no added IL-2. In some embodiments, the first cell culture medium or the second cell culture medium comprises IL-15 and / or IL-21 at a concentration of about 1 ng / mL to about 100 ng / mL. In some embodiments, the first cell culture medium or the second cell culture medium comprises IL-15 and / or IL-21 at a concentration of about 10 ng / mL. In some embodiments, the first cell culture medium comprises IL-2 and IL-21. In some embodiments, the first cell culture medium comprises IL-2 at 3000 IU / mL and IL-21 at a concentration of about 10 ng / mL. In some embodiments, the second cell culture medium comprises IL-15 and IL-21. In some embodiments, the second cell culture medium comprises IL-15 at a concentration of about 10 ng / mL and IL-21 at a concentration of about 10 ng / mL. In some embodiments, the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), and a protein kinase B (AKT) inhibitor. In some embodiments, the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, DB1 / 149057740.1 19Attorney Docket No.116983-5126-WO / AR / TW GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosine, Oridonin, Herbacetin, Tehranolide, Isoliquiritigenin, Scutellarin, and Honokiol. In some embodiments, the AKT inhibitor is AZD5363. In some embodiments, the AKT inhibitor is at a concentration of about 0.1 µM to about 10 µM. In some embodiments, the AKT inhibitor is at a concentration of about 1 µM. In some embodiments, the first expansion is performed over a period of about 11 days. In some embodiments, the second expansion is performed over a period of about 11 days.

[0059] In some embodiments, the cancer is selected from the group consisting of melanoma (including mucosal melanoma, uveal melanoma, cutaneous melanoma, choroidal melanoma, ciliary body melanoma, or iris melanoma), ovarian cancer, cervical cancer, endometrial cancer, non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple negative breast cancer, cancer caused by human papilloma virus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, and renal cell carcinoma. In some embodiments, the method further comprises the step of treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the TILs to the patient. In some embodiments, the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for three days. In some embodiments, the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for three days. In some embodiments, the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for one day. In some embodiments, the cyclophosphamide is administered with mesna. In some embodiments, the method further comprises a step of treating the patient with an IL-2 regimen starting on the day after the administration of TILs to the patient. In some embodiments, the method further comprises a step of treating the patient with an IL-2 regimen starting on the same day as administration of TILs to the patient. In some embodiments, 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. In some embodiments, the method comprises no step of treating the patient with an IL-2 DB1 / 149057740.1 20Attorney Docket No.116983-5126-WO / AR / TW regimen. In some embodiments, the therapeutic population of TILs comprises from about 2.3×1010to about 13.7×1010TILs.

[0060] Some embodiments of the present disclosure provide a packaging cell line comprising a recombinant expression vector disclosed herein, and one or more recombinant DNA molecules, each of which one or more recombinant DNA molecules encodes any one protein, any two proteins, any three proteins or all four proteins selected from the group consisting of encoding an Env protein, a Gag protein, a Pol protein, and a Rev protein, with the proviso that any protein selected from the group consisting of the Env protein, the Gag protein, the Pol protein and the Rev protein is encoded by at least one recombinant DNA molecule of the one or more recombinant DNA molecules, wherein each of the one or more recombinant DNA molecules is either integrated into the genome of the packaging cell line or comprised by a helper plasmid. In some embodiments, the one or more recombinant DNA molecules consist of a first recombinant DNA molecule. In some embodiments, the first recombinant DNA molecule is either integrated into the genome of the packaging cell line or comprised by a helper plasmid. In some embodiments, the one or more recombinant DNA molecules consist of a first recombinant DNA molecule and a second recombinant DNA molecule. In some embodiments, the first recombinant DNA molecule encodes a single protein selected from the group consisting of the Env protein, the Gag protein, the Pol protein, and the Rev protein. In some embodiments, the first recombinant DNA molecule encodes the Env protein. In some embodiments, the first recombinant DNA molecule encodes the Gag protein. In some embodiments, the first recombinant DNA molecule encodes the Pol protein. In some embodiments, the first recombinant DNA molecule encodes the Rev protein. In some embodiments, the first recombinant DNA molecule encodes two proteins selected from the group consisting of the Env protein, the Gag protein, the Pol protein, and the Rev protein. In some embodiments, the first recombinant DNA molecule encodes the Env protein and the Gag protein. In some embodiments, the first recombinant DNA molecule encodes the Env protein and the Pol protein. In some embodiments, the first recombinant DNA molecule encodes the Env protein and the Rev protein. In some embodiments, the first recombinant DNA molecule encodes the Gag protein and the Pol protein. In some embodiments, the first recombinant DNA molecule encodes the Gag protein and the Rev protein. In some embodiments, the first recombinant DNA molecule encodes the Pol protein and the Rev protein. In some embodiments, each of the first and second recombinant DNA molecules is either integrated into the genome of the packaging cell line or comprised by a helper plasmid. DB1 / 149057740.1 21Attorney Docket No.116983-5126-WO / AR / TW In some embodiments, the one or more recombinant DNA molecules consist of a first recombinant DNA molecule, a second recombinant DNA molecule, and a third recombinant DNA molecule. In some embodiments, the first recombinant DNA molecule encodes a first protein and the second recombinant DNA molecule encodes a second protein, wherein the first and second proteins are independently selected from the group consisting of the Env protein, the Gag protein, the Pol protein, and the Rev protein, with the proviso that the first and second proteins are not the same. In some embodiments, the first protein is the Env protein and the second protein is the Gag protein. In some embodiments, the first protein is the Env protein and the second protein is the Pol protein. In some embodiments, the first protein is the Env protein and the second protein is the Rev protein. In some embodiments, the first protein is the Gag protein and the second protein is the Pol protein. In some embodiments, the first protein is the Gag protein and the second protein is the Rev protein. In some embodiments, the first protein is the Pol protein and the second protein is the Rev protein. In some embodiments, the first recombinant DNA molecule encodes a first protein and the second recombinant DNA molecule encodes two proteins, wherein the first protein and each of the two proteins are independently selected from the group consisting of the Env protein, the Gag protein, the Pol protein, and the Rev protein, with the proviso that any protein is not the same as any other protein in the group consisting of the first protein and the two proteins. In some embodiments, the first protein is the Env protein, and the two proteins are the Gag protein and the Pol protein. In some embodiments, the first protein is the Env protein, and the two proteins are the Gag protein and the Rev protein. In some embodiments, the first protein is the Env protein, and the two proteins are the Pol protein and the Rev protein. In some embodiments, the first protein is the Gag protein, and the two proteins are the Env protein and the Pol protein. In some embodiments, the first protein is the Gag protein, and the two proteins are the Env protein and the Rev protein. In some embodiments, the first protein is the Gag protein, and the two proteins are the Pol protein and the Rev protein. In some embodiments, the first protein is the Pol protein, and the two proteins are the Env protein and the Gag protein. In some embodiments, the first protein is the Pol protein, and the two proteins are the Env protein and the Rev protein. In some embodiments, the first protein is the Pol protein, and the two proteins are the Gag protein and the Rev protein. In some embodiments, the first protein is the Rev protein, and the two proteins are the Env protein and the Gag protein. In some embodiments, the first protein is the Rev protein, and the two proteins are the Env protein and the Pol protein. In some embodiments, the first protein is the Rev protein, and the two proteins are the Gag protein and the Pol protein. In DB1 / 149057740.1 22Attorney Docket No.116983-5126-WO / AR / TW some embodiments, each of the first, second, and third recombinant DNA molecules is either integrated into the genome of the packaging cell line or comprised by a helper plasmid. In some embodiments, the one or more recombinant DNA molecules consist of a first recombinant DNA molecule, a second recombinant DNA molecule, a third recombinant DNA molecule, and a fourth recombinant DNA molecule. In some embodiments, each of the first, second, third, and fourth recombinant DNA molecules is either integrated into the genome of the packaging cell line or comprised by a helper plasmid. In some embodiments, the Env protein comprises a Ba-EVTR protein. In some embodiments, the nucleic acid molecule encoding TeIL-12 is integrated into the genome of the packaging cell line. In some embodiments, the recombinant DNA molecule encoding one or more the Env protein, the Gag protein, the Pol protein, and the Rev protein is under the control of an inducible promoter. In some embodiments, the packaging cell line is a 293T cell line.

[0061] Some embodiments of the present disclosure provide a method for enriching tumor- reactive tumor infiltrating lymphocytes (TILs), comprising: a) priming a population of TILs obtained from a tumor digest in the presence of IFNγ; b) enriching the population of TILs for CD137+ TILs; c) performing a first expansion by culturing the population of TILs enriched for CD137+ T cells in a first cell culture medium; and d) performing a second expansion by culturing the population of TILs enriched for CD137+ TILs in a second cell culture medium to produce a population of TILs enriched for tumor-reactive TILs. In some embodiments, the population of TILs enriched for tumor-reactive TILs comprises an increased percentage of tumor-reactive TILs in comparison to TILs expanded using a reference expansion procedure.

[0062] In some embodiments, IFNγ is present at a concentration of 200 ng / mL during the priming step. In some embodiments, the priming step is performed in the presence of IL-2, IL-15, and / or IL-21. In some embodiments, the IL-2 is at a concentration of 3000 IU / mL or lower during the priming step. In some embodiments, the IL-15 and / or IL-21 is present at a concentration of about 1 ng / mL to about 100 ng / mL during the priming step. In some embodiments, the IL-15 and / or IL-21 is present at a concentration of about 10 ng / mL during the priming step. In some embodiments, the priming step lasts for 24-48 hours. In some embodiments, the priming step lasts for about 30 hours. In some embodiments, the first expansion is conducted in the presence of feeder cells. In some embodiments, the feeder cells are T cell-depleted PBMCs. In some embodiments, the first expansion is performed in the presence of IL-2, IL-15, and / or IL-21. In some embodiments, the IL-2 is at a concentration of 3000 IU / mL or lower during the first expansion. In some embodiments, the IL-15 and / or IL- DB1 / 149057740.1 23Attorney Docket No.116983-5126-WO / AR / TW 21 is present at a concentration of about 1 ng / mL to about 100 ng / mL during the first expansion. In some embodiments, the IL-15 and / or IL-21 is present at a concentration of about 10 ng / mL during the first expansion. In some embodiments, the first expansion is performed over a period of about 3-11 days. In some embodiments, the first expansion is performed over a period of about 9 days. In some embodiments, the second cell culture medium comprises IL-15 and IL-21. In some embodiments, the second cell culture medium comprises IL-15 at a concentration of about 10 ng / mL and IL-21 at a concentration of about 10 ng / mL. In some embodiments, the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), and L-arginine. In some embodiments, the L-arginine is present at a concentration of about 1 mM to about 10 mM. In some embodiments, the L-arginine is present at a concentration of about 5 mM. In some embodiments, the second cell culture medium comprises an NAD+ booster. In some embodiments, the NAD+ booster is selected from the group consisting of L-Trp, NR, NMN, NAD+, NAM and P7C3 activator. In some embodiments, the second cell culture medium comprises an inhibitor of GSK-3α / β. In some embodiments, the inhibitor of GSK-3α / β is selected from the group consisting of SB415286, SB216763, CHIR99021, AR-AO14418, TZD8, TWS119, Lithium Chloride hydrate, BIO, and 3F8. In some embodiments, the second expansion is performed over a period of about 7- 11 days. In some embodiments, the second expansion is performed over a period of about 10 days. In some embodiments, the enriching the population of TILs for CD137+ TILs comprises contacting the population of TILs with anti-CD137 antibody immobilized on a bead. In some embodiments, the method further comprises contacting the population of TILs with an anti-CD39 antibody. In some embodiments, the method further comprises contacting the population of TILs with an anti-CD200 antibody. In some embodiments, the method further comprises contacting the population of TILs with an anti-OX40 antibody.

[0063] Some embodiments of the present disclosure provide a method for enriching tumor- reactive tumor infiltrating lymphocytes (TILs), comprising: a) priming a population of TILs obtained from a tumor digest in the presence of IFNγ; b) enriching the population of TILs for CD103+ TILs; c) performing a first expansion by culturing the population of TILs enriched for CD103+ T cells in a first cell culture medium; and d) performing a second expansion by culturing the population of TILs enriched for CD103+ T cells in a second cell culture medium to produce a population of TILs enriched for tumor-reactive TILs. In some embodiments, the population of TILs enriched for tumor-reactive TILs comprises an DB1 / 149057740.1 24Attorney Docket No.116983-5126-WO / AR / TW increased percentage of tumor-reactive TILs in comparison to TILs expanded using a reference expansion procedure.

[0064] In some embodiments, IFNγ is present at a concentration of 200 ng / mL during the priming step. In some embodiments, the priming step is performed in the presence of IL-2, IL-15, and / or IL-21. In some embodiments, the IL-2 is at a concentration of 3000 IU / mL or lower during the priming step. In some embodiments, the IL-15 and / or IL-21 is present at a concentration of about 1 ng / mL to about 100 ng / mL during the priming step. In some embodiments, the IL-15 and / or IL-21 is present at a concentration of about 10 ng / mL during the priming step. In some embodiments, the priming step lasts for 24-48 hours. In some embodiments, the priming step lasts for about 24 hours. In some embodiments, selecting CD103+ TILs comprises sorting the second population of TILs using flow cytometry. In some embodiments, step (b) comprises selecting CD103+CD31- TILs. In some embodiments, the first expansion is conducted in the presence of feeder cells. In some embodiments, the feeder cells are T cell-depleted PBMCs. In some embodiments, the first expansion is performed in the presence of IL-2, IL-15, and / or IL-21. In some embodiments, the IL-2 is at a concentration of 3000 IU / mL or lower during the first expansion. In some embodiments, the IL-15 and / or IL-21 is present at a concentration of about 1 ng / mL to about 100 ng / mL during the first expansion. In some embodiments, the IL-15 and / or IL-21 is present at a concentration of about 10 ng / mL during the first expansion. In some embodiments, the first expansion is performed over a period of about 3-11 days. In some embodiments, the first expansion is performed over a period of about 9 days. In some embodiments, the second cell culture medium comprises IL-15 and IL-21. In some embodiments, the second cell culture medium comprises IL-15 at a concentration of about 10 ng / mL and IL-21 at a concentration of about 10 ng / mL. In some embodiments, the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), and L-arginine. In some embodiments, the L-arginine is present at a concentration of about 1 mM to about 10 mM. In some embodiments, the L- arginine is present at a concentration of about 5 mM. In some embodiments, the second cell culture medium comprises an NAD+ booster. In some embodiments, the NAD+ booster is selected from the group consisting of L-Trp, NR, NMN, NAD+, NAM and P7C3 activator. In some embodiments, the second cell culture medium comprises an inhibitor of GSK-3α / β. In some embodiments, the inhibitor of GSK-3α / β is selected from the group consisting of SB415286, SB216763, CHIR99021, AR-AO14418, TZD8, TWS119, Lithium Chloride hydrate, BIO, and 3F8. In some embodiments, the second expansion is performed over a DB1 / 149057740.1 25Attorney Docket No.116983-5126-WO / AR / TW period of about 7-11 days. In some embodiments, the second expansion is performed over a period of about 10 days. Some embodiments of the present disclosure provide a method of treating a cancer in a patient in need thereof comprising: a. resecting a tumor sample from the patient; b. digesting the tumor sample to obtain a tumor digest; c. producing a population of TILs enriched for tumor-reactive TILs from the tumor digest using the methods disclosed herein; and d. administering the therapeutic population of gene-edited TILs to the patient.

[0065] In some embodiments, the cancer is selected from the group consisting of melanoma (including mucosal melanoma, uveal melanoma, cutaneous melanoma, choroidal melanoma, ciliary body melanoma, or iris melanoma), ovarian cancer, cervical cancer, endometrial cancer, non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple negative breast cancer, cancer caused by human papilloma virus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, and renal cell carcinoma. In some embodiments, the method further comprises the step of treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the TILs to the patient. In some embodiments, the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for three days. In some embodiments, the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for three days. In some embodiments, the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for one day. In some embodiments, the cyclophosphamide is administered with mesna. In some embodiments, the method further comprises a step of treating the patient with an IL-2 regimen starting on the day after the administration of TILs to the patient. In some embodiments, the method further comprises a step of treating the patient with an IL-2 regimen starting on the same day as administration of TILs to the patient. In some embodiments, 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, DB1 / 149057740.1 26Attorney Docket No.116983-5126-WO / AR / TW administered as a 15-minute bolus intravenous infusion every eight hours until tolerance. In some embodiments, the method comprises no step of treating the patient with an IL-2 regimen. In some embodiments, the therapeutic population of TILs comprises from about 2.3×1010to about 13.7×1010TILs.

[0066] Some embodiments of the present disclosure provide a method of making a population of tumor-infiltrating lymphocytes (TILs), comprising: a) performing a first expansion by culturing a population of TILs in a first cell culture medium; and b) performing a second expansion by culturing the population of TILs in a second cell culture medium, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), IL-21, IL-15, and L-arginine.

[0067] Some embodiments of the present disclosure provide a method of making a population of tumor-infiltrating lymphocytes (TILs), comprising: a) performing a first expansion by culturing a population of TILs in a first cell culture medium; and b) performing a second expansion by culturing the population of TILs in a second cell culture medium, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), IL-21, IL-15, L-arginine, and NAD+.

[0068] In some embodiments, the second cell culture medium comprises IL-15 at a concentration of about 10 ng / mL and IL-21 at a concentration of about 10 ng / mL. In some embodiments, the L-arginine is present at a concentration of about 1 mM to about 10 mM. In some embodiments, the L-arginine is present at a concentration of about 5 mM. In some embodiments, the second cell culture medium comprises an NAD+ booster. In some embodiments, the NAD+ booster is selected from the group consisting of L-Trp, NR, NMN, NAD+, NAM and P7C3 activator. In some embodiments, the NAD+ booster is NAD+. In some embodiments, the NAD+ is present at a concentration of about 50-75 µM. In some embodiments, the first expansion is performed over a period of about 3-14 days. In some embodiments, the first expansion is performed over a period of about 11 days. In some embodiments, the second expansion is performed over a period of about 7-14 days. In some embodiments, the second expansion is performed over a period of about 11 days.

[0069] Some embodiments of the present disclosure provide a therapeutic population of TILs produced by the methods disclosed herein. DB1 / 149057740.1 27Attorney Docket No.116983-5126-WO / AR / TW

[0070] Some embodiments of the present disclosure provide a pharmaceutical composition comprising the therapeutic population of TILs disclosed herein.

[0071] Some embodiments of the present disclosure provide a method of treating a cancer in a patient in need thereof comprising: a) resecting a tumor sample from the patient, wherein the tumor sample comprises a population of TILs; b) performing a first expansion by culturing the population of TILs in a first cell culture medium; c) performing a second expansion by culturing the population of TILs in a second cell culture medium, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), IL-21, IL-15, and L-arginine; and d) administering the therapeutic population of gene-edited TILs to the patient.

[0072] Some embodiments of the present disclosure provide a method of treating a cancer in a patient in need thereof comprising: a) resecting a tumor sample from the patient, wherein the tumor sample comprises a population of TILs; b) performing a first expansion by culturing the population of TILs in a first cell culture medium; c) performing a second expansion by culturing the population of TILs in a second cell culture medium, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), IL-21, IL-15, L-arginine, and NAD+; and d) administering the therapeutic population of gene-edited TILs to the patient.

[0073] In some embodiments, the second cell culture medium comprises IL-15 at a concentration of about 10 ng / mL and IL-21 at a concentration of about 10 ng / mL. In some embodiments, the L-arginine is present at a concentration of about 1 mM to about 10 mM. In some embodiments, the L-arginine is present at a concentration of about 5 mM. In some embodiments, the second cell culture medium comprises an NAD+ booster. In some embodiments, the NAD+ booster is selected from the group consisting of L-Trp, NR, NMN, NAD+, NAM and P7C3 activator. In some embodiments, the NAD+ booster is NAD+. In some embodiments, the NAD+ is present at a concentration of about 50-75 µM. In some embodiments, the first expansion is performed over a period of about 3-14 days. In some DB1 / 149057740.1 28Attorney Docket No.116983-5126-WO / AR / TW embodiments, the first expansion is performed over a period of about 11 days. In some embodiments, the second expansion is performed over a period of about 7-14 days. In some embodiments, the second expansion is performed over a period of about 11 days.

[0074] In some embodiments, the cancer is selected from the group consisting of melanoma (including mucosal melanoma, uveal melanoma, cutaneous melanoma, choroidal melanoma, ciliary body melanoma, or iris melanoma), ovarian cancer, cervical cancer, endometrial cancer, non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple negative breast cancer, cancer caused by human papilloma virus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, and renal cell carcinoma. In some embodiments, the method further comprises the step of treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the TILs to the patient. In some embodiments, the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for three days. In some embodiments, the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for three days. In some embodiments, the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for one day. In some embodiments, the cyclophosphamide is administered with mesna. In some embodiments, the method further comprises a step of treating the patient with an IL-2 regimen starting on the day after the administration of TILs to the patient. In some embodiments, the method further comprises a step of treating the patient with an IL-2 regimen starting on the same day as administration of TILs to the patient. In some embodiments, 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. In some embodiments, the method comprises no step of treating the patient with an IL-2 regimen.

[0075] Some embodiments of the present disclosure provide a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprising: DB1 / 149057740.1 29Attorney Docket No.116983-5126-WO / AR / TW (a) adding processed tumor fragments from a tumor resected from a patient into a closed system to obtain a first population of TILs; (b) 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 3-11 days to obtain the second population of TILs, and wherein the transition from step (a) to step (b) occurs without opening the system; (c) performing a second expansion by culturing the second population of TILs in a second cell culture medium comprising IL-21, IL-15, L-arginine, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-11 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic 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 (b) to step (c) occurs without opening the system; (d) harvesting the therapeutic population of TILs obtained from step (c), wherein the transition from step (c) to step (d) occurs without opening the system, and wherein the therapeutic population of TILs harvested comprises sufficient TILs for a therapeutically effective dosage of the TILs; (e) transferring the harvested TIL population from step (d) to an infusion bag, wherein the transfer from step (d) to (e) occurs without opening the system, and (f) cryopreserving the infusion bag comprising the harvested TIL population using a cryopreservation process.

[0076] Some embodiments of the present disclosure provide a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprising: (a) adding processed tumor fragments from a tumor resected from a patient into a closed system to obtain a first population of TILs; (b) 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 DB1 / 149057740.1 30Attorney Docket No.116983-5126-WO / AR / TW surface area, wherein the first expansion is performed for about 3-11 days to obtain the second population of TILs, and wherein the transition from step (a) to step (b) occurs without opening the system; (c) performing a second expansion by culturing the second population of TILs in a second cell culture medium comprising IL-21, IL-15, L-arginine, NAD+, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-11 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic 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 (b) to step (c) occurs without opening the system; (d) harvesting the therapeutic population of TILs obtained from step (c), wherein the transition from step (c) to step (d) occurs without opening the system, and wherein the therapeutic population of TILs harvested comprises sufficient TILs for a therapeutically effective dosage of the TILs; (e) transferring the harvested TIL population from step (d) to an infusion bag, wherein the transfer from step (d) to (e) occurs without opening the system, and (f) cryopreserving the infusion bag comprising the harvested TIL population using a cryopreservation process.

[0077] Some embodiments of the present disclosure provide a method for making a therapeutic population of gene-edited TILs comprising: (a) adding processed tumor fragments from a tumor resected from a patient into a closed system to obtain a first population of TILs; (b) 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 3-11 days to obtain the second population of TILs, and wherein the transition from step (a) to step (b) occurs without opening the system; DB1 / 149057740.1 31Attorney Docket No.116983-5126-WO / AR / TW (c) transducing the second population of TILs with the recombinant lentiviral particle disclosed herein to produce a population of gene-edited TILs, wherein the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion by culturing the population of gene-edited TILs in a second cell culture medium comprising IL-21, IL-15, L-arginine, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-11 days to obtain the third population of TILs, wherein the third population of TILs is the therapeutic population of gene- edited 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; (e) harvesting the therapeutic population of gene-edited TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system, and wherein the therapeutic population of gene-edited TILs harvested comprises sufficient TILs for a therapeutically effective dosage of the TILs; (f) transferring the harvested therapeutic population of gene-edited TILs from step (e) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system, and (g) cryopreserving the infusion bag comprising the harvested TIL population using a cryopreservation process.

[0078] Some embodiments of the present disclosure provide a method for making a therapeutic population of gene-edited TILs comprising: (a) adding processed tumor fragments from a tumor resected from a patient into a closed system to obtain a first population of TILs; (b) 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 3-11 days to obtain the second population of TILs, and wherein the transition from step (a) to step (b) occurs without opening the system; DB1 / 149057740.1 32Attorney Docket No.116983-5126-WO / AR / TW (c) transducing the second population of TILs with the recombinant lentiviral particle disclosed herein to produce a population of gene-edited TILs, wherein the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion by culturing the population of gene-edited TILs in a second cell culture medium comprising IL-21, IL-15, L-arginine, NAD+, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-11 days to obtain the third population of TILs, wherein the third population of TILs is the therapeutic population of gene- edited 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; (e) harvesting the therapeutic population of gene-edited TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system, and wherein the therapeutic population of gene-edited TILs harvested comprises sufficient TILs for a therapeutically effective dosage of the TILs; (f) transferring the harvested therapeutic population of gene-edited TILs from step (e) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system, and (g) cryopreserving the infusion bag comprising the harvested TIL population using a cryopreservation process.

[0079] Some embodiments of the present disclosure provide a method for making a therapeutic population of gene-edited TILs comprising: (a) processing a tumor resected from a patient to obtain a first population of TILs; (b) 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, and wherein the first expansion is performed for about 3-11 days to obtain the second population of TILs; (c) transducing the second population of TILs with the recombinant lentiviral particle disclosed herein to produce a population of gene-edited TILs; DB1 / 149057740.1 33Attorney Docket No.116983-5126-WO / AR / TW (d) performing a second expansion by culturing the population of gene-edited TILs in a second cell culture medium comprising IL-21, IL-15, L-arginine, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-11 days to obtain the third population of TILs, wherein the third population of TILs is the therapeutic population of gene- edited TILs, and wherein the second expansion is performed in a closed container providing a second gas-permeable surface area; (e) harvesting the therapeutic population of gene-edited TILs obtained from step (d), wherein the therapeutic population of gene-edited TILs harvested comprises sufficient TILs for a therapeutically effective dosage of the TILs; (f) transferring the harvested therapeutic population of gene-edited TILs from step (e) to an infusion bag, and (g) cryopreserving the infusion bag comprising the harvested TIL population using a cryopreservation process.

[0080] Some embodiments of the present disclosure provide a method for making a therapeutic population of gene-edited TILs comprising: (a) processing a tumor resected from a patient to obtain a first population of TILs; (b) 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, and wherein the first expansion is performed for about 3-11 days to obtain the second population of TILs; (c) transducing the second population of TILs with the recombinant lentiviral particle disclosed herein to produce a population of gene-edited TILs; (d) performing a second expansion by culturing the population of gene-edited TILs in a second cell culture medium comprising IL-21, IL-15, L-arginine, NAD+, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-11 days to obtain the third population of TILs, wherein the third population of TILs is the therapeutic population of gene- edited TILs, and wherein the second expansion is performed in a closed container providing a second gas-permeable surface area; DB1 / 149057740.1 34Attorney Docket No.116983-5126-WO / AR / TW (e) harvesting the therapeutic population of gene-edited TILs obtained from step (d), wherein the therapeutic population of gene-edited TILs harvested comprises sufficient TILs for a therapeutically effective dosage of the TILs; (f) transferring the harvested therapeutic population of gene-edited TILs from step (e) to an infusion bag, and (g) cryopreserving the infusion bag comprising the harvested TIL population using a cryopreservation process.

[0081] In some embodiments, the second cell culture medium comprises IL-15 at a concentration of about 10 ng / mL and IL-21 at a concentration of about 10 ng / mL. In some embodiments, the L-arginine is present at a concentration of about 1 mM to about 10 mM. In some embodiments, the L-arginine is present at a concentration of about 5 mM. In some embodiments, the second cell culture medium comprises an NAD+ booster. In some embodiments, the NAD+ booster is selected from the group consisting of L-Trp, NR, NMN, NAD+, NAM and P7C3 activator. In some embodiments, the NAD+ booster is NAD+. In some embodiments, the NAD+ is present at a concentration of about 50-75 µM. In some embodiments, the first expansion is performed over a period of about 3-14 days. In some embodiments, the first expansion is performed over a period of about 11 days. In some embodiments, the second expansion is performed over a period of about 7-14 days. In some embodiments, the second expansion is performed over a period of about 11 days.

[0082] In some embodiments, the method further comprises activating the population of TILs for 1 day or 2 days before transducing the population of TILs with the recombinant lentiviral particle. In some embodiments, the activating step comprises contacting the population of TILs with TransAct. In some embodiments, the activating step comprises contacting the population of TILs with TransAct at a ratio of 1:100. In some embodiments, the transducing step is conducted at a TIL concentration of 105cells / mL. In some embodiments, the transducing step is conducted at a multiplicity of infection (MOI) of about 10 to about 40. In some embodiments, the transducing step is conducted in the presence of RetroNectin or Vectofusin-1. In some embodiments, the transducing step comprises centrifugation. In some embodiments, the transducing step is conducted in the presence of Lentibooster. In some embodiments, the method further comprises resting the population of TILs for 2 day or 3 days after the transducing step. DB1 / 149057740.1 35Attorney Docket No.116983-5126-WO / AR / TW

[0083] Some embodiments of the present disclosure provide a therapeutic population of gene-edited TILs produced by the methods disclosed herein.

[0084] Some embodiments of the present disclosure provide a pharmaceutical composition comprising the therapeutic population of gene-edited TILs disclosed herein.

[0085] Some embodiments of the present disclosure provide a use of the therapeutic population of TILs disclosed herein, or the therapeutic population of gene-edited TILs disclosed herein, for treating cancer in a patient in need thereof, comprising administering the therapeutic population of TILs or the therapeutic population of gene-edited TILs to the patient.

[0086] In some embodiments, the cancer is selected from the group consisting of melanoma (including mucosal melanoma, uveal melanoma, cutaneous melanoma, choroidal melanoma, ciliary body melanoma, or iris melanoma), ovarian cancer, cervical cancer, endometrial cancer, non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple negative breast cancer, cancer caused by human papilloma virus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, and renal cell carcinoma. In some embodiments, the method further comprises the step of treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the TILs to the patient. In some embodiments, the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for three days. In some embodiments, the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for three days. In some embodiments, the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for one day. In some embodiments, the cyclophosphamide is administered with mesna. In some embodiments, the method further comprises a step of treating the patient with an IL-2 regimen starting on the day after the administration of TILs to the patient. In some embodiments, the method further comprises a step of treating the patient with an IL-2 regimen starting on the same day as administration of TILs to the patient. In some embodiments, 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, DB1 / 149057740.1 36Attorney Docket No.116983-5126-WO / AR / TW administered as a 15-minute bolus intravenous infusion every eight hours until tolerance. In some embodiments, the method comprises no step of treating the patient with an IL-2 regimen. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1A-1C: Summary of study to assess expression and signaling of membrane bound IL-15 / IL-21 transduced pre-REP TILs.

[0088] Figure 2A-2B: Summary of study to assess expression of mIL-15 / IL21 and CD8 and CD4 T cell subset in mIL-15 / IL-21 transduced REP TILs.

[0089] Figure 3A-3C: Summary of study to assess phenotype of mIL-15 / IL-21 transduced CD8+ REP TILs.

[0090] Figure 4A-4C: Summary of study to assess phenotype of mIL-15 / IL-21 transduced CD4+ REP TILs.

[0091] Figure 5 depicts an exemplary nucleic acid that allows for expression of a member anchored IL-12 (TeIL-12) and PD-1 shRNA in embodiments of the subject TILs provided herein.

[0092] Figure 6 depicts an exemplary workflow for the preparation of TILs expressing TeIL- 12 and / or NFAT-TeIL-12 for administration to a subject.

[0093] Figure 7A-7B: Summary of study to assess the expression of TeIL-12 and / or NFAT- TeIL-12 in REP TILs. (A) After REP harvest, surface expression of TeIL-12 on TeIL12 TIL was examined by flow assay with IL-12P70 flow Ab (B). NFAT-TeIL-12 transduced REP- TIL was stimulated with TransACT with indicated dilution or PMA.48 hours after stimulation, surface expression TeIL-12 expression was examined.

[0094] Figure 8: Summary of a study to assess IL-12 activity in TeIL-12 expressing TILs.

[0095] Figure 9A-9B: Summary of study to assess (A) expansion and (B) viability of post- REP TILs transduced to express TeIL-12 or NFAT-TeIL-12.

[0096] Figure 10A-10B: Summary of study to assess (A) the frequency of TeIL-12 and / or NFAT-TeIL-12 in a population of REP TILs from various tissues (including two lung, one head & neck, one breast and one ovarian tumor samples) and (B) viral genome copy number (VCN) per cell. DB1 / 149057740.1 37Attorney Docket No.116983-5126-WO / AR / TW

[0097] Figure 11A-11D: Summary of study to assess (A) and (B) cytotoxicity in a THP-1 based allogenic cytotoxicity assay, (C) and (D) IFN-γ production of TeIL-12 REP-TILs and NFAT-driven inducible TeIL-12 REP-TILs.

[0098] Figure 12A-12B: Summary of study to assess cytotoxicity of TeIL-12 expressing TILs was also assessed by xCelligence RTCA assay using two target cell populations (A) and (B).

[0099] Figure 13A-13C: Summary of a study to assess TIL killing efficacy. (A)Schematic of experimental design. (B) KILR® THP-1 cytotoxicity assay and IFN-g quantification, and (C) Xcellgene RTCA killing assay were performed.

[0100] Figure 14 depicts a summary of study to assess distribution of CD8+, CD4+, and CD4+ / FoxP3- T cells within a population of REP TILs transduced to express TeIL-12 or NFAT-TeIL-12.

[0101] Figure 15A-15B: Summary of study to assess T cell differentiation of (A) CD8+ and (B) CD4+ T cells, as measured by various cellular markers, within a population of REP TILs transduced to express TeIL-12 or NFAT-TeIL-12.

[0102] Figure 16A-16B: Summary of study to assess T cell exhaustion of (A) CD8+ and (B) CD4+ T cells, as measured by various cellular markers, within a population of REP TILs transduced to express TeIL-12 or NFAT-TeIL-12.

[0103] Figure 17A-17B: Summary of study to assess T cell activation of (A) CD8+ and (B) CD4+ T cells, as measured by various cellular markers, within a population of REP TILs transduced to express TeIL-12 or NFAT-TeIL-12.

[0104] Figure 18A-18B: Summary of study to assess T cell function of (A) CD8+ and (B) CD4+ T cells, as measured by various cellular markers, within a population of REP TILs transduced to express TeIL-12 or NFAT-TeIL-12.

[0105] Figure 19A-19B: Shows (A) cell expansion and (B) surface expression of TeIL-15 after the procedure: after gene transduction of TeIL-15 lentivirus, Pre-REP TIL was processed for REP expansion with feeder cell, 3000IU / ml IL-2 and aCD3 Ab OKT3 or HIT3a. OKT3 (30ng / ml) or HIT3a(30ng / ml) was added into REP culture medium in different days ( Day 0, Day 2 and Day 4) after setting REP process. After 11 days REP expansion, Post-REP-TIL was harvested and analyzed. DB1 / 149057740.1 38Attorney Docket No.116983-5126-WO / AR / TW

[0106] Figure 20A-20B: Shows (A) cell expansion and (B) surface expression of TeIL-15 / TeIL-21 after the procedure: after gene transduction of TeIL-15 / TeIL-21 lentivirus, Pre-REP TIL was processed for REP expansion with feeder cell, 3000IU / ml IL-2 and aCD3 Ab OKT3 or HIT3a. OKT3 (30ng / ml) or HIT3a(30ng / ml) was added into REP culture medium in different days ( Day 0, Day 2 and Day 4) after setting REP process. After 11 days REP expansion, Post-REP-TIL was harvested and analyzed.

[0107] Figure 21A-21B: Shows (A) cell expansion and (B) surface expression of TeIL-15 after the procedure: after gene transduction of TeIL-15 lentivirus, Pre-REP TIL was processed for 11 days REP expansion with feeder cell, 3000IU / ml IL-2 and OKT3 with indicated concentration. After 11 days REP expansion, Post-REP-TIL was harvested and analyzed.

[0108] Figure 22A-22B: Shows (A) cell expansion and (B)surface expression of TeIL-15 / TeIL-21 after the procedure: after gene transduction of TeIL-15 / TeIL-21 lentivirus, Pre-REP TIL was processed for REP expansion with feeder cell, 3000IU / ml IL-2 and OKT3 with indicated concentration. After 11 days REP expansion, Post-REP-TIL was harvested and analyzed.

[0109] Figure 23: Shows surface expression of TeIL-12 after transduction with or without TransAct.

[0110] Figure 24: Shows surface expression of TeIL-12 after transduction using Retronection or Vectofusin coated plates, and with or without centrifugation.

[0111] Figure 25: Shows surface expression of TeIL-12 after transduction using TransACT or PMA-Inomycin stimulation, and with or without Lentiboost.

[0112] Figure 26: Shows surface expression of TeIL-12 after transduction using the BaEVTR, RD114 or VSV-G Env plasmid.

[0113] Figure 27A-27D: Shows results from (A) KILR® THP-1 cell killing assay, (B) IFN production, (C) xCelligence RTCA assay, and (D) xCelligence RTCA assay after serial TransACT stimulation, of NFAT-TeIL-12 expressing TILs.

[0114] Figure 28: Shows an exemplary expression vector encoding NFAT driven TeIL-12 and EF1α driven bicistronic tCD19 and Component X.

[0115] Figure 29A-29B: Shows (A) fold expansion and (B) viability of TILs transducted with NFAT-TeIL-12 and Component X. DB1 / 149057740.1 39Attorney Docket No.116983-5126-WO / AR / TW

[0116] Figure 30A-30B: Shows (A) transduction efficiency and (B) viral copy number per cell (VCN) of TILs transducted with NFAT-TeIL-12 and Component X.

[0117] Figure 31: Shows expression of tCD19, TeIL-2, TeIL-15, IL-2, IL-15, GFP shRNA and PD-1 shRNA after different levels of TCR stimulation.

[0118] Figure 32: Shows results from xCELLgene RTCA cell killing assay of TILs expressing tCD19, TeIL-2, TeIL-15, IL-2, or IL-15.

[0119] Figure 33: Shows p-STAT activation induced by TILs expressing TeIL-2, TeIL-15, IL-2, or IL-15.

[0120] Figure 34: Shows skewed in cis proliferation of TILs expressing TeIL-2, TeIL-15, IL-2, or IL-15.

[0121] Figure 35A-35B: Shows EF-1α driven IL-2 / IL-15 improve T cell proliferation in the absence of IL-2.

[0122] Figure 36A-36B: Shows PD-1 shRNA effectively reduces PD-1 positivity in TILs measured by flow cytometry (A) or gMFI (B).

[0123] Figure 37A-37D: Shows TeIL-15 facilitates TIL survival in vitro in the absence of IL-2.

[0124] Figure 38A-38B: Shows PDX data of in vivo efficacy of NFAT-IL-12 gene engineered TIL using Gen 2 or Invigo-T + I-Arg processes.

[0125] Figure 39A-39B: Shows dynamic change of body weight (A) and body weight % related baseline (B) of NFAT-IL-12 gene engineered TIL using Gen 2 or Invigo-T + L-Arg processes.

[0126] Figure 40: Shows plasma level of IFN-γ, TNFa, CCL4 and IL-12 p70 in mice infused with Gen 2 or NFAT-IL-12 gene engineered TILs.

[0127] Figure 41: Shows tumor reactivity was enhanced when TILs were cultured with ITIL cytokines in the presence of IFNγ.

[0128] Figure 42A-42B: Shows 4-1BB expression was found to be maximal 24 hours after plating the tumor digest (A), and increase with the provision of IFNγ to the cell culture media (TS-TIL condition) (B).

[0129] Figure 43: Outlines several processes that were analyzed for their capacity to enrich tumor-reactive CD8 TILs. DB1 / 149057740.1 40Attorney Docket No.116983-5126-WO / AR / TW

[0130] Figure 44: Shows ~10x improvement in product potency of TILs made by the CD137 sorted process.

[0131] Figure 45: Shows enrichement for tumor reactive TILs made by the TS-TIL (4-1BB sort / 1x REP) process.

[0132] Figure 46: Shows both CD137 (4-1BB) sorting conditions provided the best enrichment for tumor-reactive CD8s (IFNγ +CD107a+) TILs.

[0133] Figure 47: Shows 60-fold improvement for tumor-reactive CD8s (IFNγ +CD107a+) TILs by CD137 (4-1BB) sorting condition.

[0134] Figure 48: Outlines the “TS-TIL” (4-1BB selection) process for producing tumor reactive TILs.

[0135] Figure 49: Shows a significant increase in percentage of specific tumor reactive TILs in the TS-TIL product (~7%) vs the CTRL product (~1%).

[0136] Figure 50: Shows the total number of CD8 tumor-reactive TILs generated from the TS-TIL process is, on average 7-fold, greater than the total number of CD8 tumor- reactive TILs generated from the CTRL process.

[0137] Figure 51: Shows the results for total viable cells and fold expansion as a function of REP conditions.

[0138] Figure 52: Shows the total yield of CD8+ tumor-reactive TIL (as assessed by autologous digest coculture ICS) as a function of sort and REP conditions.

[0139] Figure 53: Shows that the majority of cells was found to be Tem “effector memory” cells, with minimal differences among the TILs made from different processes.

[0140] Figure 54: Shows that TeIL-15 activated T cells in cis manner only.

[0141] Figure 55A-55D: Shows that L-Arginine enhances expansion and cell recovery post thaw during Invigo-T REP.

[0142] Figure 56A-56C: Shows that L-Arginine provides better phenotype and killing capacity.

[0143] Figure 57A-57C: Shows that NAD+ synergies with L-Arginine in boosting metabolism and providing a unique T cell signature. DB1 / 149057740.1 41Attorney Docket No.116983-5126-WO / AR / TW

[0144] Figure 58 shows the TIL manufacturing process from banked and fresh preREP TILs.

[0145] Figure 59 shows the transduction efficiency in CD3+ T cells for NFAT- TeIL12-EF1α-tCD19 (mean=30.3%) and NFAT-TeIL12-EF1α-TeIL15 (mean= 23.2%).

[0146] Figure 60A shows the transduction efficiency in CD4+ and CD8+ T cell subsets demonstrating that the expression of tCD19 in TILs transduced with NFAT-TeIL12- EF1α-tCD19 is similar between CD4+ and CD8+ cells.

[0147] Figure 60B shows the expression of TeIL15 in TILs transduced with NFAT- TeIL12-EF1α-TeIL15 is higher in CD8+ T cells compared to CD4+ T cells.

[0148] Figure 61 shows that the lentiviral vector transduced TILs exhibit low / minimal skewing of the percentages of CD4+ or CD8+ cells in the REP TIL product.

[0149] Figure 62 shows that the REP cell culture media conditions exhibit similar transduction efficiency in CD3+ T cells.

[0150] Figure 63A-63B shows that the REP cell culture media conditions exhibit similar transduction efficiency in CD4+ and CD8+ T cell subsets.

[0151] Figure 64 shows that the expansion rate and viability are enhanced with various REP cell culture media conditions.

[0152] Figure 65 shows that various REP cell culture media conditions increase the number of TILs (TVC) in untransduced TILs.

[0153] Figure 66 shows that various REP cell culture media conditions increase the viability in untransduced TILs.

[0154] Figure 67 shows that the expression level for TeIL-15 in REP TILs is enhanced by GEN2+NAD and ITARG+NAD in CD3+ cells (A), CD4+ T cells (B), and CD8+ T cells (C).

[0155] Figure 68 shows that untransduced TILs (A, C) and NFAT-TeIL-12-EF1α- TeIL-15 TILs (B, D) exhibit increased production of IFNγ (A, B) and TNFα (C, D) when cultured with autologous tumor digest (TIL+Digest) compared to TILs that did not receive any stimulation (Unstimulated).

[0156] Figure 69 shows that untransduced TILs (A, C) and NFAT-TeIL12-EF1α- TeIL15 TILs (B, D) exhibit a reduction of IFNγ (A, B) and TNFα (C, D) when cultured with DB1 / 149057740.1 42Attorney Docket No.116983-5126-WO / AR / TW autologous tumor digest (TIL+Digest) compared to co-cultures incubated with anti-HLA-I and anti-HLA-II (TIL+Digest+HLA Block).

[0157] Figure 70 shows that NFAT-TeIL12-EF1α-TeIL15 TILs produce more IFNγ when cultured with autologous tumor digest (TIL+Digest) compared to untransduced TILs, which is enhanced when the TILs underwent a GEN2+NAD or ITARG+NAD REP (A). NFAT-TeIL12-EF1α-TeIL15 TILs expanded under GEN2 conditions exhibited a 2.8-fold increase in IFNγ, whereas NFAT-TeIL12-EF1α-TeIL15 GEN2+NAD TILs and NFAT- TeIL12-EF1α-TeIL15 ITARG+NAD TILs demonstrated a 6.7-fold increase and a 5.1-fold increase in IFNγ respectively (B).

[0158] Figure 71 shows NFAT-TeIL12-EF1α-TeIL15 TILs produce more TNFα when cultured with autologous tumor digest (TIL+Digest) compared to Untransduced TILs, which is enhanced when the TILs underwent a GEN2+NAD or ITARG+NAD REP (A). NFAT-TeIL12-EF1α-TeIL15 TILs expanded under GEN2 conditions exhibited a 1.3-fold increase in TNFα, compared to NFAT-TeIL12-EF1α-TeIL15 GEN2+NAD TILs (1.8-fold), NFAT-TeIL12-EF1α-TeIL15 ITARG TILs (1.7-fold), and NFAT-TeIL12-EF1α-TeIL15 ITARG+NAD TILs (1.7-fold) (B).

[0159] Figure 72 shows TILs transduced with NFAT-TeIL12-EF1α-tCD19 or NFAT- TeIL12-EF1α-TeIL15 exhibit superior cytotoxicity against an autologous melanoma cell line in comparison to Untransduced TILs. The percentage of Caspase3 / 7+ cells at the 8:1 ET ratio (A) and 4:1 ET ratio (B) were significantly higher when tumor cells were cultured with autologous NFAT-TeIL12-EF1α-tCD19 or NFAT-TeIL12-EF1α-TeIL15 TILs in comparison to untransduced TILs.

[0160] Figure 73 shows NYESO1 TCR-selected T-cells transduced with either the single NFAT-teIL12 construct or the dual NFAT-teIL12-EF1a-TeIL15 construct demonstrated superior killing efficacy when compared to lentivirus backbone control and mock process control.

[0161] Figure 74 shows (A) total viable cells (B) fold expansion and (C) viability at the end of 22 day REP process and (D) viability 1 hour post-thaw of unmodified TIL expanded under various media formulations. Data displayed as mean ± SD. Data by Repeated measures Mixed Model, post-hoc Tukey's multiple comparisons test. * p<0.01; **p<0.001, ***p<0.0001. Abbreviations: ITARG, IL-15, IL-21, L-Arginine; NAD+, nicotinamide adenine dinucleotide. DB1 / 149057740.1 43Attorney Docket No.116983-5126-WO / AR / TW

[0162] Figure 75 shows (A) total viable cells (B) fold expansion and (C) viability at the end of 22 day REP process and (D) viability 1 hour post-thaw of tethered-cytokine TIL (NFAT-TeIL12 / TeIL15) expanded under various conditions. Data displayed as mean ± SD. Data by Repeated measures Mixed Model, post-hoc Tukey's multiple comparisons test. Ns, not significant; * p<0.01; **p<0.001, ***p<0.0001. Abbreviations: ITARG, IL-15, IL-21, L- Arginine; NAD+, nicotinamide adenine dinucleotide.

[0163] Figure 76 shows a vector map of pCMV-3Tag-4a-BaEV-TR (SEQ ID NO:165).

[0164] Figure 77 shows a vector map of pLenti- IRES-NFAT-TeIL12-EF1a-TeIL-15 (SEQ ID NO:166).

[0165] Figure 78. (A) Total viable cells (B) fold expansion and (C) viability at the end of 22-day REP process of unmodified TIL expanded under various media formulations from 41 donors across various tumor types. Data displayed as mean ± SD. Data by Repeated measures Mixed-effects Model, post-hoc Tukey's multiple comparisons test. * p<0.01; **p<0.001, ***p<0.0001. Abbreviations: ITARG, IL-15, IL-21, L-Arginine; NAD+, nicotinamide adenine dinucleotide.

[0166] Figure 79. In vitro cytotoxicity of tumor-directed TIL. Assessment of cytotoxicity of KRAS-TCR TIL products from 5 donors against KRAS (HPAC) tumor cells. Antigen-specific TILs were added to respective HPAC cells in a modified media that simulates conditions often observed in solid tumor microenvironment (TME) (with and without IL-2, (A) and (B), respectively). Data displayed as mean ± SD. Data by Repeated measures Mixed-effects Model, post-hoc Tukey's multiple comparisons test. * p<0.01; **p<0.001, ***p<0.0001. Abbreviations: ITARG, IL-15, IL-21, L-Arginine; NAD+, nicotinamide adenine dinucleotide.

[0167] Figure 80. In vitro cytotoxicity of tumor-directed TIL with membrane- tethered cytokines. Assessment of cytotoxicity of KRAS.mteth.IL12.IL15 TIL products from 5 donors against KRAS (HPAC) tumor cells in a modified media that simulates conditions often observed in solid tumor microenvironment (TME) (with and without IL-2, (A) and (B), respectively). Data displayed as mean ± SD. Data by Repeated measures Mixed-effects Model, post-hoc Tukey's multiple comparisons test. * p<0.01; **p<0.001, ***p<0.0001. Abbreviations: ITARG, IL-15, IL-21, L-Arginine; NAD+, nicotinamide adenine dinucleotide. DB1 / 149057740.1 44Attorney Docket No.116983-5126-WO / AR / TW The level of mteth.IL15 on the surface of KRAS transduced TIL detected using flow cytometry serves as a control for transduction efficiency (C),

[0168] Figure 81. TeIL-12 / TeIL-15 expressing TILs show trends of increased persistence after infusion.

[0169] Figure 82. Exemplary Gen 2 (process 2A) chart providing an overview of Steps A through F.

[0170] Figure 83A-83C. Process flow chart of an embodiment of Gen 2 (process 2A) for TIL manufacturing. DETAILED DESCRIPTION I. Introduction

[0171] Adoptive cell therapy utilizing TILs is an effective approach for inducing tumor regression in various cancers, including leukemias and melanoma. The use of adjuvants that include immunostimulatory agents has been explored to enhance adoptive cell therapies and to extend such therapies to other solid tumors. Co-administration of immunomodulators such as cytokines (e.g., interleukins), however, can lead to undesirably toxicity due to the high dosages required. Thus, supplying such adjuvants at the right time and site appears crucial to avoid such undesirable effects.

[0172] Provided herein are compositions and methods for the treatment of cancers using modified TILs, wherein the modified TILs include one or more immunomodulatory agents (e.g., cytokines) associated with their cell surface. The immunomodulatory agents associated with the TILs provide a localized immunostimulatory effect that can advantageously enhance TIL survival and / or anti-tumor activity in a patient recipient. As such, the compositions and methods disclosed herein provide effective cancer therapies. II. Definitions

[0173] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. All patents and publications referred to herein are incorporated by reference in their entireties. DB1 / 149057740.1 45Attorney Docket No.116983-5126-WO / AR / TW

[0174] The terms “co-administration,” “co-administering,” “administered in combination with,” “administering in combination with,” “simultaneous,” and “concurrent,” as used herein, 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.

[0175] The term “in vivo” refers to an event that takes place in a subject’s body.

[0176] The term “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.

[0177] The term “ex vivo” 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.

[0178] The term “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 10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold) over a period of a week, or most preferably at least about 100-fold over a period of a week. A number of rapid expansion protocols are described herein.

[0179] By “tumor infiltrating lymphocytes” or “TILs” herein is meant a population of cells originally obtained as white blood cells that have left the bloodstream of a subject and migrated into a tumor. TILs include, but are not limited to, CD8+cytotoxic T cells (lymphocytes), Th1 and Th17 CD4+T cells, natural killer cells, dendritic cells and M1 macrophages. 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 and expanded TILs DB1 / 149057740.1 46Attorney Docket No.116983-5126-WO / AR / TW (“REP TILs” or “post-REP TILs”). TIL cell populations can include genetically modified TILs.

[0180] By “population of cells” (including TILs) herein is meant a number of cells that share common traits. In general, populations generally range from 1 X 106to 1 X 1010in number, with different TIL populations comprising different numbers. For example, initial growth of primary TILs in the presence of IL-2 results in a population of bulk TILs of roughly 1 × 108cells. REP expansion is generally done to provide populations of 1.5 × 109to 1.5 × 1010cells for infusion.

[0181] By “cryopreserved TILs” herein is meant that TILs, either primary, bulk, or expanded (REP TILs), are treated and stored in the range of about -150°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.

[0182] By “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 administered to a patient.

[0183] 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 αβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into a patient.

[0184] The term “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.

[0185] The term “central memory T cell” refers to a subset of T cells that in the human are CD45R0+ and constitutively express CCR7 (CCR7hi) and CD62L (CD62hi). The surface DB1 / 149057740.1 47Attorney Docket No.116983-5126-WO / AR / TW 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.

[0186] The term “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 (CCR7lo) and are heterogeneous or low for CD62L expression (CD62Llo). 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 BLIMP1. Effector memory T cells rapidly secret high levels of inflammatory cytokines following antigenic stimulation, including interferon-γ, 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.

[0187] The term “closed system” refers to a system that is closed to the outside environment. Any closed system appropriate for cell culture methods can be employed with the methods of the present invention. 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.

[0188] The terms “fragmenting,” “fragment,” and “fragmented,” as used herein to describe processes for disrupting a tumor, 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.

[0189] The terms “peripheral blood mononuclear cells” and “PBMCs” refers to a peripheral blood cell having a round nucleus, including lymphocytes (T cells, B cells, NK cells) and monocytes. When used as an antigen presenting cell (PBMCs are a type of antigen-presenting cell), the peripheral blood mononuclear cells are preferably irradiated allogeneic peripheral blood mononuclear cells.

[0190] The term “anti-CD3 antibody” refers to an antibody or variant thereof, e.g., a monoclonal antibody and including human, humanized, chimeric or murine antibodies which DB1 / 149057740.1 48Attorney Docket No.116983-5126-WO / AR / TW 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 CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0191] The term “OKT-3” (also referred to herein as “OKT3”) 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. TABLE 1. Amino acid sequences of muromonab (exemplary OKT-3 antibody). Identifier Sequence (One-Letter Amino Acid Symbols)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. The amino acid sequence of recombinant human IL-2 suitable for use in the invention is given in Table 2 (SEQ ID NO:3). For example, the term 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 DB1 / 149057740.1 49Attorney Docket No.116983-5126-WO / AR / TW 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 (des-alanyl-1, serine-125 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 bempegaldesleukin (NKTR-214, pegylated human recombinant IL-2 as in SEQ ID NO:4 in which an average of 6 lysine residues are N6substituted with [(2,7-bis{[methylpoly(oxyethylene)]carbamoyl}-9H- fluoren-9-yl)methoxy]carbonyl), which is available from Nektar Therapeutics, South San Francisco, CA, USA, or which may be prepared by methods known in the art, such as the methods described in Example 19 of International Patent Application Publication No. WO 2018 / 132496 A1 or the method described in Example 1 of U.S. Patent Application Publication No. US 2019 / 0275133 A1, the disclosures of which are incorporated by reference herein. Bempegaldesleukin (NKTR-214) and other pegylated IL-2 molecules suitable for use in the invention are described in U.S. Patent Application Publication No. US 2014 / 0328791 A1 and International Patent Application Publication No. WO 2012 / 065086 A1, the disclosures of which are incorporated by reference herein. Alternative forms of conjugated IL-2 suitable for use in the invention are described in U.S. Patent Nos.4,766,106, 5,206,344, 5,089,261 and 4,902,502, the disclosures of which are incorporated by reference herein. Formulations of IL-2 suitable for use in the invention are described in U.S. Patent No. 6,706,289, the disclosure of which is incorporated by reference herein.

[0193] In some embodiments, an IL-2 form suitable for use in the present invention is THOR-707, available from Synthorx, Inc. The preparation and properties of THOR-707 and additional alternative forms of IL-2 suitable for use in the invention are described in U.S. Patent Application Publication Nos. US 2020 / 0181220 A1 and US 2020 / 0330601 A1, the disclosures of which are incorporated by reference herein. In some embodiments, and IL-2 form suitable for use in the invention is an interleukin 2 (IL-2) conjugate comprising: an isolated and purified IL-2 polypeptide; and a conjugating moiety that binds to the isolated and purified IL-2 polypeptide at an amino acid position selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107, wherein the numbering of the amino acid residues corresponds to SEQ ID NO:5. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72, DB1 / 149057740.1 50Attorney Docket No.116983-5126-WO / AR / TW and Y107. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from R38 and K64. In some embodiments, the amino acid position is selected from E61, E62, and E68. In some embodiments, the amino acid position is at E62. In some embodiments, the amino acid residue selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 is further mutated to lysine, cysteine, or histidine. In some embodiments, the amino acid residue is mutated to cysteine. In some embodiments, the amino acid residue is mutated to lysine. In some embodiments, the amino acid residue selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 is further mutated to an unnatural amino acid. In some embodiments, the unnatural amino acid comprises N6-azidoethoxy-L- lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO- lysine, methyltetrazine lysine, allyloxycarbonyllysine, 2-amino-8-oxononanoic acid, 2- amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p- propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L- phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4- propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3- oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenylselanyl)propanoic, or selenocysteine. In some embodiments, the IL-2 conjugate has a decreased affinity to IL-2 receptor α (IL-2Rα) subunit relative to a wild-type IL-2 polypeptide. In some embodiments, the decreased affinity is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than 99% decrease in binding affinity to IL-2Rα relative to a wild-type IL-2 polypeptide. In some embodiments, the decreased affinity is about 1-fold, 2-fold, 3-fold, 4- fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300- fold, 500-fold, 1000-fold, or more relative to a wild-type IL-2 polypeptide. In some embodiments, the conjugating moiety impairs or blocks the binding of IL-2 with IL-2Rα. In some embodiments, the conjugating moiety comprises a water-soluble polymer. In some embodiments, the additional conjugating moiety comprises a water-soluble polymer. In some embodiments, each of the water-soluble polymers independently comprises polyethylene DB1 / 149057740.1 51Attorney Docket No.116983-5126-WO / AR / TW glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N- acryloylmorpholine), or a combination thereof. In some embodiments, each of the water- soluble polymers independently comprises PEG. In some embodiments, the PEG is a linear PEG or a branched PEG. In some embodiments, each of the water-soluble polymers independently comprises a polysaccharide. In some embodiments, the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl-starch (HES). In some embodiments, each of the water-soluble polymers independently comprises a glycan. In some embodiments, each of the water-soluble polymers independently comprises polyamine. In some embodiments, the conjugating moiety comprises a protein. In some embodiments, the additional conjugating moiety comprises a protein. In some embodiments, each of the proteins independently comprises an albumin, a transferrin, or a transthyretin. In some embodiments, each of the proteins independently comprises an Fc portion. In some embodiments, each of the proteins independently comprises an Fc portion of IgG. In some embodiments, the conjugating moiety comprises a polypeptide. In some embodiments, the additional conjugating moiety comprises a polypeptide. In some embodiments, each of the polypeptides independently comprises a XTEN peptide, a glycine-rich homoamino acid polymer (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer. In some embodiments, the isolated and purified IL-2 polypeptide is modified by glutamylation. In some embodiments, the conjugating moiety is directly bound to the isolated and purified IL-2 polypeptide. In some embodiments, the conjugating moiety is indirectly bound to the isolated and purified IL-2 polypeptide through a linker. In some embodiments, the linker comprises a homobifunctional linker. In some embodiments, the homobifunctional linker comprises Lomant’s reagent dithiobis (succinimidylpropionate) DSP, 3′3′- dithiobis(sulfosuccinimidyl proprionate) (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo DST), ethylene glycobis(succinimidylsuccinate) (EGS), disuccinimidyl glutarate (DSG), N,N′-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3′- dithiobispropionimidate (DTBP), 1,4-di-(3′-(2′-pyridyldithio)propionamido)butane (DPDPB), bismaleimidohexane (BMH), aryl halide-containing compound (DFDNB), such as e.g.1,5- DB1 / 149057740.1 52Attorney Docket No.116983-5126-WO / AR / TW difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4′-difluoro-3,3′- dinitrophenylsulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3′-dimethylbenzidine, benzidine, α,α′-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid, N,N′-ethylene-bis(iodoacetamide), or N,N′-hexamethylene- bis(iodoacetamide). In some embodiments, the linker comprises a heterobifunctional linker. In some embodiments, the heterobifunctional linker comprises N-succinimidyl 3-(2- pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble-long-chain N-succinimidyl 3-(2-pyridyldithio) propionate (sulfo- LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamido]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl- 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N- hydroxysuccinimide ester (MBs), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBs), N-succinimidyl(4-iodoacteyl)aminobenzoate (sIAB), sulfosuccinimidyl(4- iodoacteyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ- maleimidobutyryloxy)succinimide ester (GMBs), N-(γ-maleimidobutyryloxy) sulfosuccinimide ester (sulfo-GMBs), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (slAXX), succinimidyl 4- (((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6-(((((4- iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino) hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive cross-linkers such as 4-(4-N- maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-1- carboxyl-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), N- hydroxysuccinimidyl-4-azidosalicylic acid (NHs-AsA), N-hydroxysulfosuccinimidyl-4- azidosalicylic acid (sulfo-NHs-AsA), sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3′-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4- azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4′-azido-2′-nitrophenyl amino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-Nos), sulfosuccinimidyl-2-(m-azido-o- nitrobenzamido)-ethyl-1,3′-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl)1,3′- dithiopropionate (sADP), N-sulfosuccinimidyl(4-azidophenyl)-1,3′-dithiopropionate (sulfo- DB1 / 149057740.1 53Attorney Docket No.116983-5126-WO / AR / TW sADP), sulfosuccinimidyl 4-(ρ-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7- azido-4-methylcoumarin-3-acetamide)ethyl-1,3′-dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumain-3-acetate (sulfo-sAMCA), p-nitrophenyl diazopyruvate (pNPDP), p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), 1-(ρ-azidosalicylamido)-4- (iodoacetamido)butane (AsIB), N-[4-(ρ-azidosalicylamido)butyl]-3′-(2′-pyridyldithio) propionamide (APDP), benzophenone-4-iodoacetamide, p-azidobenzoyl hydrazide (ABH), 4- (ρ-azidosalicylamido)butylamine (AsBA), or p-azidophenyl glyoxal (APG). In some embodiments, the linker comprises a cleavable linker, optionally comprising a dipeptide linker. In some embodiments, the dipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys. In some embodiments, the linker comprises a non-cleavable linker. In some embodiments, the linker comprises a maleimide group, optionally comprising maleimidocaproyl (mc), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo- sMCC). In some embodiments, the linker further comprises a spacer. In some embodiments, the spacer comprises p-aminobenzyl alcohol (PAB), p-aminobenzyoxycarbonyl (PABC), a derivative, or an analog thereof. In some embodiments, the conjugating moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the additional conjugating moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the IL-2 form suitable for use in the invention is a fragment of any of the IL-2 forms described herein. In some embodiments, the IL-2 form suitable for use in the invention is pegylated as disclosed in U.S. Patent Application Publication No. US 2020 / 0181220 A1 and U.S. Patent Application Publication No. US 2020 / 0330601 A1. In some embodiments, the IL-2 form suitable for use in the invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising an N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugating moiety comprising a polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:5; and the AzK substitutes for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 in reference to the amino acid positions within SEQ ID NO:5. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of one residue relative to SEQ ID NO:5. In some embodiments, the IL-2 form suitable for use in the invention lacks IL-2R alpha chain engagement but retains normal binding to the intermediate affinity IL-2R beta-gamma signaling complex. In some embodiments, the IL-2 form suitable for use in the invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising an N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugating moiety comprising a DB1 / 149057740.1 54Attorney Docket No.116983-5126-WO / AR / TW polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:5; and the AzK substitutes for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 in reference to the amino acid positions within SEQ ID NO:5. In some embodiments, the IL-2 form suitable for use in the invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising an N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugating moiety comprising a polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:5; and the AzK substitutes for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 in reference to the amino acid positions within SEQ ID NO:5. In some embodiments, the IL-2 form suitable for use in the invention is an IL-2 conjugate comprising: an IL-2 polypeptide comprising an N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugating moiety comprising a polyethylene glycol (PEG), wherein: the IL-2 polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:5; and the AzK substitutes for an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 in reference to the amino acid positions within SEQ ID NO:5.

[0194] In some embodiments, an IL-2 form suitable for use in the invention is nemvaleukin alfa, also known as ALKS-4230 (SEQ ID NO:6), which is available from Alkermes, Inc. Nemvaleukin alfa is also known as human interleukin 2 fragment (1-59), variant (Cys125>Ser51), fused via peptidyl linker (60GG61) to human interleukin 2 fragment (62-132), fused via peptidyl linker (133GSGGGS138) to human interleukin 2 receptor α-chain fragment (139-303), produced in Chinese hamster ovary (CHO) cells, glycosylated; human interleukin 2 (IL-2) (75-133)-peptide [Cys125(51)>Ser]-mutant (1-59), fused via a G2 peptide linker (60- 61) to human interleukin 2 (IL-2) (4-74)-peptide (62-132) and via a GSG3S peptide linker (133-138) to human interleukin 2 receptor α-chain (IL2R subunit alpha, IL2Rα, IL2RA) (1- 165)-peptide (139-303), produced in Chinese hamster ovary (CHO) cells, glycoform alfa. The amino acid sequence of nemvaleukin alfa is given in SEQ ID NO:6. In some embodiments, nemvaleukin alfa exhibits the following post-translational modifications: disulfide bridges at positions: 31-116, 141-285, 184-242, 269-301, 166-197 or 166-199, 168- 199 or 168-197 (using the numbering in SEQ ID NO:6), and glycosylation sites at positions: N187, N206, T212 using the numbering in SEQ ID NO:6. The preparation and properties of nemvaleukin alfa, as well as additional alternative forms of IL-2 suitable for use in the invention, is described in U.S. Patent Application Publication No. US 2021 / 0038684 A1 and DB1 / 149057740.1 55Attorney Docket No.116983-5126-WO / AR / TW U.S. Patent No.10,183,979, the disclosures of which are incorporated by reference herein. In some embodiments, an IL-2 form suitable for use in the invention is a protein having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity to SEQ ID NO:6. In some embodiments, an IL-2 form suitable for use in the invention has the amino acid sequence given in SEQ ID NO:6 or conservative amino acid substitutions thereof. In some embodiments, an IL-2 form suitable for use in the invention is a fusion protein comprising amino acids 24-452 of SEQ ID NO:7, or variants, fragments, or derivatives thereof. In some embodiments, an IL-2 form suitable for use in the invention is a fusion protein comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity to amino acids 24-452 of SEQ ID NO:7, or variants, fragments, or derivatives thereof. Other IL-2 forms suitable for use in the present invention are described in U.S. Patent No.10,183,979, the disclosures of which are incorporated by reference herein. Optionally, in some embodiments, an IL-2 form suitable for use in the invention is a fusion protein comprising a first fusion partner that is linked to a second fusion partner by a mucin domain polypeptide linker, wherein the first fusion partner is IL-1Rα or a protein having at least 98% amino acid sequence identity to IL-1Rα and having the receptor antagonist activity of IL-Rα, and wherein the second fusion partner comprises all or a portion of an immunoglobulin comprising an Fc region, wherein the mucin domain polypeptide linker comprises SEQ ID NO:8 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:8 and wherein the half-life of the fusion protein is improved as compared to a fusion of the first fusion partner to the second fusion partner in the absence of the mucin domain polypeptide linker. TABLE 2. Amino acid sequences of interleukins. Identifier Sequence (One-Letter Amino Acid Symbols)DB1 / 149057740.1Attorney Docket No.116983-5126-WO / AR / TW GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ 300 YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR 360 EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS 420 RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK 452antibody cytokine engrafted protein comprises a heavy chain variable region (VH), comprising complementarity determining regions HCDR1, HCDR2, HCDR3; a light chain variable region (VL), comprising LCDR1, LCDR2, LCDR3; and an IL-2 molecule or a fragment thereof engrafted into a CDR of the VH or the VL, wherein the antibody cytokine engrafted protein preferentially expands T effector cells over regulatory T cells. In some embodiments, the antibody cytokine engrafted protein comprises a heavy chain variable region (VH), comprising complementarity determining regions HCDR1, HCDR2, HCDR3; a light chain variable region (VL), comprising LCDR1, LCDR2, LCDR3; and an IL-2 molecule or a fragment thereof engrafted into a CDR of the VHor the VL, wherein the IL-2 molecule is a mutein, and wherein the antibody cytokine engrafted protein preferentially expands T effector cells over regulatory T cells. In some embodiments, the IL-2 regimen comprises administration of an antibody described in U.S. Patent Application Publication No. US 2020 / 0270334 A1, the disclosures of which are incorporated by reference herein. In some embodiments, the antibody cytokine engrafted protein comprises a heavy chain variable region (VH), comprising complementarity determining regions HCDR1, HCDR2, HCDR3; a light chain variable region (VL), comprising LCDR1, LCDR2, LCDR3; and an IL-2 molecule or a fragment thereof engrafted into a CDR of the VHor the VL, wherein the IL-2 molecule is a mutein, wherein the antibody cytokine engrafted protein preferentially expands T effector cells over regulatory T cells, and wherein the antibody further comprises an IgG class heavy chain and an IgG class light chain selected from the group consisting of: a IgG DB1 / 149057740.1 57Attorney Docket No.116983-5126-WO / AR / TW class light chain comprising SEQ ID NO:39 and a IgG class heavy chain comprising SEQ ID NO:38; a IgG class light chain comprising SEQ ID NO:37 and a IgG class heavy chain comprising SEQ ID NO:29; a IgG class light chain comprising SEQ ID NO:39 and a IgG class heavy chain comprising SEQ ID NO:29; and a IgG class light chain comprising SEQ ID NO:37 and a IgG class heavy chain comprising SEQ ID NO:38.

[0196] In some embodiments, an IL-2 molecule or a fragment thereof is engrafted into HCDR1 of the VH, wherein the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is engrafted into HCDR2 of the VH, wherein the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is engrafted into HCDR3 of the VH, wherein the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is engrafted into LCDR1 of the VL, wherein the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is engrafted into LCDR2 of the VL, wherein the IL-2 molecule is a mutein. In some embodiments, an IL-2 molecule or a fragment thereof is engrafted into LCDR3 of the VL, wherein the IL-2 molecule is a mutein.

[0197] The insertion of the IL-2 molecule can be at or near the N-terminal region of the CDR, in the middle region of the CDR or at or near the C-terminal region of the CDR. In some embodiments, the antibody cytokine engrafted protein comprises an IL-2 molecule incorporated into a CDR, wherein the IL2 sequence does not frameshift the CDR sequence. In some embodiments, the antibody cytokine engrafted protein comprises an IL-2 molecule incorporated into a CDR, wherein the IL-2 sequence replaces all or part of a CDR sequence. The replacement by the IL-2 molecule can be the N-terminal region of the CDR, in the middle region of the CDR or at or near the C-terminal region the CDR. A replacement by the IL-2 molecule can be as few as one or two amino acids of a CDR sequence, or the entire CDR sequences.

[0198] In some embodiments, an IL-2 molecule is engrafted directly into a CDR without a peptide linker, with no additional amino acids between the CDR sequence and the IL-2 sequence. In some embodiments, an IL-2 molecule is engrafted indirectly into a CDR with a peptide linker, with one or more additional amino acids between the CDR sequence and the IL-2 sequence.

[0199] In some embodiments, the IL-2 molecule described herein is an IL-2 mutein. In some instances, the IL-2 mutein comprising an R67A substitution. In some embodiments, the DB1 / 149057740.1 58Attorney Docket No.116983-5126-WO / AR / TW IL-2 mutein comprises the amino acid sequence SEQ ID NO:14 or SEQ ID NO:15. In some embodiments, the IL-2 mutein comprises an amino acid sequence in Table 1 in U.S. Patent Application Publication No. US 2020 / 0270334 A1, the disclosure of which is incorporated by reference herein.

[0200] In some embodiments, the antibody cytokine engrafted protein comprises an HCDR1 selected from the group consisting of SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22 and SEQ ID NO:25. In some embodiments, the antibody cytokine engrafted protein comprises an HCDR1 selected from the group consisting of SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13 and SEQ ID NO:16. In some embodiments, the antibody cytokine engrafted protein comprises an HCDR1 selected from the group consisting of HCDR2 selected from the group consisting of SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, and SEQ ID NO:26. In some embodiments, the antibody cytokine engrafted protein comprises an HCDR3 selected from the group consisting of SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, and SEQ ID NO:27. In some embodiments, the antibody cytokine engrafted protein comprises a VH region comprising the amino acid sequence of SEQ ID NO:28. In some embodiments, the antibody cytokine engrafted protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:29. In some embodiments, the antibody cytokine engrafted protein comprises a VL region comprising the amino acid sequence of SEQ ID NO:36. In some embodiments, the antibody cytokine engrafted protein comprises a light chain comprising the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine engrafted protein comprises a VH region comprising the amino acid sequence of SEQ ID NO:28 and a VLregion comprising the amino acid sequence of SEQ ID NO:36. In some embodiments, the antibody cytokine engrafted protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:29 and a light chain region comprising the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine engrafted protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:29 and a light chain region comprising the amino acid sequence of SEQ ID NO:39. In some embodiments, the antibody cytokine engrafted protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:38 and a light chain region comprising the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine engrafted protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:38 and a light chain region comprising the amino acid sequence of SEQ ID NO:39. In some embodiments, the antibody cytokine engrafted protein comprises DB1 / 149057740.1 59Attorney Docket No.116983-5126-WO / AR / TW IgG.IL2F71A.H1 or IgG.IL2R67A.H1 of U.S. Patent Application Publication No. 2020 / 0270334 A1, or variants, derivatives, or fragments thereof, or conservative amino acid substitutions thereof, or proteins with at least 80%, at least 90%, at least 95%, or at least 98% sequence identity thereto. In some embodiments, the antibody components of the antibody cytokine engrafted protein described herein comprise immunoglobulin sequences, framework sequences, or CDR sequences of palivizumab. In some embodiments, the antibody cytokine engrafted protein described herein has a longer serum half-life than a wild-type IL-2 molecule such as, but not limited to, aldesleukin or a comparable molecule. In some embodiments, the antibody cytokine engrafted protein described herein has a sequence as set forth in Table 3. TABLE 3: Sequences of exemplary palivizumab antibody-IL-2 engrafted proteins Identifier Sequence (One-Letter Amino Acid Symbols) SEQ ID NO:13 MYRMQLLSCI ALSLALVTNS APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML 60 IL-2 TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE 120 TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT 153 SEQ ID NO:14 APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTAML TFKFYMPKKA TELKHLQCLE 60 IL-2 mutein EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR 120 WITFCQSIIS TLT 133 SEQ ID NO:15 APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TAKFYMPKKA TELKHLQCLE 60 IL-2 mutein EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR 120 WITFCQSIIS TLT 133 SEQ ID NO:16 GFSLAPTSSS TKKTQLQLEH LLLDLQMILN GINNYKNPKL TAMLTFKFYM PKKATELKHL 60 HCDR1_IL-2 QCLEEELKPL EEVLNLAQSK NFHLRPRDLI SNINVIVLEL KGSETTFMCE YADETATIVE 120 FLNRWITFCQ SIISTLTSTS GMSVG 145 SEQ ID NO:17 DIWWDDKKDY NPSLKS 16 HCDR2 SEQ ID NO:18 SMITNWYFDV 10 HCDR3 SEQ ID NO:19 APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTAML TFKFYMPKKA TELKHLQCLE 60 HCDR1_IL-2 kabat EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR 120 WITFCQSIIS TLTSTSGMSV G 141 SEQ ID NO:20 DIWWDDKKDY NPSLKS 16 HCDR2 kabat SEQ ID NO:21 SMITNWYFDV 10 HCDR3 kabat SEQ ID NO:22 GFSLAPTSSS TKKTQLQLEH LLLDLQMILN GINNYKNPKL TAMLTFKFYM PKKATELKHL 60 HCDR1_IL-2 QCLEEELKPL EEVLNLAQSK NFHLRPRDLI SNINVIVLEL KGSETTFMCE YADETATIVE 120 clothia FLNRWITFCQ SIISTLTSTS GM 142 SEQ ID NO:23 WWDDK 5 HCDR2 clothia SEQ ID NO:24 SMITNWYFDV 10 HCDR3 clothia SEQ ID NO:25 GFSLAPTSSS TKKTQLQLEH LLLDLQMILN GINNYKNPKL TAMLTFKFYM PKKATELKHL 60 HCDR1_IL-2 IMGT QCLEEELKPL EEVLNLAQSK NFHLRPRDLI SNINVIVLEL KGSETTFMCE YADETATIVE 120 FLNRWITFCQ SIISTLTSTS GMS 143 SEQ ID NO:26 IWWDDKK 7 HCDR2 IMGT SEQ ID NO:27 ARSMITNWYF DV 12 HCDR3 IMGT SEQ ID NO:28 QVTLRESGPA LVKPTQTLTL TCTFSGFSLA PTSSSTKKTQ LQLEHLLLDL QMILNGINNY 60 V KNPKLTAMLT FKFYMPKKAT ELKHLQCLEE ELKPLEEVLN LAQSKNFHLR PRDLISNINV 120 IVLELKGSET TFMCEYADET ATIVEFLNRW ITFCQSIIST LTSTSGMSVG WIRQPPGKAL 180 EWLADIWWDD KKDYNPSLKS RLTISKDTSK NQVVLKVTNM DPADTATYYC ARSMITNWYF 240 DVWGAGTTVT VSS 253 SEQ ID NO:29 QMILNGINNY KNPKLTAMLT FKFYMPKKAT ELKHLQCLEE ELKPLEEVLN LAQSKNFHLR 60 Heavy chain PRDLISNINV IVLELKGSET TFMCEYADET ATIVEFLNRW ITFCQSIIST LTSTSGMSVG 120 WIRQPPGKAL EWLADIWWDD KKDYNPSLKS RLTISKDTSK NQVVLKVTNM DPADTATYYC 180 ARSMITNWYF DVWGAGTTVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFPEPV 240 TVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKR 300 VEPKSCDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV AVSHEDPEVK 360 FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALAAPIEK 420 DB1 / 149057740.1 60Attorney Docket No.116983-5126-WO / AR / TW TISKAKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT 480 PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK 533 SEQ ID NO:30 KAQLSVGYMH 10 LCDR1 kabat 7 6 3 6 60 60 120 180 60 120 180 240 360 420 480 540 583 60 120 180The term “IL-4” (also referred to herein as “IL4”) 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 naïve helper T cells (Th0 cells) to Th2 T cells. Steinke and Borish, Respir. Res.2001, 2, 66-70. 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 IgG1 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:9).

[0201] The term “IL-7” (also referred to herein as “IL7”) 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 DB1 / 149057740.1 61Attorney Docket No.116983-5126-WO / AR / TW 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:10).

[0202] The term “IL-15” (also referred to herein as “IL15”) 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 β and γ 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:11).

[0203] The term “IL-21” (also referred to herein as “IL21”) 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:12).

[0204] When “an anti-tumor effective amount”, “a tumor-inhibiting effective amount”, or “therapeutic amount” is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of DB1 / 149057740.1 62Attorney Docket No.116983-5126-WO / AR / TW 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 104to 1011cells / kg body weight (e.g., 105to 106, 105to 1010, 105to 1011, 106to 1010, 106to 1011,107to 1011, 107to 1010, 108to 1011, 108to 1010, 109to 1011, or 109to 1010cells / kg body weight), including all integer values within those ranges. TILs (including in some cases, genetically modified cytotoxic lymphocytes) compositions may also be administered multiple times at these dosages. The TILs (including, in some cases, genetically engineered TILs) can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg, et al., New Eng. J. of Med.1988, 319, 1676). 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.

[0205] The term “hematological malignancy”, “hematologic malignancy” or terms of correlative meaning refer to mammalian cancers and tumors of the hematopoietic and lymphoid tissues, including but not limited to tissues of the blood, bone marrow, lymph nodes, and lymphatic system. Hematological malignancies are also referred to as “liquid tumors.” Hematological malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), multiple myeloma, acute monocytic leukemia (aMoL), Hodgkin’s lymphoma, and non-Hodgkin’s lymphomas. The term “B cell hematological malignancy” refers to hematological malignancies that affect B cells.

[0206] The term “liquid tumor” refers to an abnormal mass of cells that is fluid in nature. Liquid tumor cancers include, but are not limited to, leukemias, myelomas, and lymphomas, as well as other hematological malignancies. TILs obtained from liquid tumors may also be referred to herein as marrow infiltrating lymphocytes (MILs). TILs obtained from liquid tumors, including liquid tumors circulating in peripheral blood, may also be referred to herein as PBLs. The terms MIL, TIL, and PBL are used interchangeably herein and differ only based on the tissue type from which the cells are derived.

[0207] The term “microenvironment,” as used herein, may refer to the solid or hematological tumor microenvironment as a whole or to an individual subset of cells within DB1 / 149057740.1 63Attorney Docket No.116983-5126-WO / AR / TW the microenvironment. The tumor microenvironment, as used herein, 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. Although 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.

[0208] In some embodiments, 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. In some embodiments, the population of TILs may be provided wherein a patient is pre-treated with nonmyeloablative chemotherapy prior to an infusion of TILs according to the present invention. In some embodiments, 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). In some embodiments, after non-myeloablative chemotherapy and TIL infusion (at day 0) according to the invention, the patient receives an intravenous infusion of IL-2 intravenously at 720,000 IU / kg every 8 hours to physiologic tolerance.

[0209] Experimental findings indicate that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes plays a key role in enhancing treatment efficacy by eliminating regulatory T cells and competing elements of the immune system (“cytokine sinks”). Accordingly, 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 TILs of the invention.

[0210] The term “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. A 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, DB1 / 149057740.1 64Attorney Docket No.116983-5126-WO / AR / TW the tissue to which it is administered, and the physical delivery system in which the compound is carried.

[0211] The terms “treatment”, “treating”, “treat”, and the like, refer 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; (b) inhibiting the disease, i.e., arresting its development or progression; and (c) relieving the disease, i.e., causing regression of the disease and / or relieving one or more disease symptoms. “Treatment” is also meant to encompass delivery of an agent in order to provide for a pharmacologic effect, even in the absence of a disease or condition. For example, “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.

[0212] The term “heterologous” 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. For instance, 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. Similarly, 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).

[0213] The terms “sequence identity,” “percent identity,” and “sequence percent identity” (or synonyms thereof, e.g., “99% identical”) in the context of two or more nucleic acids or polypeptides, refer 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. DB1 / 149057740.1 65Attorney Docket No.116983-5126-WO / AR / TW 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, while 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.

[0214] As used herein, 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.

[0215] By “tumor infiltrating lymphocytes” or “TILs” herein is meant a population of cells originally obtained as white blood cells that have left the bloodstream of a subject and migrated into a tumor. TILs include, but are not limited to, CD8+cytotoxic T cells (lymphocytes), Th1 and Th17 CD4+T cells, natural killer cells, dendritic cells and M1 macrophages. 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).

[0216] 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 αβ, CD27, CD28, CD56, CCR7, CD45RA, CD95, PD-1, and CD25. Additionally, and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon DB1 / 149057740.1 66Attorney Docket No.116983-5126-WO / AR / TW reintroduction into a patient. TILs may further be characterized by potency – for example, TILs may be considered potent if, for example, interferon (IFN) 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. TILs may be considered potent if, for example, interferon (IFNγ) 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.

[0217] The term “deoxyribonucleotide” 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.

[0218] The term “RNA” defines a molecule comprising at least one ribonucleotide residue. The term “ribonucleotide” defines a nucleotide with a hydroxyl group at the 2' position of a b-D-ribofuranose moiety. The term 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.

[0219] The terms “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. Except insofar as 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. DB1 / 149057740.1 67Attorney Docket No.116983-5126-WO / AR / TW

[0220] 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. Moreover, as used herein, 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. In general, 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.

[0221] The 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. The term “consisting of” excludes any element, step or material other than those specified in the claim and, in the latter instance, impurities ordinary associated with the specified material(s). The term “consisting essentially of” limits the scope of a claim to the specified elements, steps or material(s) and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. All 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.”

[0222] 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 DB1 / 149057740.1 68Attorney Docket No.116983-5126-WO / AR / TW 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 VHand VLregions 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). Each VHand VLis 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 (Clq) of the classical complement system.

[0223] The term “antigen” refers to a substance that induces an immune response. In some embodiments, an antigen is a molecule capable of being bound by an antibody or a TCR if presented by major histocompatibility complex (MHC) molecules. The term “antigen”, as used herein, also encompasses T cell epitopes. An antigen is additionally capable of being recognized by the immune system. In some embodiments, 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). In some embodiments, 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.

[0224] 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. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such DB1 / 149057740.1 69Attorney Docket No.116983-5126-WO / AR / TW 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.

[0225] The terms “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. Examples of 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, CLand 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 VHand CH1 domains; (iv) a Fv fragment consisting of the VLand VHdomains 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 VHor a VLdomain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VLand 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 VLand VHregions 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). Such 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. In some embodiments, a scFv protein domain comprises a VH portion and a VLportion. A scFv molecule is denoted as either VL-L-VHif the VLdomain is the N-terminal part of the scFv molecule, or as VH-L-VL if the VH domain is the N-terminal part of the scFv molecule. Methods for making scFv molecules and designing suitable peptide linkers are described in U.S. Pat. No.4,704,692, U.S. Pat. No.4,946,778, R. Raag and M. Whitlow, “Single Chain Fvs.” FASEB Vol 9:73-80 (1995) and R. E. Bird and B. W. Walker, Single Chain Antibody Variable Regions, TIBTECH, Vol 9: 132-137 (1991), the disclosures of which are incorporated by reference herein. DB1 / 149057740.1 70Attorney Docket No.116983-5126-WO / AR / TW

[0226] The term “human antibody,” as used herein, 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). The term “human antibody”, as used herein, 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.

[0227] The term “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. In some embodiments, 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.

[0228] The term “recombinant human antibody”, as used herein, 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. In certain embodiments, however, 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. DB1 / 149057740.1 71Attorney Docket No.116983-5126-WO / AR / TW

[0229] As used herein, “isotype” refers to the antibody class (e.g., IgM or IgG1) that is encoded by the heavy chain constant region genes.

[0230] The 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.”

[0231] 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.

[0232] The terms “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 immunoglobulin. For the most part, 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. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, 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. In general, 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. For further details, see Jones, et al., Nature 1986, 321, 522-525; Riechmann, et al., Nature 1988, 332, 323-329; and Presta, Curr. Op. Struct. Biol.1992, 2, 593-596. The antibodies described herein may also be modified to employ any Fc variant DB1 / 149057740.1 72Attorney Docket No.116983-5126-WO / AR / TW 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. WO 1988 / 07089 A1, WO 1996 / 14339 A1, WO 1998 / 05787 A1, WO 1998 / 23289 A1, WO 1999 / 51642 A1, WO 99 / 58572 A1, WO 2000 / 09560 A2, WO 2000 / 32767 A1, WO 2000 / 42072 A2, WO 2002 / 44215 A2, WO 2002 / 060919 A2, WO 2003 / 074569 A2, WO 2004 / 016750 A2, WO 2004 / 029207 A2, WO 2004 / 035752 A2, WO 2004 / 063351 A2, WO 2004 / 074455 A2, WO 2004 / 099249 A2, WO 2005 / 040217 A2, WO 2005 / 070963 A1, WO 2005 / 077981 A2, WO 2005 / 092925 A2, WO 2005 / 123780 A2, WO 2006 / 019447 A1, WO 2006 / 047350 A2, and WO 2006 / 085967 A2; and U.S. Patent Nos.5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; and 7,083,784; the disclosures of which are incorporated by reference herein.

[0233] The term “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.

[0234] 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). By using a 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.

[0235] The term “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 DB1 / 149057740.1 73Attorney Docket No.116983-5126-WO / AR / TW antigen, as described in U.S. Patent Nos.5,714,350 and 6,350,861. Additionally or alternatively, 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. Such altered glycosylation patterns have been demonstrated to increase the ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention to thereby produce an antibody with altered glycosylation. For example, 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. Patent Publication No.2004 / 0110704 or Yamane-Ohnuki, et al., Biotechnol. Bioeng., 2004, 87, 614-622). As another example, 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- acetylglucosamine 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, Lec 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., J. Biol. Chem.2002, 277, 26733-26740. International Patent Publication WO 99 / 54342 describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., beta(1,4)-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, 17, 176-180). Alternatively, the fucose residues of the antibody may be cleaved off using a fucosidase enzyme. For example, the fucosidase alpha-L-fucosidase removes fucosyl residues from antibodies as described in Tarentino, et al., Biochem.1975, 14, 5516-5523. DB1 / 149057740.1 74Attorney Docket No.116983-5126-WO / AR / TW

[0236] “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. Pegylation may, for example, increase the biological (e.g., serum) half life of the antibody. Preferably, 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). As used herein, the term “polyethylene glycol” is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10)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 U.S. Patent No.5,824,778, the disclosures of each of which are incorporated by reference herein.

[0237] The term “biosimilar” means a biological product, including a monoclonal antibody or protein, that is highly similar to a U.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. Furthermore, 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. The term “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 monoclonal antibodies. For example, if the reference IL-2 protein is aldesleukin (PROLEUKIN), a protein approved by drug regulatory authorities with reference to aldesleukin is a “biosimilar to” aldesleukin or is a “biosimilar thereof” of aldesleukin. In Europe, 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). 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 DB1 / 149057740.1 75Attorney Docket No.116983-5126-WO / AR / TW 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. In addition, 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. In addition, the biosimilar may be used or be intended for use to treat the same conditions as the reference medicinal product. Thus, a biosimilar as described herein may be deemed to have similar or highly similar quality characteristics to a reference medicinal product. Alternatively, or in addition, a biosimilar as described herein may be deemed to have similar or highly similar biological activity to a reference medicinal product. Alternatively, or in addition, a biosimilar as described herein may be deemed to have a similar or highly similar safety profile to a reference medicinal product. Alternatively, or in addition, a biosimilar as described herein may be deemed to have similar or highly similar efficacy to a reference medicinal product. As described herein, a biosimilar in Europe is compared to a reference medicinal product which has been authorized by the EMA. However, in some instances, 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. As used herein, 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 DB1 / 149057740.1 76Attorney Docket No.116983-5126-WO / AR / TW glycosylation pattern to the reference medicinal product. Particularly, although not exclusively, 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 pharmacodynamic (PD) profiles as compared to the reference medicinal product but is still deemed sufficiently similar to the reference medicinal product as to be authorized or considered suitable for authorization. In certain circumstances, the 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. The term “biosimilar” is also used synonymously by other national and regional regulatory agencies.

[0238] The term “recombinant lentiviral RNA molecule” refers to a single stranded RNA genome that may comprise at least a portion of a lentivirus genome, including 5’ and 3’ long terminal repeat (LTR) sequences. In some embodiments, the lentivirus genome may be modified to inhibit replication and limit pathogenicity, while retaining function. For example, the env gene, the gag gene, the pol gene, and the rev gene may be removed from the lentivirus genome that is comprised by the recombinant lentiviral RNA molecule. In some embodiments, the recombinant lentiviral RNA molecule is comprised by a lentiviral vector or a recombinant lentiviral particle as described elsewhere in this disclosure.

[0239] The term “lentiviral particle” or “lentiviral virion” refers to lentivirus, which is a subset of retrovirus. Lentiviruses can deliver significant amounts of genetic information into host cells and integrate it into the cellular genome, making genetically-engineered lentiviruses one of the most efficient tools of gene delivery. These lentiviruses contain a promoter which is used to control the expression of a transgene or shRNA but no virulence genes, making them safe to use in the laboratory.

[0240] The term “recombinant lentiviral particle” or “recombinant lentiviral virion” refers to a lentiviral particle or lentiviral virion produced by gene recombinant technologies. A recombinant lentiviral particle or lentiviral virion can be produced using any suitable method, such as by transducing or transfecting a packaging cell-line with a nucleic acid encoding the viral genome and subsequently isolating newly packaged viral particles. It is understood that DB1 / 149057740.1 77Attorney Docket No.116983-5126-WO / AR / TW the recombinant technologies may be performed at a stage upstream of production of the viral vector itself. For example, recombinant technologies may be used to produce a plasmid, and the plasmid may then be produced at a larger scale, and finally the plasmid may be introduced into a cell line for packaging to produce the viral vector.

[0241] The term “lentiviral vector” refers to a recombinant lentiviral particle comprising a recombinant lentiviral RNA molecule, which contains at least a portion of a lentivirus genome, including 5’ and 3’ LTRs, and a nucleotide sequence encoding one or more genes of interest (GOIs). The lentivirus genome may be modified to inhibit replication and limit pathogenicity, while retaining function. For example, the env gene, the gag gene, the pol gene, and the rev gene may be removed from the lentivirus genome and included in helper plasmids.

[0242] The term “transfer vector” refers to a recombinant DNA plasmid containing a nucleotide sequence encoding the recombinant lentiviral RNA molecule, which contains at least a portion of a lentivirus genome, including 5’ and 3’ LTRs, and a nucleotide sequence encoding one or more genes of interest (GOIs),, while one or more “helper plasmid(s)” or “envelope plasmid(s)” contain(s) genes for proteins that appear on the surface of the lentiviral particle or are essential for the function of the lentiviral particle. The transfer vector can be transfected into a packaging cell line along with the helper plasmid(s) to produce lentiviral vectors comprising the nucleotide sequence encoding the one or more GOIs as part of a recombinant lentiviral RNA molecule.

[0243] The term “Env” refers to the envelope glycoproteins on the surface of the lentiviral particle encoded by the env gene in the lentiviral genome. The Env protein contains a surface subunit and a transmembrane subunit. In some embodiments, the Env protein is selected from the group consisting of baboon retroviral envelope (Ba-EVTR), vesicularstomatitis-virus-G protein (VSV-G), and RD114.

[0244] The term “Gag” refers to the structural protein encoded by the gag gene in the lentiviral genome. The Gag protein is generated as a standalone protein or as a fusion protein with the Pol protein (Gag-Pol).

[0245] The term “Pol” refers to the reverse transcriptase and integrase enzymes encoded by the pol gene in the lentiviral genome. The Pol protein is generated as a fusion protein with the Gag protein (Gag-Pol). DB1 / 149057740.1 78Attorney Docket No.116983-5126-WO / AR / TW

[0246] The term “Rev” refers to the protein encoded by the rev gene in the lentiviral genome. Rev bears a leucine-rich nuclear export signal (NES) and, via association with the Rev response element (RRE), mediates nuclear-to-cytoplasmic transport of the partially spliced and unspliced RNAs, resulting in production of Gag, Gag-Pol, Env, and accessory proteins (Pollard & Malim, Annu. Rev. Microbiol., 1998, 52, 491532). III. Nucleic Acid Molecules Encoding Cytokines

[0247] Provided herein are nucleic acid molecules comprising a nucleotide sequence encoding one or more genes of interest (GOIs), e.g., cytokines selected from the group consisting of IL-12, IL-2, IL-6, IL-7, IL-9, IL-15, IL-18, IL-21, IL-23, IL-27, IL-33, IFN gamma, TNFa, IFN alpha, IFN beta, GM-CSF, GCSF, or a variant thereof. In some embodiments, the nucleic acid molecule comprising a nucleotide sequence encoding a tethered IL-12 (TeIL-12). In some embodiments, the nucleic acid molecule comprising a nucleotide sequence encoding a TeIL-12 and a tethered IL-15 (TeIL-15). In some embodiments, the nucleic acid molecule comprising a nucleotide sequence encoding a TeIL- 12 and a tethered IL-2 (TeIL-2).

[0248] In some embodiments, the nucleotide sequences encoding the one or more GOIs, e.g., cytokines selected from the group consisting of IL-12, IL-2, IL-6, IL-7, IL-9, IL-15, IL- 18, IL-21, IL-23, IL-27, IL-33, IFN gamma, TNFa, IFN alpha, IFN beta, GM-CSF, GCSF, or a variant thereof, are provided by two or more nucleic acid molecules. For example, provided herein are a nucleic acid molecule comprising a nucleotide sequence encoding IL-12 or a variant thereof; and a second nucleic acid molecule comprising a nucleotide sequence encoding IL-15 or a variant thereof. In some embodiments, provided herein are a nucleic acid molecule comprising a nucleotide sequence encoding IL-12 or a variant thereof; and a second nucleic acid molecule comprising a nucleotide sequence encoding IL-2 or a variant thereof.

[0249] In some embodiments, the cytokine is a tethered cytokine. For example, the cytokine is linked to a cell membrane anchor moiety that allows the tethering of the cytokine to the cell surface. Suitable cell membrane anchor moieties include, for example, transmembrane domains of endogenous cell surface proteins and fragments thereof. Exemplary transmembrane domains that can be used include, for example, B7-1, B7-2, and CD8a transmembrane domains and fragments thereof. In some embodiments, the cell membrane anchor moiety further includes a transmembrane and intracellular domain of an DB1 / 149057740.1 79Attorney Docket No.116983-5126-WO / AR / TW endogenous cell surface protein or fragment thereof. In some embodiments, the cell membrane anchor moiety is a B7-1, B7-2 or CD8a transmembrane-intracellular domain or fragment thereof. In certain embodiments, the cell membrane anchor moiety is a CD8a transmembrane domain having the amino acid sequence of IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO:40). In certain embodiments, the cell membrane anchor moiety is a B7-1 transmembrane-intracellular domain having the amino acid sequence of LLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO:41).

[0250] In certain embodiments, the cell membrane anchor moiety is a non-peptide cell membrane anchor moiety. In exemplary embodiments, the non-peptide cell membrane anchor moiety is a glycophosphatidylinositol (GPI) anchor. GPI anchors have a structure that includes a phosphoethanolamine linker, glycan core, and phospholipid tail. In some embodiments, the glycan core is modified with one or more side chains. In some embodiments, the glycan core is modified with one or more of the following side chains: a phosphoethanolamine group, mannose, galactose, sialic acid, or other sugars.

[0251] The membrane anchored cytokine may include linkers that allow for the linkage of components of the membrane anchored cytokine (e.g. a cytokine to a cell membrane anchor moiety). Suitable linkers include linkers that are at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid residues in length. In some embodiments, the linker is 5-10, 10-15, 15-20, 20-25, 25-30, 30- 35, 35-40, 45-50, 50-60 amino acids in length. Suitable linkers include, but are not limited: a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, or a non-helical linker. In some embodiments, the linker is a peptide linker that optionally comprises Gly and Ser. In certain embodiments, the peptide linker utilize a glycine-serine polymer, including for example (GS)n (SEQ ID NO:42), (GSGGS)n (SEQ ID NO:43), (GGGS)n (SEQ ID NO:44), (GGGGS)n (SEQ ID NO:45), (GGGGGS)n (SEQ ID NO:46), and (GGGGGGS)n (SEQ ID NO:47), where n is an integer of at least one (and generally from 3 to 10). Additional linkers that can be used with the present compositions and methods are described in U.S. Patent Publication Nos. US 2006 / 0074008, US 20050238649, and US 2006 / 0024317, each of which is incorporated by reference herein in its entirety, and particularly in pertinent parts related to linkers. In some embodiments, the peptide linker is SGGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO:48).

[0252] In some embodiments, the linker is a cleavable linker. In exemplary embodiments, the cleavable linker allows for the release of the cytokine into the tumor DB1 / 149057740.1 80Attorney Docket No.116983-5126-WO / AR / TW microenvironment. Cleavable linkers are also useful in embodiments, wherein two membrane anchored cytokines are co-expressed in the same cell. In exemplary embodiments, the linker is a self-cleaving 2A peptide. See, e.g., Liu et al., Sci. Rep.7(1):2193 (2017), which is incorporated by reference in relevant parts relating to 2A peptides. 2A peptides are viral oligopeptides that mediate cleavage of polypeptides during translation in eukaryotic cells. In some embodiments, the 2A peptide includes a C-terminus having the amino acid sequence GDVEXiNPGP (SEQ ID NO:49), wherein Xi is any naturally occurring amino acid residue. In certain embodiments, the 2A peptide is a porcine teschovirus-12A peptide (GSGATNFSLLKQAGDVEENPGP, SEQ ID NO:50). In some embodiments, the 2A peptide is an equine rhinitis A virus 2A peptide (GSGQCTNYALLKLAGDVESNPGP, SEQ ID NO:51). In certain embodiments, the 2A peptide is a foot-and-mouth disease virus 2A peptide: (GSGEGRGSLLTCGDVEENPGP, SEQ ID NO:52). In some embodiments, the cleavable linker includes a furin-cleavable sequence. Exemplary furin-cleavable sequences are described for example, Duckert et al., Protein Engineering, Design & Selection 17(1):107-112 (2004), and US Patent No.8,871,906, each of which is incorporated herein by reference, particularly in relevant parts relating to furin-cleavable sequences. In some embodiments, the linker includes a 2A peptide and a furin-cleavable sequence. In exemplary embodiments, the furin-cleavable 2A peptide includes the amino acid sequence RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO:53).

[0253] In some embodiments, the linker is a degradable linker (e.g., a disulfide linker) such that under physiological conditions, the linker degrades, thereby releasing the cytokine. In some embodiments, the cytokine is reversibly linked to functional groups through a degradable linker such that under physiological conditions, the linker degrades and releases the cytokine. Suitable degradable linkers include, but are not limited to: a protease sensitive linker that is sensitive to one or more enzymes present in biological media such as proteases in a tumor microenvironment such a matrix metalloproteases present in a tumor microenvironment or in inflamed tissue (e.g. matrix metalloproteinase 2 (MMP2) or matrix metalloproteinase 9 (MMP9)).

[0254] In other embodiments, the linker is an enzyme-sensitive linker. Exemplary cleavable linker include those that are recognized by one of the following enzymes: metalloprotease MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, plasmin, PSA, PSMA, CATHEPSIN D, CATHEPSIN K, CATHEPSIN S, ADAM10, ADAM12, ADAMTS, Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, DB1 / 149057740.1 81Attorney Docket No.116983-5126-WO / AR / TW Caspase-9, Caspase-10, Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE. See, e.g., US Patent Nos.8,541,203 and 8,580,244, each of which is incorporated by reference in its entirety and in pertinent parts related to cleavable linkers.

[0255] In certain embodiments, the membrane anchored cytokine includes a signal peptide that facilitates the translocation of the cytokine to the cell membrane. Any suitable signal peptide that facilities the localization of the cytokine to the cell membrane can be used. In some embodiments, the signal peptide does not interfere with the bioactivity of the cytokine. Exemplary signal peptide sequences include, but are not limited to: human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor signal sequence, human prolactin signal sequence, and human IgE signal sequence. In certain embodiments, the fusion protein includes a human IgE signal sequence. In exemplary embodiments, the human IgE signal sequence has the amino acid sequence MDWTWILFLVAAATRVHS (SEQ ID NO:54). In some embodiments, the human IgE signal sequence includes the amino acid sequence NIKGSPWKGSLLLLLVSNLLLCQSVAP (SEQ ID NO:55). In some embodiments, the signal peptide sequence is an IL-2 signal sequence having the amino acid sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO:56).

[0256] The nucleic acid molecule can further comprise a native or normative promoter operably linked to the nucleotide sequence encoding one or more cytokines. In some embodiments, the nucleotide sequence encoding each cytokine is operably linked to the same promoter. In some embodiments, the nucleotide sequence encoding each cytokine is operably linked to a different promoter. Preferably, the promoter is functional in T cells. The selection of a promoter, e.g., strong, weak, inducible, tissue-specific and developmental- specific, is within the ordinary skill of the artisan. Similarly, the combining of a nucleotide sequence with a promoter is also within the skill of the artisan. The promoter can be a non- viral promoter or a viral promoter, e.g., a nuclear factor of activated T-cells (NFAT) promotor, an EF-1a promoter, a cytomegalovirus (CMV) promoter, a CAG promotor, an MND promoter, or an SSFV promoter, an SV40 promoter, an RSV promoter, or a promoter found in the long-terminal repeat of the murine stem cell virus.

[0257] “NFAT promoter” as used herein means one or more NFAT responsive elements linked to a minimal promoter of any gene expressed by T-cells. Preferably, the minimal promoter of a gene expressed by T-cells is a minimal human IL-2 promoter. The NFAT responsive elements may comprise, e.g., NFATl, NFAT2, NFAT3, and / or NFAT4 responsive elements. The NFAT promoter (or functional portion or functional variant DB1 / 149057740.1 82Attorney Docket No.116983-5126-WO / AR / TW thereof) may comprise any number of binding motifs, e.g., at least two, at least three, at least four, at least five, or at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or up to twelve binding motifs. TABLE 4 – NFAT Promoter Related Sequences. Description Amino Acid Sequence TA T A TAT ATAA TT ATG A T A G C A T GGC T

[0258] In a preferred embodiment, the NFAT promoter comprises six NFAT binding motifs. See, e.g., US Patent No.8,556,882, which is incorporated by reference in its entirety and particularly for pertinent parts relating to NFAT promoters. In some embodiments, the NFAT promoter system controls expression of one or more cytokines. In certain embodiments, the cytokine is selected from the group consisting of IL-12, IL-2, IL-6, IL-7, IL-9, IL-15, IL-18, IL-21, IL-23, IL-27, IL-33, IFN gamma, TNFa, IFN alpha, IFN beta, GM- CSF, GCSF, or a variant thereof. In some embodiments, the cytokine is a tethered cytokine. In some embodiments, the NFAT promoter system controls expression of IL-12, or a variant thereof. In some embodiments, the NFAT promoter system controls expression of IL-15, or a variant thereof. In some embodiments, the NFAT promoter system controls expression of IL- 18, or a variant thereof. In some embodiments, the NFAT promoter system controls expression of TeIL-12. In some embodiments, the NFAT promoter system controls DB1 / 149057740.1 83Attorney Docket No.116983-5126-WO / AR / TW expression of TeIL-15. In some embodiments, the NFAT promoter system controls expression of TeIL-18.

[0259] As used herein, “interleukin 12”, “IL-12” and “IL12” all refer to an interleukin that is a heterodimeric cytokine encoded by the IL-12A and IL-12B genes (Genbank Accession numbers: NM_000882 (IL-12A) and NM_002187 (IL-12B)). IL-12 is composed of a bundle of four alpha helices and is involved in the differentiation of native T cells into TH1 cells. It is encoded by two separate genes, IL-12A (p35) and IL-12B (p40). The active heterodimer (referred to as 'p70'), and a homodimer of p40 are formed following protein synthesis. IL-12 binds to the IL-12 receptor, which is a heterodimeric receptor formed by IL- 12R-β1 and IL-12R-β2. IL-12 is known as a T cell-stimulating factor that can stimulate the growth and function of T cells. In particular, IL-12 can stimulate the production of interferon gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α) from T cells and natural killer (NK) cells and reduce IL-4 mediated suppression of IFN-γ. IL-12 can further mediate enhancement of the cytotoxic activity of NK cells and CD8+ cytotoxic T lymphocytes. Moreover, IL-12 can also have anti-angiogenic activity by increasing production of interferon gamma, which in turn increases the production of the chemokine inducible protein-10 (IP-10 or CXCL10). IP-10 then mediates this anti-angiogenic effect. Thus, without being bound by any particular theory of operation, it is believed that IL-12 can increase the survivability and / or anti-tumor effects of the TIL compositions provided herein.

[0260] In some embodiments, the IL-12 is a full length IL-12, a fragment or a variant of IL-12. In some embodiments, the IL-12 is a human IL-12 or a variant human IL-12. In exemplary embodiments, the IL-12 is a biological active human IL-12 variant. In some embodiments, the IL-12 includes a 1, 2, 3,4 ,5 ,67, 8, 9, or 10 mutations as compared to a wild-type IL-12.

[0261] In some embodiments, the IL-12 comprises an IL-12 p35 subunit or a variant thereof. In some embodiments, the IL-12 p35 subunit is a human IL-12 p35 subunit. In some embodiments, the IL-12 p35 subunit has the amino acid sequence of SEQ ID NO:60. In certain embodiments, the IL-12 comprises an IL-12 p40 subunit or a variant thereof. In some embodiments, the IL-12 p40 subunit has the amino acid sequence of SEQ ID NO:61. In certain embodiments, the IL-12 is a single chain IL-12 polypeptide comprising an IL-12 p35 subunit attached to an IL-12 p40 subunit. Such IL-12 single chain polypeptides advantageously retain one or more of the biological activities of wildtype IL-12. In some embodiments, the single chain IL-12 polypeptide described herein is according to the DB1 / 149057740.1 84Attorney Docket No.116983-5126-WO / AR / TW formula, from N-terminus to C-terminus, (p40)-(L)-(p35), wherein “p40” is an IL-12 p40 subunit, “p35” is IL-12 p35 subunit and L is a linker. In other embodiments, the single chain IL-12 is according to the formula from N-terminus to C-terminus, (p35)-(L)-(p40). Any suitable linker can be used in the single chain IL-12 polypeptide including those described herein. Suitable linkers can include, for example, linkers having the amino acid sequence (GGGGS)xwherein x is an integer from 1-10. Other suitable linkers include, for example, the amino acid sequence GGGGGGS. Exemplary single chain IL-12 linkers than can be used with the subject single chain IL-12 polypeptides are also described in Lieschke et al., Nature Biotechnology 15: 35-40 (1997), which is incorporated herein in its entirety by reference and particularly for its teaching of IL-12 polypeptide linkers. In an exemplary embodiment, the single chain IL-12 polypeptide is a single chain human IL-12 polypeptide (i.e., it includes a human p35 and p40 IL-12 subunit). DB1 / 149057740.1 85Attorney Docket No.116983-5126-WO / AR / TW TABLE 5 – IL-12 Related Sequences. DB1 / 149057740.1 86Attorney Docket No.116983-5126-WO / AR / TW Description Amino Acid Sequence RNLPVATPDPGMFPCLHHSQNLLRAVSNMLA T L TI D W A P P V D K S K H R P R L P V RDB1 / 149057740.1 87Attorney Docket No.116983-5126-WO / AR / TW TeIL-12-Lr1-Ar2 (nucleotide)ATGGATTGGACCTGGATTCTGTTCCTCGTGGCCGCCGCTACCCGCGTGCACTCCATCTGGGAGCTGAAGAAAGACGTGTACGTGGTGGAATTGGATTGGTACC G C G T G A T C T C T C A G C T C T A A C G T G G G T C A G G G T A C C G A T C G C A CDB1 / 149057740.1 88Attorney Docket No.116983-5126-WO / AR / TW CCAGATGTAGAGAGCGCCGCCGCAACGAGCGCCT GCGTCGTGAGAGCGTGAGGCCTGTGTGA (SEQ ID NO:63) des, p g q man IgE signal sequence peptide; amino acids 529-553: peptide linker; amino acids 554-601: membrane anchor), wherein the nucleic acid is operably linked to an NFAT promoter, an EF- 1a promoter, a CMV promotor, a CAG promotor, an MND promoter, or an SSFV promoter, as described herein. See, e.g., US Patent No.8,556,882, which is incorporated by reference in its entirety and particularly for pertinent parts relating to NFAT promoters for IL-12 expression.

[0263] In some embodiments, the nucleic acid molecule further comprises a nucleotide sequence encoding a cytokine selected from the group consisting of IL-2, IL-6, IL-7, IL-9, IL-15, IL-18, IL-21, IL-23, IL-27, IL-33, IFN gamma, TNFa, IFN alpha, IFN beta, GM-CSF, GCSF, or a variant thereof. In some embodiments, the cytokine is a tethered cytokine. In some embodiments, the cytokine is under the control of an EF1a promotor, a CMV promotor, a CAG promotor, an MND promotor, or an SSFV promoter.

[0264] As used herein, “interleukin 15”, “IL-15” and “IL15” all refer to an interleukin that binds to and signals through a complex composed of an IL-15 specific receptor alpha chain (IL-15Rα), an IL-2 / IL-15 receptor beta chain (CD122) and the common gamma chain (gamma-C, CD132) (e.g., Genbank Accession numbers: NM_00000585, NP_000576 and NP_751915 (human); and NM_001254747 and NP_001241676 (mouse)). IL-15 has been shown to stimulate T cell proliferation inside tumors. IL-15 also is able to extend the survivability of effector memory CD8+ T cells and is critical for the development of NK cells. Therefore, without being bound by any particular theory of operation, it is believed that modified TILs associated with an IL-15s described herein exhibit enhanced survival and / or anti-tumor effects.

[0265] IL-15 has a short half-life of less than 40 minutes in vivo. Modifications to IL-15 monomer can improve its in vivo pharmacokinetics in the treatment of cancers. These modifications have generally centered on improving the trans-presentation of IL-15 with the alpha subunit of IL-15 receptor, IL-15Rα. Such modifications include: 1) pre-association of IL-15 and its soluble receptor a-subunit-Fc fusion to form IL-15: IL-15Rα-Fc complex (see, e.g., Rubinstein et al., Proc Natl Acad Sci U.S.A.103:9166–71 (2006)); 2) expression of the DB1 / 149057740.1 89Attorney Docket No.116983-5126-WO / AR / TW superagonist IL-15-sIL-15Rα-sushi protein (see, e.g., Bessard et al., Molecular cancer therapeutics 8: 2736-45 (2009)); and 3) pre-association of human IL-15 mutant IL-15N72D with IL-15Rα-Fc sushi-Fc fusion complex (see, e.g., Zhu et al., Journal of Immunology 183: 3598-6007 (2009)).

[0266] In some embodiments, the IL-15 is a tethered IL-15 (TeIL-15). In some embodiments, the TeIL-15 comprises the amino acid sequence of SEQ ID NO:73 (amino acids 1-18: human IgE signal sequence peptide; amino acids 132-157: peptide linker; amino acids 158-205: membrane anchor).

[0267] In some embodiments, the IL-15 is a full length IL-15, a fragment or a variant of IL-15. In some embodiments, the IL-15 is a human IL-15 or a variant human IL-15. In exemplary embodiments, the IL-15 is a biological active human IL-15 variant. In some embodiments, the IL-15 includes a 1, 2, 3, 4 ,5, 6, 7, 8, 9, or 10 mutations as compared to a wild-type IL-15. In certain embodiments, the IL-15 includes an N72D mutation relative to a wild type human IL-15. In some embodiments, the variant IL-15 exhibits IL-15Rα binding activity.

[0268] In some embodiments, the IL-15 includes an IL-15 and an extracellular domain of an IL-15Rα. In certain embodiments, the IL-15 includes an IL-15 and an IL-15Rα fused to an Fc domain (IL-15Rα-Fc) TABLE 6 – IL-15 Related Sequences. Description Amino Acid Sequence V K G K F S K P :DB1 / 149057740.1 90Attorney Docket No.116983-5126-WO / AR / TW ITCPPPMSVEHADIWVKSYSLYSRERYICNSG Human IL-15R-alpha-Su (65aa truncated FKRKAGTSSLTECVLNKATNVAHWTTPSLK extracellular domain) CIR (SEQ ID NO: 66) K K L I V Q I V K G K G K P A I G K P A ) Q S L G Y T CDB1 / 149057740.1 91Attorney Docket No.116983-5126-WO / AR / TW CAGTCTATGCACATTGACGCAACACTTTACACCGA GAGCGATGTGCACCCCTCTTGCAAGGTGACTGCC ATGAAATGTTTCTTGCTCGAATTGCAGGTCATCTC C C G T G T T A C Gincludes a complex of human IL-15 and soluble human IL-15Rα. The combination of human IL-15 with soluble human IL-15Rα forms an IL-15 SA complex that possesses greater biological activity than human IL-15 alone. Soluble human IL-15Rα, as well as truncated versions of the extracellular domain, has been described in the art (Wei et al., 2001 J of Immunol.167: 277-282). The amino acid sequence of human IL-15Rα is set forth in SEQ ID NO: 72. In some embodiments, the IL-15SA includes a complex of human IL-15 and soluble human. IL-15Rα comprising all or a portion of the extracellular domain, without the transmembrane or cytoplasmic domain. In some embodiments, the IL-15SA includes a complex of human IL-15 and soluble human IL-15Rα that includes the full extracellular domain or a truncated form of the extracellular domain which retains IL-15 binding activity.

[0270] In some embodiments, the IL-15SA includes a complex of human IL-15 and soluble human IL-15Rα that includes a truncated form of the extracellular domain which retains IL-15 binding activity. In some embodiments, the soluble human IL-15Rα includes amino acids 1-60, 1-61, 1-62, 1-63, 1-64 or 1-65 of human IL-15Rα. In some embodiments, the soluble human IL-15Rα includes amino acids 1-80, 1-81, 1-82, 1-83, 1-84 or 1-85 of human IL-15Rα. In some embodiments, the soluble human IL-15Rα includes amino acids 1- 180, 1-181, or 1-182 of human IL-15Rα.

[0271] In some embodiments, the cytokine is an IL-15SA comprising a complex of human IL-15 and soluble human IL-15Rα comprising a truncated form of the extracellular domain which retains IL-15 binding activity and comprises a Sushi domain. The Sushi domain of IL-15Rα is described in the art as approximately 60 amino acids in length and DB1 / 149057740.1 92Attorney Docket No.116983-5126-WO / AR / TW comprises 4 cysteines. (Wei et al., 2001). Truncated forms of soluble human IL-15Rα which retain IL-15 activity and comprise a Sushi domain are useful in IL-15SA of the present disclosure.

[0272] In some embodiments, the cytokine includes a complex comprising soluble human IL-15Rα expressed as a fusion protein, such as an Fc fusion as described herein (e.g., human IgG1 Fc), with IL-15. In some embodiments, IL-15SA comprises a dimeric human IL- 15RαFc fusion protein (e.g., human IgG1 Fc) complexed with two human IL-15 molecules.

[0273] In some embodiments, the cytokine is an IL-15SA cytokine complex that includes an IL-15 molecule comprising an amino acid sequence set forth in SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO:68, or SEQ ID NO:69. In some embodiments, an IL-15SA cytokine complex comprises a soluble IL-15Rα molecule comprising a sequence of SEQ ID NO:66, SEQ ID NO: 70 or SEQ ID NO:71.

[0274] In some embodiments, the cytokine is an IL-15SA cytokine complex that includes a dimeric IL-15RαFc fusion protein complexed with two IL-15 molecules. In some embodiments, IL-15-SA comprises a dimeric IL-15RαSu (Sushi domain) / Fc (SEQ ID NO:65) and two IL-15N72D (SEQ ID NO:64) molecules (also known as ALT-803), as described in US20140134128, incorporated herein by reference. In some embodiments, the IL-15SA comprises a dimeric IL-15RαSu / Fc molecule (SEQ ID NO: 65) and two IL-15 molecules (SEQ ID NO: 67). In some embodiments, the IL-15SA comprises a dimeric IL-15RαSu / Fc molecule (SEQ ID NO: 65) and two IL-15 molecules (SEQ ID NO:68). In some embodiments, the IL-15SA comprises a dimeric IL-15RαSu / Fc molecule (SEQ ID NO:65) and two IL-15 molecules (SEQ ID NO:69).

[0275] In some embodiments, the IL-15SA includes a dimeric IL-15RαSu / Fc molecule (SEQ ID NO:65) and two IL-15 molecules having amino acid sequences selected from SEQ ID NO: 64, 67, 68, and 69.

[0276] In some embodiments, the IL-15SA includes a soluble IL-15Rα molecule (SEQ ID NO:66) and two IL-15 molecules (SEQ ID NO:64). In some embodiments, the IL- 15SA comprises a soluble IL-15Rα molecule (SEQ ID NO:66) and two IL-15 molecules (SEQ ID NO:67). In some embodiments, the IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO:66) and two IL-15 molecules (SEQ ID NO:68). In some embodiments, the IL- 15SA comprises a soluble IL-15Rα molecule (SEQ ID NO:66) and two IL-15 molecules (SEQ ID NO:69). DB1 / 149057740.1 93Attorney Docket No.116983-5126-WO / AR / TW

[0277] In some embodiments, the IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO:70) and two IL-15 molecules (SEQ ID NO:64). In some embodiments, the IL- 15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 70) and two IL-15 molecules (SEQ ID NO:67). In some embodiments, the IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 70) and two IL-15 molecules (SEQ ID NO:68). In some embodiments, the IL- 15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 70) and two IL-15 molecules (SEQ ID NO:69).

[0278] In some embodiments, the IL-15SA includes a soluble IL-15Rα molecule (SEQ ID NO:71) and two IL-15 molecules (SEQ ID NO:64). In some embodiments, the IL- 15SA comprises a soluble IL-15Rα molecule (SEQ ID NO: 71) and two IL-15 molecules (SEQ ID NO:67). In some embodiments, the IL-15SA comprises a soluble IL-15Rα molecule (SEQ ID NO:71) and two IL-15 molecules (SEQ ID NO:68). In some embodiments, the IL- 15SA comprises a soluble IL-15Rα molecule (SEQ ID NO:71) and two IL-15 molecules (SEQ ID NO:69).

[0279] In some embodiments, the IL-15SA comprises a dimeric IL-15RαSu / Fc (SEQ ID NO:65) molecule and two IL-15 molecules (SEQ ID NO:68). In some embodiments, the IL-15SA includes a dimeric IL-15RαSu / Fc (SEQ ID NO:65) molecule and two IL-15 molecules (SEQ ID NO:69).

[0280] In some embodiments, the IL-15SA includes SEQ ID NO:65 and SEQ ID NO:66. In some embodiments IL-15SA comprises SEQ ID NO:67 or SEQ ID NO:68. In some embodiments the IL-15SA comprises SEQ ID NO:67 and SEQ ID NO:65. In some embodiments the IL-15SA comprises SEQ ID NO:68 and SEQ ID NO:65. In some embodiments the IL-15SA comprises SEQ ID NO:69 and SEQ ID NO:65. In some embodiments, the IL-15SA comprises SEQ ID NO:67 and SEQ ID NO:66. In some embodiments the IL-15SA comprises SEQ ID NO:68 and SEQ ID NO:66.

[0281] As used herein, “interleukin 18”, “IL-18,” “IL18,” “IGIF,” “IL-1g,” “interferon-gamma inducing factor,” and “IL1F4,” all refer to an interleukin that is a heterodimeric cytokine encoded by the IL-18 gene (e.g., Genbank Accession numbers: NM_001243211, NM_001562 and NM_001386420). IL-18, structurally similar to IL-1β, is a member of IL-1 superfamily of cytokines. This cytokine, which is expressed by many human lymphoid and nonlymphoid cells, has an important role in inflammatory processes. IL-18 in combination with IL-12 can activate cytotoxic T cells (CTLs), as well as natural DB1 / 149057740.1 94Attorney Docket No.116983-5126-WO / AR / TW killer (NK) cells, to produce IFN-γ and, therefore, contributes to tumor immunity. Thus, without being bound by any particular theory of operation, it is believed that IL-18 can enhance the anti-tumor effects of the TIL compositions provided herein.

[0282] In some embodiments, the IL-18 is a tethered IL-18 (TeIL-18). In some embodiments, the TeIL-18 comprises the amino acid sequence of SEQ ID NO:132 (amino acids 1-18: human IgE signal sequence peptide; amino acids 176-200: peptide linker; amino acids 201-248: membrane anchor). In some embodiments, the TeIL-18 comprises the amino acid sequence of SEQ ID NO:134 (amino acids 1-18: human IgE signal sequence peptide; amino acids 175-199: peptide linker; amino acids 200-247: membrane anchor).

[0283] In some embodiments, the IL-18 is a full length IL-18, a fragment or a variant of IL-18. In some embodiments, the IL-18 is a human IL-18 or a variant human IL-18. In exemplary embodiments, the IL-18 is a biological active human IL-18 variant. In some embodiments, the IL-18 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mutations as compared to a wild-type IL-18 (SEQ ID NO:75). In some embodiments, the bioactive variant is a decoy resistant IL-18 variant (“DR-IL18,” or “DR-IL-18”) that provides IL-18 signaling activity even in the presence of an inhibitory molecule such as IL-18 binding protein (IL-18BP). Exemplary IL-18 variants that can be included in the subject modified TILs described herein are shown below in Table 7. Additional IL-18 variants that can be included in the subject modified TILs are described in WO 2022 / 094473, which is incorporated by reference in its entirety and particular with respect to disclosures relating to variant DR-IL-18.

[0284] In some embodiments, the variant IL-18 includes a stability mutation pair selection from: C38S / C68S, C38S / C68G, C38S / C68A, C38S / C68D, and C38S / C68N [relative to the human wild-type IL-18 - SEQ ID NO: 75]. In some embodiments, the variant IL-18 includes mutations at amino acid positions M51 (e.g., M51E, M51R, M51K, M51T, M51D, or M51N), K53 (e.g., K53G, K53S, K53T, or K53R), Q56 (e g., Q56G, Q56R, Q56L, Q56E, Q56A, Q56V, or Q56K), D110 (e.g., D110S, D110N, D110G, D110K, D110H, D110Q, or D110E) and N111 (e.g., N111G, N111R, N111S, N111D, N111H, or N111Y) in addition to a stabilizing mutation pair selected from: C38S / C68S, C38S / C68G, C38S / C68A, C38S / C68D, and C38S / C68N [relative to the human wild type IL-18 - SEQ ID NO: 75], In some such cases the stabilized IL-18 variant polypeptide additionally includes a mutation at amino acid position S105 (e.g., S105D, S105A, S105N, S105R, S105D, or S105K); and in DB1 / 149057740.1 95Attorney Docket No.116983-5126-WO / AR / TW some cases, further includes mutations at amino acid positions P57 (e.g., P57A, P57L, P57G, or P57K) and M60 (e.g., M60L, M60R, M60K, or M60Q). TABLE 7 – IL-18 Related Sequences. Description Amino Acid Sequence YFGKLESKLSVIRNLND VLFID GNRPLFEV T D T E T K E L E T K E T K E TDB1 / 149057740.1 96Attorney Docket No.116983-5126-WO / AR / TW Description Amino Acid Sequence ISVKDEKISTLSCENKIISFKEMNPPDNIKDTK E T K E T K E T D V T T D T DDB1 / 149057740.1 97Attorney Docket No.116983-5126-WO / AR / TW Description Amino Acid Sequence YFGKLESQLSVIRNLNDQVLFIDQGNRPLFET D V T A D D T D TIS D I S D I SDB1 / 149057740.1 98Attorney Docket No.116983-5126-WO / AR / TW Description Amino Acid Sequence KERDLFKLILKKEDELGDRSIMFTTQHED D I S D I S T D T D T D D I S D I SDB1 / 149057740.1Attorney Docket No.116983-5126-WO / AR / TW Description Amino Acid Sequence DIIFFKRRVPGHNHKMQFESSSYEGYFLACE D I S T D D I S D I S D I S T D T KDB1 / 149057740.1Attorney Docket No.116983-5126-WO / AR / TW Description Amino Acid Sequence RFGKLESRLSVIRNLNDQVLFIDQGNRPLFEDI S T K E V T C V T C V T C T C T C V T CDB1 / 149057740.1Attorney Docket No.116983-5126-WO / AR / TW Description Amino Acid Sequence T K E T D T C T C T K V T C V T C V T CDB1 / 149057740.1 102Attorney Docket No.116983-5126-WO / AR / TW Description Amino Acid Sequence EKERDLFKLILKKEDELGDRSIMFTVQNED V T D T K E T C T D T C T C T C L FIDB1 / 149057740.1 103Attorney Docket No.116983-5126-WO / AR / TW Description Amino Acid Sequence ISMYKDSQPRGMAVTISVKCEKISTLSCENKIISF F G C G C C C G C A G G C G Q L FI E Y Q N C A ADB1 / 149057740.1 104Attorney Docket No.116983-5126-WO / AR / TW Description Amino Acid Sequence CCATCTTTATCATCTCCAAGTACTCCGATTCTC A C G G G G C T DAccession numbers: NM_001207006 and NP_001193935 (human); and NM_0001291041 and NP_001277970 (mouse)) all refer to a member of a cytokine that binds to IL-21 receptor and has potent regulatory effects on cells of the immune system, including natural killer (NK) cells and cytotoxic cells and binds to IL-21 receptor that can destroy virally infected or cancerous cells. Thus, without being bound by any particular theory of operation, it is believed that IL-21 can increase the survivability and / or anti-tumor effects of the TIL compositions provided herein.

[0286] In some embodiments, the IL-21 is a tethered IL-21. In some embodiments, the tethered IL-21 comprises the amino acid sequence of SEQ ID NO: 137 (amino acids 1-18: human IgE signal sequence peptide; amino acids 152-197: peptide linker; amino acids 198- 245: membrane anchor).

[0287] In some embodiments, the IL-21 is a human IL-21 (SEQ ID NO: 136). In some embodiments, the IL-21 associated with the modified TIL is a full length IL-21, a fragment or a variant of IL-21. In some embodiments, the IL-21 is a human IL-21 or a variant human IL-21. In exemplary embodiments, the IL-21 is a biological active human IL- 21 variant. In some embodiments, the IL-21 includes a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations as compared to a wild-type IL-21. DB1 / 149057740.1 105Attorney Docket No.116983-5126-WO / AR / TW TABLE 8 – IL-21 Related Sequences. Description Amino Acid Sequence QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEQS P S G RAccession numbers: NM_000586 and NP_000577 (human) all refer to a member of a cytokine that binds to IL-2 receptor. IL-2 enhances activation-induced cell death (AICD). IL- 2 also promotes the differentiation of T cells into effector T cells and into memory T cells when the initial T cell is also stimulated by an antigen, thus helping the body fight off infections. Together with other polarizing cytokines, IL-2 stimulates naive CD4+ T cell differentiation into Th1 and Th2 lymphocytes and impedes differentiation into Th17 and follicular Th lymphocytes. IL-2 also increases the cell killing activity of both natural killer cells and cytotoxic T cells. Thus, without being bound by any particular theory of operation, it is believed that IL-2 can increase the survivability and / or anti-tumor effects of the TIL compositions provided herein.

[0289] In some embodiments, the IL-2 is a tethered IL-2. In some embodiments, the tethered IL-2 comprises the amino acid sequence of SEQ ID NO: 139 (amino acids 1-20: human IL-2 signal peptide; amino acids 154-178: peptide linker; amino acids 179-226: membrane anchor).

[0290] In some embodiments, the IL-2 is a human IL-2 (SEQ ID NO: 138). In some embodiments, the IL-2 associated with the modified TIL is a full length IL-2, a fragment or a variant of IL-2. In some embodiments, the IL-2 is a human IL-2 or a variant human IL-2. In exemplary embodiments, the IL-2 is a biological active human IL-2 variant. In some DB1 / 149057740.1 106Attorney Docket No.116983-5126-WO / AR / TW embodiments, the IL-2 includes a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations as compared to a wild-type IL-2. TABLE 9 – IL-2 Related Sequences. Description Amino Acid Sequence MYRM LLSCIALSLALVTNSAPTSSSTKKTQF M Q L L V IS S S T T A G G G A G A C A G C G C C C Ccomprises a nucleotide sequence that encodes a truncated CD-19 (tCD19).

[0292] In some embodiments, the nucleic acid molecule provided herein further comprises a nucleotide sequence that encodes an shRNA. In some embodiments, the shRNA inhibits the expression of an immune checkpoint gene. DB1 / 149057740.1 107Attorney Docket No.116983-5126-WO / AR / TW

[0293] Non-limiting examples of immune checkpoint genes that may be silenced or inhibited by the shRNA include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, BAFF (BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11, and BCOR. For example, immune checkpoint genes that may be silenced or inhibited the shRNA may be selected from the group comprising PD-1, CTLA-4, LAG-3, TIM-3, Cish, CBL-B, TIGIT, TET2, TGFβ, and PKA. BAFF (BR3) is described in Bloom, et al., J. Immunother., 2018, in press. According to another example, immune checkpoint genes that may be silenced or inhibited the shRNA may be selected from the group comprising PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, TET2, CISH, TGFβR2, PRA, CBLB, BAFF (BR3), and combinations thereof. Exemplary PD-1 shRNA sequences are provided in the table below. TABLE 9 – PD-1 shRNA sequences Construct DNA sequence PD-1 GGATTTCCAGTGGCGAGAGAACTCGAGTTCTCTCGCCACTGGAAATCC (SEQ D Dfurther comprises long terminal repeat (LTR) sequences, for example, a 5’ LTR sequence and DB1 / 149057740.1 108Attorney Docket No.116983-5126-WO / AR / TW a 3’ LTR sequence (Fig.28). The LTR sequences are essential for viral genome integration into the host cell genome and viral gene expression in the host cell.

[0295] In an embodiment of the invention, the nucleic acid molecule disclosed herein is in the form of a recombinant lentiviral RNA molecule. For example, the recombinant lentiviral RNA molecule may comprise at least a portion of a lentivirus genome, including 5’ and 3’ LTRs. In some embodiments, the lentivirus genome may be modified to inhibit replication and limit pathogenicity, while retaining function. For example, the env gene, the gag gene, the pol gene, and the rev gene may be removed from the lentivirus genome and included in helper plasmids. In some embodiments, the recombinant lentiviral RNA molecule is comprised by a lentiviral vector or a recombinant lentiviral particle as described elsewhere in this disclosure.

[0296] In another embodiment of the invention, the nucleic acid molecule disclosed herein is in the form of a recombinant lentiviral proviral DNA molecule. For example, the recombinant lentiviral proviral DNA molecule is comprised by a host cell, e.g., a gene-edited TIL as disclosed herein. In some embodiments, the recombinant lentiviral proviral DNA molecule is integrated into the genome of the host cell, e.g., a gene-edited TIL as disclosed herein. In some embodiments, recombinant lentiviral proviral DNA molecule is comprised by a packaging cell line as described elsewhere in this disclosure. In some embodiments, the recombinant lentiviral proviral DNA molecule is integrated into the genome of the packaging cell line. TABLE 10 – Exemplary nucleic acid molecule sequences C DNA sequence on G G T A G G TDB1 / 149057740.1 109Attorney Docket No.116983-5126-WO / AR / TW - CCAGGTCAAGGAGTTCGGTGATGCAGGACAGTACACGTGTCACAAGGGCG tC GGGAGGTCTTGTCCCATAGCCTGCTGCTCCTGCACAAGAAGGAAGACGGC D ATCTGGTCTACGGATATCCTTAAAGACCAGAAGGAGCCCAAGAACAAAAC G G A G G T C G G G G C C G A T T G G C T G G A T A A G C C CDB1 / 149057740.1 110Attorney Docket No.116983-5126-WO / AR / TW TTGTCTTTGGAGCTGAAAGATGACCGGCCGGCTCGTGACATGTGGGTGAT GGAGACCGGCCTGCTGCTGCCGCGCGCCACGGCCCAGGATGCGGGGAAGT ACTACTGTCACCGCGGCAACCTGACCATGTCGTTCCACTTGGAGATCACCG A G G T A G G T G C C G G A G G T G G G C C G T T G GDB1 / 149057740.1 111Attorney Docket No.116983-5126-WO / AR / TW GTCGTGAGAGCGTGAGGCCTGTGTGAGAATTCCGCCCCCCCCCCCCCCCG GGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTC GGCAATTGATCCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAA T G G A A A C C C T G A T C A C G G G G TDB1 / 149057740.1 112Attorney Docket No.116983-5126-WO / AR / TW E TCTGTTCCTCGTGGCCGCCGCTACCCGCGTGCACTCCATCTGGGAGCTGAA F1 GAAAGACGTGTACGTGGTGGAATTGGATTGGTACCCCGACGCCCCAGGCG - AGATGGTGGTTCTGACTTGCGACACGCCGGAGGAGGACGGCATCACCTGG T G C C G G A G G T C G G G G C C G A T T G G C T G G A T A A G CDB1 / 149057740.1 113Attorney Docket No.116983-5126-WO / AR / TW TGGGCCAAGGACCGCCCTGAGATCTGGGAAGGAGAGCCTCCGTGCGTGCC CCCGCGTGATTCGCTCAACCAGAGCCTGTCCCAGGACCTCACGATGGCAC CCGGTAGCACCCTGTGGTTATCCTGCGGCGTCCCCCCCGACTCCGTGTCCC T G A A T A C T T A G G T A G G T G C C G G A G G TDB1 / 149057740.1 114Attorney Docket No.116983-5126-WO / AR / TW CGCGACTGTCATTTGTCGGAAGAACGCGAGCATCTCCGTACGGGCACAGG ATCGCTATTACTCCAGCAGTTGGTCAGAGTGGGCTTCGGTTCCGTGTTCTG GAGGAGGTGGCGGCGGCTCACGCAACCTGCCCGTGGCGACGCCGGACCCG G C C G A T T G G C T G G A T A A G C C C T G A T A G TDB1 / 149057740.1 115Attorney Docket No.116983-5126-WO / AR / TW G G T A G G T G C C G G A G G T G G G C C G T T G G TDB1 / 149057740.1 116Attorney Docket No.116983-5126-WO / AR / TW ATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCG CCAGAACACAGGCTTGATCACGCGTGCCACCATGGATTGGACCTGGATCC TGTTCCTGGTGGCTGCAGCCACCCGTGTTCATTCCCAGGTCACCGACATCA T G GIV. Recombinant Expression Vectors, Lentiviral Expression Systems, Packaging Cell Line

[0298] In an embodiment of the invention, the inventive nucleic acid molecule disclosed herein is carried in a recombinant expression vector. Accordingly, an embodiment of the invention provides a recombinant expression vector comprising any of the inventive nucleic acid molecules described herein with respect to other aspects of the invention.

[0299] In some embodiments, the nucleotide sequences encoding the one or more GOIs, e.g., cytokines selected from the group consisting of IL-12, IL-2, IL-6, IL-7, IL-9, IL-15, IL- 18, IL-21, IL-23, IL-27, IL-33, IFN gamma, TNFa, IFN alpha, IFN beta, GM-CSF, GCSF, or a variant thereof, are provided by two or more recombinant expression vectors. For example, provided herein are a recombinant expression vector comprising a nucleotide sequence encoding IL-12 or a variant thereof; and a second recombinant expression vector comprising a nucleotide sequence encoding IL-15 or a variant thereof. In some embodiments, provided herein are a recombinant expression vector comprising a nucleotide sequence encoding IL-12 or a variant thereof; and a second recombinant expression vector comprising a nucleotide sequence encoding IL-2 or a variant thereof.

[0300] For purposes herein, the term "recombinant expression vector" means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell. The vectors of the invention are not naturally-occurring as a whole. However, parts of the vectors can be naturally-occurring. The recombinant DB1 / 149057740.1 117Attorney Docket No.116983-5126-WO / AR / TW expression vector can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides. The recombinant expression vectors can comprise naturally-occurring or non-naturally-occurring internucleotide linkages, or both types of linkages. Preferably, the non-naturally occurring or altered nucleotides or intemucleotide linkages do not hinder the transcription or replication of the vector. The vector may contain regulatory nucleic acid sequences which provide for expression of the inventive nucleic acid.

[0301] The recombinant expression vector can be any suitable recombinant expression vector. Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses. For example, the vector can be selected from the pUC series (Fermentas Life Sciences, Glen Bumie, MD), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA).

[0302] Bacteriophage vectors, such as λGT10, λGT11, λZap II (Stratagene), λEMBL4, and λNMI 149, also can be used. Examples of plant expression vectors useful in the context of the invention include pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expression vectors useful in the context of the invention include pEUK-Cl, pMAM, and pMAMneo (Clontech).

[0303] In some embodiments, the recombinant expression vector is a viral vector. Suitable viral vectors include, without limitation, lentiviral, retroviral, alphaviral, vaccinial, adenoviral, adenoassociated viral, herpes viral, and fowl pox viral vectors, and preferably have a native or engineered capacity to transform T cells.

[0304] The recombinant expression vectors can be prepared using standard recombinant DNA techniques described in, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, (4th Ed.) Cold Spring Harbor Laboratory Press, New York (2012). Constructs of expression vectors, which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell. Replication systems can be derived, e.g., from ColEl, 2 µ plasmid, λ, SV40, bovine papilloma virus, and the like.

[0305] The recombinant expression vector can comprise regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the DB1 / 149057740.1 118Attorney Docket No.116983-5126-WO / AR / TW type of host (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate, and taking into consideration whether the vector is DNA- or RNA-based.

[0306] The recombinant expression vector can include one or more marker genes, which allow for selection of transformed or transfected hosts. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like. Suitable marker genes for the recombinant expression vectors include, for instance, neomycin / G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0307] The recombinant expression vector can comprise a native or normative promoter operably linked to the nucleic acid molecule. Preferably, the promoter is functional in T cells. The selection of a promoter, e.g., strong, weak, inducible, tissue-specific and developmental-specific, is within the ordinary skill of the artisan. Similarly, the combining of a nucleotide sequence with a promoter is also within the skill of the artisan. The promoter can be a non-viral promoter or a viral promoter, e.g., an NFAT promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, or a promoter found in the long- terminal repeat of the murine stem cell virus.

[0308] Further provided herein is a lentiviral expression system comprising a transfer vector comprising the nucleic acid molecules disclosed herein which can be transcribed into a recombinant lentiviral RNA molecule for packaging into a lentiviral particle, and one or more helper vectors or helper plasmids encoding an Env protein, a Gag protein, a Pol protein, and a Rev protein. In some embodiments, the Env protein is selected from the group consisting of Ba-EVTR, VSV-G, and RD114.

[0309] In some embodiments, the recombinant lentiviral RNA molecule may comprise at least a portion of a lentivirus genome, including 5’ and 3’ LTRs. In some embodiments, the lentivirus genome may be modified to inhibit replication and limit pathogenicity, while retaining function. For example, the env gene, the gag gene, the pol gene, and the rev gene may be removed from the lentivirus genome and included in helper plasmids. In some embodiments, the recombinant lentiviral RNA molecule is comprised by a lentiviral vector or recombinant lentiviral particle as described elsewhere in this disclosure. DB1 / 149057740.1 119Attorney Docket No.116983-5126-WO / AR / TW

[0310] In some embodiments, two helper vectors or helper plasmids encode an Env protein, a Gag protein, a Pol protein, and a Rev protein. In some embodiments, three helper vectors or helper plasmids encode an Env protein, a Gag protein, a Pol protein, and a Rev protein. In some embodiments, four helper vectors or helper plasmids encode an Env protein, a Gag protein, a Pol protein, and a Rev protein. Any combination of helper vectors or helper plasmids can be used to encode an Env protein, a Gag protein, a Pol protein, and a Rev protein. For example, in some embodiments, one helper vectors or helper plasmids encode an Env protein, a Gag protein, and a Pol protein. In some embodiments, one helper vectors or helper plasmids encode a Gag protein, a Pol protein, and a Rev protein. In some embodiments, one helper vectors or helper plasmids encode a Gag protein, and a Pol protein. In some embodiments, one helper vectors or helper plasmids encode en Env protein, and a Rev protein.

[0311] A packaging cell line can be used to package a nucleic acid, e.g., an RNA encoding a transgene, into a lentiviral vector. Accordingly, the systems and methods described herein may comprise, e.g., a lentiviral packaging cell line comprising at least one plasmid adapted for the production of a lentiviral vector, e.g., a lentiviral vector optionally comprising a transgene. Various lentiviral components useful for the production of a lentiviral vector are known in the art. See for example Zufferey et al., 1997, Nat. Biotechnol. 15:871-875 and Dull et al, 1998, J. Virol.72(11): 8463 -8471. The different functions suitable for the production of a lentiviral vector can be provided to the packaging cells in a lentiviral packaging system comprising one or more nucleic acids (e.g., plasmids), e.g., at least one, two, three, or four plasmids, wherein one plasmid encodes a retroviral envelope protein (Env plasmid), one plasmid encodes one or more retroviral packaging proteins, e.g., Gag and Pol proteins (packaging plasmid or Gag-Pol plasmid), one plasmid encodes a lentiviral Rev protein (Rev plasmid) and one or more plasmids comprising at least one gene of interest (GOI) expression cassette (transfer vector). In some embodiments, the lentiviral packaging system further comprises, or a method described herein comprises use of, at least one, two, three, or four plasmids. In some embodiments, the lentiviral packaging system further comprises, or a method described herein comprises use of, a fifth plasmid. In certain embodiments, a method described herein comprises transfecting five plasmids into the packaging cell, wherein the fifth plasmid does not encode a protein of the lentiviral vector packaging system. In some embodiments, the lentiviral packaging system comprises one or more nucleic acids (e.g., plasmids), e.g., five plasmids, wherein one plasmid encodes an DB1 / 149057740.1 120Attorney Docket No.116983-5126-WO / AR / TW expression vector, one plasmid encodes a Tat (e.g., pcDNATat), one plasmid encodes a Rev protein (e.g., pHCMV-Rev), one plasmid encodes a gagpol (e.g., pHCMV-gagpol), and one plasmid encodes VSV-G (e.g., pVSVG), e.g., as described in Rout-Pitt et al., J Biol. Methods 5(2): 1-9, 2018). In some embodiments, a plasmid may comprise a dual gene expression cassette, e.g., a bicistronic cassette, e.g., a bicistronic construct encoding two transgenes of interest. In some embodiments, the first transgene of interest encodes a first cytokine, e.g., TeIL-12, and the second transgene of interest encodes a second cytokine, e.g., IL-15. In some embodiments the retroviral packaging proteins are derived from a lentivirus, e.g., lentiviral packaging proteins, e.g., lentiviral gag and pol proteins.

[0312] In some embodiments, the lentiviral gag protein is a wild-type lentiviral gag protein, and in other embodiments it has one or more sequence modifications relative to the wild-type sequence. In some embodiments, the lentiviral pol protein is a wild-type lentiviral pol protein, and in other embodiments it has one or more sequence modifications relative to the wild-type sequence. In some embodiments, the rev protein is a wild-type rev protein, and in other embodiments it has one or more sequence modifications relative to the wild-type sequence. In some embodiments, the lentiviral vector packaging system may be a pseudotyped lentiviral vector packaging system, comprising a modified envelope protein, e.g., an envelope protein derived from a different virus or a chimeric envelope protein, e.g., the Env plasmid may encode a Ba-EVTR Env protein.

[0313] In some embodiments, a lentiviral vector is generated using a packaging system comprising pMDLgpRRE, pRSV-Rev and pMD.G plasmids (Dull et al., supra), but using a kanamycin resistance marker, e.g., a marker that confers resistance to both kanamycin and neomycin, e.g., neomycin phosphotransferase II instead of an ampicillin gene.

[0314] Therefore, further provided here is a packaging cell line comprising a recombination expression vector disclosed herein, for example, a transfer vector, comprising the nucleic acid molecule comprising a nucleotide sequence encoding a tethered IL-12 (TeIL- 12) and optionally a cytokine selected from the group consisting of IL-2, IL-6, IL-7, IL-9, IL- 15, IL-18, IL-21, IL-23, IL-27, IL-33, IFN gamma, TNFa, IFN alpha, IFN beta, GM-CSF, GCSF, or a variant thereof. In some embodiments, the cytokine is a tethered cytokine. In some embodiments, the nucleic acid molecule comprising a nucleotide sequence encoding a TeIL-12 and a tethered IL-15 (TeIL-15). In some embodiments, the nucleic acid molecule comprising a nucleotide sequence encoding a TeIL-12 and a tethered IL-18 (TeIL-18). DB1 / 149057740.1 121Attorney Docket No.116983-5126-WO / AR / TW

[0315] In some embodiments, the nucleic acid molecule further comprises a nucleic acid sequence encoding an shRNA. In some embodiments, the shRNA inhibits the expression of an immune checkpoint gene. In some embodiments, the shRNA inhibits the expression of PD-1.

[0316] Non-limiting examples of immune checkpoint genes that may be silenced or inhibited by the shRNA include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, BAFF (BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11, and BCOR. For example, immune checkpoint genes that may be silenced or inhibited the shRNA may be selected from the group comprising PD-1, CTLA-4, LAG-3, TIM-3, Cish, CBL-B, TIGIT, TET2, TGFβ, and PKA. BAFF (BR3) is described in Bloom, et al., J. Immunother., 2018, in press. According to another example, immune checkpoint genes that may be silenced or inhibited the shRNA may be selected from the group comprising PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, TET2, CISH, TGFβR2, PRA, CBLB, BAFF (BR3), and combinations thereof.

[0317] In some embodiments, the packaging cell line is a human cell line. In some embodiments, the packaging cell line is a 293T cell line.

[0318] In some embodiments, the packaging cell line is a stable packaging cell line produced by the EuLV® system (Shenzhen Eureka Biotechnology Co., Limited, Shenzhen, China) described in Xue et al., Cell & Gene Therapy Insights 2022; 8(2), 199–209, the content of which is incorporated by reference in its entirety.

[0319] Any suitable protocols known in the art may be used in developing the stable packaging cell line. For example, Broussau et al., Mol. Thera., 2008, 16:500-507 describes an inducible packaging cell line, 293SF-PacLV, for production of lentiviral vectors in serum- free culture, the content of which is hereby incorporated by reference in its entirety. A cell line derived from 293SF cells, expressing the repressor (CymR) of the cumate switch and the reverse transactivator (rtTA2S-M2) of the tetracycline (Tet) switch, is established first. Clones stably expressing the Gag / Pol and Rev genes of human immunodeficiency virus-1, and the glycoprotein of vesicular stomatitis virus (VSV-G), are generated next. Expression of DB1 / 149057740.1 122Attorney Docket No.116983-5126-WO / AR / TW Rev and VSV-G is tightly regulated by the cumate and Tet switches in the 293SF-PacLV cell line. Two approaches are used to generate the packaging cell lines. In the first approach (Two-step), a stable clone expressing the Gag / Pol and Rev genes is first generated and characterized before producing a clone expressing VSV-G and additional Rev. In the second approach (One-shot), all the LV components (Gag / Pol, rev, and VSV-G) are added simultaneously through a single transfection event.

[0320] Two-step approach: A cell line derived from a clone of 293 cells (293SF) adapted to grow in suspension and in serum-free medium that expresses CymR is generated. 293SF were transfected with pMPGBFP / CMV5-CymR / tk-neo and a resistant pool of cells is isolated in the presence of neomycin. Clones are then isolated by limiting dilution of the pool. The clones are maintained for 6 weeks in the absence of selective pressure in order to test their stability. The CymR function is analyzed using an adenoviral vector (AdV) expressing β-galactosidase (β-gal) regulated by the CMV5-CuO promoter. The best β-gal On / Off ratio in the presence and absence of cumate is used in order to select the clone producing the optimal quantity of CymR. Clone G, which showed an On / Off ratio of 14, is selected as a recipient for the rtTA2S-M2 transactivator.293SF-CymR-G cells are transfected with plasmid pUDHrtTA2S-M2.hygro. A pool of hygromycin-resistant cells is generated, and clones are obtained by limiting dilution of the pool in the absence of selection. The functioning of CymR and rtTA2S-M2 produced by the clones is tested using an AdV encoding the green fluorescent protein (GFP) regulated by the TR5 and CuO promoters. The clone with the best On / Off ratio is chosen for the development of the packaging cell lines.

[0321] This is done by transfecting it with pMPG-RSV-Rev / CMV-Gag / polRRE, a plasmid that encodes the Rev, Gag, and pol genes of human immunodeficiency virus-1, as well as the resistance for phleomycin as a fusion protein with GFP. Phleomycin-resistant clones are isolated in 96-well plates. The clones with the best GFP expression level are analyzed for LV production by transient transfection with pCSII-CMV5-GFPq and VSV-G. The titers obtained with the best clones are 10- to 55-fold lower than those obtained when the same clones are transfected with plasmids encoding Rev regulated by Rous sarcoma virus (RSV) (pRSV-Rev). Two clones (#19 and #64) are then subcloned by limiting dilution and analyzed for LV production by transient transfection. As observed for the parent clones, the LV titer obtained is significantly higher in the presence of additional Rev.

[0322] Because the previous results indicate that the quantity of Rev produced by the 293SF-Rev-Gag-Pol cells is not optimal, two of the best subclones (#19-17 and #64-8) are DB1 / 149057740.1 123Attorney Docket No.116983-5126-WO / AR / TW co-transfected with plasmids encoding the puromycin resistance (pPuro), Rev (pkCMV5- CuO-Rev) and VSV-G (pTR5-CuO-VSVg-IRES-GFPq) to generate a cell line expressing VSV-G and more Rev. VSV-G is regulated by the cumate switch and the Tet switch, whereas Rev is regulated by the cumate switch only. The puromycin-resistant clones are first screened for VSV-G expression by measuring GFP (through the IRES-GFP) after induction with Dox and cumate. The LV production is then analyzed by transient transfection with pCSII-CMV5- GFPq. Co-transfection of the best clones with pRSV-Rev increases the LV titer by only twofold to fourfold, which is lower than the earlier level. The three best clones are subcloned in the absence of selective pressure. LV production is analyzed by transient transfection as described earlier. The production from the six best subclones can be improved by only twofold to threefold after the addition of Rev, thereby indicating that the quantity of Rev is nearly optimal. The efficacy of the double switch is investigated by measuring the increase of GFP expression (from the stably integrated VSV-G-IRES-GFPq cassette) after induction of clone #16-22. An induction factor over 2,500 is observed in the presence of cumate and Dox. GFP expression in the presence of the two inducers is much higher than when each inducer is used separately. In the presence of cumate only, the GFP expression level increases by a factor of 4.5, thereby indicating that the cumate switch improves the tightness of the Tet switch by this factor.

[0323] One-shot approach: Because Rev is required for the efficient expression of the protease from the Gag gene, which has been reported to be cytotoxic, even a relatively small amount of Rev could be detrimental to the cells. For this reason, a packaging cell line expressing Rev under very tight regulation is generated. pTR5-CuO-Rev, which contains the Rev coding sequence doubly regulated by the Tet switch and the cumate switch is constructed. The 293SF-CymR-rtTA2S-M2 cell line is co-transfected with plasmids encoding Rev, Gag, Pol, VSV-G, and the resistance for puromycin. Stable clones are selected in the presence of puromycin. Clones having the highest GFP induction factor after addition of Dox and cumate are analyzed for LV production by transfection with transfer vector pCSII- CMV5-GFPq. The two best clones are then subcloned without selection and analyzed for LV production. The transfection of the best subclones with pCSIICMV5-GFPq and pRSV-Rev does not increase the amount of LVs produced, thereby suggesting that the quantity of Rev produced by the clones is not limiting.

[0324] The LV production levels obtained in the best clones when using the Two-step (#16-22) and One-shot (#29-6) approaches is then compared. Titers of 8.6 x 106and 2.6 x 107DB1 / 149057740.1 124Attorney Docket No.116983-5126-WO / AR / TW TU / ml are obtained for #16-22 and #29-6, respectively. The amount of p24 produced after induction is also evaluated by enzyme-linked immunosorbent assay (ELISA). The amounts of p24 produced are 39 and 571 ng / ml by #16-22 and #29-6, respectively. The fact that the specific activity (TU / ng of p24) is 219,000 for #16-22 and 45,000 for #29-6 indicates that the latter clone produced more defective or empty virions.

[0325] Briefly, Ba-EVTR, gag / pol, and rev are stably inserted into 293T cells to obtain packaging cell populations, and the optimal packaging cell line is obtained through monoclonal and titer screening. Then, a transfer vector comprising the nucleic acid molecules disclosed herein, i.e., a transfer vector encoding the GOI, for example, a transfer vector comprising the nucleic acid molecule comprising a nucleotide sequence encoding a tethered IL-12 (TeIL-12) and optionally a cytokine selected from the group consisting of IL-2, IL-6, IL-7, IL-9, IL-15, IL-18, IL-21, IL-23, IL-27, IL-33, IFN gamma, TNFa, IFN alpha, IFN beta, GM-CSF, GCSF, or a variant thereof, is integrated into the optimal packaging cell line. In some embodiments, the cytokine is a tethered cytokine. In some embodiments, the nucleic acid molecule comprising a nucleotide sequence encoding a TeIL-12 and a tethered IL-15 (TeIL-15). In some embodiments, the nucleic acid molecule comprising a nucleotide sequence encoding a TeIL-12 and a tethered IL-18 (TeIL-18).

[0326] In some embodiments, the nucleic acid molecule further comprises a nucleic acid sequence encoding an shRNA. In some embodiments, the shRNA inhibits the expression of an immune checkpoint gene. In some embodiments, the shRNA inhibits the expression of PD-1.

[0327] Non-limiting examples of immune checkpoint genes that may be silenced or inhibited by the shRNA include PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, BAFF (BR3), CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11, and BCOR. For example, immune checkpoint genes that may be silenced or inhibited the shRNA may be selected from the group comprising PD-1, CTLA-4, LAG-3, TIM-3, Cish, CBL-B, TIGIT, TET2, TGFβ, and PKA. BAFF (BR3) is described in Bloom, et al., J. Immunother., 2018, in press. According to another example, immune checkpoint genes that may be silenced or inhibited the shRNA DB1 / 149057740.1 125Attorney Docket No.116983-5126-WO / AR / TW may be selected from the group comprising PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, TET2, CISH, TGFβR2, PRA, CBLB, BAFF (BR3), and combinations thereof.

[0328] In some embodiments, the packaging cell line comprises a recombinant DNA encoding an Env protein, a Gag protein, a Pol protein, and a Rev protein. In some embodiments, the recombinant DNA encoding one or more of the Env protein, the Gag protein, the Pol protein, and the Rev protein is integrated into the genome of the packaging cell line. In some embodiments, the recombinant DNA encoding the Env protein, the Gag protein, and the Pol protein is integrated into the genome of the packaging cell line. In some embodiments, the recombinant DNA encoding the Env protein, the Gag protein, and the Rev protein is integrated into the genome of the packaging cell line. In some embodiments, the recombinant DNA encoding the Env protein, the Pol protein, and the Rev protein is integrated into the genome of the packaging cell line. In some embodiments, the recombinant DNA encoding the Gag protein, the Pol protein, and the Rev protein is integrated into the genome of the packaging cell line. In some embodiments, the Env protein is selected from the group consisting of Ba-EVTR, VSV-G, and RD114. In some embodiments, the Env protein is a Ba-EVTR Env protein. In some embodiments, the recombinant DNA encoding one or more of the Env protein, the Gag protein, the Pol protein, and the Ba-EVTR Rev protein is integrated into the genome of the packaging cell line. In some embodiments, the recombinant DNA encoding the Ba-EVTR Env protein, the Gag protein, and the Pol protein is integrated into the genome of the packaging cell line. In some embodiments, the recombinant DNA encoding the Ba-EVTR Env protein, the Gag protein, and the Rev protein is integrated into the genome of the packaging cell line. In some embodiments, the recombinant DNA encoding the Ba-EVTR Env protein, the Pol protein, and the Rev protein is integrated into the genome of the packaging cell line.

[0329] In some embodiments, the recombinant DNA encoding the Env protein, the Gag protein, the Pol protein, and the Rev protein is integrated into the genome of the packaging cell line. In some embodiments, the nucleic acid molecule disclosed herein, e.g., the recombinant lentiviral proviral DNA molecule, is integrated into the genome of the packaging cell line.

[0330] In some embodiments, the different functions for production of a lentiviral vector are provided to a plurality of host cells, e.g., mammalian cells, e.g., HEK293 cells, e.g., Expi293F cells (e.g., plurality of Expi293F cells growing in suspension under serum-free conditions) by transfection, e.g., transient or stable transfection, of a lentiviral packaging DB1 / 149057740.1 126Attorney Docket No.116983-5126-WO / AR / TW system adapted for producing lentiviral vectors. In some embodiments, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of host cells, e.g., HEK293 cells, e.g., Expi293F cells are transfected. Methods for transfection or infection are well known by those of skill in the art. In some embodiments, at least 0.3pg, at least 0.4pg. at least 0.5pg, at least 0.6pg. at least 0.7pg, at least 0.8pg cells, at least 0.9pg, or at least 1.0 pg of lentiviral packaging system is provided per million cells for transfection. In some embodiments, a transfection reagent is used for transfecting the host cells, e.g., mammalian cells, e.g., HEK293 cells, e.g., Expi293F cells. In some embodiments, a transfection reagent is used. Transfection reagents are well known in the art and are available from commercial suppliers. Examples of transfection reagents include but are not limited to, Lipofectamine™ (Invitrogen), Polifectamine, LentiTran (Origene), PEIpro® (Polyplus), FectoVIR® - AAV (Polyplus), and ProFection® (Promega). V. Recombinant Lentiviral Particle And Method Of Making

[0331] This disclosure provides, in some embodiments, method of making a recombinant lentiviral particle. The following general steps may be used. First, culturing a population of packaging cells in a cell culture medium. Once sufficient numbers of packaging cells are obtained, the desired nucleic acids, such as a lentiviral vector comprising a nucleic acid molecule encoding the cytokines disclosed herein, can be introduced into the packaging cells. Additional nucleic acids that may be introduced into the packaging cells include plasmids that promote packaging, e.g., plasmids encoding viral gag, pol, env, and rev. The packaging cells then begin to produce recombinant lentiviral particles. After transfection, a nuclease such as benzonase may be added to the culture media.

[0332] In some embodiments, a stable packaging cell line with the VSV-G, gag / pol, and rev integrated into the genome can be used for the production of the recombinant lentiviral particles. In some embodiments, the stable packaging cell line further comprises the lentiviral vector comprising a nucleic acid molecule encoding the cytokines disclosed herein integrated into the genome.

[0333] Any suitable protocols known in the art may be used for producing the recombinant lentiviral particles using a stable packaging cell line. For example, Broussau et al., supra, describes lentiviral particle production using a stable packaging cell line, the content of which is hereby incorporated by reference in its entirety. DB1 / 149057740.1 127Attorney Docket No.116983-5126-WO / AR / TW

[0334] Neither the preparation of large quantities of plasmid nor the transfection procedure would be necessary if a cell line (a producer) that synthetizes all the essential viral functions including the viral RNA are available. Using such a cell line, LV production can be initiated solely by adding the inducers, such as Dox and cumate. The ability of packaging cells to generate stable producers is evaluated. Clones #29-6 and #16-22 are transduced with a conditional-SIN-LV, produced by transfecting packaging cells with the transfer vector pLVR2-GFP23. Clones are isolated by limiting dilution of the pool of transduced cells. The clones having the highest GFP expression following induction with Dox are expended. The production in serum-free suspension culture of the best clones from parent cells #16-22 (16- 22-22) and #29-6 (#29-6-14) is tested. The medium is changed on a daily basis and the number of infectious particles is determined by flow cytometry.

[0335] The amount of p24 produced is also analyzed by ELISA. The behavior of the two clones is quite similar, except for the absolute amount of LVs produced, which is two to three times higher for clones #29-6-14. The number of infectious particles produced increases every day until it reaches a maximum at day 4. It then decreases progressively. The highest titer (3.4 x 107TU / ml) is obtained at day 4 when #29-6-14 is used. The quantity of p24 produced by the cells at different time points follows the same pattern. On day 1, the specific activity of the viral preparation (TU / ng of p24) is 54,000 and 166,000 for #16-22-22 and #29- 6-14, respectively, and decreases progressively. At the end of the production (day 6 or 7) the specific activity is lower by a factor of 3 to 4, thereby indicating that more defective particles are produced at later time points.

[0336] The relative stability of four different producer clones derived from packaging cells #29-6 is evaluated. The LV production is compared after 4 weeks and 18 weeks of culture in the absence of selective pressure. The titers obtained with these four clones does not decrease significantly after 18 weeks of culture. These data indicate that, using the packaging cells described in this study, it is possible to obtain producers whose long-term stability is more than adequate for a large-scale production process. In addition, tests to detect the presence of replication-competent lentiviruses are carried out by infecting 293 cells using concentrated stock of LVs. No replication-competent lentiviruses are detected by ELISA analysis for P24, and no VSV-G is detected by polymerase chain reaction (PCR).

[0337] Because subcloning and analysis of individual clones is a time-consuming process, whether high levels of LVs can be obtained using a pool of cells is investigated. For this experiment, packaging cells #29-6 are transduced with a conditional-SIN-LV produced DB1 / 149057740.1 128Attorney Docket No.116983-5126-WO / AR / TW by transfecting packaging cells with pTet07-CSII-CMVGFPq. The production efficiency of the pool of producers is then tested in serum-free suspension culture in shake flasks. The medium is changed on a daily basis and the number of infectious particles is determined by flow cytometry. The production pattern of the pool is very similar to that of the two clones, except for the fact that the highest titer (1.9 x 107TU / ml) is obtained at day 3 instead of day 4, and that the duration of useful production is one day shorter.

[0338] Without wishing to be bound by theory, in some embodiments, the cell culture medium is a source of contaminating nucleic acids to the final lentiviral preparation, e.g., the culture medium may contain packaging cell DNA from lysed packaging cells. Accordingly, addition of benzonase to the cell culture medium may degrade the contaminating nucleic acids, allowing for improved purification of the recombinant lentiviral particles.

[0339] Next, recombinant lentiviral particles can be harvested from the packaging cell culture to begin purification of the recombinant lentiviral particles. In some embodiments, harvesting of recombinant lentiviral particles comprises separating the supernatant or cell culture media from the packaging cell. In some embodiments, the packaging cell is not lysed before clarification. In some embodiments, the packaging cells may be lysed, and the lysate may be clarified.

[0340] Naturally occurring lentiviruses are a genus of viruses of the Retroviridae family, characterized by a long incubation period. Lentiviruses can typically deliver a significant amount of genetic information into the DNA of the host cell. Examples of lentiviruses include HIV (human immunodeficiency vims; including HIV type 1, and HIV type 2), the etiologic agent of the human acquired immunodeficiency syndrome (AIDS); visna-maedi, which causes encephalitis (visna) or pneumonia (maedi) in sheep, the caprine arthritis-encephalitis vims, which causes immune deficiency, arthritis, and encephalopathy in goats; equine infectious anemia vims, which causes autoimmune hemolytic anemia, and encephalopathy in horses; feline immunodeficiency vims (FIV), which causes immune deficiency in cats; bovine immune deficiency vims (BIV), which causes lymphadenopathy, lymphocytosis, and possibly central nervous system infection in cattle; and simian immunodeficiency vims (SIV), which cause immune deficiency and encephalopathy in sub- human primates. Diseases caused by these vimses are characterized by a long incubation period and protracted course. Usually, the viruses latently infect monocytes and macrophages, from which they spread to other cells. HIV, FIV, and SIV also readily infect T lymphocytes (i.e., T-cells). DB1 / 149057740.1 129Attorney Docket No.116983-5126-WO / AR / TW

[0341] Further provided herein is a recombinant lentiviral particle comprising the recombinant lentiviral RNA molecule disclosed herein. In some embodiments, the recombinant lentiviral particle is produced by the packaging cell line disclosed herein.

[0342] In some embodiments, the recombinant lentiviral particle comprises the recombinant lentiviral vector disclosed herein. In some embodiments, the recombinant lentiviral vector comprises the recombinant lentiviral RNA molecule disclosed herein. In some embodiments, the recombinant lentiviral particle further comprises a capsid enclosing the recombinant RNA viral genome. In some embodiments, the recombinant lentiviral particle further comprises an Env protein selected from the group consisting of Ba-EVTR, VSV-G, and RD114. In some embodiments, the recombinant lentiviral particle further comprises a reverse transcriptase. Add section on making non-gene-edited TILs VI. Method Of Making Gene-Edited TILs

[0343] In some embodiments of the present invention directed to methods for expanding TIL populations, the methods comprise one or more steps of gene-editing at least a portion of the TILs in order to enhance their therapeutic effect. As used herein, “gene-editing,” “gene editing,” and “genome editing” refer to a type of genetic modification in which DNA is permanently modified in the genome of a cell, e.g., DNA is inserted, deleted, modified or replaced within the cell’s genome. In some embodiments, gene-editing causes the expression of a DNA sequence to be silenced (sometimes referred to as a gene knockout) or inhibited / reduced (sometimes referred to as a gene knockdown). In other embodiments, gene-editing causes the expression of a DNA sequence to be enhanced (e.g., by causing over- expression). In accordance with embodiments of the present invention, gene-editing technology is used to enhance the effectiveness of a therapeutic population of TILs.

[0344] In some embodiments of the present invention, provided herein is a method of making a population of gene-edited TILs, comprising: a) performing a first expansion by culturing a population of TILs in a first cell culture medium; b) performing a second expansion by culturing the population of TILs in a second cell culture medium; and c) at any time, transducing the population of TILs with the recombinant lentiviral particle disclosed herein to produce the population of gene-edited TILs, wherein the population of gene-edited TILs expresses one or more cytokines, such as IL-12, IL-2, IL-6, IL-7, IL-9, IL-15, IL-18, IL- DB1 / 149057740.1 130Attorney Docket No.116983-5126-WO / AR / TW 21, IL-23, IL-27, IL-33, IFN gamma, TNFa, IFN alpha, IFN beta, GM-CSF, GCSF, or a variant thereof.

[0345] Cell surface markers PD-1, CD39, and CD103 represent the leading biomarkers to identify tumor-specific T cells but these TILs are terminally differentiated. Up-regulation of CD137 (4-1BB; TNFSR9) on recently activated CD8+ T-cells has been used to identify tumor antigen-specific T-cells (Draghi A., et al. Front Immunol 2021;12:705422, the content of which is hereby incorporated by reference in its entirety) which are antigen specific and tend to have a fitter / younger phenotype (c-fos and c-jun expression). CD137 through costimulation effects provides antigen-primed T cells with augmented survival, proliferation and effector functions as well as metabolic advantages.

[0346] Therefore, the present invention provides a method which enriches tumor-specific CD8 TILs (preserving the breadth of the TCR tumor-reactive repertoire), while providing CD4 TILs in the same process. In some embodiments, the method comprises priming tumors and use of CD137 as a marker to enrich and expand tumor-specific T cells without knowledge of epitope specificities. See, e.g., Fig.48. A. Obtain Patient Tumor Sample

[0001] In general, TILs are initially obtained from a patient tumor sample (“primary TILs”) and then expanded into a larger population for further manipulation as described herein.

[0002] 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. In some embodiments, multilesional sampling is used. In some embodiments, 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 (i.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). In general, 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 any cancer type, including, but not limited to, breast, pancreatic, prostate, colorectal, lung, brain, renal, stomach, and skin (including but not limited to squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, the cancer is selected from endometrial DB1 / 149057740.1 131Attorney Docket No.116983-5126-WO / AR / TW cancer, cervical cancer, head and neck cancer (including, for example, head and neck squamous cell carcinoma (HNSCC)), glioblastoma (GBM), gastrointestinal cancer, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple negative breast cancer, and non-small cell lung carcinoma.. In some embodiments, useful TILs are obtained from a melanoma.

[0003] Once obtained, the tumor sample is generally fragmented using sharp dissection into small pieces of between 1 to about 8 mm3, with from about 2-3 mm3being particularly useful. In some embodiments, 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). 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. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, 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 A1, 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. B. Tumor Fragmentation and / or Digest

[0004] As indicated above, in some embodiments, the TILs are derived from solid tumors. In some embodiments, the solid tumors are not fragmented. In some embodiments, the solid tumors are not fragmented and are subjected to enzymatic digestion as whole tumors. In some embodiments, the tumors are digested in in an enzyme mixture comprising collagenase, dNase, and neutral protease. In some embodiments, the tumors are digested in in an enzyme mixture comprising collagenase, dNase, and neutral protease for 1-2 hours. In some embodiments, the tumors are digested in in an enzyme mixture comprising collagenase, dNase, and neutral protease for 1-2 hours at 37°C, 5% CO2. In some embodiments, the tumors are digested in in an enzyme mixture comprising collagenase, dNase, and neutral protease for DB1 / 149057740.1 132Attorney Docket No.116983-5126-WO / AR / TW 1-2 hours at 37°C, 5% CO2 with rotation. In some embodiments, the tumors are digested overnight with constant rotation. In some embodiments, the tumors are digested overnight at 37°C, 5% CO2 with constant rotation. In some embodiments, the whole tumor is combined with the enzymes to form a tumor digest reaction mixture.

[0005] In some embodiments, the tumor is reconstituted with the lyophilized enzymes in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

[0006] In some embodiments, the enzyme mixture comprises collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working stock for the collagenase is a 100 mg / ml 10X working stock.

[0007] In some embodiments, the enzyme mixture comprises DNAse. In some embodiments, the working stock for the DNAse is a 10,000 IU / ml 10X working stock.

[0008] In some embodiments, the enzyme mixture comprises hyaluronidase. In some embodiments, the working stock for the hyaluronidase is a 10-mg / ml 10X working stock.

[0009] In some embodiments, the enzyme mixture comprises 10 mg / ml collagenase, 1000 IU / ml DNAse, and 1 mg / ml hyaluronidase.

[0010] In some embodiments, the enzyme mixture comprises 10 mg / ml collagenase, 500 IU / ml DNAse, and 1 mg / ml hyaluronidase.

[0011] In some embodiments, the enzyme mixture comprises neutral protease. In some embodiments, the working stock for the neutral protease is reconstituted at a concentration of 175 DMC U / mL.

[0012] In some embodiments, the enzyme mixture comprises neutral protease, dNase, and collagenase.

[0013] In some embodiments, the enzyme mixture comprises 10 mg / ml collagenase, 1000 IU / ml dNase, and 0.31 DMC U / ml neutral protease. In some embodiments, the enzyme mixture comprises 10 mg / ml collagenase, 500 IU / ml dNase, and 0.31 DMC U / ml neutral protease.

[0014] In general, the harvested cell suspension is called a “primary cell population” or a “freshly harvested” cell population.

[0015] In some embodiments, fragmentation includes physical fragmentation, including for example, dissection as well as digestion. In some embodiments, the fragmentation is physical DB1 / 149057740.1 133Attorney Docket No.116983-5126-WO / AR / TW fragmentation. In some embodiments, the fragmentation is dissection. In some embodiments, the fragmentation is by digestion. In some embodiments, TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from patients. In some embodiments, TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from patients prior to gene-editing.

[0016] In some embodiments, where the tumor is a solid tumor, the tumor undergoes physical fragmentation after the tumor sample is obtained. In some embodiments, the fragmentation occurs before cryopreservation. In some embodiments, the fragmentation 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, 50, 60, 70, 80, 90, 100 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 mm3. In some embodiments, the multiple fragments comprise about 30 to about 60 fragments with a total volume of about 1300 mm3to about 1500 mm3. In some embodiments, the multiple fragments comprise about 50 fragments with a total volume of about 1350 mm3. 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 embodiments, the multiple fragments comprise about 4 fragments. In some embodiments, the multiple fragments comprise about to about 100 fragments.

[0017] In some embodiments, the TILs are obtained from tumor fragments. In some embodiments, the tumor fragment is obtained by sharp dissection. In some embodiments, the tumor fragment is between about 1 mm3and 10 mm3. In some embodiments, the tumor fragment is between about 1 mm3and 8 mm3. In some embodiments, the tumor fragment is about 1 mm3. In some embodiments, the tumor fragment is about 2 mm3. In some embodiments, the tumor fragment is about 3 mm3. In some embodiments, the tumor fragment is about 4 mm3. In some embodiments, the tumor fragment is about 5 mm3. In some embodiments, the tumor fragment is about 6 mm3. In some embodiments, the tumor fragment is about 7 mm3. In some embodiments, the tumor fragment is about 8 mm3. In some embodiments, the tumor fragment is about 9 mm3. In some embodiments, the tumor fragment DB1 / 149057740.1 134Attorney Docket No.116983-5126-WO / AR / TW is about 10 mm3. In some embodiments, the tumors are 1-4 mm x 1-4 mm x 1-4 mm. In some embodiments, the tumors are 1 mm x 1 mm x 1 mm. In some embodiments, 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 mm x 4 mm x 4 mm.

[0018] In some embodiments, 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.

[0019] In some embodiments, 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. In some embodiments, 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. 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. In some embodiments, after the third mechanical disruption if large pieces of tissue were present, 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. In some embodiments, at the end of the final incubation if the cell suspension contained a large number of red blood cells or dead cells, a density gradient separation using Ficoll can be performed to remove these cells.

[0020] In some embodiments, the harvested cell suspension prior to the first expansion step is called a “primary cell population” or a “freshly harvested” cell population.

[0021] In some embodiments, cells can be optionally frozen after sample harvest and stored frozen prior to entry into the expansion described in further detail below. DB1 / 149057740.1 135Attorney Docket No.116983-5126-WO / AR / TW

[0022] In some embodiments, the TILs are obtained by thawing a cryopreserved tumor digest comprising a first population of TILs from a tumor resected from a subject. In some embodiments, the first population of TILs is obtained from a tumor resected from a subject by processing a tumor sample obtained from the subject into a tumor digest. In some embodiments, the tumor digest or tumor fragments comprise a first population of TILs and are obtained from a tumor that was resected from the subject.

[0023] In some embodiments, the methods disclosed herein comprise adding a tumor digest or tumor fragments into a closed system, wherein the tumor digest or tumor fragments comprise a first population of TILs and are obtained from a tumor that was resected from the subject. In some embodiments, the methods disclosed herein comprise performing a first expansion by thawing a cryopreserved tumor digest comprising a first population of TILs from a tumor that was resected from the subject. In some embodiments, the methods disclosed herein comprise obtaining a first population of TILs from a tumor resected from a patient by processing a tumor sample obtained from the patient into a tumor digest or multiple tumor fragments.

[0024] In some embodiments, a tumor lysate can be further obtained from the tumor digest through several freeze-thaw cycles or mass spectrometry procedures.

[0025] In some embodiments, the tumor fragments and / or tumor digest and / or tumor lysate can be...

Claims

Attorney Docket No.116983-5126-WO / AR / TW WHAT IS CLAIMED IS:

1. A nucleic acid molecule comprising a nucleotide sequence encoding a tethered IL-12 (TeIL-12), wherein the TeIL-12 is under the control of a nuclear factor of activated T- cells (NFAT)-responsive promoter.

2. The nucleic acid molecule of claim 1, wherein the TeIL-12 comprises a membrane anchor, and a human IL-12 p40 subunit fused to a human IL-12 p35 subunit.

3. The nucleic acid molecule of claim 2, wherein the human IL-12 p35 subunit has the amino acid sequence of SEQ ID NO:60 and the human IL-12 p40 subunit has the amino acid sequence of SEQ ID NO:

61.

4. The nucleic acid molecule of any one of claims 1-3, wherein the TeIL-12 comprises an amino acid sequence set forth in SEQ ID NO:

62.

5. The nucleic acid molecule of any one of claims 1-4, wherein the nucleotide sequence encoding the TeIL-12 is set forth in SEQ ID NO:

162.

6. The nucleic acid molecule of any one of claims 1-5, further comprising a nucleotide sequence encoding a cytokine selected from the group consisting of IL-2, IL-6, IL-7, IL- 9, IL-15, IL-18, IL-21, IL-23, IL-27, IL-33, IFN gamma, TNFa, IFN alpha, IFN beta, GM-CSF, GCSF, or a variant thereof.

7. The nucleic acid molecule of claim 6, wherein the cytokine is under the control of a constitutive promoter.

8. The nucleic acid molecule of claim 7, wherein the constitutive promoter is selected from the group consisting of an EF1α promotor, a CMV promotor, a CAG promotor, an MND promotor, and an SSFV promoter.

9. The nucleic acid molecule of claim 7, wherein the constitutive promoter is an EF1α promotor.

10. The nucleic acid molecule of any one of claims 6-9, wherein the cytokine is IL-15 or a variant thereof.

11. The nucleic acid molecule of claim 10, wherein the IL-15 is a human IL-15.

12. The nucleic acid molecule of claim 11, wherein the human IL-15 has the amino acid sequence of SEQ ID NO:

64.

13. The nucleic acid molecule of any one of claims 10-12, wherein the IL-15 is a tethered IL- DB1 / 149057740.1 351Attorney Docket No.116983-5126-WO / AR / TW 15 (TeIL-15).

14. The nucleic acid molecule of claim 13, wherein the TeIL-15 comprises a membrane anchor, and a human IL-15.

15. The nucleic acid molecule of claim 14, wherein the TeIL-15 has the amino acid sequence of SEQ ID NO:

73.

16. The nucleic acid molecule of any one of claims 6-9, wherein the cytokine is IL-2 or a variant thereof.

17. The nucleic acid molecule of claim 16, wherein the IL-2 is a human IL-2.

18. The nucleic acid molecule of claim 17, wherein the human IL-2 has the amino acid sequence of SEQ ID NO:

138.

19. The nucleic acid molecule of any one of claims 16-18, wherein the IL-2 is a tethered IL-2 (TeIL-2).

20. The nucleic acid molecule of claim 19, wherein the TeIL-2 has the amino acid sequence of SEQ ID NO:

139.

21. The nucleic acid molecule of any one of claims 1-20, comprising the nucleic acid sequence set forth in SEQ ID NO:148-150.

22. The nucleic acid molecule of any one of claims 1-21, further comprising a nucleotide sequence encoding an shRNA.

23. The nucleic acid molecule of claim 22, wherein the shRNA inhibits the expression of an immune checkpoint gene.

24. The nucleic acid molecule of claim 23, wherein the immune checkpoint gene is selected from the group consisting of PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), CISH, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11, and BCOR.

25. The nucleic acid molecule of claim 23, wherein the immune checkpoint gene is PD-1.

26. The nucleic acid molecule of claim 25, wherein the PD-1 shRNA comprises a nucleic DB1 / 149057740.1 352Attorney Docket No.116983-5126-WO / AR / TW acid sequence set forth in SEQ ID NO:141-147.

27. The nucleic acid molecule of any one of claims 1-26, comprising the nucleic acid sequence set forth in SEQ ID NO:

151.

28. A recombinant expression vector comprising the nucleic acid molecule of any one of claims 1-27.

29. The recombinant expression vector of claim 28, wherein the vector is a transfer vector.

30. The recombinant expression vector of claim 29, wherein the transfer vector is derived from the human immunodeficiency virus-1 (HIV-1) and is replication deficient.

31. The recombinant expression vector of claim 30, wherein the transfer vector is a pLenti- IRES-EGFP vector.

32. A lentiviral expression system comprising the recombinant expression vector of claim 30 or 31 and one or more helper plasmids encoding an Env protein, a Gag protein, a Pol protein, and a Rev protein.

33. The lentiviral expression system of claim 32, comprising a helper plasmid encoding the Env protein, the Gag protein, the Pol protein, and the Rev protein.

34. The lentiviral expression system of claim 32, comprising a helper plasmid encoding the Env protein, and a helper plasmid encoding the Gag protein, the Pol protein, and the Rev protein.

35. The lentiviral expression system of claim 32, comprising a helper plasmid encoding the Env protein, the Gag protein, and the Pol protein, and a helper plasmid encoding the Rev protein.

36. The lentiviral expression system of claim 32, comprising a helper plasmid encoding the Env protein and the Rev protein, and a helper plasmid encoding the Gag protein and the Pol protein.

37. The lentiviral expression system of any one of claims 32-36, wherein the Env protein is selected from the group consisting of Ba-EVTR, VSV-G, and RD114.

38. The lentiviral expression system of claim 37, wherein the Env protein comprises a Ba- EVTR protein.

39. A method of making a recombinant lentiviral particle, comprising: DB1 / 149057740.1 353Attorney Docket No.116983-5126-WO / AR / TW a) culturing a population of packaging cells in a cell culture medium; b) contacting the population of packaging cells with the lentiviral expression system of any one of claims 32-38; and c) harvesting the supernatant of the cell culture medium, wherein the supernatant comprises the recombinant lentiviral particle.

40. A recombinant lentiviral RNA molecule comprising the nucleic acid molecule of any one of claims 1-27.

41. A recombinant lentiviral proviral DNA comprising the nucleic acid molecule of any one of claims 1-27.

42. A recombinant lentiviral particle comprising the recombinant lentiviral RNA molecule of claim 40.

43. The recombinant lentiviral particle of claim 42, produced by the packaging cell line of any one of claims 240-284.

44. The recombinant lentiviral particle of claim 42 or 43, comprising an Env protein selected from the group consisting of Ba-EVTR, VSV-G, and RD114.

45. The recombinant lentiviral particle of any one of claims 42-44, further comprising a reverse transcriptase.

46. The recombinant lentiviral particle of any one of claims 42-45, further comprising a capsid enclosing the recombinant lentiviral RNA molecule.

47. A gene-edited tumor infiltrating lymphocyte (TIL) comprising the recombinant lentiviral proviral DNA of claim 41.

48. The gene-edited TIL of claim 47, wherein the recombinant lentiviral proviral DNA is integrated into the genome of the gene-edited TIL.

49. A gene-edited tumor infiltrating lymphocyte (TIL) expressing an exogenous tethered IL- 12 (TeIL-12).

50. The gene-edited TIL of claim 49, wherein the TeIL-12 comprises a membrane anchor, and a human IL-12 p40 subunit fused to a human IL-12 p35 subunit.

51. The gene-edited TIL of claim 50, wherein the human IL-12 p35 subunit has the amino acid sequence of SEQ ID NO:60 and the human IL-12 p40 subunit has the amino acid DB1 / 149057740.1 354Attorney Docket No.116983-5126-WO / AR / TW sequence of SEQ ID NO:

61.

52. The gene-edited TIL of claim 51, wherein the TeIL-12 comprises an amino acid sequence set forth in SEQ ID NO:

62.

53. The gene-edited TIL of any one of claims 49-52, further expressing a cytokine selected from the group consisting of IL-2, IL-6, IL-7, IL-9, IL-15, IL-18, IL-21, IL-23, IL-27, IL- 33, IFN gamma, TNFa, IFN alpha, IFN beta, GM-CSF, GCSF, and a variant thereof.

54. The gene-edited TIL of claim 53, wherein the cytokine is IL-15 or a variant thereof.

55. The gene-edited TIL of claim 54, wherein the IL-15 is a human IL-15.

56. The gene-edited TIL of claim 55, wherein the human IL-15 has the amino acid sequence of SEQ ID NO:

64.

57. The gene-edited TIL of any one of claims 54-56, wherein the IL-15 is a tethered IL-15 (TeIL-15).

58. The gene-edited TIL of claim 57, wherein the TeIL-15 comprises a membrane anchor, and a human IL-15.

59. The gene-edited TIL of claim 57, wherein the TeIL-15 has the amino acid sequence of SEQ ID NO:

73.

60. A method of making a population of gene-edited tumor-infiltrating lymphocytes (TILs), comprising: a) performing a first expansion by culturing a population of TILs in a first cell culture medium; b) performing a second expansion by culturing the population of TILs in a second cell culture medium; and c) at any time, transducing the population of TILs with the recombinant lentiviral particle of any one of claims 42–46 to produce the population of gene-edited TILs, wherein the population of gene-edited TILs expresses a TeIL-12.

61. The method of claim 60, further comprising activating the population of TILs for 1 day or 2 days before transducing the population of TILs with the recombinant lentiviral particle.

62. The method of claim 61, wherein the activating step comprises contacting the population of TILs with a cytokine selected from the group consisting of IL-2, IL-15, IL-21, IL-7, DB1 / 149057740.1 355Attorney Docket No.116983-5126-WO / AR / TW and a combination thereof.

63. The method of claim 62, wherein the activating step comprises contacting the population of TILs with 20ng / mL IL-15.

64. The method of claim 62, wherein the activating step comprises contacting the population of TILs with 10 ng / mL IL-7.

65. The method of claim 62, wherein the activating step comprises contacting the population of TILs with TransAct.

66. The method of claim 62, wherein the activating step comprises contacting the population of TILs with TransAct at a ratio of 1:

100.

67. The method of any one of claims 60-66, wherein the transducing step is conducted at a TIL concentration of 105cells / mL.

68. The method of any one of claims 60-67, wherein the transducing step is conducted at a multiplicity of infection (MOI) of about 10 to about 40.

69. The method of any one of claims 60-68, wherein the transducing step is conducted in the presence of RetroNectin or Vectofusin-1.

70. The method of any one of claims 60-69, wherein the transducing step comprises centrifugation.

71. The method of any one of claims 60-70, wherein the transducing step is conducted in the presence of Lentibooster.

72. The method of any one of claims 60-71, wherein the transducing step is conducted after the first expansion step and before the second expansion step.

73. The method of any one of claims 61-72, wherein the activating step is conducted after the first expansion step and before the second expansion step.

74. The method of any one of claims 60-73, further comprising resting the population of TILs for 2 day or 3 days after the transducing step.

75. The method of any one of claims 60-74, wherein the population of TILs is obtained from a tumor digest.

76. The method of any one of claims 60-75, further comprising a priming step.

77. The method of claim 76, wherein the priming step is performed before the first expansion DB1 / 149057740.1 356Attorney Docket No.116983-5126-WO / AR / TW step.

78. The method of claim 76 or 77, wherein the priming step is performed in the presence of IFNγ.

79. The method of claim 78, wherein IFNγ is present at a concentration of 200 ng / mL during the priming step.

80. The method of claim any one of claims 76-79, wherein the priming step is performed in the presence of IL-2, IL-15, and / or IL-21.

81. The method of claim 80, wherein the IL-2 is at a concentration of 3000 IU / mL or lower during the priming step.

82. The method of claim 80 or 81, wherein the IL-15 and / or IL-21 is present at a concentration of about 1 ng / mL to about 100 ng / mL during the priming step.

83. The method of claim 80 or 81, wherein the IL-15 and / or IL-21 is present at a concentration of about 10 ng / mL during the priming step.

84. The method of any one of claims 76-83, wherein the priming step lasts for 24-48 hours.

85. The method of any one of claims 76-83, wherein the priming step lasts for about 30 hours.

86. The method of any one of claims 60-85, further comprising an enrichment step.

87. The method of claim 86, wherein the enrichment step is performed after the priming step and before the first expansion step.

88. The method of claim 86 or 87, wherein the population of TILs is enriched for CD137+ T cells.

89. The method of claim 86 or 87, wherein the population of TILs is enriched for CD137+ and CD39+T cells.

90. The method of claim 86 or 87, wherein the population of TILs is enriched for CD137+ and CD200+T cells.

91. The method of claim 86 or 87, wherein the population of TILs is enriched for CD137+, CD200+, CD39+, and / or OX40+ T cells.

92. The method of any one of claims 60-91, wherein the first expansion is conducted in the presence of feeder cells. DB1 / 149057740.1 357Attorney Docket No.116983-5126-WO / AR / TW 93. The method of claim 92, wherein the feeder cells are T cell-depleted PBMCs.

94. The method of any one of claims 60-93, wherein the first expansion is performed over a period of about 3-11 days.

95. The method of any one of claims 60-93, wherein the first expansion is performed over a period of about 9 days.

96. The method of any one of claims 60-95, wherein the second cell culture medium comprises IL-15 and IL-21.

97. The method of claim 96, wherein the second cell culture medium comprises IL-15 at a concentration of about 10 ng / mL and IL-21 at a concentration of about 10 ng / mL.

98. The method of any one of claims 60-97, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), and L-arginine.

99. The method of claim 98, wherein the L-arginine is present at a concentration of about 1 mM to about 10 mM.

100. The method of claim 98, wherein the L-arginine is present at a concentration of about 5 mM.

101. The method of any one of claims 60-100, wherein the second cell culture medium comprises an NAD+ booster.

102. The method of claim 101, wherein the NAD+ booster is selected from the group consisting of L-Trp, NR, NMN, NAD+, NAM and P7C3 activator.

103. The method of any one of claims 60-102, wherein the second cell culture medium comprises an inhibitor of GSK-3α / β.

104. The method of claim 103, wherein the inhibitor of GSK-3α / β is selected from the group consisting of SB415286, SB216763, CHIR99021, AR-AO14418, TZD8, TWS119, Lithium Chloride hydrate, BIO, and 3F8.

105. The method of any one of claims 60-104, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), and a protein kinase B (AKT) inhibitor.

106. The method of claim 105, wherein the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC- 0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosine, Oridonin, Herbacetin, DB1 / 149057740.1 358Attorney Docket No.116983-5126-WO / AR / TW Tehranolide, Isoliquiritigenin, Scutellarin, and Honokiol.

107. The method of claim 105, wherein the AKT inhibitor is AZD5363.

108. The method of any one of claims 105-107, wherein the AKT inhibitor is at a concentration of about 0.1 µM to about 10 µM.

109. The method of any one of claims 105-107, wherein the AKT inhibitor is at a concentration of about 1 µM.

110. The method of any one of claims 60-109, wherein the second expansion is performed over a period of about 7-11 days.

111. The method of any one of claims 60-109, wherein the second expansion is performed over a period of about 10 days.

112. A method of making a population of gene-edited tumor-infiltrating lymphocytes (TILs), comprising: a) priming a tumor digest comprising a population of TILs in the presence of IFNγ; b) enriching the population of TILs for CD137+ T cells; c) performing a first expansion by culturing the population of TILs enriched for CD137+ T cells in a first cell culture medium; d) transducing the population of TILs enriched for CD137+ T cells with a recombinant lentiviral particle comprising a nucleic acid sequence encoding TeIL-12 to produce the population of gene-edited TILs; and e) performing a second expansion by culturing the population of gene-edited TILs in a second cell culture medium.

113. The method of claim 112, wherein the TeIL-12 is under the control of an NFAT promoter.

114. The method of claim 112 or 113, wherein the TeIL-12 comprises an amino acid sequence set forth in SEQ ID NO:

62.

115. The method of claim 112 or 113, wherein the nucleotide sequence encoding the TeIL- 12 is set forth in SEQ ID NO:

162.

116. The method of any one of claims 112-115, further comprising activating the population of TILs for 1 day or 2 days before transducing the population of TILs with the DB1 / 149057740.1 359Attorney Docket No.116983-5126-WO / AR / TW recombinant lentiviral particle.

117. The method of claim 116, wherein the activating step comprises contacting the population of TILs with TransAct at a ratio of 1:

100.

118. The method of any one of claims 112-117, wherein the transducing step is conducted at a TIL concentration of 105cells / mL.

119. The method of any one of claims 112-118, wherein the transducing step is conducted at a multiplicity of infection (MOI) of about 10 to about 40.

120. The method of any one of claims 112-119, wherein the transducing step is conducted in the presence of RetroNectin or Vectofusin-1.

121. The method of any one of claims 112-120, wherein the transducing step comprises centrifugation.

122. The method of any one of claims 112-121, wherein the transducing step is conducted in the presence of Lentibooster.

123. The method of any one of claims 112-122, further comprising resting the population of TILs for 2 day or 3 days after the transducing step.

124. The method of any one of claims 112-123, wherein IFNγ is present at a concentration of 200 ng / mL during the priming step.

125. The method of any one of claims 112-124, wherein the priming step is performed in the presence of IL-2, IL-15, and / or IL-21.

126. The method of claim 125, wherein the IL-2 is at a concentration of 3000 IU / mL or lower during the priming step.

127. The method of claim 125 or 126, wherein the IL-15 and / or IL-21 is present at a concentration of about 10 ng / mL during the priming step.

128. The method of any one of claims 112-127, wherein the priming step lasts for about 30 hours.

129. The method of any one of claims 112-128, wherein the first expansion is conducted in the presence of feeder cells.

130. The method of claim 129, wherein the feeder cells are T cell-depleted PBMCs.

131. The method of any one of claims 112-130, wherein the first expansion is performed in DB1 / 149057740.1 360Attorney Docket No.116983-5126-WO / AR / TW the presence of IL-2, IL-15, and / or IL-21.

132. The method of claim 131, wherein the IL-2 is at a concentration of 3000 IU / mL or lower during the priming step.

133. The method of claim 130 or 131, wherein the IL-15 and / or IL-21 is present at a concentration of about 10 ng / mL during the first expansion.

134. The method of any one of claims 112-133, wherein the first expansion is performed over a period of about 9 days.

135. The method of any one of claims 112-134, wherein the second cell culture medium comprises IL-15 and IL-21.

136. The method of claim 135, wherein the second cell culture medium comprises IL-15 at a concentration of about 10 ng / mL and IL-21 at a concentration of about 10 ng / mL.

137. The method of any one of claims 112-136, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), and L-arginine.

138. The method of claim 137, wherein the L-arginine is present at a concentration of about 5 mM.

139. The method of any one of claims 112-138, wherein the second cell culture medium comprises an NAD+ booster.

140. The method of claim 139, wherein the NAD+ booster is selected from the group consisting of L-Trp, NR, NMN, NAD+, NAM and P7C3 activator.

141. The method of any one of claims 112-140, wherein the second cell culture medium comprises an inhibitor of GSK-3α / β.

142. The method of claim 141, wherein the inhibitor of GSK-3α / β is selected from the group consisting of SB415286, SB216763, CHIR99021, AR-AO14418, TZD8, TWS119, Lithium Chloride hydrate, BIO, and 3F8.

143. The method of any one of claims 112-142, wherein the second expansion is performed over a period of about 10 days.

144. A method of making a population of gene-edited tumor-infiltrating lymphocytes (TILs), comprising: a) priming a tumor digest comprising a population of TILs in the presence of IFNγ; DB1 / 149057740.1 361Attorney Docket No.116983-5126-WO / AR / TW b) enriching the population of TILs for CD137+ T cells; c) performing a first expansion by culturing the population of TILs enriched for CD137+ T cells in a first cell culture medium; d) transducing the population of TILs enriched for CD137+ T cells with a recombinant lentiviral particle comprising a nucleic acid sequence encoding TeIL-12 and TeIL-15 to produce the population of gene-edited TILs; and e) performing a second expansion by culturing the population of gene-edited TILs in a second cell culture medium.

145. The method of claim 144, wherein the TeIL-12 is under the control of an NFAT promoter.

146. The method of claim 144 or 145, wherein the TeIL-15 is under the control of a constitutive promoter.

147. The method of claim 146, wherein the constitutive promoter is an EF1α promotor.

148. The method of any one of claims 145-147, wherein the NFAT promoter and the constitutive promoter are introduced in the same nucleic acid of the coding sequence.

149. The method of any one of claims 144-148, wherein the TeIL-12 comprises an amino acid sequence set forth in SEQ ID NO:

62.

150. The method of any one of claims 144-148, wherein the nucleotide sequence encoding the TeIL-12 is set forth in SEQ ID NO:

162.

151. The method of any one of claims 144-150, wherein the TeIL-15 has the amino acid sequence of SEQ ID NO:

73.

152. The method of any one of claims 144-151, further comprising activating the population of TILs for 1 day or 2 days before transducing the population of TILs with the recombinant lentiviral particle.

153. The method of claim 152, wherein the activating step comprises contacting the population of TILs with TransAct at a ratio of 1:

100.

154. The method of any one of claims 144-153, further comprising resting the population of TILs for 2 day or 3 days after the transducing step.

155. The method of any one of claims 144-154, wherein IFNγ is present at a concentration DB1 / 149057740.1 362Attorney Docket No.116983-5126-WO / AR / TW of 200 ng / mL during the priming step.

156. The method of any one of claims 144-155, wherein the priming step is performed in the presence of IL-2, IL-15, and / or IL-21.

157. The method of claim 156, wherein the IL-2 is at a concentration of 3000 IU / mL or lower during the priming step.

158. The method of claim 156 or 157, wherein the IL-15 and / or IL-21 is present at a concentration of about 10 ng / mL during the priming step.

159. The method of any one of claims 144-158, wherein the priming step lasts for about 30 hours.

160. The method of any one of claims 144-159, wherein the first expansion is conducted in the presence of feeder cells.

161. The method of claim 160, wherein the feeder cells are T cell-depleted PBMCs.

162. The method of any one of claims 144-161, wherein the first expansion is performed over a period of about 9 days.

163. The method of any one of claims 144-162, wherein the second cell culture medium comprises IL-15 and IL-21.

164. The method of claim 163, wherein the second cell culture medium comprises IL-15 at a concentration of about 10 ng / mL and IL-21 at a concentration of about 10 ng / mL.

165. The method of any one of claims 144-164, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), and L-arginine.

166. The method of claim 165, wherein the L-arginine is present at a concentration of about 5 mM.

167. The method of any one of claims 144-166, wherein the second cell culture medium comprises an NAD+ booster.

168. The method of claim 167, wherein the NAD+ booster is selected from the group consisting of L-Trp, NR, NMN, NAD+, NAM and P7C3 activator.

169. The method of any one of claims 144-168, wherein the second cell culture medium comprises an inhibitor of GSK-3α / β.

170. The method of claim 169, wherein the inhibitor of GSK-3α / β is selected from the DB1 / 149057740.1 363Attorney Docket No.116983-5126-WO / AR / TW group consisting of SB415286, SB216763, CHIR99021, AR-AO14418, TZD8, TWS119, Lithium Chloride hydrate, BIO, and 3F8.

171. The method of any one of claims 144-170, wherein the second expansion is performed over a period of about 10 days.

172. A therapeutic population of gene-edited TILs produced by the method of any one of claims 60-171.

173. The therapeutic population of gene-edited TILs of claim 172, wherein about 15-60% of the population of gene-edited TILs expresses the TeIL-12.

174. The therapeutic population of gene-edited TILs of claim 172, wherein more than 30% of the population of gene-edited TILs expresses the TeIL-12.

175. The therapeutic population of gene-edited TILs of claim 172, wherein more than 50% of the population of gene-edited TILs expresses the TeIL-12.

176. The therapeutic population of gene-edited TILs of any one of claims 172-175, wherein about 15-60% of the population of gene-edited TILs expresses the TeIL-15.

177. The therapeutic population of gene-edited TILs of any one of claims 172-175, wherein more than 30% of the population of gene-edited TILs expresses the TeIL-15.

178. The therapeutic population of gene-edited TILs of any one of claims 172-175, wherein more than 50% of the population of gene-edited TILs expresses the TeIL-15.

179. The therapeutic population of gene-edited TILs of any one of claims 172-178, wherein about 15-60% of the population of gene-edited TILs expresses the TeIL-2.

180. The therapeutic population of gene-edited TILs of any one of claims 172-178, wherein more than 30% of the population of gene-edited TILs expresses the TeIL-2.

181. The therapeutic population of gene-edited TILs of any one of claims 172-178, wherein more than 50% of the population of gene-edited TILs expresses the TeIL-2.

182. The therapeutic population of gene-edited TILs of any one of claims 172-181, wherein more than 30% of the population of gene-edited TILs has reduced expression of PD-1.

183. The therapeutic population of gene-edited TILs of any one of claims 172-181, wherein more than 40% of the population of gene-edited TILs has reduced expression of PD-1. DB1 / 149057740.1 364Attorney Docket No.116983-5126-WO / AR / TW 184. The therapeutic population of gene-edited TILs of any one of claims 172-181, wherein more than 60% of the population of gene-edited TILs has reduced expression of PD-1.

185. A pharmaceutical composition comprising the therapeutic population of gene-edited TILs of any one of claims 172-184.

186. A method of treating a cancer in a patient in need thereof comprising: a) resecting a tumor sample from the patient, wherein the tumor sample comprises a population of TILs; b) performing a first expansion by culturing the population of TILs in a first cell culture medium; c) performing a second expansion by culturing the population of TILs in a second cell culture medium; d) at any time, transducing the population of TILs with the recombinant lentiviral particle of any one of claims 42–46 to produce a therapeutic population of gene-edited TILs; and e) administering the therapeutic population of gene-edited TILs to the patient.

187. The method of claim 186, further comprising activating the population of TILs for 1 day or 2 days before transducing the population of TILs with the recombinant lentiviral particle.

188. The method of claim 187, wherein the activating step comprises contacting the population of TILs with a cytokine selected from the group consisting of IL-2, IL-15, IL- 21, IL-7, and a combination thereof.

189. The method of claim 187, wherein the activating step comprises contacting the population of TILs with TransAct.

190. The method of any one of claims 186-189, wherein the transducing step is conducted at a TIL concentration of 105cells / mL.

191. The method of any one of claims 186-190, wherein the transducing step is conducted at a multiplicity of infection (MOI) of about 10 to about 40.

192. The method of any one of claims 186-191, wherein the transducing step is conducted in the presence of RetroNectin or Vectofusin-1. DB1 / 149057740.1 365Attorney Docket No.116983-5126-WO / AR / TW 193. The method of any one of claims 186-192, wherein the transducing step comprises centrifugation.

194. The method of any one of claims 186-193, wherein the transducing step is conducted in the presence of Lentibooster.

195. The method of any one of claims 186-194, wherein the transducing step is conducted after the first expansion step and before the second expansion step.

196. The method of any one of claims 187-195, wherein the activating step is conducted after the first expansion step and before the second expansion step.

197. The method of any one of claims 186-196, further comprising resting the population of TILs for 2 day or 3 days after the transducing step.

198. The method of any one of claims 186-197, wherein the population of TILs is obtained from a tumor digest.

199. The method of claim 198, further comprising a priming step, wherein the tumor digest is primed in the presence of IL-2, IL-15, IL-21, and IFNγ.

200. The method of claim 199, wherein IFNγ is present at a concentration of 200 ng / mL during the priming step.

201. The method of claim 199, wherein the IL-2 is at a concentration of 3000 IU / mL or lower during the priming step.

202. The method of any one of claims 199-201, wherein the IL-15 and / or IL-21 is present at a concentration of about 1 ng / mL to about 100 ng / mL during the priming step.

203. The method of any one of claims 199-201, wherein the IL-15 and / or IL-21 is present at a concentration of about 10 ng / mL during the priming step.

204. The method of any one of claims 199-203, wherein the priming step lasts for 24-48 hours.

205. The method of any one of claims 199-203, wherein the priming step lasts for about 30 hours.

206. The method of any one of claims 186-205, further comprising an enrichment step, wherein the population of TILs is enriched for CD137+ T cells.

207. The method of any one of claims 186-205, further comprising an enrichment step, DB1 / 149057740.1 366Attorney Docket No.116983-5126-WO / AR / TW wherein the population of TILs is enriched for CD137+ and CD39+T cells.

208. The method of any one of claims 186-205, further comprising an enrichment step, wherein the population of TILs is enriched for CD137+ and CD200+T cells.

209. The method of any one of claims 186-205, further comprising an enrichment step, wherein the population of TILs is enriched for CD137+, CD200+, CD39+, and / or OX40+ T cells.

210. The method of any one of claims 186-209, wherein the first expansion is conducted in the presence of feeder cells.

211. The method of claim 210, wherein the feeder cells are T cell-depleted PBMCs.

212. The method of any one of claims 186-211, wherein the first expansion is performed over a period of about 3-11 days.

213. The method of any one of claims 186-211, wherein the first expansion is performed over a period of about 9 days.

214. The method of any one of claims 186-213, wherein the second cell culture medium comprises IL-15 and IL-21.

215. The method of claim 214, wherein the second cell culture medium comprises IL-15 at a concentration of about 10 ng / mL and IL-21 at a concentration of about 10 ng / mL.

216. The method of any one of claims 186-215, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), and L-arginine.

217. The method of claim 216, wherein the L-arginine is present at a concentration of about 1 mM to about 10 mM.

218. The method of claim 216, wherein the L-arginine is present at a concentration of about 5 mM.

219. The method of any one of claims 186-218, wherein the second cell culture medium comprises an NAD+ booster.

220. The method of claim 219, wherein the NAD+ booster is selected from the group consisting of L-Trp, NR, NMN, NAD+, NAM and P7C3 activator.

221. The method of any one of claims 186-220, wherein the second cell culture medium comprises an inhibitor of GSK-3α / β. DB1 / 149057740.1 367Attorney Docket No.116983-5126-WO / AR / TW 222. The method of claim 221, wherein the inhibitor of GSK-3α / β is selected from the group consisting of SB415286, SB216763, CHIR99021, AR-AO14418, TZD8, TWS119, Lithium Chloride hydrate, BIO, and 3F8.

223. The method of any one of claims 186-222, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), and a protein kinase B (AKT) inhibitor.

224. The method of claim 223, wherein the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC- 0068, AT7867, CCT128930, MK-2206, BAY 1125976, Perifosine, Oridonin, Herbacetin, Tehranolide, Isoliquiritigenin, Scutellarin, and Honokiol.

225. The method of claim 223, wherein the AKT inhibitor is AZD5363.

226. The method of any one of claims 223-225, wherein the AKT inhibitor is at a concentration of about 0.1 µM to about 10 µM.

227. The method of any one of claims 223-225, wherein the AKT inhibitor is at a concentration of about 1 µM.

228. The method of any one of claims 186-227, wherein the second expansion is performed over a period of about 7-11 days.

229. The method of any one of claims 186-227, wherein the second expansion is performed over a period of about 10 days.

230. The method of any one of claims 186-227, wherein the cancer is selected from the group consisting of melanoma (including mucosal melanoma, uveal melanoma, cutaneous melanoma, choroidal melanoma, ciliary body melanoma, or iris melanoma), ovarian cancer, cervical cancer, endometrial cancer, non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple negative breast cancer, cancer caused by human papilloma virus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, and renal cell carcinoma.

231. The method of any one of claims 186-230, further comprising the step of treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the TILs to the patient.

232. The method of claim 231, wherein the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day for DB1 / 149057740.1 368Attorney Docket No.116983-5126-WO / AR / TW two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for three days.

233. The method of claim 231, wherein the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for three days.

234. The method of claim 231, wherein the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for one day.

235. The method of any one of claims 232-234, wherein the cyclophosphamide is administered with mesna.

236. The method of any one of claims 186-235, further comprising a step of treating the patient with an IL-2 regimen starting on the day after the administration of TILs to the patient.

237. The method of any one of claims 186-235, further comprising a step of treating the patient with an IL-2 regimen starting on the same day as administration of TILs to the patient.

238. The method of claim 236 or 237, wherein 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.

239. The method of any one of claims 186-235, comprising no step of treating the patient with an IL-2 regimen.

240. A packaging cell line comprising the recombinant expression vector of any one of claims 28-31, and one or more recombinant DNA molecules, each of which one or more recombinant DNA molecules encodes any one protein, any two proteins, any three proteins or all four proteins selected from the group consisting of encoding an Env protein, a Gag protein, a Pol protein, and a Rev protein, with the proviso that any protein selected from the group consisting of the Env protein, the Gag protein, the Pol protein and the Rev protein is encoded by at least one recombinant DNA molecule of the one or more DB1 / 149057740.1 369Attorney Docket No.116983-5126-WO / AR / TW recombinant DNA molecules, wherein each of the one or more recombinant DNA molecules is either integrated into the genome of the packaging cell line or comprised by a helper plasmid.

241. The packaging cell line of claim 240, wherein the one or more recombinant DNA molecules consist of a first recombinant DNA molecule.

242. The packaging cell line of claim 241, wherein the first recombinant DNA molecule is either integrated into the genome of the packaging cell line or comprised by a helper plasmid.

243. The packaging cell line of claim 241, wherein the one or more recombinant DNA molecules consist of a first recombinant DNA molecule and a second recombinant DNA molecule.

244. The packaging cell line of claim 243, wherein the first recombinant DNA molecule encodes a single protein selected from the group consisting of the Env protein, the Gag protein, the Pol protein, and the Rev protein.

245. The packaging cell line of claim 244, wherein the first recombinant DNA molecule encodes the Env protein.

246. The packaging cell line of claim 244, wherein the first recombinant DNA molecule encodes the Gag protein.

247. The packaging cell line of claim 244, wherein the first recombinant DNA molecule encodes the Pol protein.

248. The packaging cell line of claim 244, wherein the first recombinant DNA molecule encodes the Rev protein.

249. The packaging cell line of claim 243, wherein the first recombinant DNA molecule encodes two proteins selected from the group consisting of the Env protein, the Gag protein, the Pol protein, and the Rev protein.

250. The packaging cell line of claim 249, wherein the first recombinant DNA molecule encodes the Env protein and the Gag protein.

251. The packaging cell line of claim 249, wherein the first recombinant DNA molecule encodes the Env protein and the Pol protein.

252. The packaging cell line of claim 249, wherein the first recombinant DNA molecule DB1 / 149057740.1 370Attorney Docket No.116983-5126-WO / AR / TW encodes the Env protein and the Rev protein.

253. The packaging cell line of claim 249, wherein the first recombinant DNA molecule encodes the Gag protein and the Pol protein.

254. The packaging cell line of claim 249, wherein the first recombinant DNA molecule encodes the Gag protein and the Rev protein.

255. The packaging cell line of claim 249, wherein the first recombinant DNA molecule encodes the Pol protein and the Rev protein.

256. The packaging cell line of any one of claims 243-255, wherein each of the first and second recombinant DNA molecules is either integrated into the genome of the packaging cell line or comprised by a helper plasmid.

257. The packaging cell line of claim 240, wherein the one or more recombinant DNA molecules consist of a first recombinant DNA molecule, a second recombinant DNA molecule, and a third recombinant DNA molecule.

258. The packaging cell line of claim 257, wherein the first recombinant DNA molecule encodes a first protein and the second recombinant DNA molecule encodes a second protein, wherein the first and second proteins are independently selected from the group consisting of the Env protein, the Gag protein, the Pol protein, and the Rev protein, with the proviso that the first and second proteins are not the same.

259. The packaging cell line of claim 258, wherein the first protein is the Env protein and the second protein is the Gag protein.

260. The packaging cell line of claim 258, wherein the first protein is the Env protein and the second protein is the Pol protein.

261. The packaging cell line of claim 258, wherein the first protein is the Env protein and the second protein is the Rev protein.

262. The packaging cell line of claim 258, wherein the first protein is the Gag protein and the second protein is the Pol protein.

263. The packaging cell line of claim 258, wherein the first protein is the Gag protein and the second protein is the Rev protein.

264. The packaging cell line of claim 258, wherein the first protein is the Pol protein and the second protein is the Rev protein. DB1 / 149057740.1 371Attorney Docket No.116983-5126-WO / AR / TW 265. The packaging cell line of claim 257, wherein the first recombinant DNA molecule encodes a first protein and the second recombinant DNA molecule encodes two proteins, wherein the first protein and each of the two proteins are independently selected from the group consisting of the Env protein, the Gag protein, the Pol protein, and the Rev protein, with the proviso that any protein is not the same as any other protein in the group consisting of the first protein and the two proteins.

266. The packaging cell line of claim 265, wherein the first protein is the Env protein, and the two proteins are the Gag protein and the Pol protein.

267. The packaging cell line of claim 265, wherein the first protein is the Env protein, and the two proteins are the Gag protein and the Rev protein.

268. The packaging cell line of claim 265, wherein the first protein is the Env protein, and the two proteins are the Pol protein and the Rev protein.

269. The packaging cell line of claim 265, wherein the first protein is the Gag protein, and the two proteins are the Env protein and the Pol protein.

270. The packaging cell line of claim 265, wherein the first protein is the Gag protein, and the two proteins are the Env protein and the Rev protein.

271. The packaging cell line of claim 265, wherein the first protein is the Gag protein, and the two proteins are the Pol protein and the Rev protein.

272. The packaging cell line of claim 265, wherein the first protein is the Pol protein, and the two proteins are the Env protein and the Gag protein.

273. The packaging cell line of claim 265, wherein the first protein is the Pol protein, and the two proteins are the Env protein and the Rev protein.

274. The packaging cell line of claim 265, wherein the first protein is the Pol protein, and the two proteins are the Gag protein and the Rev protein.

275. The packaging cell line of claim 265, wherein the first protein is the Rev protein, and the two proteins are the Env protein and the Gag protein.

276. The packaging cell line of claim 265, wherein the first protein is the Rev protein, and the two proteins are the Env protein and the Pol protein.

277. The packaging cell line of claim 265, wherein the first protein is the Rev protein, and the two proteins are the Gag protein and the Pol protein. DB1 / 149057740.1 372Attorney Docket No.116983-5126-WO / AR / TW 278. The packaging cell line of any one of claims 257-277, wherein each of the first, second, and third recombinant DNA molecules is either integrated into the genome of the packaging cell line or comprised by a helper plasmid .

279. The packaging cell line of claim 240, wherein the one or more recombinant DNA molecules consist of a first recombinant DNA molecule, a second recombinant DNA molecule, a third recombinant DNA molecule, and a fourth recombinant DNA molecule.

280. The packaging cell line of claim 279, wherein each of the first, second, third, and fourth recombinant DNA molecules is either integrated into the genome of the packaging cell line or comprised by a helper plasmid.

281. The packaging cell line of any one of claims 240-280, wherein the Env protein comprises a Ba-EVTR protein.

282. The packaging cell line of any one of claims 240-281, wherein the nucleic acid molecule encoding TeIL-12 is integrated into the genome of the packaging cell line.

283. The packaging cell line of any one of claims 240-282, wherein the recombinant DNA molecule encoding one or more the Env protein, the Gag protein, the Pol protein, and the Rev protein is under the control of an inducible promoter.

284. The packaging cell line of any one of claims 240-283, comprising a 293T cell line.

285. A method for enriching tumor-reactive tumor infiltrating lymphocytes (TILs), comprising: a. priming a population of TILs obtained from a tumor digest in the presence of IFNγ; b. enriching the population of TILs for CD137+ TILs; c. performing a first expansion by culturing the population of TILs enriched for CD137+ T cells in a first cell culture medium; and d. performing a second expansion by culturing the population of TILs enriched for CD137+ TILs in a second cell culture medium to produce a population of TILs enriched for tumor-reactive TILs.

286. The method of claim 285, wherein the population of TILs enriched for tumor-reactive TILs comprises an increased percentage of tumor-reactive TILs in comparison to TILs expanded using a reference expansion procedure. DB1 / 149057740.1 373Attorney Docket No.116983-5126-WO / AR / TW 287. The method of claim 285 or 286, wherein step (a) is performed for about 30 hours.

288. The method of any one of claims 285-287, wherein step (c) is performed for about 9 days.

289. The method of any one of claims 285-288, wherein step (d) is performed for about 10 days.

290. The method of any one of claims 285 -289, wherein the first cell culture medium comprises IL-2 at a concentration of 3000 IU / mL, IL-15 at a concentration of 10 ng / mL, and IL-21 at a concentration of 10 ng / mL.

291. The method of any one of claims 285-289, wherein the first cell culture medium comprises IL-2 at a concentration of 6000 IU / mL.

292. The method of any one of claims 285-291, wherein step (c) is performed in the presence of iPBMC.

293. The method of claim 292, wherein the iPBMC is depleted of T cells.

294. The method of any one of claims 285-293, wherein the IFNγ is present at a concentration of 200 ng / mL.

295. The method of any one of claims 285-294, wherein the second cell culture medium comprises IL-2 at a concentration of 3000 IU / mL, IL-21 at a concentration of 10 ng / mL, and IL-15 at a concentration of 10 ng / mL.

296. The method of any one of claims 285-295, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), and L-arginine.

297. The method of claim 296, wherein the L-arginine is present at a concentration of about 1 mM to about 10 mM.

298. The method of claim 296, wherein the L-arginine is present at a concentration of about 5 mM.

299. The method of any one of claims 285-298, wherein the second cell culture medium comprises an NAD+ booster.

300. The method of claim 299, wherein the NAD+ booster is selected from the group consisting of L-Trp, NR, NMN, NAD+, NAM and P7C3 activator.

301. The method of any one of claims 285-300, wherein the second cell culture medium DB1 / 149057740.1 374Attorney Docket No.116983-5126-WO / AR / TW comprises an inhibitor of GSK-3α / β.

302. The method of claim 301, wherein the inhibitor of GSK-3α / β is selected from the group consisting of SB415286, SB216763, CHIR99021, AR-AO14418, TZD8, TWS119, Lithium Chloride hydrate, BIO, and 3F8.

303. The method of any one of claims 285-302, wherein the enriching the population of TILs for CD137+ TILs comprises contacting the population of TILs with anti-CD137 antibody immobilized on a bead.

304. The method of any one of claims 285-303, further comprising contacting the population of TILs with an anti-CD39 antibody.

305. The method of any one of claims 285-304, further comprising contacting the population of TILs with an anti-CD200 antibody.

306. A method for enriching tumor-reactive tumor infiltrating lymphocytes (TILs), comprising: a. priming a population of TILs obtained from a tumor digest in the presence of IFNγ; b. enriching the population of TILs for CD103+ TILs; c. performing a first expansion by culturing the population of TILs enriched for CD103+ T cells in a first cell culture medium; and d. performing a second expansion by culturing the population of TILs enriched for CD103+ T cells in a second cell culture medium to produce a population of TILs enriched for tumor-reactive TILs.

307. The method of claim 306, wherein the population of TILs enriched for tumor-reactive TILs comprises an increased percentage of tumor-reactive TILs in comparison to TILs expanded using a reference expansion procedure.

308. The method of claim 306 or 307, wherein step (a) is performed for about 24 hours.

309. The method of any one of claims 306-308, wherein selecting CD103+ TILs comprises sorting the second population of TILs using flow cytometry.

310. The method of any one of claims 306-309, wherein step (b) comprises selecting CD103+CD31- TILs. DB1 / 149057740.1 375Attorney Docket No.116983-5126-WO / AR / TW 311. The method of any one of claims 306-310, wherein step (c) is performed for about 9 days.

312. The method of any one of claims 306-311, wherein step (d) is performed for about 10 days.

313. The method of any one of claims 306-312, wherein the first cell culture medium comprises IL-2 at a concentration of 3000 IU / mL, IL-15 at a concentration of 10 ng / mL, and IL-21 at a concentration of 10 ng / mL.

314. The method of any one of claims 306-313, wherein the first cell culture medium comprises IL-2 at a concentration of 6000 IU / mL.

315. The method of any one of claims 306-314, wherein step (c) is performed in the presence of iPBMC.

316. The method of claim 315, wherein the iPBMC is depleted of T cells.

317. The method of any one of claims 306-316, wherein the IFNγ is present at a concentration of 200 ng / mL.

318. The method of any one of claims 306-317, wherein the second cell culture medium comprises IL-2 at a concentration of 3000 IU / mL, IL-21 at a concentration of 10 ng / mL, and IL-15 at a concentration of 10 ng / mL.

319. The method of any one of claims 306-318, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), and L-arginine.

320. The method of claim 319, wherein the L-arginine is present at a concentration of about 1 mM to about 10 mM.

321. The method of claim 319, wherein the L-arginine is present at a concentration of about 5 mM.

322. The method of any one of claims 306-321, wherein the second cell culture medium comprises an NAD+ booster.

323. The method of claim 322, wherein the NAD+ booster is selected from the group consisting of L-Trp, NR, NMN, NAD+, NAM and P7C3 activator.

324. The method of any one of claims 306-323, wherein the second cell culture medium comprises an inhibitor of GSK-3α / β. DB1 / 149057740.1 376Attorney Docket No.116983-5126-WO / AR / TW 325. The method of claim 324, wherein the inhibitor of GSK-3α / β is selected from the group consisting of SB415286, SB216763, CHIR99021, AR-AO14418, TZD8, TWS119, Lithium Chloride hydrate, BIO, and 3F8.

326. A method of treating a cancer in a patient in need thereof comprising: a. resecting a tumor sample from the patient; b. digesting the tumor sample to obtain a tumor digest; c. producing a population of TILs enriched for tumor-reactive TILs from the tumor digest using the method of any one of claims 285-325; and d. administering the therapeutic population of gene-edited TILs to the patient.

327. The method of claim 326, wherein the cancer is selected from the group consisting of melanoma (including mucosal melanoma, uveal melanoma, cutaneous melanoma, choroidal melanoma, ciliary body melanoma, or iris melanoma), ovarian cancer, cervical cancer, endometrial cancer, non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple negative breast cancer, cancer caused by human papilloma virus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, and renal cell carcinoma.

328. The method of claim 326 or 327, further comprising the step of treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the TILs to the patient.

329. The method of claim 328, wherein the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for three days.

330. The method of claim 328, wherein the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for three days.

331. The method of claim 328, wherein the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for one day. DB1 / 149057740.1 377Attorney Docket No.116983-5126-WO / AR / TW 332. The method of any one of claims 329-331, wherein the cyclophosphamide is administered with mesna.

333. The method of any one of claims 326-332, further comprising a step of treating the patient with an IL-2 regimen starting on the day after the administration of TILs to the patient.

334. The method of any one of claims 326-332, further comprising a step of treating the patient with an IL-2 regimen starting on the same day as administration of TILs to the patient.

335. The method of claim 333 or 334, wherein 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.

336. The method of any one of claims 326-332, comprising no step of treating the patient with an IL-2 regimen.

337. A method of making a population of tumor-infiltrating lymphocytes (TILs), comprising: a) performing a first expansion by culturing a population of TILs in a first cell culture medium; and b) performing a second expansion by culturing the population of TILs in a second cell culture medium, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), IL-21, IL-15, and L-arginine.

338. The method of claim 337, wherein the second cell culture medium comprises IL-15 at a concentration of about 10 ng / mL and IL-21 at a concentration of about 10 ng / mL.

339. The method of claim 337 or 338, wherein the L-arginine is present at a concentration of about 1 mM to about 10 mM.

340. The method of claim 337 or 338, wherein the L-arginine is present at a concentration of about 5 mM.

341. The method of any one of claims 337-340, wherein the second cell culture medium comprises an NAD+ booster.

342. The method of claim 341, wherein the NAD+ booster is selected from the group DB1 / 149057740.1 378Attorney Docket No.116983-5126-WO / AR / TW consisting of L-Trp, NR, NMN, NAD+, NAM and P7C3 activator.

343. The method of claim 341, wherein the NAD+ booster is NAD+.

344. The method of claim 343, wherein the NAD+ is present at a concentration of about 50-75 µM.

345. The method of any one of claims 337-344, wherein the first expansion is performed over a period of about 3-14 days.

346. The method of any one of claims 337-344, wherein the first expansion is performed over a period of about 11 days.

347. The method of any one of claims 337-345, wherein the second expansion is performed over a period of about 7-14 days.

348. The method of any one of claims 337-345, wherein the second expansion is performed over a period of about 11 days.

349. A therapeutic population of TILs produced by the method of any one of claims 337- 348.

350. A pharmaceutical composition comprising the therapeutic population of TILs of claim 349.

351. A method of treating a cancer in a patient in need thereof comprising: a) resecting a tumor sample from the patient, wherein the tumor sample comprises a population of TILs; b) performing a first expansion by culturing the population of TILs in a first cell culture medium; c) performing a second expansion by culturing the population of TILs in a second cell culture medium, wherein the second cell culture medium comprises OKT-3, antigen presenting cells (APCs), IL-21, IL-15, and L-arginine; and d) administering the therapeutic population of gene-edited TILs to the patient.

352. The method of claim 351, wherein the second cell culture medium comprises IL-15 at a concentration of about 10 ng / mL and IL-21 at a concentration of about 10 ng / mL.

353. The method of claim 351 or 352, wherein the L-arginine is present at a concentration of about 1 mM to about 10 mM. DB1 / 149057740.1 379Attorney Docket No.116983-5126-WO / AR / TW 354. The method of claim 351 or 352, wherein the L-arginine is present at a concentration of about 5 mM.

355. The method of any one of claims 351-354, wherein the second cell culture medium comprises an NAD+ booster.

356. The method of claim 355, wherein the NAD+ booster is selected from the group consisting of L-Trp, NR, NMN, NAD+, NAM and P7C3 activator.

357. The method of claim 355, wherein the NAD+ booster is NAD+.

358. The method of claim 357, wherein the NAD+ is present at a concentration of about 50-75 µM.

359. The method of any one of claims 351-358, wherein the first expansion is performed over a period of about 3-14 days.

360. The method of any one of claims 351-358, wherein the first expansion is performed over a period of about 11 days.

361. The method of any one of claims 351-360, wherein the second expansion is performed over a period of about 7-14 days.

362. The method of any one of claims 351-360, wherein the second expansion is performed over a period of about 11 days.

363. The method of any one of claims 351-362, wherein the cancer is selected from the group consisting of melanoma (including mucosal melanoma, uveal melanoma, cutaneous melanoma, choroidal melanoma, ciliary body melanoma, or iris melanoma), ovarian cancer, cervical cancer, endometrial cancer, non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple negative breast cancer, cancer caused by human papilloma virus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, and renal cell carcinoma.

364. The method of any one of claims 351-363, further comprising the step of treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the TILs to the patient.

365. The method of claim 364, wherein the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for three days. DB1 / 149057740.1 380Attorney Docket No.116983-5126-WO / AR / TW 366. The method of claim 364, wherein the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for three days.

367. The method of claim 364, wherein the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for one day.

368. The method of any one of claims 365-367, wherein the cyclophosphamide is administered with mesna.

369. The method of any one of claims 351-368, further comprising a step of treating the patient with an IL-2 regimen starting on the day after the administration of TILs to the patient.

370. The method of any one of claims 351-368, further comprising a step of treating the patient with an IL-2 regimen starting on the same day as administration of TILs to the patient.

371. The method of claim 369 or 370, wherein 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.

372. The method of any one of claims 351-368, comprising no step of treating the patient with an IL-2 regimen.

373. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprising: (a) adding processed tumor fragments from a tumor resected from a patient into a closed system to obtain a first population of TILs; (b) 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 3-11 days to obtain DB1 / 149057740.1 381Attorney Docket No.116983-5126-WO / AR / TW the second population of TILs, and wherein the transition from step (a) to step (b) occurs without opening the system; (c) performing a second expansion by culturing the second population of TILs in a second cell culture medium comprising IL-21, IL-15, L-arginine, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-11 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic 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 (b) to step (c) occurs without opening the system; (d) harvesting the therapeutic population of TILs obtained from step (c), wherein the transition from step (c) to step (d) occurs without opening the system, and wherein the therapeutic population of TILs harvested comprises sufficient TILs for a therapeutically effective dosage of the TILs; (e) transferring the harvested TIL population from step (d) to an infusion bag, wherein the transfer from step (d) to (e) occurs without opening the system, and (f) cryopreserving the infusion bag comprising the harvested TIL population using a cryopreservation process.

374. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprising: (a) adding processed tumor fragments from a tumor resected from a patient into a closed system to obtain a first population of TILs; (b) 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 3-11 days to obtain the second population of TILs, and wherein the transition from step (a) to step (b) occurs without opening the system; (c) performing a second expansion by culturing the second population of TILs in a second cell culture medium comprising IL-21, IL-15, L-arginine, NAD+, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the DB1 / 149057740.1 382Attorney Docket No.116983-5126-WO / AR / TW second expansion is performed for about 7-11 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic 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 (b) to step (c) occurs without opening the system; (d) harvesting the therapeutic population of TILs obtained from step (c), wherein the transition from step (c) to step (d) occurs without opening the system, and wherein the therapeutic population of TILs harvested comprises sufficient TILs for a therapeutically effective dosage of the TILs; (e) transferring the harvested TIL population from step (d) to an infusion bag, wherein the transfer from step (d) to (e) occurs without opening the system, and (f) cryopreserving the infusion bag comprising the harvested TIL population using a cryopreservation process.

375. A method for making a therapeutic population of gene-edited TILs comprising: (a) adding processed tumor fragments from a tumor resected from a patient into a closed system to obtain a first population of TILs; (b) 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 3-11 days to obtain the second population of TILs, and wherein the transition from step (a) to step (b) occurs without opening the system; (c) transducing the second population of TILs with the recombinant lentiviral particle of any one of claims 42–46 to produce a population of gene-edited TILs, wherein the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion by culturing the population of gene-edited TILs in a second cell culture medium comprising IL-21, IL-15, L-arginine, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-11 days to obtain the third population of TILs, wherein the third population of TILs is the therapeutic population of gene- edited TILs, wherein the second expansion is performed in a closed container DB1 / 149057740.1 383Attorney Docket No.116983-5126-WO / AR / TW providing a second gas-permeable surface area, and wherein the transition from step (c) to step (d) occurs without opening the system; (e) harvesting the therapeutic population of gene-edited TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system, and wherein the therapeutic population of gene-edited TILs harvested comprises sufficient TILs for a therapeutically effective dosage of the TILs; (f) transferring the harvested therapeutic population of gene-edited TILs from step (e) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system, and (g) cryopreserving the infusion bag comprising the harvested TIL population using a cryopreservation process.

376. A method for making a therapeutic population of gene-edited TILs comprising: (a) adding processed tumor fragments from a tumor resected from a patient into a closed system to obtain a first population of TILs; (b) 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 3-11 days to obtain the second population of TILs, and wherein the transition from step (a) to step (b) occurs without opening the system; (c) transducing the second population of TILs with the recombinant lentiviral particle of any one of claims 42–46 to produce a population of gene-edited TILs, wherein the transition from step (b) to step (c) occurs without opening the system; (d) performing a second expansion by culturing the population of gene-edited TILs in a second cell culture medium comprising IL-21, IL-15, L-arginine, NAD+, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-11 days to obtain the third population of TILs, wherein the third population of TILs is the therapeutic population of gene- edited 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; DB1 / 149057740.1 384Attorney Docket No.116983-5126-WO / AR / TW (e) harvesting the therapeutic population of gene-edited TILs obtained from step (d), wherein the transition from step (d) to step (e) occurs without opening the system, and wherein the therapeutic population of gene-edited TILs harvested comprises sufficient TILs for a therapeutically effective dosage of the TILs; (f) transferring the harvested therapeutic population of gene-edited TILs from step (e) to an infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system, and (g) cryopreserving the infusion bag comprising the harvested TIL population using a cryopreservation process.

377. A method for making a therapeutic population of gene-edited TILs comprising: (a) processing a tumor resected from a patient to obtain a first population of TILs; (b) 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, and wherein the first expansion is performed for about 3-11 days to obtain the second population of TILs; (c) transducing the second population of TILs with the recombinant lentiviral particle of any one of claims 42–46 to produce a population of gene-edited TILs; (d) performing a second expansion by culturing the population of gene-edited TILs in a second cell culture medium comprising IL-21, IL-15, L-arginine, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-11 days to obtain the third population of TILs, wherein the third population of TILs is the therapeutic population of gene- edited TILs, and wherein the second expansion is performed in a closed container providing a second gas-permeable surface area; (e) harvesting the therapeutic population of gene-edited TILs obtained from step (d), wherein the therapeutic population of gene-edited TILs harvested comprises sufficient TILs for a therapeutically effective dosage of the TILs; (f) transferring the harvested therapeutic population of gene-edited TILs from step (e) to an infusion bag, and DB1 / 149057740.1 385Attorney Docket No.116983-5126-WO / AR / TW (g) cryopreserving the infusion bag comprising the harvested TIL population using a cryopreservation process.

378. A method for making a therapeutic population of gene-edited TILs comprising: (a) processing a tumor resected from a patient to obtain a first population of TILs; (b) 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, and wherein the first expansion is performed for about 3-11 days to obtain the second population of TILs; (c) transducing the second population of TILs with the recombinant lentiviral particle of any one of claims 42–46 to produce a population of gene-edited TILs; (d) performing a second expansion by culturing the population of gene-edited TILs in a second cell culture medium comprising IL-21, IL-15, L-arginine, NAD+, OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 7-11 days to obtain the third population of TILs, wherein the third population of TILs is the therapeutic population of gene- edited TILs, and wherein the second expansion is performed in a closed container providing a second gas-permeable surface area; (e) harvesting the therapeutic population of gene-edited TILs obtained from step (d), wherein the therapeutic population of gene-edited TILs harvested comprises sufficient TILs for a therapeutically effective dosage of the TILs; (f) transferring the harvested therapeutic population of gene-edited TILs from step (e) to an infusion bag, and (g) cryopreserving the infusion bag comprising the harvested TIL population using a cryopreservation process.

379. The method of any one of claims 373-378, wherein the second cell culture medium comprises IL-15 at a concentration of about 10 ng / mL and IL-21 at a concentration of about 10 ng / mL.

380. The method of any one of claims 373-379, wherein the L-arginine is present at a concentration of about 1 mM to about 10 mM.

381. The method of any one of claims 373-379, wherein the L-arginine is present at a DB1 / 149057740.1 386Attorney Docket No.116983-5126-WO / AR / TW concentration of about 5 mM.

382. The method of any one of claims 374, 376, 378-381, wherein the NAD+ is present at a concentration of about 50-75 µM.

383. The method of any one of claims 373-382, wherein the first expansion is performed over a period of about 3-11 days.

384. The method of any one of claims 373-382, wherein the first expansion is performed over a period of about 11 days.

385. The method of any one of claims 373-384, wherein the second expansion is performed over a period of about 7-11 days.

386. The method of any one of claims 373-384, wherein the second expansion is performed over a period of about 11 days.

387. The method of any one of claims 373-386, further comprising activating the population of TILs for 1 day or 2 days before transducing the population of TILs with the recombinant lentiviral particle.

388. The method of claim 387, wherein the activating step comprises contacting the population of TILs with TransAct.

389. The method of claim 387, wherein the activating step comprises contacting the population of TILs with TransAct at a ratio of 1:

100.

390. The method of any one of claims 373-389, wherein the transducing step is conducted at a TIL concentration of 105cells / mL.

391. The method of any one of claims 373-390, wherein the transducing step is conducted at a multiplicity of infection (MOI) of about 10 to about 40.

392. The method of any one of claims 373-391, wherein the transducing step is conducted in the presence of RetroNectin or Vectofusin-1.

393. The method of any one of claims 373-392, wherein the transducing step comprises centrifugation.

394. The method of any one of claims 373-393, wherein the transducing step is conducted in the presence of Lentibooster.

395. The method of any one of claims 373-394, further comprising resting the population DB1 / 149057740.1 387Attorney Docket No.116983-5126-WO / AR / TW of TILs for 2 day or 3 days after the transducing step.

396. A therapeutic population of gene-edited TILs produced by the method of any one of claims 373-395.

397. A pharmaceutical composition comprising the therapeutic population of gene-edited TILs of claim 396.

398. Use of the therapeutic population of TILs of claim 349, or the therapeutic population of gene-edited TILs of any one of claims 172-184 and 396, for treating cancer in a patient in need thereof, comprising administering the therapeutic population of TILs or the therapeutic population of gene-edited TILs to the patient.

399. The use of claim 398, wherein the cancer is selected from the group consisting of melanoma (including mucosal melanoma, uveal melanoma, cutaneous melanoma, choroidal melanoma, ciliary body melanoma, or iris melanoma), ovarian cancer, cervical cancer, endometrial cancer, non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple negative breast cancer, cancer caused by human papilloma virus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, and renal cell carcinoma.

400. The use of claim 398 or 399, further comprising the step of treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the TILs to the patient.

401. The use of claim 400, wherein the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for three days.

402. The use of claim 400, wherein the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for three days.

403. The use of claim 400, wherein the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg / m2 / day and fludarabine at a dose of 25 mg / m2 / day for two days followed by administration of fludarabine at a dose of 25 mg / m2 / day for one day. DB1 / 149057740.1 388Attorney Docket No.116983-5126-WO / AR / TW 404. The use of any one of claims 401-403, wherein the cyclophosphamide is administered with mesna.

405. The use of any one of claims 398-404, further comprising a step of treating the patient with an IL-2 regimen starting on the day after the administration of TILs to the patient.

406. The use of any one of claims 398-404, further comprising a step of treating the patient with an IL-2 regimen starting on the same day as administration of TILs to the patient.

407. The use of claim 405 or 406, wherein 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.

408. The method of any one of claims 398-404, comprising no step of treating the patient with an IL-2 regimen. DB1 / 149057740.1 389

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