Methods of gene-editing using programmable nucleases

Abstract

Some embodiments disclosed herein provide a method for gene-editing a population of cells using a programmable nuclease, the method comprising introducing an RNA molecule associated with the programmable nuclease into the population of cells in a volume, wherein the RNA molecule is present at a fixed concentration not dependent on the number of cells in the volume, to generate a population of gene-edited cells. Further provided are methods for preparing gene-edited tumor infiltrating lymphocytes (TILs) having reduced expression of one or two target genes. In some embodiments, the gene-edited TILs are prepared by introducing one or more programmable nucleases into the TILs.

Description

Attorney Docket No.: 116983-5129-WO METHODS OF GENE-EDITING USING PROGRAMMABLE NUCLEASES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.63 / 581,593,filed September 8, 2023, the disclosure of which is herein incorporated by reference in its entirety. BACKGROUND OF THE INVENTION

[0002] Gene editing technologies based on programmable nucleases have significantly broadened our ability to make precise and direct changes in the genomic DNA of various species, including human cells. Early studies utilize zincfinger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) for genome editing. ZFNs and TALENs depend on protein engineering of DNA- binding domains to recognize and edit specific DNA sequences. With the recent development of the clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 system, gene editing has become efficient, convenient and programmable, leading to promising translational studies and clinical trials for both genetic and non-genetic diseases. Guo et al., Front. Bioeng. Biotechnol.2023, 11,1143157.

[0003] Delivery of programmable nucleases into the target cell is one of the pressing challenges in transforming the technology into medicine. In vitro-transcribed (IVT) mRNA-mediated delivery of nucleases has several advantages, such as transient expression with efficient in vitro delivery, no genomic integration, and high editing efficiency. Zhang et al., Mol. Therapy 2019, 27,736-746.

[0004] A major concern in the applications of a programmable nuclease is about its off-target effects, namely the deposition of unexpected, unwanted, or even adverse alterations to the genome. The current inventors unexpectedly discovered a method for minimizing the off-target effects of a programmable nuclease, while maintaining high efficiency in editing desired targets. BRIEF SUMMARY OF THE INVENTION

[0005] Some embodiments of the present invention provide a method for gene-editing a population of cells using a programmable nuclease, the method comprising introducing an RNA molecule associated with the programmable nuclease into the population of cells in a volume, wherein the RNA molecule is present at afixed concentration not dependent on the number of cells in the volume, to generate a population of gene-edited cells. In some embodiments, the programmable nuclease is selected from the group consisting of a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease (ZFN), a CRISPR-associated (Cas) nuclease, and a meganuclease (MN). 1DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO In some embodiments, the RNA molecule is an mRNA molecule encoding the programmable nuclease. In some embodiments, thefixed concentration of the mRNA molecule is from about 1 µg / mL to about 100 µg / mL. In some embodiments, thefixed concentration of the mRNA molecule is from about 10 µg / mL to about 50 µg / mL. In some embodiments, thefixed concentration of the mRNA molecule is about 10 µg / mL. In some embodiments, thefixed concentration of the mRNA molecule is about 12.5 µg / mL. In some embodiments, thefixed concentration of the mRNA molecule is about 20 µg / mL. In some embodiments, thefixed concentration of the mRNA molecule is about 30 µg / mL. In some embodiments, thefixed concentration of the mRNA molecule is about 40 µg / mL. In some embodiments, thefixed concentration of the mRNA molecule is about 50 µg / mL. In some embodiments, the RNA molecule is a gRNA molecule. In some embodiments, the volume is about 1 mL to about 100 mL. In some embodiments, the volume comprises about 105to about 109cells.

[0006] In some embodiments, the RNA molecule is introduced via a transfection technique selected from the group consisting of electroporation, nanoparticle mediated transfection, magnetic bead- based transfection, and calcium phosphate precipitation. In some embodiments, the RNA molecule is introduced via electroporation. In some embodiments, the volume comprises an electroporation buffer. In some embodiments, the electroporation buffer is CTS™ Xenon™ Genome Editing Buffer. In some embodiments, the electroporation is conducted using the Xenon™ electroporator with the settings 2300 V, 2 ms pulse width, 3 pulses. In some embodiments, the method further comprises activating the population of cells for 1 day or 2 days before introducing the RNA molecule. In some embodiments, the activating step comprises contacting the population of cells with TransAct. In some embodiments, the activating step comprises contacting the population of cells with TransAct at a ratio of 1:100. In some embodiments, the method further comprises resting the population of cells for 1 day or 2 days after introducing the RNA molecule.

[0007] In some embodiments, the programmable nuclease cuts a DNA sequence in a target gene. In some embodiments, the target gene is selected from the group consisting of an immune checkpoint gene, a TCR gene, a PCSK9 gene, a hATTR gene, aSOD1 gene, a TAU gene, a LRRK2 gene, a SMN2 gene, a TTR gene, a Factor XI gene, an APOL1 gene, and a CCR5 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 immune checkpoint gene is 2 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO selected from the group consisting of PD-1, TIGIT, CTLA-4, LAG-3, and CISH. In some embodiments, the expression of the target gene is reduced. In some embodiments, the expression of the target gene is reduced by at least 50%. In some embodiments, the expression of the target gene is reduced by at least 80%. In some embodiments, the cells are T cells. In some embodiments, the T cells are tumor infiltrating lymphocytes (TILs). In some embodiments, the T cells express a chimeric antigen receptor (CAR). In some embodiments, the CAR binds to an antigen chosen from: CD19, CD20, CD22, BCMA, mesothelin, EGFRvIII, GD2, Tn antigen, sTn antigen, Tn-O-Glycopeptides, sTn-O-Glycopeptides, PSMA, CD97, TAG72, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, leguman, GD3, CD171, IL-11Ra, PSCA, MAD-CT-1, MAD-CT-2, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, folate receptor alpha, ERBBs (e.g., ERBB2), Her2 / neu, MUC1, EGFR, NCAM, Ephrin B2, CAIX, LMP2, sLe, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, FAP, Legumain, HPV E6 or E7, ML-IAP, CLDN6, TSHR, GPRC5D, ALK, Polysialic acid, Fos-related antigen, neutrophil elastase, TRP-2, CYP1B1, sperm protein 17, beta human chorionic gonadotropin, AFP, thyroglobulin, PLAC1, globoH, RAGE1, MN-CA IX, human telomerase reverse transcriptase, intestinal carboxyl esterase, mut hsp 70-2, NA-17, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, NY-ESO-1, GPR20, Ly6k, OR51E2, TARP, GFRα4, or a peptide of any of these antigens presented on MHC.

[0008] Some embodiments of the present invention provide a therapeutic population of gene- edited cells produced by the method disclosed herein. In some embodiments, the gene-edited cells have reduced expression of a target gene. In some embodiments, the target gene is selected from the group consisting of an immune checkpoint gene, a TCR gene, a PCSK9 gene, a hATTR gene, aSOD1 gene, a TAU gene, a LRRK2 gene, a SMN2 gene, a TTR gene, a Factor XI gene, an APOL1 gene, and a CCR5 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 immune checkpoint gene is selected from the group consisting of PD-1, TIGIT, CTLA-4, LAG-3, and CISH. In some embodiments, the expression of the target gene is reduced. In some embodiments, the expression of the target gene is reduced by at least 50%. In some embodiments, the expression of the target gene is reduced by at least 60%. In some embodiments, the expression of the target gene is reduced by at least 80%. In some embodiments, the cells are T cells. In some embodiments, the T cells are tumor infiltrating lymphocytes (TILs). In some 3 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO embodiments, the T cells express a chimeric antigen receptor (CAR). In some embodiments, the CAR binds to an antigen chosen from: CD19, CD20, CD22, BCMA, mesothelin, EGFRvIII, GD2, Tn antigen, sTn antigen, Tn-O-Glycopeptides, sTn-O-Glycopeptides, PSMA, CD97, TAG72, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, leguman, GD3, CD171, IL-11Ra, PSCA, MAD-CT-1, MAD-CT-2, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, folate receptor alpha, ERBBs (e.g., ERBB2), Her2 / neu, MUC1, EGFR, NCAM, Ephrin B2, CAIX, LMP2, sLe, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, FAP, Legumain, HPV E6 or E7, ML-IAP, CLDN6, TSHR, GPRC5D, ALK, Polysialic acid, Fos-related antigen, neutrophil elastase, TRP-2, CYP1B1, sperm protein 17, beta human chorionic gonadotropin, AFP, thyroglobulin, PLAC1, globoH, RAGE1, MN-CA IX, human telomerase reverse transcriptase, intestinal carboxyl esterase, mut hsp 70-2, NA-17, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, NY-ESO-1, GPR20, Ly6k, OR51E2, TARP, GFRα4, or a peptide of any of these antigens presented on MHC.

[0009] Some embodiments of the present invention provide a method for gene-editing a population of TILs using a programmable nuclease, comprising: (a) performing afirst expansion of the population of TILs in a cell culture medium comprising IL-2, wherein thefirst expansion is performed for about 3-14 days; and (b) performing a second expansion of the population of TILs in a cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs), wherein the second expansion is performed for about 7-14 days, wherein the method further comprises introducing afirst RNA molecule associated with the programmable nuclease into the population of TILs in a volume, wherein the RNA molecule is present at afixed concentration not dependent on the number of TILs in the volume, and wherein thefirst RNA molecule is introduced before step (a), after step (a) and before step (b), or after step (b).

[0010] In some embodiments, the method further comprises introducing a second RNA molecule associated with a second programmable nuclease into the population of TILs. In some embodiments, the introducing thefirst and / or second RNA molecule is performed after thefirst expansion and before the second expansion. In some embodiments, the method further comprises resting the population of TILs for 1 day or 2 days after introducing thefirst RNA molecule. In some embodiments, the method further comprises activating the population of cells for 1 day or 2 days before introducing thefirst RNA molecule. In some embodiments, the activating step comprises contacting the population of cells with TransAct. In some embodiments, the activating step comprises contacting the population of cells with TransAct at a ratio of 1:100. In some embodiments, thefirst RNA molecule is afirst mRNA molecule encoding thefirst programmable nuclease. In some embodiments, the second RNA molecule is a second mRNA molecule encoding the second programmable nuclease. In some embodiments, thefirst and / or second programmable nuclease is 4 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO selected from the group consisting of a transcription activator-like effector nuclease (TALEN), a zinc- finger nuclease (ZFN), a CRISPR-associated (Cas) nuclease, and a meganuclease (MN). In some embodiments, thefixed concentration of thefirst and / or second mRNA molecule is from about 1 µg / mL to about 100 µg / mL. In some embodiments, thefixed concentration of thefirst and / or second mRNA molecule is from about 10 µg / mL to about 50 µg / mL. In some embodiments, thefixed concentration of thefirst and / or second mRNA molecule is about 10 µg / mL. In some embodiments, thefixed concentration of thefirst and / or second mRNA molecule is about 12.5 µg / mL. In some embodiments, thefixed concentration of thefirst and / or second mRNA molecule is about 20 µg / mL. In some embodiments, thefixed concentration of thefirst and / or second mRNA molecule is about 10 µg / mL. In some embodiments, thefixed concentration of thefirst and / or second mRNA molecule is about 40 µg / mL. In some embodiments, thefixed concentration of thefirst and / or second mRNA molecule is about 50 µg / mL.

[0011] In some embodiments, thefirst programmable nuclease cuts a DNA sequence in afirst target gene. In some embodiments, the second programmable nuclease cuts a DNA sequence in a second target gene. In some embodiments, thefirst and / or second target gene is selected from the group consisting of an immune checkpoint gene, a TCR gene, a PCSK9 gene, a hATTR gene, aSOD1 gene, a TAU gene, a LRRK2 gene, a SMN2 gene, a TTR gene, a Factor XI gene, an APOL1 gene, and a CCR5 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 immune checkpoint gene is selected from the group consisting of PD-1, TIGIT, CTLA-4, LAG-3, and CISH. In some embodiments, thefirst target gene is PD-1 and the second target gene is CTLA-4. In some embodiments, thefirst target gene is PD-1 and the second target gene is TIGIT. In some embodiments, thefirst target gene is PD-1 and the second target gene is LAG-3. In some embodiments, thefirst target gene is PD-1 and the second target gene is CISH. In some embodiments, thefirst and / or second RNA molecule is a gRNA molecule. In some embodiments, the first and / or second RNA molecule is introduced via a transfection technique selected from the group consisting of electroporation, nanoparticle mediated transfection, magnetic bead-based transfection, and calcium phosphate precipitation. In some embodiments, thefirst and / or second RNA molecule is introduced via electroporation. In some embodiments, the volume comprises an electroporation 5 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO buffer. In some embodiments, the electroporation buffer is CTS™ Xenon™ Genome Editing Buffer. In some embodiments, the electroporation is conducted using the Xenon™ electroporator with the settings 2300 V, 2 ms pulse width, 3 pulses. In some embodiments, the volume is about 1 mL to about 100 mL. In some embodiments, the volume comprises about 105to about 109cells. In some embodiments, thefirst expansion is performed for about 3-11 days. In some embodiments, the second expansion is performed for about 7-11 days. In some embodiments, the IL-2 is present at an initial concentration of between 1000 IU / mL and 6000 IU / mL in the cell culture medium in thefirst expansion. In some embodiments, in the second expansion step, the IL-2 is present at an initial concentration of between 1000 IU / mL and 6000 IU / mL and the OKT-3 antibody is present at an initial concentration of about 30 ng / mL.

[0012] Some embodiments of the present invention provide a method for gene-editing a population of TILs using a programmable nuclease, comprising: (a) culturing afirst population of TILs in afirst cell culture medium comprising IL-2 for about 5-7 days to produce a second population of TILs; (b) activating the second population of TILs for about 2-4 days, to produce a third population of TILs; (c) introducing an RNA molecule associated with the programmable nuclease into the third population of TILs in a volume, wherein the RNA molecule is present at afixed concentration not dependent on the number of TILs in the volume, to produce a fourth population of TILs, wherein the programmable nuclease cuts the DNA sequence of a target gene; and (d) culturing the fourth population of TILs in a second cell culture medium comprising antigen presenting cells (APCs), OKT-3, and IL-2 for about 7- 11 days, to produce sixth population of TILs having reduced expression of the target gene.

[0013] 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 programmable nuclease is selected from the group consisting of a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease (ZFN), a CRISPR-associated (Cas) nuclease, and a meganuclease (MN). In some embodiments, the RNA molecule is an mRNA molecule encoding the programmable nuclease. In some embodiments, thefixed concentration of the mRNA molecule is from about 1 µg / mL to about 100 µg / mL. In some embodiments, thefixed concentration of the mRNA molecule is from about 10 µg / mL to about 50 µg / mL. In some embodiments, thefixed concentration of the mRNA molecule is about 10 µg / mL. In some embodiments, thefixed concentration of the mRNA molecule is about 12.5 µg / mL. In some embodiments, thefixed concentration of the mRNA molecule is about 20 µg / mL. In some embodiments, thefixed concentration of the mRNA molecule is about 30 µg / mL. In some 6 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO embodiments, thefixed concentration of the mRNA molecule is about 40 µg / mL. In some embodiments, thefixed concentration of the mRNA molecule is about 50 µg / mL.

[0014] In some embodiments, the programmable nuclease cuts a DNA sequence in a target gene. In some embodiments, the target gene is selected from the group consisting of an immune checkpoint gene, a TCR gene, a PCSK9 gene, a hATTR gene, aSOD1 gene, a TAU gene, a LRRK2 gene, a SMN2 gene, a TTR gene, a Factor XI gene, an APOL1 gene, and a CCR5 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 immune checkpoint gene is selected from the group consisting of PD-1, TIGIT, CTLA-4, LAG-3, and CISH.

[0015] Some embodiments of the present invention provide a method for gene-editing a population of TILs using two programmable nucleases, comprising: (a) culturing afirst population of TILs in afirst cell culture medium comprising IL-2 for about 5-7 days to produce a second population of TILs; (b) activating the second population of TILs for about 2-4 days, to produce a third population of TILs; (c) introducing afirst RNA molecule associated with afirst programmable nuclease into the third population of TILs in a volume, wherein thefirst RNA molecule is present at afixed concentration not dependent on the number of TILs in the volume, to produce a fourth population of TILs, wherein the first programmable nuclease cuts the DNA sequence of afirst target gene; (d) resting the fourth population of TILs in thefirst cell culture medium comprising IL-2 for about 2 to 3 days; (e) introducing a second RNA molecule associated with a second programmable nuclease into the fourth population of TILs in a volume, wherein the second RNA molecule is present at afixed concentration not dependent on the number of TILs in the volume, to produce afifth population of TILs, wherein the second programmable nuclease cuts the DNA sequence of a second target gene; and (f) culturing thefifth population of TILs in a second cell culture medium comprising antigen presenting cells (APCs), OKT-3, and IL-2 for about 7-11 days, to produce sixth population of TILs having reduced expression of thefirst target gene and the second target gene.

[0016] 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, thefirst RNA molecule is afirst mRNA molecule encoding thefirst programmable nuclease. In some embodiments, the second RNA molecule is a 7 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO second mRNA molecule encoding the second programmable nuclease. In some embodiments, the first and / or second programmable nuclease is selected from the group consisting of a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease (ZFN), a CRISPR-associated (Cas) nuclease, and a meganuclease (MN). In some embodiments, thefixed concentration of thefirst and / or second mRNA molecule is from about 1 µg / mL to about 100 µg / mL. In some embodiments, thefixed concentration of thefirst and / or second mRNA molecule is from about 10 µg / mL to about 50 µg / mL. In some embodiments, thefixed concentration of thefirst and / or second mRNA molecule is about 10 µg / mL. In some embodiments, thefixed concentration of thefirst and / or second mRNA molecule is about 12.5 µg / mL. In some embodiments, thefixed concentration of thefirst and / or second mRNA molecule is about 20 µg / mL. In some embodiments, thefixed concentration of the first and / or second mRNA molecule is about 10 µg / mL. In some embodiments, thefixed concentration of thefirst and / or second mRNA molecule is about 40 µg / mL. In some embodiments, thefixed concentration of thefirst and / or second mRNA molecule is about 50 µg / mL.

[0017] In some embodiments, thefirst programmable nuclease cuts a DNA sequence in afirst target gene. In some embodiments, the second programmable nuclease cuts a DNA sequence in a second target gene. In some embodiments, thefirst and / or second target gene is selected from the group consisting of an immune checkpoint gene, a TCR gene, a PCSK9 gene, a hATTR gene, aSOD1 gene, a TAU gene, a LRRK2 gene, a SMN2 gene, a TTR gene, a Factor XI gene, an APOL1 gene, and a CCR5 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 immune checkpoint gene is selected from the group consisting of PD-1, TIGIT, CTLA-4, LAG-3, and CISH. In some embodiments, thefirst target gene is PD-1 and the second target gene is CTLA-4. In some embodiments, thefirst target gene is PD-1 and the second target gene is TIGIT. In some embodiments, thefirst target gene is PD-1 and the second target gene is LAG-3. In some embodiments, thefirst target gene is PD-1 and the second target gene is CISH. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1: shows PD-1 on-target hyperbolafit options. 8 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO

[0019] Figures 2A-2F: show PD-1 off-target signals for Candidates 3, 1, 19, 9, 17, and 4, respectively.

[0020] Figure 3: shows TIGIT on-target hyperbolafit options.

[0021] Figures 4A-4B: show TIGIT off-target signals for Candidates 1 and 2, respectively. DETAILED DESCRIPTION OF THE INVENTION I. Definitions

[0022] 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.

[0023] The term “gene editing” refers to altering the DNA sequence of a cell.

[0024] The term “programmable nuclease” refers to a protein that can, either alone or in combination with another molecule, alter the DNA sequence of a cell with sequence specificity, for example, a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease (ZFN), a CRISPR associated (Cas) nuclease, a meganuclease, a nickase or a natural or engineered variant, family-member, orthologue, fragment or fusion construct thereof.

[0025] The term “in vivo” refers to an event that takes place in a subject's body.

[0026] 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.

[0027] 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.

[0028] 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. 9 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO

[0029] 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 (“REP TILs” or “post-REP TILs”). TIL cell populations can include genetically modified TILs.

[0030] 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.

[0031] 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.

[0032] 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 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.

[0033] 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 10 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO 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.

[0034] 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.

[0035] 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.

[0036] 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 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.

[0037] 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).11 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO

[0038] The term “IL-2” (also referred to herein as “IL2”) refers to the T cell growth factor known as interleukin-2, and includes all forms of IL-2 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-2 is described, e.g., in Nelson, J. Immunol.2004, 172, 3983-88 and Malek, Annu. Rev. Immunol.2008, 26, 453-79, the disclosures of which are incorporated by reference herein. 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 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 N6 substituted 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 12 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO 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.

[0039] 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, 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- 13 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO 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 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 14 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO embodiments, the linker comprises a homobifunctional linker. In some embodiments, the homobifunctional linker comprises Lomant's reagent dithiobis (succinimidylpropionate) DSP, 3ʹ3ʹ- 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,Nʹ- disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3ʹ-dithiobispropionimidate (DTBP), 1,4-di-(3ʹ-(2ʹ- pyridyldithio)propionamido)butane (DPDPB), bismaleimidohexane (BMH), aryl halide-containing compound (DFDNB), such as e.g.1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4ʹ-difluoro-3,3ʹ-dinitrophenylsulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3ʹ-dimethylbenzidine, benzidine, α,αʹ-p-diaminodiphenyl, diiodo-p- xylene sulfonic acid, N,Nʹ-ethylene-bis(iodoacetamide), or N,Nʹ-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- 15 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO azidosalicylamido)ethyl-1,3ʹ-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4ʹ-azido-2ʹ- nitrophenyl amino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4ʹ-azido-2ʹ- nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3ʹ-dithiopropionate (sAND), N-succinimidyl- 4(4-azidophenyl)1,3ʹ-dithiopropionate (sADP), N-sulfosuccinimidyl(4-azidophenyl)-1,3ʹ- dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(ρ-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3ʹ-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]-3ʹ-(2ʹ-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 16 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO 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 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.

[0040] 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 17 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO Publication No. US 2021 / 0038684 A1 and 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 afirst fusion partner that is linked to a second fusion partner by a mucin domain polypeptide linker, wherein thefirst 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 thefirst fusion partner to the second fusion partner in the absence of the mucin domain polypeptide linker. TABLE 2. Amino acid sequences of interleukins.18 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO

[0041] In some embodiments, an IL-2 form suitable for use in the invention includes a 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 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 19 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO IL-2 molecule or a fragment thereof engrafted into a CDR of the VH or 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 VH or 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 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.

[0042] 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.

[0043] 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 20 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO 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.

[0044] 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.

[0045] 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 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.

[0046] 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 VL region 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 21 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO 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 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 EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR 120 kabat WITFCQSIIS TLTSTSGMSV G 141 SEQ ID NO:20 DIWWDDKKDY NPSLKS 16 22 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO 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 QCLEEELKPL EEVLNLAQSK NFHLRPRDLI SNINVIVLEL KGSETTFMCE YADETATIVE 120 IMGT 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 TISKAKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT 480 PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK 533 SEQ ID NO:30 KAQLSVGYMH 10 LCDR1 kabat SEQ ID NO:31 DTSKLAS 7 LCDR2 kabat SEQ ID NO:32 FQGSGYPFT 9 LCDR3 kabat SEQ ID NO:33 QLSVGY 6 23 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO LCDR1 chothia SEQ ID NO:34 DTS 3 LCDR2 chothia SEQ ID NO:35 GSGYPF 6 LCDR3 chothia SEQ ID NO:36 DIQMTQSPST LSASVGDRVT ITCKAQLSVG YMHWYQQKPG KAPKLLIYDT SKLASGVPSR 60 V FSGSGSGTEF TLTISSLQPD DFATYYCFQG SGYPFTFGGG TKLEIK 106 SEQ ID NO:37 DIQMTQSPST LSASVGDRVT ITCKAQLSVG YMHWYQQKPG KAPKLLIYDT SKLASGVPSR 60 Light chain FSGSGSGTEF TLTISSLQPD DFATYYCFQG SGYPFTFGGG TKLEIKRTVA APSVFIFPPS 120 DEQLKSGTAS VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL 180 SKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC 213 SEQ ID NO:38 QVTLRESGPA LVKPTQTLTL TCTFSGFSLA PTSSSTKKTQ LQLEHLLLDL QMILNGINNY 60 Light chain KNPKLTRMLT AKFYMPKKAT ELKHLQCLEE ELKPLEEVLN LAQSKNFHLR PRDLISNINV 120 IVLELKGSET TFMCEYADET ATIVEFLNRW ITFCQSIIST LTSTSGMSVG WIRQPPGKAL 180 EWLADIWWDD KKDYNPSLKS RLTISKDTSK NQVVLKVTNM DPADTATYYC ARSMITNWYF 240 DVWGAGTTVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFPEPV TVSWNSGALT 300 SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKR VEPKSCDKTH 360 TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV AVSHEDPEVK FNWYVDGVEV 420 HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALAAPIEK TISKAKGQPR 480 EPQVYTLPPS REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF 540 FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK 583 SEQ ID NO:39 DIQMTQSPST LSASVGDRVT ITCKAQLSVG YMHWYQQKPG KAPKLLIYDT SKLASGVPSR 60 Light chain FSGSGSGTEF TLTISSLQPD DFATYYCFQG SGYPFTFGGG TKLEIKRTVA APSVFIFPPS 120 DEQLKSGTAS VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL 180 SKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC 213

[0047] The 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). 24 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO

[0048] 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 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).

[0049] 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).

[0050] 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). 25 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO

[0051] 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).

[0052] 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. 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.

[0053] 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 26 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO specifically bind to the antigen of the reference antibody. The term variant also includes pegylated antibodies or proteins.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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, 27 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO 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.” II. Methods for Gene-Editing a Population of Cells

[0058] Some embodiments disclosed herein provide a method for gene-editing a population of cells using a programmable nuclease, the method comprising introducing an RNA molecule associated with the programmable nuclease into the population of cells in a volume, wherein the RNA molecule is present at afixed concentration not dependent on the number of cells in the volume, to generate a population of gene-edited cells.

[0059] 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 some embodiments, the DNA sequence exhibiting enhanced expression is an endogenous DNA sequence. In some embodiments, the DNA sequence exhibiting expression is an exogenous DNA sequence that originates from outside the edited cell. In accordance with embodiments of the present invention, gene-editing technology is used to enhance the effectiveness of a therapeutic population of cells, such as T cells.

[0060] According to an embodiment, the gene-editing process may comprise the use of a programmable nuclease that mediates the generation of a double-strand or single-strand break at one or more immune checkpoint genes. Such programmable nucleases enable precise genome editing by introducing breaks at specific genomic loci, i.e., they rely on the recognition of a specific DNA sequence within the genome to target a nuclease domain to this location and mediate the generation of a double-strand break at the target sequence. A double-strand break in the DNA subsequently recruits endogenous repair machinery to the break site to mediate genome editing by either non-homologous end-joining (NHEJ) or homology-directed repair (HDR). Thus, the repair of 28 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO the break can result in the introduction of insertion / deletion mutations that disrupt (e.g., silence, repress, or enhance) the target gene product.

[0061] In some embodiments, the programmable nuclease is selected from the group consisting of a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease (ZFN), a CRISPR- associated (Cas) nuclease, and a meganuclease (MN).

[0062] In some embodiments, the RNA molecule is an mRNA molecule encoding the programmable nuclease. The mRNA molecule may be introduced at afixed concentration, for example, from about 0.1 µg / mL to about 1,000 µg / mL. In some embodiments, the mRNA molecule may be introduced at a fixed concentration, for example, from about 1 µg / mL to about 100 µg / mL. In some embodiments, the mRNA molecule may be introduced at afixed concentration, for example, from about 10 µg / mL to about 50 µg / mL. In some embodiments, the mRNA molecule may be introduced at afixed concentration, for example, at about 1 µg / mL, about 2 µg / mL, about 3 µg / mL, about 4 µg / mL, about 5 µg / mL, about 6 µg / mL, about 7 µg / mL, about 8 µg / mL, about 9 µg / mL, about 10 µg / mL, about 20 µg / mL, about 30 µg / mL, about 40 µg / mL, about 50 µg / mL, about 60 µg / mL, about 70 µg / mL, about 80 µg / mL, about 90 µg / mL, about 100 µg / mL. In some embodiments, the mRNA molecule may be introduced at afixed concentration of about 10 µg / mL. In some embodiments, the mRNA molecule may be introduced at afixed concentration of about 12.5 µg / mL. In some embodiments, the mRNA molecule may be introduced at afixed concentration of about 20 µg / mL. In some embodiments, the mRNA molecule may be introduced at afixed concentration of about 30 µg / mL. In some embodiments, the mRNA molecule may be introduced at afixed concentration of about 40 µg / mL. In some embodiments, the mRNA molecule may be introduced at afixed concentration of about 50 µg / mL.

[0063] In some embodiments, the RNA molecule may be a guide RNA (gRNA) molecule for a CRISPR / Cas gene-editing system.

[0064] The volume for introducing the RNA molecule into the population of cells may vary, for example, from about 1 µL to about 1 L. In some embodiments, the volume for introducing the RNA molecule into the population of cells may be from about 1 mL to about 100 mL. In some embodiments, the volume may comprise from about 103to about 1010cells, or more. In some embodiments, the volume may comprise from about 104to about 109cells. In some embodiments, the volume may comprise from about 105to about 109cells. 29 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO

[0065] In some embodiments, the population of gene-edited cells comprises a signal of less than 0.4% mutation rate at an off target site. In some embodiments, the population of gene-edited cells comprises a signal of less than 0.5% mutation rate at an off target site.

[0066] In some embodiments, the RNA molecule is introduced via a transfection technique selected from the group consisting of electroporation, nanoparticle mediated transfection, magnetic bead-based transfection, and calcium phosphate precipitation. In some embodments, the RNA molecule is introduced via electroporation. Electroporation methods are known in the art and are described, e.g., in Tsong, Biophys. J.1991, 60, 297-306, and U.S. Patent Application Publication No. 2014 / 0227237 A1, the disclosures of each of which are incorporated by reference herein. Other electroporation methods known in the art, such as those described in U.S. Patent Nos.5,019,034; 5,128,257; 5,137,817; 5,173,158; 5,232,856; 5,273,525; 5,304,120; 5,318,514; 6,010,613 and 6,078,490, the disclosures of which are incorporated by reference herein, may be used. In some embodiments, the electroporation method is a sterile electroporation method. In some embodiments, the electroporation method is a pulsed electroporation method. In some embodiments, the electroporation method is a pulsed electroporation method comprising the steps of treating cells with pulsed electricalfields to alter, manipulate, or cause defined and controlled, permanent or temporary changes in the cells, comprising the step of applying a sequence of at least three single, operator-controlled, independently programmed, DC electrical pulses, havingfield strengths equal to or greater than 100 V / cm, to the cells, wherein the sequence of at least three DC electrical pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses differ from each other in pulse amplitude; (2) at least two of the at least three pulses differ from each other in pulse width; and (3) afirst pulse interval for afirst set of two of the at least three pulses is different from a second pulse interval for a second set of two of the at least three pulses. In some embodiments, the electroporation method is a pulsed electroporation method comprising the steps of treating cells with pulsed electricalfields to alter, manipulate, or cause defined and controlled, permanent or temporary changes in the cells, comprising the step of applying a sequence of at least three single, operator-controlled, independently programmed, DC electrical pulses, havingfield strengths equal to or greater than 100 V / cm, to the cells, wherein at least two of the at least three pulses differ from each other in pulse amplitude. In some embodiments, the electroporation method is a pulsed electroporation method comprising the steps of treating cells with pulsed electricalfields to alter, manipulate, or cause defined and controlled, permanent or temporary changes in the cells, comprising the step of applying a sequence of at least three single, operator-controlled, independently programmed, DC electrical pulses, havingfield 30 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO strengths equal to or greater than 100 V / cm, to the cells, wherein at least two of the at least three pulses differ from each other in pulse width. In some embodiments, the electroporation method is a pulsed electroporation method comprising the steps of treating cells with pulsed electricalfields to alter, manipulate, or cause defined and controlled, permanent or temporary changes in the cells, comprising the step of applying a sequence of at least three single, operator-controlled, independently programmed, DC electrical pulses, havingfield strengths equal to or greater than 100 V / cm, to the cells, wherein afirst pulse interval for afirst set of two of the at least three pulses is different from a second pulse interval for a second set of two of the at least three pulses. In some embodiments, the electroporation method is a pulsed electroporation method comprising the steps of treating cells with pulsed electricalfields to induce pore formation in the cells, comprising the step of applying a sequence of at least three DC electrical pulses, havingfield strengths equal to or greater than 100 V / cm, to cells, wherein the sequence of at least three DC electrical pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses differ from each other in pulse amplitude; (2) at least two of the at least three pulses differ from each other in pulse width; and (3) afirst pulse interval for afirst set of two of the at least three pulses is different from a second pulse interval for a second set of two of the at least three pulses, such that induced pores are sustained for a relatively long period of time, and such that viability of the cells is maintained.

[0067] In some embodiments, the volume comprises an electroporation buffer. In some embodiments, the electroporation buffer is CTS™ Xenon™ Genome Editing Buffer. In some embodiments, the electroporation is conducted using the Xenon™ electroporator with the settings 2300 V, 2 ms pulse width, 3 pulses. In some embodiments, the electroporation is conducted using the Neon™ electroporator. An example of a suitableflow electroporation system suitable for use with some embodiments of the present invention is the commercially-available MaxCyte STX system. There are several alternative commercially-available electroporation instruments which may be suitable for use with the present invention, such as the AgilePulse system or ECM 830 available from BTX-Harvard Apparatus, Cellaxess Elektra (Cellectricon), Nucleofector (Lonza / Amaxa), GenePulser MXcell (BIORAD), iPorator-96 (Primax) or siPORTer96 (Ambion).

[0068] In some embodiments, the RNA molecule is introduced via calcium phosphate transfection. Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating, and endocytosis) are known in the art and are described in Graham and van der Eb, Virology 1973, 52, 456-467; Wigler, et al., Proc. Natl. Acad. Sci.1979, 76, 1373-1376; and Chen and Okayarea, Mol. Cell. Biol.1987, 7, 2745-2752; and in U.S. Patent No.5,593,875, the disclosures of each of which are incorporated by reference herein. In some embodiments, the RNA molecule is 31 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO introduced via liposomal transfection. Liposomal transfection methods, such as methods that employ a 1:1 (w / w) liposome formulation of the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-n,n,n- trimethylammonium chloride (DOTMA) and dioleoyl phophotidylethanolamine (DOPE) infiltered water, are known in the art and are described in Rose, et al., Biotechniques 1991, 10, 520-525 and Felgner, et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 7413-7417 and in U.S. Patent Nos.5,279,833; 5,908,635; 6,056,938; 6,110,490; 6,534,484; and 7,687,070, the disclosures of each of which are incorporated by reference herein. In some embodiments, the RNA molecule is introduced via transfection using methods described in U.S. Patent Nos.5,766,902; 6,025,337; 6,410,517; 6,475,994; and 7,189,705; the disclosures of each of which are incorporated by reference herein.

[0069] The population of cells may be activated before introducing the RNA molecule, for example, for 1 day, 2 days, 3 days, 4 days, 5 days, or more. In some embodiments, the method further comprises activating the population of cells for 1 day or 2 days before introducing the RNA molecule. In some embodiments, the method further comprises activating the population of cells for 1 day before introducing the RNA molecule. In some embodiments, the method further comprises activating the population of cells for 2 days before introducing the RNA molecule. In some embodiments, the method further comprises activating the population of cells for 3 days before introducing the RNA molecule.

[0070] The population of cells may be rested after introducing the RNA molecule, for example, for 1 day, 2 days, 3 days, 4 days, 5 days, or more. In some embodiments, the method further comprises resting the population of cells for 1 day or 2 days after introducing the RNA molecule. In some embodiments, the method further comprises resting the population of cells for 1 day after introducing the RNA molecule. In some embodiments, the method further comprises resting the population of cells for 2 days after introducing the RNA molecule. In some embodiments, the method further comprises resting the population of cells for 3 days after introducing the RNA molecule.

[0071] In some embodiments, the step of activating the population of cells is performed using anti-CD3 agonist and anti-CD28 agonist, such as TransAct. In some embodiments, the step of activating the population of cells is performed using TransAct at 1:10 dilution, at 1:17.5 dilution, at 1:20 dilution, at 1:25 dilution, at 1:30 dilution, at 1:40 dilution, at 1:50 dilution, at 1:60 dilution, at 1:70 dilution, at 1:80 dilution, at 1:90 dilution, at 1:100 dilution, at 1:200 dilution, at 1:300 dilution, at 1:400 dilution, at 1:500 dilution, or at 1:1,000 dilution.

[0072] In some embodiments, the programmable nuclease cuts a DNA sequence in a target gene. For example, the programmable nuclease cuts a DNA sequence in an immune checkpoint gene, a TCR 32 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO gene, a PCSK9 gene, a hATTR gene, aSOD1 gene, a TAU gene, a LRRK2 gene, a SMN2 gene, a TTR gene, a Factor XI gene, an APOL1 gene, a CCR5 gene, etc. exemplary immune checkpoint genes include 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.

[0073] In some embodiments, the introducing the RNA molecule results in decreased and / or reduced expression of the target gene. For example, the introducing the RNA molecule reduces the expression of target gene by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%.

[0074] In some embodiments, the introducing the RNA molecule results in decreased and / or reduced expression of a gene selected from the group consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF (BR3), and combinations thereof. In some embodiments, the introducing the RNA molecule results in decreased and / or reduced expression of two genes selected from the group consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF (BR3), and combinations thereof. In some embodiments, the introducing the RNA molecule results in decreased and / or reduced expression of PD-1 and one gene selected from the group consisting of LAG3, TIM3, CTLA-4, TIGIT, CISH, TGFβR2, PKA, CBLB, BAFF (BR3), and combinations thereof. In some embodiments, the introducing the RNA molecule results in decreased and / or reduced expression of PD-1, LAG-3, CISH, CTLA-4, TIGIT, CBLB, TIM3, and combinations thereof. In some embodiments, the introducing the RNA molecule results in decreased and / or reduced expression of PD-1 and one of LAG3, CISH, CTLA-4, TIGIT, CBLB, TIM3, and combinations thereof. In some embodiments, the introducing the RNA molecule results in decreased and / or reduced expression of PD-1 and CTLA-4. In some embodiments, the introducing the RNA molecule results in decreased and / or reduced expression of PD-1 and TIGIT. In some embodiments, the introducing the RNA molecule results in decreased and / or reduced expression of PD-1 and LAG3. In some embodiments, the introducing the RNA molecule results in decreased and / or reduced expression of PD-1 and CISH. In some embodiments, the introducing the RNA molecule results in decreased and / or reduced expression of PD-1 and CBLB. In some embodiments, the introducing the RNA molecule results in decreased and / or reduced expression of CTLA-4 and TIGIT. In some embodiments, the introducing the RNA molecule results in decreased and / or reduced expression of CTLA-4 and LAG3. In some embodiments, the introducing the RNA molecule results in decreased and / or reduced expression of 33 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO CTLA-4 and CISH. In some embodiments, the introducing the RNA molecule results in decreased and / or reduced expression of CTLA-4 and CBLB. In some embodiments, the introducing the RNA molecule results in decreased and / or reduced expression of CTLA-4 and TIM3. In some embodiments, the introducing the RNA molecule results in decreased and / or reduced expression of TIGIT and LAG3. In some embodiments, the introducing the RNA molecule results in decreased and / or reduced expression of TIGIT and CISH. In some embodiments, the introducing the RNA molecule results in decreased and / or reduced expression of TIGIT and CBLB. In some embodiments, the introducing the RNA molecule results in decreased and / or reduced expression of TIGIT and TIM3. In some embodiments, the introducing the RNA molecule results in decreased and / or reduced expression of LAG3 and CISH. In some embodiments, the introducing the RNA molecule results in decreased and / or reduced expression of LAG3 and CBLB. In some embodiments, the introducing the RNA molecule results in decreased and / or reduced expression of CISH and CBLB. In some embodiments, the introducing the RNA molecule results in decreased and / or reduced expression of TIM3 and PD-1. In some embodiments, the introducing the RNA molecule results in decreased and / or reduced expression of TIM3 and LAG3. In some embodiments, the introducing the RNA molecule results in decreased and / or reduced expression of TIM3 and CISH. In some embodiments, the introducing the RNA molecule results in decreased and / or reduced expression of TIM3 and CBLB.

[0075] In some embodiments, there is a reduction in expression of about 5%, about 10%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, there is a reduction in expression of at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, there is a reduction in expression of at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, there is a reduction in expression of at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, there is a reduction in expression of at least about 85%, about 90%, or about 95%. In some embodiments, there is a reduction in expression of at least about 80%. In some embodiments, there is a reduction in expression of at least about 85%, In some embodiments, there is a reduction in expression of at least about 90%. In some embodiments, there is a reduction in expression of at least about 95%. In some embodiments, there is a reduction in expression of at least about 99%.

[0076] In some embodiments, the population of gene-edited cells comprises at least 10% knockout (KO) efficiency of the target gene. In some embodiments, the population of gene-edited cells comprises at least 20% knockout (KO) efficiency of the target gene. In some embodiments, the 34 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO population of gene-edited cells comprises at least 30% knockout (KO) efficiency of the target gene. In some embodiments, the population of gene-edited cells comprises at least 40% knockout (KO) efficiency of the target gene. In some embodiments, the population of gene-edited cells comprises at least 50% knockout (KO) efficiency of the target gene. In some embodiments, the population of gene- edited cells comprises at least 60% knockout (KO) efficiency of the target gene. In some embodiments, the population of gene-edited cells comprises at least 70% knockout (KO) efficiency of the target gene. In some embodiments, the population of gene-edited cells comprises at least 80% knockout (KO) efficiency of the target gene. In some embodiments, the population of gene-edited cells comprises at least 90% knockout (KO) efficiency of the target gene. In some embodiments, the population of gene-edited cells comprises at least 95% knockout (KO) efficiency of the target gene. In some embodiments, the population of gene-edited cells comprises at least 98% knockout (KO) efficiency of the target gene. In some embodiments, the population of gene-edited cells comprises at least 99% knockout (KO) efficiency of the target gene. A. Programmable Nucleases

[0077] According to an embodiment, the gene-editing process may comprise the use of a programmable nuclease that mediates the generation of a double-strand or single-strand break at one or more immune checkpoint genes. Such programmable nucleases enable precise genome editing by introducing breaks at specific genomic loci, i.e., they rely on the recognition of a specific DNA sequence within the genome to target a nuclease domain to this location and mediate the generation of a double-strand break at the target sequence. A double-strand break in the DNA subsequently recruits endogenous repair machinery to the break site to mediate genome editing by either non-homologous end-joining (NHEJ) or homology-directed repair (HDR). Thus, the repair of the break can result in the introduction of insertion / deletion mutations that disrupt (e.g., silence, repress, or enhance) the target gene product.

[0078] Major classes of nucleases that have been developed to enable site-specific genomic editing include zincfinger nucleases (ZFNs), transcription activator-like nucleases (TALENs), CRISPR- associated nucleases (e.g., CRISPR / Cas9), and meganucleases (MNs). These nuclease systems can be broadly classified into two categories based on their mode of DNA recognition: ZFNs, TALENs and MNs achieve specific DNA binding via protein-DNA interactions, whereas CRISPR systems, such as Cas9, are targeted to specific DNA sequences by a short RNA guide molecule that base-pairs directly with the target DNA and by protein-DNA interactions. See, e.g., Cox et al., Nature Medicine, 2015, Vol.21, No.2. 35 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO

[0079] Non-limiting examples of gene-editing methods that may be used in accordance with TIL expansion methods of the present invention include CRISPR methods, TALE methods, ZFN methods, and MN methods, embodiments of which are described in more detail below. According to an embodiment, a method for expanding TILs into a therapeutic population may be carried out in accordance with any embodiment of the methods described herein (e.g., process 2A) or as described in PCT / US2017 / 058610, PCT / US2018 / 012605, or PCT / US2018 / 012633, wherein the method further comprises gene-editing at least a portion of the TILs by one or more of a CRISPR method, a TALE method, a ZFN method, or an MN method, in order to generate TILs that can provide an enhanced therapeutic effect. According to an embodiment, gene-edited TILs can be evaluated for an improved therapeutic effect by comparing them to non-modified TILs in vitro, e.g., by evaluating in vitro effector function, cytokine profiles, etc. compared to unmodified TILs.

[0080] In some embodiments, of the present invention, electroporation is used for delivery of a gene editing system, such as CRISPR, TALEN, ZFN, and MN. In some embodiments, of the present invention, the electroporation system is aflow electroporation system. An example of a suitableflow electroporation system suitable for use with some embodiments of the present invention is the commercially-available MaxCyte STX system. There are several alternative commercially-available electroporation instruments which may be suitable for use with the present invention, such as the AgilePulse system or ECM 830 available from BTX-Harvard Apparatus, Cellaxess Elektra (Cellectricon), Nucleofector (Lonza / Amaxa), GenePulser MXcell (BIORAD), iPorator-96 (Primax) or siPORTer96 (Ambion). In some embodiments, of the present invention, the electroporation system forms a closed, sterile system with the remainder of the TIL expansion method. In some embodiments, of the present invention, the electroporation system is a pulsed electroporation system as described herein, and forms a closed, sterile system with the remainder of the TIL expansion method.

[0081] In some embodiments of the present invention, nanoparticle mediated transfection (e.g., cationic polymer based transfection, cationic lipid-based transfection, polyethylenimine (PEI)-based transfection, etc.), magnetic bead based transfection, calcium phosphate precipitation based transfection, or microinjection is used for delivery of a gene editing system, such as CRISPR, TALEN, ZFN, and MN. 1. CRISPR Methods

[0082] A method for expanding TILs into a therapeutic population may be carried out in accordance with any embodiment of the methods described herein (e.g., process 2A) or as described in PCT / US2017 / 058610, PCT / US2018 / 012605, or PCT / US2018 / 012633, wherein the method further 36 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO comprises gene-editing at least a portion of the TILs by a CRISPR method (e.g., CRISPR / Cas9 or CRISPR / Cpf1). According to particular embodiments, the use of a CRISPR method during the TIL expansion process causes expression of one or more immune checkpoint genes to be silenced or reduced in at least a portion of the therapeutic population of TILs. Alternatively, the use of a CRISPR method during the TIL expansion process causes expression of one or more immune checkpoint genes to be enhanced in at least a portion of the therapeutic population of TILs.

[0083] CRISPR stands for “Clustered Regularly Interspaced Short Palindromic Repeats.” A method of using a CRISPR system for gene editing is also referred to herein as a CRISPR method. CRISPR systems can be divided into two main classes, Class 1 and Class 2, which are further classified into different types and sub-types. The classification of the CRISPR systems is based on the effector Cas proteins that are capable of cleaving specific nucleic acids. In Class 1 CRISPR systems the effector module consists of a multi-protein complex, whereas Class 2 systems only use one effector protein. Class 1 CRISPR includes Types I, III, and IV and Class 2 CRISPR includes Types II, V, and VI. While any of these types of CRISPR systems may be used in accordance with the present invention, there are three types of CRISPR systems which incorporate RNAs and Cas proteins that are preferred for use in accordance with the present invention: Types I (exemplified by Cas3), II (exemplified by Cas9), and III (exemplified by Cas10). The Type II CRISPR is one of the most well-characterized systems.

[0084] CRISPR technology was adapted from the natural defense mechanisms of bacteria and archaea (the domain of single-celled microorganisms). These organisms use CRISPR-derived RNA and various Cas proteins, including Cas9, to foil attacks by viruses and other foreign bodies by chopping up and destroying the DNA of a foreign invader. A CRISPR is a specialized region of DNA with two distinct characteristics: the presence of nucleotide repeats and spacers. Repeated sequences of nucleotides are distributed throughout a CRISPR region with short segments of foreign DNA (spacers) interspersed among the repeated sequences. In the type II CRISPR / Cas system, spacers are integrated within the CRISPR genomic loci and transcribed and processed into short CRISPR RNA (crRNA). These crRNAs anneal to trans-activating crRNAs (tracrRNAs) and direct sequence-specific cleavage and silencing of pathogenic DNA by Cas proteins. Target recognition by the Cas9 protein requires a “seed” sequence within the crRNA and a conserved dinucleotide-containing protospacer adjacent motif (PAM) sequence upstream of the crRNA-binding region. The CRISPR / Cas system can thereby be retargeted to cleave virtually any DNA sequence by redesigning the crRNA. Thus, according to certain embodiments, Cas9 serves as an RNA-guided DNA endonuclease that cleaves DNA upon crRNA-tracrRNA recognition. The crRNA and tracrRNA in the native system can be simplified into a single guide RNA (sgRNA) of approximately 100 nucleotides for use in genetic 37 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO engineering. The sgRNA is a synthetic RNA that includes a scaffold sequence necessary for Cas- binding and a user-defined approximately 17- to 20-nucleotide spacer that defines the genomic target to be modified. Thus, a user can change the genomic target of the Cas protein by changing the target sequence present in the sgRNA. The CRISPR / Cas system is directly portable to human cells by co-delivery of plasmids expressing the Cas9 endo-nuclease and the RNA components (e.g., sgRNA). Different variants of Cas proteins may be used to reduce targeting limitations (e.g., orthologs of Cas9, such as Cpf1).

[0085] According to some embodiments, an engineered, programmable, non-naturally occurring Type II CRISPR-Cas system comprises a Cas9 protein and at least one guide RNA that targets and hybridizes to a target sequence of a DNA molecule in a TIL, wherein the DNA molecule encodes and the TIL expresses at least one immune checkpoint molecule and the Cas9 protein cleaves the DNA molecules, whereby expression of the at least one immune checkpoint molecule is altered; and, wherein the Cas9 protein and the guide RNA do not naturally occur together. According to an embodiment, the expression of two or more immune checkpoint molecules is altered. According to an embodiment, the guide RNA(s) comprise a guide sequence fused to a tracr sequence. For example, the guide RNA may comprise crRNA-tracrRNA or sgRNA. According to aspects of the present invention, the terms "guide RNA", "single guide RNA" and "synthetic guide RNA" may be used interchangeably and refer to the polynucleotide sequence comprising the guide sequence, which is the approximately 17-20 bp sequence within the guide RNA that specifies the target site.

[0086] Variants of Cas9 having improved on-target specificity compared to Cas9 may also be used in accordance with embodiments of the present invention. Such variants may be referred to as high- fidelity Cas-9s. According to an embodiment, a dual nickase approach may be utilized, wherein two nickases targeting opposite DNA strands generate a DSB within the target DNA (often referred to as a double nick or dual nickase CRISPR system). For example, this approach may involve the mutation of one of the two Cas9 nuclease domains, turning Cas9 from a nuclease into a nickase. Non-limiting examples of high-fidelity Cas9s include eSpCas9, SpCas9-HF1 and HypaCas9. Such variants may reduce or eliminate unwanted changes at non-target DNA sites. See, e.g., Slaymaker IM, et al. Science.2015 Dec 1, Kleinstiver BP, et al. Nature.2016 Jan 6, and Ran et al., Nat Protoc.2013 Nov; 8(11):2281-2308, the disclosures of which are incorporated by reference herein.

[0087] Additionally, according to particular embodiments, Cas9 scaffolds may be used that improve gene delivery of Cas9 into cells and improve on-target specificity, such as those disclosed in U.S. Patent Application Publication No.2016 / 0102324, which is incorporated by reference herein. For example, Cas9 scaffolds may include a RuvC motif as defined by (D-[I / L]-G-X-X-S-X-G-W-A) and / or 38 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO a HNH motif defined by (Y-X-X-D-H-X-X-P-X-S-X-X-X-D-X-S), where X represents any one of the 20 naturally occurring amino acids and [I / L] represents isoleucine or leucine. The HNH domain is responsible for nicking one strand of the target dsDNA and the RuvC domain is involved in cleavage of the other strand of the dsDNA. Thus, each of these domains nick a strand of the target DNA within the protospacer in the immediate vicinity of PAM, resulting in blunt cleavage of the DNA. These motifs may be combined with each other to create more compact and / or more specific Cas9 scaffolds. Further, the motifs may be used to create a split Cas9 protein (i.e., a reduced or truncated form of a Cas9 protein or Cas9 variant that comprises either a RuvC domain or a HNH domain) that is divided into two separate RuvC and HNH domains, which can process the target DNA together or separately.

[0088] According to particular embodiments, a CRISPR method comprises silencing or reducing the expression of one or more immune checkpoint genes in TILs by introducing a Cas9 nuclease and a guide RNA (e.g., crRNA-tracrRNA or sgRNA) containing a sequence of approximately 17-20 nucleotides specific to a target DNA sequence of the immune checkpoint gene(s). The guide RNA may be delivered as RNA or by transforming a plasmid with the guide RNA-coding sequence under a promoter. The CRISPR / Cas enzymes introduce a double-strand break (DSB) at a specific location based on a sgRNA-defined target sequence. DSBs may be repaired in the cells by non-homologous end joining (NHEJ), a mechanism which frequently causes insertions or deletions (indels) in the DNA. Indels often lead to frameshifts, creating loss of function alleles; for example, by causing premature stop codons within the open reading frame (ORF) of the targeted gene. According to certain embodiments, the result is a loss-of-function mutation within the targeted immune checkpoint gene.

[0089] Alternatively, DSBs induced by CRISPR / Cas enzymes may be repaired by homology-directed repair (HDR) instead of NHEJ. While NHEJ-mediated DSB repair often disrupts the open reading frame of the gene, homology directed repair (HDR) can be used to generate specific nucleotide changes ranging from a single nucleotide change to large insertions. According to an embodiment, HDR is used for gene editing immune checkpoint genes by delivering a DNA repair template containing the desired sequence into the TILs with the sgRNA(s) and Cas9 or Cas9 nickase. The repair template preferably contains the desired edit as well as additional homologous sequence immediately upstream and downstream of the target gene (often referred to as left and right homology arms).

[0090] According to particular embodiments, an enzymatically inactive version of Cas9 (deadCas9 or dCas9) may be targeted to transcription start sites in order to repress transcription by blocking initiation. Thus, targeted immune checkpoint genes may be repressed without the use of a DSB. A 39 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO dCas9 molecule retains the ability to bind to target DNA based on the sgRNA targeting sequence. According to an embodiment of the present invention, a CRISPR method comprises silencing or reducing the expression of one or more immune checkpoint genes by inhibiting or preventing transcription of the targeted gene(s). For example, a CRISPR method may comprise fusing a transcriptional repressor domain, such as a Kruppel-associated box (KRAB) domain, to an enzymatically inactive version of Cas9, thereby forming, e.g., a dCas9-KRAB, that targets the immune checkpoint gene’s transcription start site, leading to the inhibition or prevention of transcription of the gene. Preferably, the repressor domain is targeted to a window downstream from the transcription start site, e.g., about 500 bp downstream. This approach, which may be referred to as CRISPR interference (CRISPRi), leads to robust gene knockdown via transcriptional reduction of the target RNA.

[0091] According to particular embodiments, an enzymatically inactive version of Cas9 (deadCas9 or dCas9) may be targeted to transcription start sites in order to activate transcription. This approach may be referred to as CRISPR activation (CRISPRa). According to an embodiment, a CRISPR method comprises increasing the expression of one or more immune checkpoint genes by activating transcription of the targeted gene(s). According to such embodiments, targeted immune checkpoint genes may be activated without the use of a DSB. A CRISPR method may comprise targeting transcriptional activation domains to the transcription start site; for example, by fusing a transcriptional activator, such as VP64, to dCas9, thereby forming, e.g., a dCas9-VP64, that targets the immune checkpoint gene’s transcription start site, leading to activation of transcription of the gene. Preferably, the activator domain is targeted to a window upstream from the transcription start site, e.g., about 50-400 bp downstream.

[0092] Additional embodiments of the present invention may utilize activation strategies that have been developed for potent activation of target genes in mammalian cells. Non-limiting examples include co-expression of epitope-tagged dCas9 and antibody-activator effector proteins (e.g., the SunTag system), dCas9 fused to a plurality of different activation domains in series (e.g., dCas9-VPR) or co-expression of dCas9-VP64 with a modified scaffold gRNA and additional RNA- binding helper activators (e.g., SAM activators).

[0093] According to other embodiments, a CRISPR-mediated genome editing method referred to as CRISPR assisted rational protein engineering (CARPE) may be used in accordance with embodiments of the present invention, as disclosed in US Patent No.9,982,278, which is incorporated by reference herein. CARPE involves the generation of “donor” and “destination” libraries that incorporate directed mutations from single-stranded DNA (ssDNA) or double-stranded 40 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO DNA (dsDNA) editing cassettes directly into the genome. Construction of the donor library involves cotransforming rationally designed editing oligonucleotides into cells with a guide RNA (gRNA) that hybridizes to a target DNA sequence. The editing oligonucleotides are designed to couple deletion or mutation of a PAM with the mutation of one or more desired codons in the adjacent gene. This enables the entire donor library to be generated in a single transformation. The donor library is retrieved by amplification of the recombinant chromosomes, such as by a PCR reaction, using a synthetic feature from the editing oligonucleotide, namely, a second PAM deletion or mutation that is simultaneously incorporated at the 3’ terminus of the gene. This covalently couples the codon target mutations directed to a PAM deletion. The donor libraries are then co-transformed into cells with a destination gRNA vector to create a population of cells that express a rationally designed protein library.

[0094] According to other embodiments, methods for trackable, precision genome editing using a CRISPR-mediated system referred to as Genome Engineering by Trackable CRISPR Enriched Recombineering (GEn-TraCER) may be used in accordance with embodiments of the present invention, as disclosed in US Patent No.9,982,278, which is incorporated by reference herein. The GEn-TraCER methods and vectors combine an editing cassette with a gene encoding gRNA on a single vector. The cassette contains a desired mutation and a PAM mutation. The vector, which may also encode Cas9, is the introduced into a cell or population of cells. This activates expression of the CRISPR system in the cell or population of cells, causing the gRNA to recruit Cas9 to the target region, where a dsDNA break occurs, allowing integration of the PAM mutation.

[0095] Examples of systems, methods, and compositions for altering the expression of a target gene sequence by a CRISPR method, and which may be used in accordance with embodiments of the present invention, are described in U.S. Patent Nos.8,697,359; 8,993,233; 8,795,965; 8,771,945; 8,889,356; 8,865,406; 8,999,641; 8,945,839; 8,932,814; 8,871,445; 8,906,616; and 8,895,308, which are incorporated by reference herein. Resources for carrying out CRISPR methods, such as plasmids for expressing CRISPR / Cas9 and CRISPR / Cpf1, are commercially available from companies such as GenScript.

[0096] In some embodiments, genetic modifications of populations of TILs, as described herein, may be performed using the CRISPR / Cpf1 system as described in U.S. Patent No. US 9,790,490, the disclosure of which is incorporated by reference herein. The CRISPR / Cpf1 system is functionally distinct from the CRISPR-Cas9 system in that Cpf1-associated CRISPR arrays are processed into mature crRNAs without the need for an additional tracrRNA. The crRNAs used in the CRISPR / Cpf1 41 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO system have a spacer or guide sequence and a direct repeat sequence. The Cpf1p-crRNA complex that is formed using this method is sufficient by itself to cleave the target DNA. 2. TALE Methods

[0097] A method for expanding TILs into a therapeutic population may be carried out in accordance with any embodiment of the methods described herein (e.g., process 2A) or as described in PCT / US2017 / 058610, PCT / US2018 / 012605, or PCT / US2018 / 012633, wherein the method further comprises gene-editing at least a portion of the TILs by a TALE method. According to particular embodiments, the use of a TALE method during the TIL expansion process causes expression of one or more immune checkpoint genes to be silenced or reduced in at least a portion of the therapeutic population of TILs. Alternatively, the use of a TALE method during the TIL expansion process causes expression of one or more immune checkpoint genes to be enhanced in at least a portion of the therapeutic population of TILs.

[0098] TALE stands for “Transcription Activator-Like Effector” proteins, which include TALENs (“Transcription Activator-Like Effector Nucleases”). A method of using a TALE system for gene editing may also be referred to herein as a TALE method. TALEs are naturally occurring proteins from the plant pathogenic bacteria genus Xanthomonas, and contain DNA-binding domains composed of a series of 33–35-amino-acid repeat domains that each recognizes a single base pair. TALE specificity is determined by two hypervariable amino acids that are known as the repeat-variable di-residues (RVDs). Modular TALE repeats are linked together to recognize contiguous DNA sequences. A specific RVD in the DNA-binding domain recognizes a base in the target locus, providing a structural feature to assemble predictable DNA-binding domains. The DNA binding domains of a TALE are fused to the catalytic domain of a type IIS FokI endonuclease to make a targetable TALE nuclease. To induce site- specific mutation, two individual TALEN arms, separated by a 14-20 base pair spacer region, bring FokI monomers in close proximity to dimerize and produce a targeted double-strand break.

[0099] Several large, systematic studies utilizing various assembly methods have indicated that TALE repeats can be combined to recognize virtually any user-defined sequence. Strategies that enable the rapid assembly of custom TALE arrays include Golden Gate molecular cloning, high- throughput solid-phase assembly, and ligation-independent cloning techniques. Custom-designed TALE arrays are also commercially available through Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, KY, USA), and Life Technologies (Grand Island, NY, USA). Additionally web-based tools, such as TAL Effector-Nucleotide Target 2.0, are available that enable the design of custom TAL effector repeat arrays for desired targets and also provides predicted TAL 42 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO effector binding sites. See Doyle, et al., Nucleic Acids Research, 2012, Vol.40, W117-W122. Examples of TALE and TALEN methods suitable for use in the present invention are described in U.S. Patent Application Publication Nos. US 2011 / 0201118 A1; US 2013 / 0117869 A1; US 2013 / 0315884 A1; US 2015 / 0203871 A1 and US 2016 / 0120906 A1, the disclosures of which are incorporated by reference herein.

[0100] According to an embodiment of the present invention, a TALE method comprises silencing or reducing the expression of one or more immune checkpoint genes by inhibiting or preventing transcription of the targeted gene(s). For example, a TALE method may include utilizing KRAB-TALEs, wherein the method comprises fusing a transcriptional Kruppel-associated box (KRAB) domain to a DNA binding domain that targets the gene’s transcription start site, leading to the inhibition or prevention of transcription of the gene.

[0101] According to another embodiment, a TALE method comprises silencing or reducing the expression of one or more immune checkpoint genes by introducing mutations in the targeted gene(s). For example, a TALE method may include fusing a nuclease effector domain, such as Fokl, to the TALE DNA binding domain, resulting in a TALEN. Fokl is active as a dimer; hence, the method comprises constructing pairs of TALENs to position the FOKL nuclease domains to adjacent genomic target sites, where they introduce DNA double strand breaks. A double strand break may be completed following correct positioning and dimerization of Fokl. Once the double strand break is introduced, DNA repair can be achieved via two different mechanisms: the high-fidelity homologous recombination pair (HRR) (also known as homology-directed repair or HDR) or the error-prone non- homologous end joining (NHEJ). Repair of double strand breaks via NHEJ preferably results in DNA target site deletions, insertions or substitutions, i.e., NHEJ typically leads to the introduction of small insertions and deletions at the site of the break, often inducing frameshifts that knockout gene function. According to particular embodiments, the TALEN pairs are targeted to the most 5’ exons of the genes, promoting early frame shift mutations or premature stop codons. The genetic mutation(s) introduced by TALEN are preferably permanent. Thus, according to some embodiments, the method comprises silencing or reducing expression of an immune checkpoint gene by utilizing dimerized TALENs to induce a site-specific double strand break that is repaired via error-prone NHEJ, leading to one or more mutations in the targeted immune checkpoint gene.

[0102] According to additional embodiments, TALENs are utilized to introduce genetic alterations via HRR, such as non-random point mutations, targeted deletion, or addition of DNA fragments. The introduction of DNA double strand breaks enables gene editing via homologous recombination in the presence of suitable donor DNA. According to an embodiment, the method comprises co-delivering 43 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO dimerized TALENs and a donor plasmid bearing locus-specific homology arms to induce a site-specific double strand break and integrate one or more transgenes into the DNA.

[0103] According to another embodiment, a TALEN that is a hybrid protein derived from FokI and AvrXa7, as disclosed in U.S. Patent Publication No.2011 / 0201118, may be used in accordance with embodiments of the present invention. This TALEN retains recognition specificity for target nucleotides of AvrXa7 and the double-stranded DNA cleaving activity of FokI. The same methods can be used to prepare other TALEN having different recognition specificity. For example, compact TALENs may be generated by engineering a core TALE scaffold having different sets of RVDs to change the DNA binding specificity and target a specific single dsDNA target sequence. See U.S. Patent Publication No.2013 / 0117869. A selection of catalytic domains can be attached to the scaffold to effect DNA processing, which may be engineered to ensure that the catalytic domain is capable of processing DNA near the single dsDNA target sequence when fused to the core TALE scaffold. A peptide linker may also be engineered to fuse the catalytic domain to the scaffold to create a compact TALEN made of a single polypeptide chain that does not require dimerization to target a specific single dsDNA sequence. A core TALE scaffold may also be modified by fusing a catalytic domain, which may be a TAL monomer, to its N-terminus, allowing for the possibility that this catalytic domain might interact with another catalytic domain fused to another TAL monomer, thereby creating a catalytic entity likely to process DNA in the proximity of the target sequences. See U.S. Patent Publication No.2015 / 0203871. This architecture allows only one DNA strand to be targeted, which is not an option for classical TALEN architectures.

[0104] According to an embodiment of the present invention, conventional RVDs may be used create TALENs that are capable of significantly reducing gene expression. In some embodiments, four RVDs, NI, HD, NN, and NG, are used to target adenine, cytosine, guanine, and thymine, respectively. These conventional RVDs can be used to, for instance, create TALENs targeting the PD-1 gene. Examples of TALENs using conventional RVDs include the T3v1 and T1 TALENs disclosed in Gautron et al., Molecular Therapy: Nucleic Acids Dec.2017, Vol.9:312-321 (Gautron), which is incorporated by reference herein. The T3v1 and T1 TALENs target the second exon of the PDCD1 locus where the PD-L1 binding site is located and are able to considerably reduce PD-1 production.

[0105] According to another embodiment, TALENs are modified using non-conventional RVDs to improve their activity and specificity for a target gene, such as disclosed in Gautron. Naturally occurring RVDs only cover a small fraction of the potential diversity repertoire for the hypervariable amino acid locations. Non-conventional RVDs provide an alternative to natural RVDs and have novel intrinsic targeting specificity features that can be used to exclude the targeting of off-site targets 44 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO (sequences within the genome that contain a few mismatches relative to the targeted sequence) by TALEN. Non-conventional RVDs may be identified by generating and screening collections of TALEN containing alternative combinations of amino acids at the two hypervariable amino acid locations at defined positions of an array as disclosed in Juillerat, et al., Scientific Reports 5, Article Number 8150 (2015), which is incorporated by reference herein. Next, non-conventional RVDs may be selected that discriminate between the nucleotides present at the position of mismatches, which can prevent TALEN activity at off-site sequences while still allowing appropriate processing of the target location. The selected non-conventional RVDs may then be used to replace the conventional RVDs in a TALEN. Examples of TALENs where conventional RVDs have been replaced by non-conventional RVDs include the T3v2 and T3v3 PD-1 TALENs produced by Gautron. These TALENs had increased specificity when compared to TALENs using conventional RVDs.

[0106] According to additional embodiments, TALEN may be utilized to introduce genetic alterations to silence or reduce the expression of two genes. For instance, two separate TALEN may be generated to target two different genes and then used together. The molecular events generated by the two TALEN at their respective loci and potential off-target sites may be characterized by high- throughput DNA sequencing. This enables the analysis of off-target sites and identification of the sites that might result from the use of both TALEN. Based on this information, appropriate conventional and non-conventional RVDs may be selected to engineer TALEN that have increased specificity and activity even when used together. For example, Gautron discloses the combined use of T3v4 PD-1 and TRAC TALEN to produce double knockout CAR T cells, which maintained a potent in vitro anti-tumor function.

[0107] According to some embodiments, TALENs may be specifically designed, which allows higher rates of DSB events within the target cell(s) that are able to target a specific selection of genes. See U.S. Patent Publication No.2013 / 0315884. The use of such rare cutting endonucleases increases the chances of obtaining double inactivation of target genes in transfected cells, allowing for the production of engineered cells, such as T-cells. Further, additional catalytic domains can be introduced with the TALEN to increase mutagenesis and enhance target gene inactivation. The TALENs described in U.S. Patent Publication No.2013 / 0315884 were successfully used to engineer T- cells to make them suitable for immunotherapy. TALENs may also be used to inactivate various immune checkpoint genes in T-cells, including the inactivation of at least two genes in a single T-cell. See U.S. Patent Publication No.2016 / 0120906. Additionally, TALENs may be used to inactivate genes encoding targets for immunosuppressive agents and T-cell receptors, as disclosed in U.S. Patent Publication No.2018 / 0021379, which is incorporated by reference herein. Further, TALENs may be 45 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO used to inhibit the expression of beta 2-microglobulin (B2M) and / or class II major histocompatibility complex transactivator (CIITA), as disclosed in U.S. Patent Publication No.2019 / 0010514, which is incorporated by reference herein.

[0108] Non-limiting examples of genes that may be silenced or inhibited by permanently gene- editing TILs via a TALE method include PD-1, TGIT, TET2, TGFβR2, PRA, BAFF (BR3), 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, SOCS1, ANKRD11, BCOR, and any combinations thereof (e.g., two more more of the above immune checkpoints, such as PD-1 and LAG3, PD-1 and TIM3, TIM3 and CTLA-4, etc.).

[0109] Non-limiting examples of TALE-nucleases targeting the PD-1 gene are provided in the following table. In these examples, the targeted genomic sequences contain two 17-base pair (bp) long sequences (referred to as half targets, shown in upper case letters) separated by a 15-bp spacer (shown in lower case letters). Each half target is recognized by repeats of half TALE-nucleases listed in Table 4. Thus, according to particular embodiments, TALE-nucleases according to the invention recognize and cleave the target sequence selected from the group consisting of: SEQ ID NO: 40 and SEQ ID NO: 41. TALEN sequences and gene-editing methods are also described in Gautron, discussed above. TABLE 4 – TALEN PD-1 Sequences.46 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO47 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO48 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO49 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO50 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO51 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO52 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO53 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO

[0110] Examples of systems, methods, and compositions for altering the expression of a target gene sequence by a TALE method, and which may be used in accordance with embodiments of the present invention, are described in U.S. Patent No.8,586,526, which is incorporated by reference herein. These disclosed examples include the use of a non-naturally occurring DNA-binding polypeptide that has two or more TALE-repeat units containing a repeat RVD, an N-cap polypeptide made of residues of a TALE protein, and a C-cap polypeptide made of a fragment of a full length C- terminus region of a TALE protein.

[0111] Examples of TALEN designs and design strategies, activity assessments, screening strategies, and methods that can be used to efficiently perform TALEN-mediated gene integration and inactivation, and which may be used in accordance with embodiments of the present invention, are described in Valton, et al., Methods, 2014, 69, 151-170, which is incorporated by reference herein. 3. Zinc Finger Methods

[0112] A method for expanding TILs into a therapeutic population may be carried out in accordance with any embodiment of the methods described herein (e.g., process 2A) or as described 54 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO in PCT / US2017 / 058610, PCT / US2018 / 012605, or PCT / US2018 / 012633, wherein the method further comprises gene-editing at least a portion of the TILs by a zincfinger or zincfinger nuclease method. According to particular embodiments, the use of a zincfinger method during the TIL expansion process causes expression of one or more immune checkpoint genes to be silenced or reduced in at least a portion of the therapeutic population of TILs. Alternatively, the use of a zincfinger method during the TIL expansion process causes expression of one or more immune checkpoint genes to be enhanced in at least a portion of the therapeutic population of TILs.

[0113] An individual zincfinger contains approximately 30 amino acids in a conserved ββα configuration. Several amino acids on the surface of the α-helix typically contact 3 bp in the major groove of DNA, with varying levels of selectivity. Zincfingers have two protein domains. Thefirst domain is the DNA binding domain, which includes eukaryotic transcription factors and contain the zincfinger. The second domain is the nuclease domain, which includes the FokI restriction enzyme and is responsible for the catalytic cleavage of DNA.

[0114] The DNA-binding domains of individual ZFNs typically contain between three and six individual zincfinger repeats and can each recognize between 9 and 18 base pairs. If the zincfinger domains are specific for their intended target site then even a pair of 3-finger ZFNs that recognize a total of 18 base pairs can, in theory, target a single locus in a mammalian genome. One method to generate new zinc-finger arrays is to combine smaller zinc-finger "modules" of known specificity. The most common modular assembly process involves combining three separate zincfingers that can each recognize a 3 base pair DNA sequence to generate a 3-finger array that can recognize a 9 base pair target site. Alternatively, selection-based approaches, such as oligomerized pool engineering (OPEN) can be used to select for new zinc-finger arrays from randomized libraries that take into consideration context-dependent interactions between neighboringfingers. Engineered zincfingers are available commercially; Sangamo Biosciences (Richmond, CA, USA) has developed a propriety platform (CompoZr®) for zinc-finger construction in partnership with Sigma–Aldrich (St. Louis, MO, USA).

[0115] Examples of systems, methods, and compositions for altering the expression of a target gene sequence by a zincfinger method, which may be used in accordance with embodiments of the present invention, are described in U.S. Patent Nos.6,534,261, 6,607,882, 6,746,838, 6,794,136, 6,824,978, 6,866,997, 6,933,113, 6,979,539, 7,013,219, 7,030,215, 7,220,719, 7,241,573, 7,241,574, 7,585,849, 7,595,376, 6,903,185, and 6,479,626, which are incorporated by reference herein. 55 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO

[0116] Other examples of systems, methods, and compositions for altering the expression of a target gene sequence by a zincfinger method, which may be used in accordance with embodiments of the present invention, are described in Beane, et al., Mol. Therapy, 2015, 231380-1390, the disclosure of which is incorporated by reference herein. 4. MN Methods

[0117] Naturally-occurring meganucleases (MNs) recognize 15-40 base-pair cleavage sites and are commonly grouped into four families: the LAGLIDADG family, the GTY-YIG family, the His-Cyst box family and the HNH family. Exemplary homing endonucleases include I-Scel, I-Ceul, PI-PspI, ΡΙ-Sce, 1- SceIV, I-Csml, I-Panl, I-Ppol, I-SceII, I-Crel, I-Tevl, I-TevII and I-TevIII. Their recognition sequences are known. See also U.S. Pat. No.5,420,032; U.S. Pat. No.6,833,252; Belfort et al. (1997) Nucleic Acids Res.25:3379-3388; Dujon et al. (1989) Gene 82: 115-118; Perler et al. (1994) Nucleic Acids Res.22, 1125-1127; Jasin (1996) Trends Genet.12:224-228; Gimble et al. (1996) J. Mol. Biol.263: 163-180; Argast et al. (1998) J. Mol. Biol.280:345-353 and the New England Biolabs catalogue. Thus, any meganuclease domain (or functional portion thereof) may be combined with any DNA-binding domain (e.g., ZFP, TALE) to form a nuclease. Furthermore, the nuclease domain may also bind to DNA independent of the DNA-binding domain. DNA-binding domains from naturally-occurring meganucleases, primarily from the LAGLIDADG family, have been used to promote site-specific genome modification in plants, yeast, Drosophila, mammalian cells and mice, but this approach has been limited to the modification of either homologous genes that conserve the meganuclease recognition sequence (Monet et al. (1999), Biochem. Biophysics. Res. Common.255: 88-93) or to pre-engineered genomes into which a recognition sequence has been introduced (Route et al. (1994), Mol. Cell. Biol.14: 8096-106; Chilton et al. (2003), Plant Physiology.133: 956-65; Puchta et al. (1996), Proc. Natl. Acad. Sci. USA 93: 5055-60; Rong et al. (2002), Genes Dev.16: 1568-81 ; Gouble et al. (2006), J. Gene Med.8(5):616-622). Accordingly, attempts have been made to engineer meganucleases to exhibit novel binding specificity at medically or biotechnologically relevant sites (Porteus et al. (2005), Nat. Biotechnol.23: 967-73; Sussman et al. (2004), J. Mol. Biol.342: 31-41 ; Epinat et al. (2003), Nucleic Acids Res.31 : 2952-62; Chevalier et al. (2002) Molec. Cell 10:895-905; Epinat et al. (2003) Nucleic Acids Res.31 :2952-2962; Ashworth et al. (2006) Nature 441 :656-659; Paques et al. (2007) Current Gene Therapy 7:49-66; U.S. Patent Publication Nos.2007 / 0117128; 2006 / 0206949; 2006 / 0153826; 2006 / 0078552; and 2004 / 0002092). In addition, naturally-occurring or engineered DNA-binding domains from meganucleases have also been operably linked with a cleavage domain from a heterologous nuclease (e.g., Fokl) (also known as mega TALs). In other embodiments, the nuclease is a zincfinger nuclease (ZFN). ZFNs comprise a zincfinger protein that 56 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO has been engineered to bind to a target site in a gene of choice and cleavage domain or a cleavage half-domain. III. Methods for Preparing Gene-Edited TILs

[0118] Embodiments of the present invention are directed to methods for preparing gene-edited tumor infiltrating lymphocytes (TILs) having reduced expression of one or two target genes. In some embodiments, the gene-edited TILs are prepared by introducing one or more programmable nucleases into the TILs.

[0119] In some embodiments, the programmable nuclease cuts a DNA sequence in a target gene. For example, the programmable nuclease cuts a DNA sequence in an immune checkpoint gene, a TCR gene, a PCSK9 gene, a hATTR gene, aSOD1 gene, a TAU gene, a LRRK2 gene, a SMN2 gene, a TTR gene, a Factor XI gene, an APOL1 gene, a CCR5 gene, etc. exemplary immune checkpoint genes include 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. A. Overview: TIL Expansion + Gene-Editing

[0120] Embodiments disclosed herein provide a method for expanding TILs into a therapeutic population that further comprises gene-editing at least a portion of the TILs to produce TILs having reduced expression of one or more target genes. According to particular embodiments, the introduction of one or more RNA molecules associated with programmable nucleases during the TIL expansion process causes expression of one or more target genes to be silenced or reduced in at least a portion of the therapeutic population of TILs.

[0121] 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 accordance with embodiments of the present invention, gene-editing technology is used to enhance the effectiveness of a therapeutic population of TILs. 57 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO

[0122] A method for expanded TILs having reduced expression of one or more target genes may be carried out in accordance with any embodiment of the methods described herein or by modifying the methods described in WO 2012 / 129201 A1, WO 2018 / 081473 A1, WO 2018 / 129332 A1, or WO 2018 / 182817 A1, the contents of which are herein incorporated by reference in their entireties, to incorporate steps for reducing the expression of one or more target genes in TILs as described herein. Briefly, in some embodiments, the method for expanding TILs comprises afirst expansion step of culturing a population of TILs in afirst cell culture medium comprising IL-2 for about 3-14 days (the “pre-REP” step), an activation step, a step of introducing afirst RNA molecule associated with a programmable nuclease, and a second expansion step of culturing a population of TILs after the second introducing step in a second cell culture medium comprising antigen presenting cells (APCs), OKT-3, and IL-2 for about 7-14 days (the “REP” step). In some embodiments, the method for expanding TILs comprises afirst expansion step of culturing a population of TILs in afirst cell culture medium comprising IL-2 for about 3-14 days (the “pre-REP” step), an activation step, a step of introducing afirst RNA molecule associated with a programmable nuclease, a resting step, a step of introducing a second RNA molecule associated with a programmable nuclease, and a second expansion step of culturing a population of TILs after the second introducing step in a second cell culture medium comprising antigen presenting cells (APCs), OKT-3, and IL-2 for about 7-14 days (the “REP” step).

[0123] In some embodiments, the method comprises: (a) performing afirst expansion of the population of TILs in a cell culture medium comprising IL-2, wherein thefirst expansion is performed for about 3-14 days; and (b) performing a second expansion of the population of TILs in a cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs), wherein the second expansion is performed for about 7-14 days, wherein the method further comprises introducing afirst RNA molecule associated with the programmable nuclease into the population of TILs in a volume, wherein the RNA molecule is present at afixed concentration not dependent on the number of TILs in the volume, and wherein thefirst RNA molecule is introduced before step (a), after step (a) and before step (b), or after step (b). B. Obtain Patient Tumor Sample 58 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO

[0124] 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, wherein the expanded TILs have been genetically modified via gene editing by introducing one or more RNA molecules associated with programmable nucleases.

[0125] 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 skin tissue. In some embodiments, useful TILs are obtained from a melanoma.

[0126] 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.

[0127] 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 59 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO 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 1-2 hours at 37°C, 5% CO2with rotation. In some embodiments, the tumors are digested overnight with constant rotation. In some embodiments, the tumors are digested overnight at 37°C, 5% CO2with constant rotation. In some embodiments, the whole tumor is combined with the enzymes to form a tumor digest reaction mixture.

[0128] In some embodiments, the tumor is reconstituted with the lyophilized enzymes in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] In some embodiments, the enzyme mixture comprises 10 mg / ml collagenase, 1000 IU / ml DNAse, and 1 mg / ml hyaluronidase.

[0133] In some embodiments, the enzyme mixture comprises 10 mg / ml collagenase, 500 IU / ml DNAse, and 1 mg / ml hyaluronidase.

[0134] 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.

[0135] In some embodiments, the enzyme mixture comprises neutral protease, DNase, and collagenase. 60 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO

[0136] 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.

[0137] In general, the harvested cell suspension is called a “primary cell population” or a “freshly harvested” cell population.

[0138] In some embodiments, fragmentation includes physical fragmentation, including for example, dissection as well as digestion. In some embodiments, the fragmentation is physical 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 by introducing one or more RNA molecules associated with programmable nucleases.

[0139] 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 thefirst expansion. In some embodiments, the tumor is fragmented and 30 or 40 fragments or pieces are placed in each container for thefirst expansion. In some embodiments, the tumor is fragmented and 40 fragments or pieces are placed in each container for thefirst 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.

[0140] 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 61 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO 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 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.

[0141] 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.

[0142] 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% CO2and 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.

[0143] In some embodiments, the harvested cell suspension prior to thefirst expansion step is called a “primary cell population” or a “freshly harvested” cell population. 62 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO

[0144] 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. C. First Expansion

[0145] After dissection or digestion of tumor fragments, the resulting cells are cultured in serum containing IL-2 under conditions that favor the growth of TILs over tumor and other cells. In some embodiments, the tumor digests are incubated in 2 mL wells in media comprising inactivated human AB serum with 6000 IU / mL of IL-2. This primary cell population is cultured for a period of days, generally from 3 to 14 days, resulting in a bulk TIL population, generally about 1 × 108bulk TIL cells, wherein the expanded TILs will be genetically modified via gene editing by introducing one or more RNA molecules associated with programmable nucleases. In some embodiments, this primary cell population is cultured for a period of 3 to 9 days, resulting in a bulk TIL population, generally about 1 × 108bulk TIL cells, wherein the expanded TILs will be genetically modified via gene editing by introducing one or more RNA molecules associated with programmable nucleases. In some embodiments, this primary cell population is cultured for a period of 5 to 7 days, resulting in a bulk TIL population, generally about 1 × 108bulk TIL cells, wherein the expanded TILs will be genetically modified via gene editing by introducing one or more RNA molecules associated with programmable nucleases. In some embodiments, this primary cell population is cultured for a period of about 7 days, resulting in a bulk TIL population, generally about 1 × 108bulk TIL cells, wherein the expanded TILs will be genetically modified via gene editing by introducing one or more RNA molecules associated with programmable nucleases.

[0146] In embodiments where TIL cultures are initiated in 24-well plates, for example, using Costar 24-well cell culture cluster,flat bottom (Corning Incorporated, Corning, NY, each well can be seeded with 1 × 106tumor digest cells or one tumor fragment in 2 mL of complete medium (CM) with IL-2 (6000 IU / mL; Chiron Corp., Emeryville, CA), wherein the expanded TILs will be genetically modified via gene editing by introducing one or more RNA molecules associated with programmable nucleases. In some embodiments, the tumor fragment is between about 1 mm3and 10 mm3.

[0147] In some embodiments, thefirst expansion culture medium is referred to as “CM”, an abbreviation for culture media. In some embodiments, CM for Step B consists of RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg / mL gentamicin. In embodiments where cultures are initiated in gas-permeableflasks with a 40 mL capacity and a 10 cm2gas-permeable silicon bottom (for example, G-Rex10; Wilson Wolf Manufacturing, New Brighton, MN, eachflask may be loaded with 10–40 × 106viable tumor digest cells or 5–30 tumor fragments in 63 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO 10–40 mL of CM with IL-2. Both the G-Rex10 and 24-well plates may be incubated in a humidified incubator at 37°C in 5% CO2and 5 days after culture initiation, half the media may be removed and replaced with fresh CM and IL-2 and after day 5, half the media may be changed every 2–3 days.

[0148] After preparation of the tumor fragments, the resulting cells (i.e., fragments) are cultured in serum containing IL-2 under conditions that favor the growth of TILs over tumor and other cells, wherein the TILs whose growth is favored will be genetically modified via gene editing by introducing one or more RNA molecules associated with programmable nucleases. In some embodiments, the tumor digests are incubated in 2 mL wells in media comprising inactivated human AB serum (or, in some cases, as outlined herein, in the presence of aAPC cell population) with 6000 IU / mL of IL-2. This primary cell population is cultured for a period of days, generally from 10 to 14 days, resulting in a bulk TIL population, generally about 1×108bulk TIL cells. In some embodiments, the growth media during thefirst expansion comprises IL-2 or a variant thereof. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments the IL-2 stock solution has a specific activity of 20-30×106IU / mg for a 1 mg vial. In some embodiments the IL-2 stock solution has a specific activity of 20×106IU / mg for a 1 mg vial. In some embodiments the IL-2 stock solution has a specific activity of 25×106IU / mg for a 1 mg vial. In some embodiments the IL-2 stock solution has a specific activity of 30×106IU / mg for a 1 mg vial. In some embodiments, the IL- 2 stock solution has afinal concentration of 4-8×106IU / mg of IL-2. In some embodiments, the IL- 2 stock solution has afinal concentration of 5-7×106IU / mg of IL-2. In some embodiments, the IL- 2 stock solution has afinal concentration of 6×106IU / mg of IL-2. In some embodiments, thefirst expansion culture media comprises about 10,000 IU / mL of IL-2, about 9,000 IU / mL of IL-2, about 8,000 IU / mL of IL-2, about 7,000 IU / mL of IL-2, about 6000 IU / mL of IL-2 or about 5,000 IU / mL of IL-2. In some embodiments, thefirst expansion culture media comprises about 9,000 IU / mL of IL-2 to about 5,000 IU / mL of IL-2. In some embodiments, thefirst expansion culture media comprises about 8,000 IU / mL of IL-2 to about 6,000 IU / mL of IL-2. In some embodiments, thefirst expansion culture media comprises about 7,000 IU / mL of IL-2 to about 6,000 IU / mL of IL-2. In some embodiments, thefirst expansion culture media comprises about 6,000 IU / mL of IL-2. In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU / mL of IL-2. In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU / mL of IL-2. In some embodiments, the cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL, about 7500 IU / mL, or 64 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO about 8000 IU / mL of IL-2. In some embodiments, the cell culture medium comprises between 1000 and 2000 IU / mL, between 2000 and 3000 IU / mL, between 3000 and 4000 IU / mL, between 4000 and 5000 IU / mL, between 5000 and 6000 IU / mL, between 6000 and 7000 IU / mL, between 7000 and 8000 IU / mL, between 1000 and 5000 IU / mL, or about 8000 IU / mL of IL-2.

[0149] In some embodiments, the cell culture medium comprises OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng / mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, or about 1 µg / mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises between 0.1 ng / mL and 1 ng / mL, between 1 ng / mL and 5 ng / mL, between 5 ng / mL and 10 ng / mL, between 10 ng / mL and 20 ng / mL, between 20 ng / mL and 30 ng / mL, between 30 ng / mL and 40 ng / mL, between 40 ng / mL and 50 ng / mL, and between 50 ng / mL and 100 ng / mL of OKT-3 antibody. In some embodiments, the cell culture medium does not comprise OKT-3 antibody. In some embodiments, the OKT-3 antibody is muromonab (see Table 1).

[0150] In some embodiments, the cell culture medium comprises one or more TNFRSF agonists in a cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of urelumab, utomilumab, EU-101, a fusion protein, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of between 0.1 µg / mL and 100 µg / mL. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of between 20 µg / mL and 40 µg / mL.

[0151] In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium further comprises IL-2 at an initial concentration of about 3000 IU / mL and OKT-3 antibody at an initial concentration of about 30 ng / mL, and wherein the one or more TNFRSF agonists comprises a 4-1BB agonist.

[0152] In some embodiments, thefirst TIL expansion can proceed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days, wherein the expanded TILs will be genetically modified via TALEN gene editing by introducing sequentially into the TILs nucleic acids, such as mRNAs, encoding TALEN systems targeting PD-1 and TIGIT. In some 65 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO embodiments, thefirst TIL expansion can proceed for 1 day to 14 days. In some embodiments, the first TIL expansion can proceed for 2 days to 14 days. In some embodiments, thefirst TIL expansion can proceed for 3 days to 14 days. In some embodiments, thefirst TIL expansion can proceed for 4 days to 14 days. In some embodiments, thefirst TIL expansion can proceed for 5 days to 14 days. In some embodiments, thefirst TIL expansion can proceed for 6 days to 14 days. In some embodiments, thefirst TIL expansion can proceed for 7 days to 14 days. In some embodiments, thefirst TIL expansion can proceed for 8 days to 14 days. In some embodiments, thefirst TIL expansion can proceed for 9 days to 14 days. In some embodiments, thefirst TIL expansion can proceed for 10 days to 14 days. In some embodiments, thefirst TIL expansion can proceed for 11 days to 14 days. In some embodiments, thefirst TIL expansion can proceed for 12 days to 14 days. In some embodiments, the first TIL expansion can proceed for 13 days to 14 days. In some embodiments, thefirst TIL expansion can proceed for 14 days. In some embodiments, thefirst TIL expansion can proceed for 1 day to 11 days. In some embodiments, thefirst TIL expansion can proceed for 2 days to 11 days. In some embodiments, thefirst TIL expansion can proceed for 3 days to 11 days. In some embodiments, the first TIL expansion can proceed for 4 days to 11 days. In some embodiments, thefirst TIL expansion can proceed for 5 days to 11 days. In some embodiments, thefirst TIL expansion can proceed for 6 days to 11 days. In some embodiments, thefirst TIL expansion can proceed for 7 days to 11 days. In some embodiments, thefirst TIL expansion can proceed for 8 days to 11 days. In some embodiments, thefirst TIL expansion can proceed for 9 days to 11 days. In some embodiments, thefirst TIL expansion can proceed for 10 days to 11 days. In some embodiments, thefirst TIL expansion can proceed for 11 days. In some embodiments, thefirst TIL expansion can proceed for 5 days to 7 days. In some embodiments, thefirst TIL expansion can proceed for 6 days to 7 days. In some embodiments, thefirst TIL expansion can proceed for 7 days to 12 days. In some embodiments, the first TIL expansion can proceed for 8 days to 12 days. In some embodiments, thefirst TIL expansion can proceed for 9 days to 12 days. In some embodiments, thefirst TIL expansion can proceed for 10 days to 12 days. In some embodiments, thefirst TIL expansion can proceed for 7 days. In some embodiments, thefirst TIL expansion can proceed for 9 days.

[0153] In some embodiments, thefirst expansion is performed in a closed system bioreactor. In some embodiments, a closed system is employed for the TIL expansion, as described herein. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor employed is for example a G-REX -10 or a G-REX -100. In some embodiments, the closed system bioreactor is a single bioreactor. 66 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO

[0154] In some embodiments, thefirst cell culture medium comprises 6000 IU / mL of IL-2. In some embodiments, thefirst cell culture medium comprises 3000 IU / mL of IL-2. In some embodiments, thefirst cell culture medium comprises 2000 IU / mL of IL-2. In some embodiments, thefirst cell culture medium comprises 1000 IU / mL of IL-2. D. Activation

[0155] In some embodiments, after thefirst expansion (pre-REP) step the TILs are activated by adding anti-CD3 agonist and anti-CD28 agonist, such as TransAct, to the culture medium and culturing for about 1 to 3 days, wherein the TILs will be genetically modified via gene editing by introducing one or more RNA molecules associated with programmable nucleases.

[0156] In some embodiments, the step of activating the second population of TILs (obtained from thefirst expansion or pre-REP step) can be performed for a period that is, is about, is less than, is more than, 1 day, 2 days, 3 days, or a range that is between any of the above values. For example, in some embodiments, the step of activating the second population of TILs is performed for about 1 day. In some embodiments, the step of activating the second population of TILs is performed for about 2 days. In some embodiments, the step of activating the second population of TILs is performed for about 3 days.

[0157] In some embodiments, the step of activating the second population of TILs (obtained from thefirst expansion or pre-REP step) is performed using anti-CD3 agonist and anti-CD28 agonist, such as TransAct. In some embodiments, the step of activating the second population of TILs is performed using TransAct at 1:10 dilution, at 1:17.5 dilution, at 1:20 dilution, at 1:25 dilution, at 1:30 dilution, at 1:40 dilution, at 1:50 dilution, at 1:60 dilution, at 1:70 dilution, at 1:80 dilution, at 1:90 dilution, or at 1:100 dilution.

[0158] In some embodiments, the step of activating the second population of TILs (obtained from thefirst expansion or pre-REP step) can be performed by adding the anti-CD3 agonist and anti-CD28 agonist, such as TransAct, to thefirst cell culture medium. In some embodiments, the step of activating the second population of TILs can be performed by replacing thefirst cell culture medium with a cell culture medium comprising the anti-CD3 agonist and anti-CD28 agonist, such as TransAct. E. Gene Editing

[0159] In some embodiments, the activation step is followed by one or more steps of genetically modifying TILs by introducing into the TILs one or more RNA molecules associated with programmable nucleases. In some embodiments, the gene modification step is performed by 67 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO genetically modifying the TILs obtained from the activation step by intorducing into the TILs one RNA molecule associated with a programmable nuclease. In some embodiments, the gene modification steps are performed by genetically modifying the TILs obtained from the activation step by intorducing into the TILs afirst RNA molecule associated with afirst programmable nuclease, followed by intorducing into the TILs a second RNA molecule associated with a second programmable nuclease.

[0160] According to an embodiment, the gene-editing process may comprise the use of a programmable nuclease that mediates the generation of a double-strand or single-strand break at one or more immune checkpoint genes. Such programmable nucleases enable precise genome editing by introducing breaks at specific genomic loci, i.e., they rely on the recognition of a specific DNA sequence within the genome to target a nuclease domain to this location and mediate the generation of a double-strand break at the target sequence. A double-strand break in the DNA subsequently recruits endogenous repair machinery to the break site to mediate genome editing by either non-homologous end-joining (NHEJ) or homology-directed repair (HDR). Thus, the repair of the break can result in the introduction of insertion / deletion mutations that disrupt (e.g., silence, repress, or enhance) the target gene product.

[0161] In some embodiments, the programmable nuclease is selected from the group consisting of a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease (ZFN), a CRISPR-associated (Cas) nuclease, and a meganuclease (MN).

[0162] In some embodiments, the RNA molecule is an mRNA molecule encoding the programmable nuclease. The mRNA molecule may be introduced at afixed concentration, for example, from about 0.1 µg / mL to about 1,000 µg / mL. In some embodiments, the mRNA molecule may be introduced at afixed concentration, for example, from about 1 µg / mL to about 100 µg / mL. In some embodiments, the mRNA molecule may be introduced at afixed concentration, for example, from about 10 µg / mL to about 50 µg / mL. In some embodiments, the mRNA molecule may be introduced at afixed concentration, for example, at about 1 µg / mL, about 2 µg / mL, about 3 µg / mL, about 4 µg / mL, about 5 µg / mL, about 6 µg / mL, about 7 µg / mL, about 8 µg / mL, about 9 µg / mL, about 10 µg / mL, about 20 µg / mL, about 30 µg / mL, about 40 µg / mL, about 50 µg / mL, about 60 µg / mL, about 70 µg / mL, about 80 µg / mL, about 90 µg / mL, about 100 µg / mL. In some embodiments, the mRNA molecule may be introduced at afixed concentration of about 10 µg / mL. In some embodiments, the mRNA molecule may be introduced at afixed concentration of about 12.5 µg / mL. In some embodiments, the mRNA molecule may be introduced at afixed concentration of about 20 µg / mL. In some embodiments, the mRNA molecule may be introduced at afixed concentration of 68 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO about 30 µg / mL. In some embodiments, the mRNA molecule may be introduced at afixed concentration of about 40 µg / mL. In some embodiments, the mRNA molecule may be introduced at afixed concentration of about 50 µg / mL.

[0163] In some embodiments, the invention provides a method for post-transcriptional modification of the transcribed mRNA to add a cap by further treating the mRNA with an enzyme to form a 5’ capped mRNA. See Ensinger, et al., PNAS, 1975, 72(7) 2525-2529; Moss, et al., Virology, 1976, 72(2), 341-351, the contents of which are herein incorporated by reference in their entireties. Alternatively, capped transcripts can be produced by using a cap analog during the in vitro transcription reaction. See Ishikawa, et al., Nucl. Acids Symp. Series, 2009, 53, 129-131; Sikorski, et al., Nucl. Acid Res., 2020, 48(4), 1607-1626; Stepinski, et al., RNA, 2001, 7(10), 1486-1495, the contents of which are herein incorporated by reference in their entireties. In some embodiments, the invention provides a process for in vitro transcription in which 5’ capped mRNA transcripts can be produced by using a cap analog during the in vitro transcription reaction, without any post- transcriptional modification. In some embodiments, the mRNA sequence may be produced in vitro using the CleanCap® AG technology by TriLink Biotechnologies, which is described in Henderson, et al., Current Protocols, 2021, 1, e39. doi: 10.1002 / cpz1.39; and PCT Patent Publication No. WO 2017053297 A1, the contents of which are herein incorporated by reference in their entireties. In some embodiments, the mRNA sequence may be transcribed from linearized plasmid DNA using the “Basic Protocol 1: IVT WITH CleanCap” described in Henderson, et al., supra.

[0164] In some embodiments, the invention provides a DNA template for transcription of an mRNA molecule, and further comprising a 5’ un-transcribed region (UTR) compatible with the CleanCap® AG technology having the sequence of AGCTAGCGCCGCCACC (SEQ ID NO: 50). In some embodiments, the DNA template for the mRNA molecule comprises a T7 RNA polymerase promotor sequence of TAATACGACTCACTATA (SEQ ID NO: 51) before the 5’ UTR.

[0165] Electroporation methods are known in the art and are described, e.g., in Tsong, Biophys. J. 1991, 60, 297-306, and U.S. Patent Application Publication No.2014 / 0227237 A1, the disclosures of each of which are incorporated by reference herein. Other electroporation methods known in the art, such as those described in U.S. Patent Nos.5,019,034; 5,128,257; 5,137,817; 5,173,158; 5,232,856; 5,273,525; 5,304,120; 5,318,514; 6,010,613 and 6,078,490, the disclosures of which are incorporated by reference herein, may be used. In some embodiments, the electroporation method is a sterile electroporation method. In some embodiments, the electroporation method is a pulsed electroporation method. In some embodiments, the electroporation method is a pulsed electroporation method comprising the steps of treating TILs with pulsed electricalfields to alter, 69 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO manipulate, or cause defined and controlled, permanent or temporary changes in the TILs, comprising the step of applying a sequence of at least three single, operator-controlled, independently programmed, DC electrical pulses, havingfield strengths equal to or greater than 100 V / cm, to the TILs, wherein the sequence of at least three DC electrical pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses differ from each other in pulse amplitude; (2) at least two of the at least three pulses differ from each other in pulse width; and (3) afirst pulse interval for afirst set of two of the at least three pulses is different from a second pulse interval for a second set of two of the at least three pulses. In some embodiments, the electroporation method is a pulsed electroporation method comprising the steps of treating TILs with pulsed electricalfields to alter, manipulate, or cause defined and controlled, permanent or temporary changes in the TILs, comprising the step of applying a sequence of at least three single, operator-controlled, independently programmed, DC electrical pulses, havingfield strengths equal to or greater than 100 V / cm, to the TILs, wherein at least two of the at least three pulses differ from each other in pulse amplitude. In some embodiments, the electroporation method is a pulsed electroporation method comprising the steps of treating TILs with pulsed electricalfields to alter, manipulate, or cause defined and controlled, permanent or temporary changes in the TILs, comprising the step of applying a sequence of at least three single, operator-controlled, independently programmed, DC electrical pulses, havingfield strengths equal to or greater than 100 V / cm, to the TILs, wherein at least two of the at least three pulses differ from each other in pulse width. In some embodiments, the electroporation method is a pulsed electroporation method comprising the steps of treating TILs with pulsed electricalfields to alter, manipulate, or cause defined and controlled, permanent or temporary changes in the TILs, comprising the step of applying a sequence of at least three single, operator-controlled, independently programmed, DC electrical pulses, havingfield strengths equal to or greater than 100 V / cm, to the TILs, wherein afirst pulse interval for afirst set of two of the at least three pulses is different from a second pulse interval for a second set of two of the at least three pulses. In some embodiments, the electroporation method is a pulsed electroporation method comprising the steps of treating TILs with pulsed electricalfields to induce pore formation in the TILs, comprising the step of applying a sequence of at least three DC electrical pulses, havingfield strengths equal to or greater than 100 V / cm, to TILs, wherein the sequence of at least three DC electrical pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses differ from each other in pulse amplitude; (2) at least two of the at least three pulses differ from each other in pulse width; and (3) afirst pulse interval for a first set of two of the at least three pulses is different from a second pulse interval for a second set of two of the at least three pulses, such that induced pores are sustained for a relatively long period of 70 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO time, and such that viability of the TILs is maintained. In some embodiments, a method of genetically modifying a population of TILs includes the step of calcium phosphate transfection. Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating, and endocytosis) are known in the art and are described in Graham and van der Eb, Virology 1973, 52, 456-467; Wigler, et al., Proc. Natl. Acad. Sci.1979, 76, 1373-1376; and Chen and Okayarea, Mol. Cell. Biol.1987, 7, 2745-2752; and in U.S. Patent No.5,593,875, the disclosures of each of which are incorporated by reference herein. In some embodiments, a method of genetically modifying a population of TILs includes the step of liposomal transfection. Liposomal transfection methods, such as methods that employ a 1:1 (w / w) liposome formulation of the cationic lipid N-[1-(2,3- dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoyl phophotidylethanolamine (DOPE) infiltered water, are known in the art and are described in Rose, et al., Biotechniques 1991, 10, 520-525 and Felgner, et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 7413- 7417 and in U.S. Patent Nos.5,279,833; 5,908,635; 6,056,938; 6,110,490; 6,534,484; and 7,687,070, the disclosures of each of which are incorporated by reference herein. In some embodiments, a method of genetically modifying a population of TILs includes the step of transfection using methods described in U.S. Patent Nos.5,766,902; 6,025,337; 6,410,517; 6,475,994; and 7,189,705; the disclosures of each of which are incorporated by reference herein.

[0166] In some embodiments of the present invention, electroporation is used for delivery of the desired RNA molecules. In some embodiments of the present invention, the electroporation system is aflow electroporation system. An example of a suitableflow electroporation system suitable for use with some embodiments of the present invention is the commercially-available MaxCyte STX system. There are several alternative commercially-available electroporation instruments which may be suitable for use with the present invention, such as the CTS Xenon Electroporation System or the Neon Transfection System available from Thermo-Fisher, the AgilePulse system or ECM 830 available from BTX-Harvard Apparatus, Cellaxess Elektra (Cellectricon), Nucleofector (Lonza / Amaxa), GenePulser MXcell (BIORAD), iPorator-96 (Primax) or siPORTer96 (Ambion). In some embodiments of the present invention, the electroporation system forms a closed, sterile system with the remainder of the TIL expansion method. In some embodiments of the present invention, the electroporation system is a pulsed electroporation system as described herein, and forms a closed, sterile system with the remainder of the TIL expansion method. 1. Resting Step

[0167] In some embodiments, the two steps of genetic modification of TILs with RNA molecules are separated by a resting step. According to some embodiments, the resting step 71 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO comprises incubating the fourth population of TILs at about 30-40 °C with about 5% CO2. According to some embodiments, the resting step is carried out at about 30°C, about 30.5°C, about 31°C, about 31.5°C, about 32°C, about 32.5°C, about 33°C, about 33.5°C, about 34°C, about 34.5°C, about 35°C, about 35.5°C, about 36°C, about 36.5°C, about 37°C, about 37.5°C, about 38°C, about 38.5°C, about 39°C, about 39.5°C, about 40°C. According to some embodiments, the resting step is carried out for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 2 days, about 3 days, about 4 days, or longer. According to some embodiments, the resting step comprises incubating the fourth population of TILs in a cell culture medium comprising IL-2. According to some embodiments, the resting step comprises incubating the fourth population of TILs in a cell culture medium comprising IL-2 at 300 IU / mL, 1,000 IU / mL, 2,000 IU / mL, 3,000 IU / mL, or 6,000 IU / mL. According to some embodiments, the resting step comprises incubating the fourth population of TILs in CM1 with 1,000 IU / mL IL-2. According to some embodiments, the resting step comprises incubating the third or fourth population of TILs in a cell culture medium comprising IL-2 for about 15 hours to about 23 hours at about 30°C with about 5% CO2. According to some embodiments, the resting step comprises incubating the fourth population of TILs in a cell culture medium comprising IL-2 for about 1 day to about 3 days at 37°C with about 5% CO2. According to some embodiments, the resting step comprises incubating the fourth population of TILs in a cell culture medium comprising IL-2 for about 2 days at 37°C with about 5% CO2.

[0168] In some embodiments, each of the two steps of genetic modification of TILs with RNA molecules is followed by an overnight resting step. According to some embodiments, the overnight resting step comprises incubating the fourth orfifth population of TILs in a cell culture medium comprising IL-2 at about 25-37 °C with about 5% CO2. According to some embodiments, the overnight resting step is carried out at about 25°C, about 25.5°C, about 26°C, about 26.5°C, about 27°C, about 27.5°C, about 28°C, about 28.5°C, about 29°C, about 29.5°C, about 30°C, about 30.5°C, about 31°C, about 31.5°C, about 32°C, about 32.5°C, about 33°C, about 33.5°C, about 34°C, about 34.5°C, about 35°C, about 35.5°C, about 36°C, about 36.5°C, and about 37°C. According to some embodiments, the overnight resting step comprises incubating the fourth orfifth population of TILs in a cell culture medium comprising IL-2 at about 30 °C with about 5% CO2.

[0169] In some embodiments, each of the two steps of genetic modification of TILs with RNA molecules is followed by an overnight resting step, separated by a resting step of about 1-3 days 72 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO between the two electroporation steps. According to some embodiments, the overnight resting step comprises incubating the fourth orfifth population of TILs in a cell culture medium comprising IL-2 at about 25-37 °C with about 5% CO2and the resting step between the two genetic modification steps comprises incubating the fourth population of TILs for about 1-3 days at about 30-40 °C with about 5% CO2. According to some embodiments, the overnight resting step comprises incubating the fourth orfifth population of TILs in a cell culture medium comprising IL-2 at about 30 °C with about 5% CO2and the resting step between the two genetic modification steps comprises incubating the fourth population of TILs for about 1-3 days at about 37 °C with about 5% CO2. According to some embodiments, the overnight resting step comprises incubating the fourth orfifth population of TILs in a cell culture medium comprising IL-2 at about 30 °C with about 5% CO2and the resting step between the two genetic modification steps comprises incubating the fourth population of TILs for about 1 day at about 37 °C with about 5% CO2. According to some embodiments, the overnight resting step comprises incubating the fourth orfifth population of TILs in a cell culture medium comprising IL-2 at about 30 °C with about 5% CO2and the resting step between the two genetic modification steps comprises incubating the fourth population of TILs for about 2 days at about 37 °C with about 5% CO2. According to some embodiments, the overnight resting step comprises incubating the fourth orfifth population of TILs in a cell culture medium comprising IL-2 at about 30 °C with about 5% CO2and the resting step between the two genetic modification steps comprises incubating the fourth population of TILs for about 3 days at about 37 °C with about 5% CO2. F. Second Expansion

[0170] In some embodiments, the TIL cell population is expanded in number after initial bulk processing, pre-REP expansion, and genetic modification, wherein the expanded TILs have been genetically modified. This further expansion is referred to herein as the second expansion, which can include expansion processes generally referred to in the art as a rapid expansion process (REP). The second expansion is generally accomplished using a culture media comprising a number of components, including feeder cells, a cytokine source, and an anti-CD3 agonist antibody, in a gas- permeable container.

[0171] In some embodiments, the second expansion or second TIL expansion (which can include expansions sometimes referred to as REP) of TIL can be performed using any TILflasks or containers known by those of skill in the art, wherein the expanded TILs have been genetically modified. In some embodiments, the second TIL expansion can proceed for 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the second TIL expansion can proceed for 73 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO about 7 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 7 days to about 12 days. In some embodiments, the second TIL expansion can proceed for about 7 days to about 10 days. In some embodiments, the second TIL expansion can proceed for about 7 days to about 9 days. In some embodiments, the second TIL expansion can proceed for about 8 days to about 9 days. In some embodiments, the second TIL expansion can proceed for about 9 days. In some embodiments, the second TIL expansion can proceed for about 10 days. In some embodiments, the second TIL expansion can proceed for about 11 days.

[0172] In some embodiments, the second expansion can be performed in a gas permeable container using the methods of the present disclosure (including for example, expansions referred to as REP). For example, TILs can be rapidly expanded using non-specific T-cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). The non-specific T-cell receptor stimulus can include, for example, an anti-CD3 agonist antibody, such as about 30 ng / ml of OKT3, a mouse monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA) or UHCT-1 (commercially available from BioLegend, San Diego, CA, USA). TILs can be expanded to induce further stimulation of the TILs in vitro by including one or more antigens during the second expansion, including antigenic portions thereof, such as epitope(s), of the cancer, which can be optionally expressed from a vector, such as a human leukocyte antigen A2 (HLA-A2) binding peptide, e.g., 0.3 μΜ MART-1 :26-35 (27 L) or gpl 00:209-217 (210M), optionally in the presence of a T-cell growth factor, such as 300 IU / mL IL-2 or IL-15. Other suitable antigens may include, e.g., NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic portions thereof. TIL may also be rapidly expanded by re-stimulation with the same antigen(s) of the cancer pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, the TILs can be further re-stimulated with, e.g., example, irradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, the re-stimulation occurs as part of the second expansion. In some embodiments, the second expansion occurs in the presence of irradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0173] In some embodiments, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000 IU / mL of IL-2. In some embodiments, the cell culture medium comprises about 1000 IU / mL, about 1500 IU / mL, about 2000 IU / mL, about 2500 IU / mL, about 3000 IU / mL, about 3500 IU / mL, about 4000 IU / mL, about 4500 IU / mL, about 5000 IU / mL, about 5500 IU / mL, about 6000 IU / mL, about 6500 IU / mL, about 7000 IU / mL, about 7500 IU / mL, or about 8000 IU / mL of IL-2. In some embodiments, the cell culture medium comprises 74 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO between 1000 and 2000 IU / mL, between 2000 and 3000 IU / mL, between 3000 and 4000 IU / mL, between 4000 and 5000 IU / mL, between 5000 and 6000 IU / mL, between 6000 and 7000 IU / mL, between 7000 and 8000 IU / mL, or between 8000 IU / mL of IL-2.

[0174] In some embodiments, the cell culture medium comprises OKT-3 antibody. In some embodiments, the cell culture medium comprises about 30 ng / mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, or about 1 µg / mL of OKT-3 antibody. In some embodiments, the cell culture medium comprises between 0.1 ng / mL and 1 ng / mL, between 1 ng / mL and 5 ng / mL, between 5 ng / mL and 10 ng / mL, between 10 ng / mL and 20 ng / mL, between 20 ng / mL and 30 ng / mL, between 30 ng / mL and 40 ng / mL, between 40 ng / mL and 50 ng / mL, and between 50 ng / mL and 100 ng / mL of OKT-3 antibody. In some embodiments, the cell culture medium does not comprise OKT-3 antibody. In some embodiments, the OKT-3 antibody is muromonab.

[0175] In some embodiments, the cell culture medium comprises one or more TNFRSF agonists in a cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of urelumab, utomilumab, EU-101, a fusion protein, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of between 0.1 µg / mL and 100 µg / mL. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a concentration in the cell culture medium of between 20 µg / mL and 40 µg / mL.

[0176] In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium further comprises IL-2 at an initial concentration of about 3000 IU / mL and OKT-3 antibody at an initial concentration of about 30 ng / mL, and wherein the one or more TNFRSF agonists comprises a 4-1BB agonist.

[0177] In some embodiments the antigen-presenting feeder cells (APCs) are PBMCs. In some embodiments, the ratio of TILs to PBMCs and / or antigen-presenting cells in the rapid expansion and / or the second expansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500. In some 75 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO embodiments, the ratio of TILs to PBMCs in the rapid expansion and / or the second expansion is between 1 to 50 and 1 to 300. In some embodiments, the ratio of TILs to PBMCs in the rapid expansion and / or the second expansion is between 1 to 100 and 1 to 200.

[0178] In some embodiments, REP and / or the second expansion is performed inflasks with the bulk TILs being mixed with a 100- or 200-fold excess of inactivated feeder cells, 30 mg / mL OKT3 anti- CD3 antibody and 3000 IU / mL IL-2 in 150 ml media. Media replacement is done (generally 2 / 3 media replacement via respiration with fresh media) until the cells are transferred to an alternative growth chamber. Alternative growth chambers include G-REXflasks and gas permeable containers as more fully discussed below.

[0179] In some embodiments, the second expansion (which can include processes referred to as the REP process) is shortened to 7-14 days, as discussed in the examples, wherein the TILs expanded by such a second expansion have been genetically modified. In some embodiments, the second expansion is shortened to 9 days.

[0180] In some embodiments, REP and / or the second expansion may be performed using T-175 flasks and gas permeable bags as previously described (Tran, et al., J. Immunother.2008, 31, 742-51; Dudley, et al., J. Immunother.2003, 26, 332-42) or gas permeable cultureware (G-Rexflasks), wherein the TILs expanded by such a second expansion have been genetically modified. In some embodiments, the second expansion (including expansions referred to as rapid expansions) is performed in T-175flasks, and about 1 x 106TILs suspended in 150 mL of media may be added to each T-175flask. The TILs may be cultured in a 1 to 1 mixture of CM and AIM-V medium, supplemented with 3000 IU per mL of IL-2 and 30 ng per ml of anti-CD3. The T-175flasks may be incubated at 37° C in 5% CO2,wherein the TILs expanded by such a second expansion have been genetically modified. Half the media may be exchanged on day 5 using 50 / 50 medium with 3000 IU per mL of IL-2. In some embodiments, on day 7 cells from two T-175flasks may be combined in a 3 L bag and 300 mL of AIM V with 5% human AB serum and 3000 IU per mL of IL-2 was added to the 300 ml of TIL suspension. The number of cells in each bag was counted every day or two and fresh media was added to keep the cell count between 0.5 and 2.0 x 106cells / mL.

[0181] In some embodiments, the second expansion (which can include expansions referred to as REP) may be performed in 500 mL capacity gas permeableflasks with 100 cm gas-permeable silicon bottoms (G-Rex 100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA), 5 × 106or 10 × 106TIL may be cultured with PBMCs in 400 mL of 50 / 50 medium, supplemented with 5% human AB serum, 3000 IU per mL of IL-2 and 30 ng per ml of anti-CD3 76 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO (OKT3), wherein the TILs expanded by such a second expansion have been genetically modified. The G-Rex 100flasks may be incubated at 37°C in 5% CO2. On day 5, 250 mL of supernatant may be removed and placed into centrifuge bottles and centrifuged at 1500 rpm (491 × g) for 10 minutes. The TIL pellets may be re-suspended with 150 mL of fresh medium with 5% human AB serum, 3000 IU per mL of IL-2, and added back to the original G-Rex 100flasks. When TIL are expanded serially in G-Rex 100flasks, on day 7 the TIL in each G-Rex 100 may be suspended in the 300 mL of media present in eachflask and the cell suspension may be divided into 3100 mL aliquots that may be used to seed 3 G-Rex 100flasks. Then 150 mL of AIM-V with 5% human AB serum and 3000 IU per mL of IL-2 may be added to eachflask. The G-Rex 100flasks may be incubated at 37° C in 5% CO2and after 4 days 150 mL of AIM-V with 3000 IU per mL of IL-2 may be added to each G-REX 100flask. The cells may be harvested on day 14 of culture.

[0182] In some embodiments, the second expansion (which can include expansions referred to as REP) may be performed in 500 mL capacity gas permeableflasks with 100 cm gas-permeable silicon bottoms (G-REX-100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA), 5 × 106or 10 × 106TIL may be cultured with PBMCs in 400 mL of 50 / 50 medium, supplemented with 5% human AB serum, 3000 IU per mL of IL-2 and 30 ng per mL of anti- CD3 (OKT3). The G-REX-100 (or G-REX100M)flasks may be incubated at 37°C in 5% CO2. On day 5, 250 mL of supernatant may be removed and placed into centrifuge bottles and centrifuged at 1500 rpm (491 × g) for 10 minutes. The TIL pellets may be re-suspended with 150 mL of fresh medium with 5% human AB serum, 6000 IU per mL of IL-2, and added back to the original GREX-100flasks. When TIL are expanded serially in GREX-100flasks, on day 10 or 11 the TILs can be moved to a larger flask, such as a GREX-500 (or G-REX500M). The cells may be harvested on day 14 of culture. The cells may be harvested on day 15 of culture. The cells may be harvested on day 16 of culture. In some embodiments, media replacement is done until the cells are transferred to an alternative growth chamber. In some embodiments, 2 / 3 of the media is replaced by aspiration of spent media and replacement with an equal volume of fresh media. In some embodiments, alternative growth chambers include GREXflasks and gas permeable containers as more fully discussed below. In some embodiments, the process employed varying centrifugation speeds (400g, 300g, 200g for 5 minutes) and varying numbers of repetitions.

[0183] In some embodiments, the second expansion (including expansions referred to as REP) is performed inflasks with the bulk TILs being mixed with a 100- or 200-fold excess of inactivated feeder cells, 30 mg / mL OKT3 anti-CD3 antibody and 3000 IU / mL IL-2 in 150 ml media. In some embodiments, media replacement is done until the cells are transferred to an alternative growth 77 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO chamber, wherein the TILs expanded by such a second expansion have been genetically modified. In some embodiments, 2 / 3 of the media is replaced by aspiration of spent media followed by infusion with fresh media. In some embodiments, alternative growth chambers include G-REXflasks and gas permeable containers as more fully discussed below.

[0184] In some embodiments, the second expansion culture medium (e.g., sometimes referred to as CM2 or the second cell culture medium), comprises IL-2, OKT-3, as well as the antigen- presenting feeder cells (APCs), as discussed in more detail below.

[0185] In some embodiments, the second expansion is performed in a closed system bioreactor. In some embodiments, a closed system is employed for the TIL expansion, as described herein. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor employed is for example a G-REX -10 or a G-REX -100. In some embodiments, the closed system bioreactor is a single bioreactor.

[0186] In some embodiments, the steps of the method are completed within a period of about 22 days. In some embodiments, the steps of the method are completed within a period of about 8 days. In some embodiments, the steps of the method are completed within a period of about 9 days. In some embodiments, the steps of the method are completed within a period of about 10 days. In some embodiments, the steps of the method are completed within a period of about 11 days. In some embodiments, the steps of the method are completed within a period of about 12 days. In some embodiments, the steps of the method are completed within a period of about 13 days. In some embodiments, the steps of the method are completed within a period of about 14 days. In some embodiments, the steps of the method are completed within a period of about 15 days. In some embodiments, the steps of the method are completed within a period of about 16 days. In some embodiments, the steps of the method are completed within a period of about 17 days. In some embodiments, the steps of the method are completed within a period of about 18 days. In some embodiments, the steps of the method are completed within a period of about 19 days. In some embodiments, the steps of the method are completed within a period of about 20 days. In some embodiments, the steps of the method are completed within a period of about 21 days. In some embodiments, the steps of the method are completed within a period of about 22 days. In some embodiments, the steps of the method are completed within a period of about 23 days. In some embodiments, the steps of the method are completed within a period of about 24 days. In some embodiments, the steps of the method are completed within a period of about 25 days. In some embodiments, the steps of the method are completed within a period of about 26 days. In some embodiments, the steps of the method are completed within a period of 78 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO about 27 days. In some embodiments, the steps of the method are completed within a period of about 28 days. In some embodiments, the steps of the method are completed within a period of about 29 days. In some embodiments, the steps of the method are completed within a period of about 30 days. In some embodiments, the steps of the method are completed within a period of about 31 days.

[0187] In some embodiments, the antigen presenting cells (APCs) are PBMCs. According to some embodiments, the PBMCs are irradiated. According to some embodiments, the PBMCs are allogeneic. According to some embodiments, the PBMCs are irradiated and allogeneic. According to some embodiments, the antigen-presenting cells are artificial antigen-presenting cells.

[0188] In some embodiments, the IL-2 is present at an initial concentration of between 1000 IU / mL and 6000 IU / mL in the cell culture medium in thefirst expansion. In some embodiments, the IL-2 is present at an initial concentration of between 1500 IU / mL and 6000 IU / mL in the cell culture medium in thefirst expansion. In some embodiments, the IL-2 is present at an initial concentration of between 2000 IU / mL and 6000 IU / mL in the cell culture medium in thefirst expansion. In some embodiments, the IL-2 is present at an initial concentration of between 2500 IU / mL and 6000 IU / mL in the cell culture medium in thefirst expansion. In some embodiments, the IL-2 is present at an initial concentration of between 3000 IU / mL and 6000 IU / mL in the cell culture medium in thefirst expansion. In some embodiments, the IL-2 is present at an initial concentration of between 3500 IU / mL and 6000 IU / mL in the cell culture medium in thefirst expansion. In some embodiments, the IL-2 is present at an initial concentration of between 4000 IU / mL and 6000 IU / mL in the cell culture medium in thefirst expansion. In some embodiments, the IL-2 is present at an initial concentration of between 4500 IU / mL and 6000 IU / mL in the cell culture medium in thefirst expansion. In some embodiments, the IL-2 is present at an initial concentration of between 5000 IU / mL and 6000 IU / mL in the cell culture medium in thefirst expansion. In some embodiments, the IL-2 is present at an initial concentration of between 5500 IU / mL and 6000 IU / mL in the cell culture medium in thefirst expansion. In some embodiments, the IL-2 is present at an initial concentration of between 1000 IU / mL and 5000 IU / mL in the cell culture medium in thefirst expansion. In some embodiments, the IL-2 is present at an initial concentration of between 1500 IU / mL and 5000 IU / mL in the cell culture medium in thefirst expansion. In some embodiments, the IL-2 is present at an initial concentration of between 2000 IU / mL and 5000 IU / mL in the cell culture medium in thefirst expansion. In some embodiments, the IL-2 is present at an initial concentration of between 2500 IU / mL and 5000 IU / mL in the cell culture medium in thefirst expansion. In some embodiments, the IL-2 is present at an initial concentration of between 3000 IU / mL and 5000 IU / mL in the cell culture medium in thefirst 79 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO expansion. In some embodiments, the IL-2 is present at an initial concentration of between 3500 IU / mL and 5000 IU / mL in the cell culture medium in thefirst expansion. In some embodiments, the IL-2 is present at an initial concentration of between 4000 IU / mL and 5000 IU / mL in the cell culture medium in thefirst expansion. In some embodiments, the IL-2 is present at an initial concentration of between 4500 IU / mL and 5000 IU / mL in the cell culture medium in thefirst expansion. In some embodiments, the IL-2 is present at an initial concentration of between 1000 IU / mL and 4000 IU / mL in the cell culture medium in thefirst expansion. In some embodiments, the IL-2 is present at an initial concentration of between 1500 IU / mL and 4000 IU / mL in the cell culture medium in thefirst expansion. In some embodiments, the IL-2 is present at an initial concentration of between 2000 IU / mL and 4000 IU / mL in the cell culture medium in thefirst expansion. In some embodiments, the IL-2 is present at an initial concentration of between 2500 IU / mL and 4000 IU / mL in the cell culture medium in thefirst expansion. In some embodiments, the IL-2 is present at an initial concentration of between 3000 IU / mL and 4000 IU / mL in the cell culture medium in thefirst expansion. In some embodiments, the IL-2 is present at an initial concentration of between 3500 IU / mL and 4000 IU / mL in the cell culture medium in thefirst expansion. In some embodiments, the IL-2 is present at an initial concentration of between 1000 IU / mL and 3000 IU / mL in the cell culture medium in thefirst expansion. In some embodiments, the IL-2 is present at an initial concentration of between 1500 IU / mL and 3000 IU / mL in the cell culture medium in thefirst expansion. In some embodiments, the IL-2 is present at an initial concentration of between 2000 IU / mL and 3000 IU / mL in the cell culture medium in thefirst expansion. In some embodiments, the IL-2 is present at an initial concentration of between 2500 IU / mL and 3000 IU / mL in the cell culture medium in thefirst expansion. In some embodiments, the IL-2 is present at an initial concentration of between 1000 IU / mL and 2000 IU / mL in the cell culture medium in thefirst expansion. In some embodiments, the IL-2 is present at an initial concentration of between 1500 IU / mL and 2000 IU / mL in the cell culture medium in thefirst expansion.

[0189] In some embodiments, the second expansion step, the IL-2 is present at an initial concentration of between 1000 IU / mL and 6000 IU / mL and the OKT-3 antibody is present at an initial concentration of about 30 ng / mL.

[0190] In some embodiments, thefirst cell culture medium and / or the second cell culture medium further comprises a 4-1BB agonist and / or an OX40 agonist.

[0191] In some embodiments, thefirst expansion is performed using a gas permeable container. In some embodiments, the second expansion is performed using a gas permeable container. 80 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO

[0192] In some embodiments, thefirst cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof. In some embodiments, the second cell culture medium and / or third culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof. 1. Feeder Cells and Antigen Presenting Cells

[0193] In some embodiments, the second expansion procedures described herein require an excess of feeder cells during REP TIL expansion and / or during the second expansion. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units from healthy blood donors. The PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation.

[0194] In general, the allogeneic PBMCs are inactivated, either via irradiation or heat treatment, and used in the REP procedures, as described in the examples, which provides an exemplary protocol for evaluating the replication incompetence of irradiate allogeneic PBMCs.

[0195] In some embodiments, PBMCs are considered replication incompetent and accepted for use in the TIL expansion procedures described herein if the total number of viable cells on day 14 is less than the initial viable cell number put into culture on day 0 of the REP and / or day 0 of the second expansion (i.e., the start day of the second expansion).

[0196] In some embodiments, PBMCs are considered replication incompetent and accepted for use in the TIL expansion procedures described herein if the total number of viable cells, cultured in the presence of OKT3 and IL-2, on day 7 and day 14 has not increased from the initial viable cell number put into culture on day 0 of the REP and / or day 0 of the second expansion (i.e., the start day of the second expansion). In some embodiments, the PBMCs are cultured in the presence of 30 ng / mL OKT3 antibody and 3000 IU / mL IL-2.

[0197] In some embodiments, PBMCs are considered replication incompetent and accepted for use in the TIL expansion procedures described herein if the total number of viable cells, cultured in the presence of OKT3 and IL-2, on day 7 and day 14 has not increased from the initial viable cell number put into culture on day 0 of the REP and / or day 0 of the second expansion (i.e., the start day of the second expansion). In some embodiments, the PBMCs are cultured in the presence of 5-60 ng / mL OKT3 antibody and 1000-6000 IU / mL IL-2. In some embodiments, the PBMCs are cultured in the presence of 10-50 ng / mL OKT3 antibody and 2000-5000 IU / mL IL-2. In some embodiments, the PBMCs are cultured in the presence of 20-40 ng / mL OKT3 antibody and 2000-4000 IU / mL IL-2. In 81 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO some embodiments, the PBMCs are cultured in the presence of 25-35 ng / mL OKT3 antibody and 2500-3500 IU / mL IL-2.

[0198] In some embodiments, the antigen-presenting feeder cells are PBMCs. In some embodiments, the antigen-presenting feeder cells are artificial antigen-presenting feeder cells. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1 to 50 and 1 to 300. In some embodiments, the ratio of TILs to antigen-presenting feeder cells in the second expansion is between 1 to 100 and 1 to 200.

[0199] In some embodiments, the second expansion procedures described herein require a ratio of about 2.5x109feeder cells to about 100x106TIL. In other embodiments, the second expansion procedures described herein require a ratio of about 2.5x109feeder cells to about 50x106TIL. In yet other embodiments, the second expansion procedures described herein require about 2.5x109feeder cells to about 25x106TIL.

[0200] In some embodiments, the second expansion procedures described herein require an excess of feeder cells during the second expansion. In many embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs) obtained from standard whole blood units from healthy blood donors. The PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation. In some embodiments, artificial antigen-presenting (aAPC) cells are used in place of PBMCs.

[0201] In some embodiments, artificial antigen presenting cells are used in the second expansion as a replacement for, or in combination with, PBMCs. 2. Cytokines and Other Additives

[0202] The expansion methods described herein generally use culture media with high doses of a cytokine, in particular IL-2, as is known in the art.

[0203] Alternatively, using combinations of cytokines for the rapid expansion and or second expansion of TILs is possible, with combinations of two or more of IL-2, IL-15 and IL-21 as is described in U.S. Patent Application Publication No. US 2017 / 0107490 A1, the disclosure of which is incorporated by reference herein. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, 82 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO IL-15 and IL-21 and IL-2, IL-15 and IL-21, with the latterfinding particular use in many embodiments. The use of combinations of cytokines specifically favors the generation of lymphocytes, and in particular T-cells as described therein. G. Harvest TILs

[0204] After the second expansion step, cells can be harvested. TILs can be harvested in any appropriate and sterile manner, including for example by centrifugation. Methods for TIL harvesting are well known in the art and any such know methods can be employed with the present process. In some embodiments, TILs are harvested using an automated system.

[0205] Cell harvesters and / or cell processing systems are commercially available from a variety of sources, including, for example, Fresenius Kabi, Tomtec Life Science, Perkin Elmer, and Inotech Biosystems International, Inc. Any cell based harvester can be employed with the present methods. In some embodiments, the cell harvester and / or cell processing systems is a membrane-based cell harvester. In some embodiments, cell harvesting is via a cell processing system, such as the LOVO system (manufactured by Fresenius Kabi). The term “LOVO cell processing system” also refers to any instrument or device manufactured by any vendor that can pump a solution comprising cells through a membrane orfilter such as a spinning membrane or spinningfilter in a sterile and / or closed system environment, allowing for continuousflow and cell processing to remove supernatant or cell culture media without pelletization. In some embodiments, the cell harvester and / or cell processing system can perform cell separation, washing,fluid-exchange, concentration, and / or other cell processing steps in a closed, sterile system.

[0206] In some embodiments, the harvest is performed from a closed system bioreactor. In some embodiments, a closed system is employed for the TIL expansion, as described herein. In some embodiments, a single bioreactor is employed. In some embodiments, the single bioreactor employed is for example a G-REX-10 or a G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor.

[0207] In some embodiments, the closed system is accessed via syringes under sterile conditions in order to maintain the sterility and closed nature of the system. In some embodiments, a closed system as described in the Examples is employed. H. Final Formulation and Transfer to Infusion Container

[0759] After the steps as outlined in detailed above and herein are complete, TILs are transferred to a container for use in administration to a patient, such as an infusion bag or sterile vial. In some 83 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO embodiments, once a therapeutically sufficient number of TILs are obtained using the expansion methods described above, they are transferred to a container, such as an infusion bag, for use in administration to a patient. In some embodiments, the TILs are cryopreserved in the infusion bag. In some embodiments, the TILs are cryopreserved prior to placement in an infusion bag. In some embodiments, the TILs are cryopreserved and not placed in an infusion bag. In some embodiments, cryopreservation is performed using a cryopreservation medium. In some embodiments, the cryopreservation media contains dimethylsulfoxide (DMSO). This is generally accomplished by putting the TIL population into a freezing solution, e.g.85% complement inactivated AB serum and 15% dimethyl sulfoxide (DMSO). The cells in solution are placed into cryogenic vials and stored for 24 hours at -80 °C, with optional transfer to gaseous nitrogen freezers for cryopreservation. See, Sadeghi, et al., Acta Oncologica 2013, 52, 978-986.

[0760] When appropriate, the cells are removed from the freezer and thawed in a 37 °C water bath until approximately 4 / 5 of the solution is thawed. The cells are generally resuspended in complete media and optionally washed one or more times. In some embodiments, the thawed TILs can be counted and assessed for viability as is known in the art.

[0208] In some embodiments, a population of TILs is cryopreserved using CS10 cryopreservation media (CryoStor 10, BioLife Solutions). In some embodiments, a population of TILs is cryopreserved using a cryopreservation media containing dimethylsulfoxide (DMSO). In some embodiments, a population of TILs is cryopreserved using a 1:1 (vol:vol) ratio of CS10 and cell culture media. In some embodiments, a population of TILs is cryopreserved using about a 1:1 (vol:vol) ratio of CS10 and cell culture media, further comprising additional IL-2.

[0209] In some embodiments, TILs are administered to a patient as a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs expanded by methods described in the present disclosure may be administered by any suitable route as known in the art. In some embodiments, the T-cells are administered as a single intra-arterial or intravenous infusion, which preferably lasts approximately 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration. EXAMPLES

[0210] The embodiments encompassed herein are now described with reference to the following examples. These examples are provided for the purpose of illustration only and the disclosure encompassed herein should in no way be construed as being limited to these examples, 84 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO but rather should be construed to encompass any and all variations which become evident as a result of the teachings provided herein. EXAMPLE 1: PREPARATION OF MEDIA FOR PRE-REP AND REP PROCESSES

[0211] This example describes the procedure for the preparation of tissue culture media for use in protocols involving the culture of tumor infiltrating lymphocytes (TIL) derived from various solid tumors. This media can be used for preparation of any of the TILs described in the present application and other examples.

[0212] Preparation of CM1. Removed the following reagents from cold storage and warm them in a 37°C water bath: (RPMI1640, Human AB serum, 200 mM L-glutamine). Prepared CM1 medium according to Table 5 below by adding each of the ingredients into the top section of a 0.2 µmfilter unit appropriate to the volume to befiltered. Store at 4°C. TABLE 5. Preparation of CM1

[0213] On the day of use, prewarmed required amount of CM1 in 37°C water bath and add 6000 IU / mL IL-2.

[0214] Additional supplementation may be performed as needed according to Table 6. TABLE 6. Additional supplementation of CM1, as needed.85 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WOPreparation of CM2

[0215] Removed prepared CM1 from refrigerator or prepare fresh CM1. Removed AIM-V® from refrigerator and prepared the amount of CM2needed by mixing prepared CM1 with an equal volume of AIM-V® in a sterile media bottle. Added 3000 IU / mL IL-2 to CM2 medium on the day of usage. Made sufficient amount of CM2with 3000 IU / mL IL-2 on the day of usage. Labeled the CM2 media bottle with its name, the initials of the preparer, the date it wasfiltered / prepared, the two- week expiration date and store at 4°C until needed for tissue culture. Preparation of CM3

[0216] Prepared CM3 on the day it was required for use. CM3 was the same as AIM-V® medium, supplemented with 3000 IU / mL IL-2 on the day of use. Prepared an amount of CM3 sufficient to experimental needs by adding IL-2 stock solution directly to the bottle or bag of AIM-V. Mixed well by gentle shaking. Label bottle with “3000 IU / mL IL-2” immediately after adding to the AIM-V. If there was excess CM3, stored it in bottles at 4°C labeled with the media name, the initials of the preparer, the date the media was prepared, and its expiration date (7 days after preparation). Discarded media supplemented with IL-2 after 7 days storage at 4°C. Preparation of CM4

[0217] CM4 was the same as CM3, with the additional supplement of 2mM G1utaMAXTM (final concentration). For every 1L of CM3, add 10 mL of 200 mM GlutaMAXTM. Prepare an amount of CM4 sufficient to experimental needs by adding IL-2 stock solution and GlutaMAXTM stock solution directly to the bottle or bag of AIM-V. Mixed well by gentle shaking. Labeled bottle with “3000 IL / mL IL-2 and GlutaMAX” immediately after adding to the AIM-V. If there was excess CM4, stored it in bottles at 4°C labeled with the media name, “G1utaMAX”, and its expiration date (7 days after preparation). Discarded media supplemented with IL-2 after more than 7-days storage at 4°C. EXAMPLE 2: EXEMPLARY GEN 2 PRODUCTION OF A CRYOPRESERVED TIL CELL THERAPY

[0218] This examples describes the the cGMP manufacture of Iovance Biotherapeutics, Inc. TIL Cell Therapy Process in G-REX Flasks according to current Good Tissue Practices and current Good Manufacturing Practices. This example describes an exemplary cGMP manufacture of TIL Cell Therapy Process in G-REX Flasks according to current Good Tissue Practices and current Good Manufacturing Practices. 86 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO TABLE 7. Process Expansion Exemplary Plan.TABLE 8. Flask Volumes.

[0219] Day 0 CM1 Media Preparation. In the BSC added reagents to RPMI 1640 Media bottle. Added the following reagents t Added per bottle: Heat Inactivated Human AB Serum (100.0 mL); GlutaMax™ (10.0 mL); Gentamicin sulfate, 50 mg / mL (1.0 mL); 2-mercaptoethanol (1.0 mL)

[0220] Removed unnecessary materials from BSC. Passed out media reagents from BSC, left Gentamicin Sulfate and HBSS in BSC for Formulated Wash Media preparation.

[0221] Thawed IL-2 aliquot. Thawed one 1.1 mL IL-2 aliquot (6x106IU / mL) (BR71424) until all ice had melted. Recorded IL-2: Lot # and Expiry

[0222] Transferred IL-2 stock solution to media. In the BSC, transferred 1.0 mL of IL-2 stock solution to the CM1 Day 0 Media Bottle prepared. Added CM1 Day 0 Media 1 bottle and IL-2 (6x106IU / mL) 1.0 mL.

[0223] Passed G-REX100MCS into BSC. Aseptically passed G-REX100MCS (W3013130) into the BSC.

[0224] Pumped all Complete CM1 Day 0 Media into G-REX100MCSflask. Tissue Fragments Conical or GRex100MCS. 87 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO

[0225] Day 0 Tumor Wash Media Preparation. In the BSC, added 5.0 mL Gentamicin (W3009832 or W3012735) to 1 x 500 mL HBSS Media (W3013128) bottle. Added per bottle: HBSS (500.0 mL); Gentamicin sulfate, 50 mg / mL (5.0 mL). Filtered HBSS containing gentamicin prepared through a 1L 0.22-micronfilter unit (W1218810).

[0226] Day 0 Tumor Processing. Obtained tumor specimen and transferred into suite at 2-8 ºC immediately for processing. Aliquoted tumor wash media. Tumor wash 1 is performed using 8” forceps (W3009771). The tumor is removed from the specimen bottle and transferred to the “Wash 1” dish prepared. This is followed by tumor wash 2 and tumor wash 3. Measured and assessed tumor. Assessed whether > 30% of entire tumor area observed to be necrotic and / or fatty tissue. Clean up dissection if applicable. If tumor was large and >30% of tissue exterior was observed to be necrotic / fatty, performed “clean up dissection” by removing necrotic / fatty tissue while preserving tumor inner structure using a combination of scalpel and / or forceps. Dissect tumor. Using a combination of scalpel and / or forceps, cut the tumor specimen into even, appropriately sized fragments (up to 6 intermediate fragments). Transferred intermediate tumor fragments. Dissected tumor fragments into pieces approximately 3x3x3mm in size. Stored Intermediate Fragments to prevent drying. Repeated intermediate fragment dissection. Determined number of pieces collected. If desirable tissue remains, selected additional favorable tumor pieces from the “favorable intermediate fragments” 6-well plate tofill the drops for a maximum of 50 pieces.

[0227] Prepared conical tube. Transferred tumor pieces to 50 mL conical tube. Prepared BSC for G-REX100MCS. Removed G-REX100MCS from incubator. Aseptically passed G-REX100MCSflask into the BSC. Added tumor fragments to G-REX100MCSflask. Evenly distributed pieces.

[0228] Incubated G-REX100MCS at the following parameters: Incubated G-REXflask: Temperature LED Display: 37.0±2.0 ºC; CO2Percentage: 5.0±1.5 %CO2. Calculations: Time of incubation; lower limit = time of incubation + 252 hours; upper limit = time of incubation + 276 hours.

[0229] After process was complete, discarded any remaining warmed media and thawed aliquots of IL-2.

[0230] Day 11 – Media Preparation. Monitored incubator. Incubator parameters: Temperature LED Display: 37.0±2.0 ºC; CO2Percentage: 5.0±1.5 %CO2.

[0231] Warmed 3× 1000 mL RPMI 1640 Media (W3013112) bottles and 3× 1000 mL AIM-V (W3009501) bottles in an incubator for ≥ 30 minutes. Removed RPMI 1640 Media from incubator. 88 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO Prepared RPMI 1640 Media. Filter Media. Thawed 3 x 1.1 mL aliquots of IL-2 (6x106IU / mL) (BR71424). Removed AIM-V Media from the incubator. Add IL-2 to AIM-V. Aseptically transferred a 10 L Labtainer Bag and a repeater pump transfer set into the BSC.

[0232] Prepared 10 L Labtainer media bag. Prepared Baxa pump. Prepared 10L Labtainer media bag. Pumped media into 10 L Labtainer. Removed pumpmatic from Labtainer bag.

[0233] Mixed media. Gently massaged the bag to mix. Sample media per sample plan. Removed 20.0 mL of media and place in a 50 mL conical tube. Prepared cell count dilution tubes. In the BSC, added 4.5 mL of AIM-V Media that had been labelled with “For Cell Count Dilutions” and lot number to four 15 mL conical tubes. Transferred reagents from the BSC to 2-8°C. Prepared 1 L Transfer Pack. Outside of the BSC weld (per Process Note 5.11) a 1L Transfer Pack to the transfer set attached to the “Complete CM2Day 11 Media” bag prepared. Prepared feeder cell transfer pack. Incubated Complete CM2Day 11 Media.

[0234] Day 11 - TIL Harvest. Preprocessing table. Incubator parameters: Temperature LED display: 37.0±2.0 ºC; CO2Percentage: 5.0±1.5 % CO2. Removed G-REX100MCS from incubator. Prepared 300 mL Transfer Pack. Welded transfer packs to G-REX100MCS.

[0235] Prepareflask for TIL Harvest and initiation of TIL Harvest. TIL Harvested. Using the GatheRex, transferred the cell suspension through the bloodfilter into the 300 mL transfer pack. Inspect membrane for adherent cells.

[0236] Rinsedflask membrane. Closed clamps on G-REX100MCS. Ensured all clamps are closed. Heat sealed the TIL and the “Supernatant” transfer pack. Calculated volume of TIL suspension. Prepared Supernatant Transfer Pack for Sampling.

[0237] Pulled Bac-T Sample. In the BSC, draw up approximately 20.0 mL of supernatant from the 1L “Supernatant” transfer pack and dispense into a sterile 50 mL conical tube.

[0238] Inoculated BacT per Sample Plan. Removed a 1.0 mL sample from the 50 mL conical labeled BacT prepared using an appropriately sized syringe and inoculated the anaerobic bottle.

[0239] Incubated TIL. Placed TIL transfer pack in incubator until needed. Performed cell counts and calculations. Determined the Average of Viable Cell Concentration and Viability of the cell counts performed. Viability ÷ 2. Viable Cell Concentration ÷ 2. Determined Upper and Lower Limit for counts. Lower Limit: Average of Viable Cell Concentration x 0.9. Upper Limit: Average of 89 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO Viable Cell Concentration x 1.1. Confirmed both counts within acceptable limits. Determined an average Viable Cell Concentration from all four counts performed.

[0240] Adjusted Volume of TIL Suspension: Calculate the adjusted volume of TIL suspension after removal of cell count samples. Total TIL Cell Volume (A). Volume of Cell Count Sample Removed (4.0 mL) (B) Adjusted Total TIL Cell Volume C=A-B.

[0241] Calculated Total Viable TIL Cells. Average Viable Cell Concentration*: Total Volume; Total Viable Cells: C = A x B.

[0242] Calculation forflow cytometry: if the Total Viable TIL Cell count from was ≥ 4.0x107, calculated the volume to obtain 1.0×107cells for theflow cytometry sample.

[0243] Total viable cells required forflow cytometry: 1.0×107cells. Volume of cells required forflow cytometry: Viable cell concentration divided by 1.0×107cells A.

[0244] Calculated the volume of TIL suspension equal to 2.0×108viable cells. As needed, calculated the excess volume of TIL cells to remove and removed excess TIL and placed TIL in incubator as needed. Calculated total excess TIL removed, as needed.

[0245] Calculated amount of CS-10 media to add to excess TIL cells with the target cell concentration for freezing is 1.0×108cells / mL. Centrifuged excess TILs, as needed. Observed conical tube and added CS-10.

[0246] Filled Vials. Aliquoted 1.0 mL cell suspension, into appropriately sized cryovials. Aliquoted residual volume into appropriately sized cryovial. If volume is ≤0.5 mL, add CS10 to vial until volume is 0.5 mL.

[0247] Calculated the volume of cells required to obtain 1x107cells for cryopreservation. Removed sample for cryopreservation. Placed TIL in incubator.

[0248] Cryopreservation of sample. Observed conical tube and added CS-10 slowly and record volume of 0.5 mL of CS10 added.

[0249] Day 11 - Feeder Cells. Obtained feeder cells. Obtained 3 bags of feeder cells with at least two different lot numbers from LN2 freezer. Kept cells on dry ice until ready to thaw. Prepared water bath or cryotherm. Thawed feeder cells at 37.0 ± 2.0°C in the water bath or cytotherm for ~3-5 minutes or until ice has just disappeared. Removed media from incubator. Pooled thawed feeder 90 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO cells. Added feeder cells to transfer pack. Dispensed the feeder cells from the syringe into the transfer pack. Mixed pooled feeder cells and labeled transfer pack.

[0250] Calculated total volume of feeder cell suspension in transfer pack. Removed cell count samples. Using a separate 3 mL syringe for each sample, pulled 4x1.0 mL cell count samples from Feeder Cell Suspension Transfer Pack using the needless injection port. Aliquoted each sample into the cryovials labeled. Performed cell counts and determine multiplication factors, elected protocols and entered multiplication factors. Determined the average of viable cell concentration and viability of the cell counts performed. Determined upper and lower limit for counts and confirm within limits.

[0251] Adjusted volume of feeder cell suspension. Calculated the adjusted volume of feeder cell suspension after removal of cell count samples. Calculated total viable feeder cells. Obtained additional feeder cells as needed. Thawed additional feeder cells as needed. Placed the 4th feeder cell bag into a zip top bag and thaw in a 37.0 ± 2.0°C water bath or cytotherm for ~3-5 minutes and pooled additional feeder cells. Measured volume. Measured the volume of the feeder cells in the syringe and recorded below (B). Calculated the new total volume of feeder cells. Added feeder cells to transfer pack.

[0252] Prepared dilutions as needed, adding 4.5 mL of AIM-V Media to four 15 mL conical tubes. Prepared cell counts. Using a separate 3 mL syringe for each sample, removed 4 x 1.0 mL cell count samples from Feeder Cell Suspension transfer pack, using the needless injection port. Performed cell counts and calculations. Determined an average viable cell concentration from all four counts performed. Adjusted volume of feeder cell suspension and calculated the adjusted volume of feeder cell suspension after removal of cell count samples. Total Feeder Cell Volume minues 4.0 mL removed. Calculated the volume of Feeder Cell Suspension that was required to obtain 5x109viable feeder cells. Calculated excess feeder cell volume. Calculated the volume of excess feeder cells to remove. Removed excess feeder cells.

[0253] Using a 1.0 mL syringe and 16G needle, drew up 0.15 mL of OKT3 and added OKT3. Heat sealed the feeder cell suspension transfer pack.

[0254] Day 11 G-REX Fill and Seed Set up G-REX500MCS. Removed “Complete CM2Day 11 Media”, from incubator and pumped media into G-REX500MCS. Pumped 4.5L of media into the G- REX500MCS,filling to the line marked on theflask. Heat sealed and incubatedflask as needed. Welded the Feeder Cell suspension transfer pack to the G-REX500MCS. Added Feeder Cells to G- 91 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO REX500MCS. Heat sealed. Welded the TIL Suspension transfer pack to theflask. Added TIL to G- REX500MCS. Heat sealed. Incubated G-REX500MCS at 37.0±2.0 ºC, CO2Percentage: 5.0±1.5 %CO2.

[0255] Calculated incubation window. Performed calculations to determine the proper time to remove G-REX500MCS from incubator on Day 16. Lower limit: Time of incubation + 108hours. Upper limit: Time of incubation + 132 hours.

[0256] Day 11 Excess TIL Cryopreservation. Applicable: Froze Excess TIL Vials. Verified the CRF has been set up prior to freeze. Perform Cryopreservation. Transferred vials from Controlled Rate Freezer to the appropriate storage. Upon completion of freeze, transfer vials from CRF to the appropriate storage container. Transferred vials to appropriate storage. Recorded storage location in LN2.

[0257] Day 16 Media Preparation. Pre-warmed AIM-V Media. Calculated time Media was warmed for media bags 1, 2, and 3. Ensured all bags have been warmed for a duration between 12 and 24 hours. Setup 10L Labtainer for Supernatant. Attached the larger diameter end of afluid pump transfer set to one of the female ports of a 10L Labtainer bag using the Luer connectors. Setup 10L Labtainer for Supernatant and label. Setup 10L Labtainer for Supernatant. Ensure all clamps were closed prior to removing from the BSC. NOTE: Supernatant bag was used during TIL Harvest, which may be performed concurrently with media preparation.

[0258] Thawed IL-2. Thawed 5×1.1 mL aliquots of IL-2 (6×106IU / mL) (BR71424) per bag of CTS AIM V media until all ice had melted. Aliquoted 100.0 mL GlutaMax™. Added IL-2 to GlutaMax™. Prepared CTS AIM V media bag for formulation. Prepared CTS AIM V media bag for formulation. Stage Baxa Pump. Prepared to formulate media. Pumped GlutaMax™ +IL-2 into bag. Monitored parameters: Temperature LED Display: 37.0±2.0 ºC, CO2Percentage: 5.0±1.5% CO2. Warmed Complete CM4 Day 16 Media. Prepared Dilutions.

[0259] Day 16 REP Spilt. Monitored Incubator parameters: Temperature LED display: 37.0±2.0 ºC, CO2Percentage: 5.0±1.5 %CO2. Removed G-REX500MCS from the incubator. Prepared and labeled 1 L Transfer Pack as TIL Suspension and weighed 1L.

[0260] Volume Reduction of G-REX500MCS. Transferred ~4.5L of culture supernatant from the G-REX500MCS to the 10L Labtainer.

[0261] Preparedflask for TIL harvest. After removal of the supernatant, closed all clamps to the red line. 92 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO

[0262] Initiation of TIL Harvest. Vigorously tapflask and swirl media to release cells and ensure all cells have detached.

[0263] TIL Harvest. Released all clamps leading to the TIL suspension transfer pack. Using the GatheRex transferred the cell suspension into the TIL Suspension transfer pack. NOTE: Be sure to maintain the tilted edge until all cells and media are collected. Inspected membrane for adherent cells. Rinsedflask membrane. Closed clamps on G-REX500MCS. Heat sealed the Transfer Pack containing the TIL. Heat sealed the 10L Labtainer containing the supernatant. Recorded weight of Transfer Pack with cell suspension and calculate the volume suspension. Prepared transfer pack for sample removal. Removed testing samples from cell supernatant.

[0264] Sterility & BacT testing sampling. Removed a 1.0 mL sample from the 15 mL conical labeled BacT prepared. Removed Cell Count Samples. In the BSC, using separate 3 mL syringes for each sample, removed 4x1.0 mL cell count samples from “TIL Suspension” transfer pack.

[0265] Removed mycoplasma samples. Using a 3 mL syringe, removed 1.0 mL from TIL Suspension transfer pack and place into 15 mL conical labeled “Mycoplasma diluent” prepared.

[0266] Prepared transfer pack for seeding. Placed TIL in incubator. Removed cell suspension from the BSC and place in incubator until needed. Performed cell counts and calculations. Diluted cell count samples initially by adding 0.5 mL of cell suspension into 4.5 mL of AIM-V media prepared which gave a 1:10 dilution. Determined the average of viable cell concentration and viability of the cell counts performed. Determined upper and lower limit for counts. Note: dilution may be adjusted according based off the expected concentration of cells. Determined an average viable cell concentration from all four counts performed. Adjusted volume of TIL suspension. Calculated the adjusted volume of TIL suspension after removal of cell count samples. Total TIL cell volume minus 5.0 mL removed for testing.

[0267] Calculated total viable TIL cells. Calculated the total number offlasks to seed. NOTE: The maximum number of G-REX500MCSflasks to seed wasfive. If the calculated number offlasks to seed exceededfive, onlyfive were seeded using the entire volume of cell suspension available.

[0268] Calculate number offlasks for subculture. Calculated the number of media bags required in addition to the bag prepared. Prepared one 10L bag of “CM4 Day 16 Media” for every two G-REX-500Mflask needed as calculated. Proceeded to seed thefirst GREX-500Mflask(s) while additional media is prepared and warmed. Prepared and warmed the calculated number of additional media bags determined. Filled G-REX500MCS. Prepared to pump media and pumped 4.5L 93 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO of media into G-REX500MCS. Heat Sealed. Repeated Fill. Incubatedflask. Calculated the target volume of TIL suspension to add to the new G-REX500MCSflasks. If the calculated number offlasks exceedsfive onlyfive will be seeded, USING THE ENTIRE VOLUME OF CELL SUSPENSION. Prepared Flasks for Seeding. Removed G-REX500MCS from the incubator. Prepared G-REX500MCS for pumping. Closed all clamps on except largefilter line. Removed TIL from incubator. Prepared cell suspension for seeding. Sterile welded (per Process Note 5.11) “TIL Suspension” transfer pack to pump inlet line. Placed TIL suspension bag on a scale.

[0269] Seededflask with TIL Suspension. Pump the volume of TIL suspension calculated intoflask. Heat sealed. Filled remainingflasks.

[0270] Monitored Incubator. Incubator parameters: Temperature LED Display: 37.0±2.0 ºC, CO2Percentage: 5.0±1.5 % CO2. Incubated Flasks.

[0271] Determined the time range to remove G-REX500MCS from incubator on Day 22.

[0272] Day 22 Wash Buffer Preparation. Prepared 10 L Labtainer Bag. In BSC, attach a 4” plasma transfer set to a 10L Labtainer Bag via luer connection. Prepared 10 L Labtainer Bag. Closed all clamps before transferring out of the BSC. NOTE: Prepared one 10L Labtainer Bag for every two G-REX500MCSflasks to be harvested. Pumped Plasmalyte into 3000 mL bag and removed air from 3000 mL Origen bag by reversing the pump and manipulating the position of the bag. Added human albumin 25% to 3000 mL Bag. Obtain afinal volumeof 120.0 mL of human albumin 25%.

[0273] Prepared IL-2 diluent. Using a 10 mL syringe, removed 5.0 mL of LOVO Wash Buffer using the needleless injection port on the LOVO Wash Buffer bag. Dispensed LOVO wash buffer into a 50 mL conical tube.

[0274] CRF blank bag LOVO wash buffer aliquotted. Using a 100 mL syringe, drew up 70.0 mL of LOVO Wash Buffer from the needleless injection port.

[0275] Thawed one 1.1 mL of IL-2 (6x106IU / mL), until all ice has melted. Added 50 µL IL-2 stock (6×106IU / mL) to the 50 mL conical tube labeled “IL-2 Diluent.”

[0276] Cryopreservation preparation. Placed 5 cryo-cassettes at 2-8°C to precondition them forfinal product cryopreservation.

[0277] Prepared cell count dilutions. In the BSC, added 4.5 mL of AIM-V Media that has been labelled with lot number and “For Cell Count Dilutions” to 4 separate 15 mL conical tubes. Prepared cell counts. Labeled 4 cryovials with vial number (1-4). Kept vials under BSC to be used. 94 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO

[0278] Day 22 TIL Harvest. Monitored Incubator. Incubator Parameters Temperature LED display: 37 ± 2.0°C, CO2Percentage: 5%±1.5%. Removed G-REX500MCS Flasks from Incubator. Prepared TIL collection bag and labeled. Sealed off extra connections. Volume Reduction: Transferred ~4.5L of supernatant from the G-REX500MCS to the Supernatant bag.

[0279] Preparedflask for TIL harvest. Initiated collection of TIL. Vigorously tapflask and swirl media to release cells. Ensure all cells have detached. Initiated collection of TIL. Released all clamps leading to the TIL suspension collection bag. TIL Harvest. Using the GatheRex, transferred the TIL suspension into the 3000 mL collection bag. Inspect membrane for adherent cells. Rinsed flask membrane. Closed clamps on G- Rex500MCS and ensured all clamps are closed. Transferred cell suspension into LOVO source bag. Closed all clamps. Heat Sealed. Removed 4x1.0 mL Cell Counts Samples

[0280] Performed Cell Counts. Performed cell counts and calculations utilizing NC-200 and Process Note 5.14. Diluted cell count samples initially by adding 0.5 mL of cell suspension into 4.5 mL of AIM-V media prepared. This gave a 1:10 dilution. Determined the average viability, viable cell concentration, and total nucleated cell concentration of the cell counts performed. Determined Upper and Lower Limit for counts. Determined the average viability, viable cell concentration, and total nucleated cell concentration of the cell counts performed. Weighed LOVO source bag. Calculated total viable TIL Cells. Calculated total nucleated cells.

[0281] Prepared Mycoplasma Diluent. Removed 10.0 mL from one supernatant bag via luer sample port and placed in a 15 mL conical.

[0282] Performed “TIL G-REX Harvest” protocol and determined thefinal product target volume. Loaded disposable kit. Removedfiltrate bag. Entered Filtrate capacity. Placed Filtrate container on benchtop. Attached PlasmaLyte. Verified that the PlasmaLyte was attached and observed that the PlasmaLyte is moving. Attached Source container to tubing and verified Source container was attached. Confirmed PlasmaLyte was moving.

[0283] Final Formulation and Fill. Target volume / bag calculation. Calculated volume of CS-10 and LOVO wash buffer to formulate blank bag. Prepared CRF Blank.

[0284] Calculated the volume of IL-2 to add to the Final Product. Final IL-2 Concentration desired (IU / mL) – 300IU / mL. IL-2 working stock: 6 × 104 IU / mL. Assembled connect apparatus. Sterile welded a 4S-4M60 to a CC2 cell connection. Sterile welded the CS750 cryobags to the harness prepared. Welded CS-10 bags to spikes of the 4S-4M60. Prepared TIL with IL-2. Using an 95 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO appropriately sized syringe, removed amount of IL-2 determined from the “IL-26x104” aliquot. Labeled forumlated TIL Bag. Added the formulated TIL bag to the apparatus. Added CS10. Switched Syringes. Drew ~10 mL of air into a 100 mL syringe and replaced the 60 mL syringe on the apparatus. Added CS10. Prepared CS-750 bags. Dispensed cells.

[0285] Removed air fromfinal product bags and take retain. Once the lastfinal product bag wasfilled, closed all clamps. Drew 10 mL of air into a new 100 mL syringe and replace the syringe on the apparatus. Dispensed retain into a 50 mL conical tube and label tube as “Retain” and lot number. Repeat air removal step for each bag.

[0286] Preparedfinal product for cryopreservation, including visual inspection. Held the cryobags on cold pack or at 2-8°C until cryopreservation.

[0287] Removed cell count sample. Using an appropriately sized pipette, remove 2.0 mL of retain and place in a 15 mL conical tube to be used for cell counts. Performed cell counts and calculations. NOTE: Diluted only one sample to appropriate dilution to verify dilution is sufficient. Diluted additional samples to appropriate dilution factor and proceed with counts. Determined the Average of Viable Cell Concentration and Viability of the cell counts performed. Determined Upper and Lower Limit for counts. NOTE: Dilution may be adjusted according based off the expected concentration of cells. Determined the Average of Viable Cell Concentration and Viability. Determined Upper and Lower Limit for counts. Calculated IFN-γ. Heat Sealed Final Product bags.

[0288] Labeled and collected samples per exemplary sample plan below. TABLE 9. Sample plan.96 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO

[0289] Sterility and BacT testing. Testing Sampling. In the BSC, remove a 1.0 mL sample from the retained cell suspension collected using an appropriately sized syringe and inoculate the anaerobic bottle. Repeat the above for the aerobic bottle.

[0290] Final Product Cryopreservation. Prepared controlled rate freezer (CRF). Verified the CRF had been set up. Set up CRF probes. Placedfinal product and samples in CRF. Determined the time needed to reach 4 ºC ± 1.5 ºC and proceed with the CRF run. CRF completed and stored. Stopped the CRF after the completion of the run. Remove cassettes and vials from CRF. Transferred cassettes and vials to vapor phase LN2 for storage. Recorded storage location.

[0291] Post-Processing and analysis offinal drug product included the following tests: (Day 22) Determination of CD3+ cells on Day 22 REP byflow cytometry; (Day 22) Gram staining method (GMP); (Day 22) Bacterial endotoxin test by Gel Clot LAL Assay (GMP); (Day 16) BacT Sterility Assay (GMP); (Day 16) Mycoplasma DNA detection by TD-PCR (GMP); Acceptable appearance attributes; (Day 22) BacT sterility assay (GMP)(Day 22); (Day 22) IFN-gamma assay. Other potency assay as described herein are also employed to analyze TIL products. EXAMPLE 3: PD-1 TIGIT On / Off Target mRNA Titration Detailed protocol (for Experiment #10)

[0292] PreREP TILs were thawed and counted. TILs were activated for 2 days using 5 mL of GMP TransAct (Miltenyi Biotec cat # 170-076-156) in 100 mL of CM1. After activation, TILs were counted and split between the electroporation conditions (below). TILs were washed 1x with PBS and 1x with of CTS™ Xenon™ Genome Editing Buffer (Thermo Fisher, cat # A4998001). TILs were resuspended in CTS™ Xenon™ Genome Editing Buffer and mRNA (volumes based on the conditions below, 1 mL total volume). Each condition was transferred to a CTS Xenon SingleShot Electroporation Chamber (Thermo Fisher, cat # A50305) and electroporated using the Xenon electroporator with the settings 2300 V, 2 ms pulse width, 3 pulses. TILs were rested in CM2 overnight at 30°C. REPs were set up after the overnight rest using 50e6 iPBMCs, 30 ng / ml MACS® GMP CD3 pure (Miltenyi Biotec, cat # 170-076-116), 4e5 TILs, and 100 ml CM2 per condition. After 5 days the scale up was performed by splitting the sample and adding CM4 (100mL total). TILs were harvested after 4 days and frozen in CS10. 97 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO

[0293] For small scale and full scale runs, PD-1 and TIGIT TALEN mRNA was added at 2 ug / 1e6 cells. For Experiments #6-9, thawed REP TILs (in contrast to thawed preREP TILs) were used for electroporation. Detailed protocol (for Experiment #10) Day 0 – Thaw and activate 1) Frozen preREP vials (D11) TILs were thawed into warm CM1 and counted 2) Cells were transferred into a Grex100Mflask in 100 mL of CM1 3) 1 bottle of TransAct (5 mL) was added to theflask 4) Flask was placed in the incubator for 2 days of stimulation at 37°C Day 2 – PD-1 electroporation 1) Cells were harvested from theflask by resuspending the cell suspension and passing through a 70 um cell strainer into a 250mL conical tube 2) Cell counts were taken 3) Sample was split into the following conditions, each in a 50 ml conical tube:98 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO TALEN with 5M cells (2ug / M cells)99 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO 4) Cells were washed with PBS and centrifuged 400g x 5 min 5) Cells were washed with 10 mL of CTS™ Xenon™ Genome Editing Buffer (Thermo Fisher, cat # A4998001) and centrifuged 400g x 5 min 6) Cells were resuspended in CTS™ Xenon™ Genome Editing Buffer and mRNA (total of 1mL) and were electroporated using the Xenon electroporator with the settings 2300 V, 2 ms pulse width, 3 pulses 7) Cells were rested in CM2 at 30°C overnight Day 3 - REP 1) Cells were collected and counted 2) 4e5 TIL were plated in a GREX 6well plate, 1 well per condition in 100 mL of CM2 3) 50e6 iPBMCs + 30 ng / ml of OKT3 were added to each well 4) Cells were incubated at 37°C Day 8 – Scale up 1) Each well was volume reduced by ~80mL 2) Each well was resuspended and transferred to a 50 ml conical tube 3) ½ of the sample was replated in the Grex 6well plate 4) CM4 was added to each well to 100mL total volume Day 12 – Harvest 1) Each well was volume reduced by ~80mL 2) Each well was resuspended and transferred to a 50 ml conical tube 1) Cell counts were taken 2) Cells were frozen at ~10e6 cells / ml in 1 ml of CS10 DNA Sequencing

[0294] DNA was isolated from harvested TILs using the QIAamp DNA Blood Mini Kit (Qiagen, Cat #: 51106), followed by the library prep for Next Gen Sequencing using the CleanPlex Custom Panel Kit (Paragon Genomics, Cat #: 937001). All DNA samples were sequenced on Illumina NextSeq2000 using either NextSeq 1000 / 2000 P1 Reagents - 300 Cycles (Illumina, Cat# 20050264) or NextSeq 1000 / 2000 P2 Reagents - 300 Cycles (Illumina, Cat# 20046813), using paired end 2 x 150bp reads. Sequencing Analysis

[0295] Targeted DNA sequencing analysis was performed using the R package ampliCan1 (v1.22.1). Local alignment of the fastq reads to expected amplicon sequences was done using the amplicanAlignfunction with default parameters. Alignment events observed in overlapping primer or primer-dimer infected reads werefiltered out. Insertions and deletions (indels) were then quantified per sample. Further analysis was performed via a custom python script to further summarize and normalize the data. TALEN associated edits at each targeted region of interest were 100 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO calculated by the subtraction of frequency of indels in non-edited control samples from the signals observed in TALEN treated samples.

[0296] Observed on-target and candidate off-target edits in individual samples from various experiments were analyzed further relative to their corresponding mRNA concentration used for electroporation. Individual values converted to µg mRNA per mL solution were plotted and nonlinear regressions were calculated using hyperbolic interpolation using GraphPad Prism. In each generated curve, the point of inflection demonstrates where an increase in mRNA concentration no longer substantially increases the edit rate. The optimal mRNA concentration maximizes the edit for the on- target sites, and minimizes signals observed for the candidate off-target sites.

[0297] Fig.1 shows PD-1 on-target hyperbolafit options. Observed PD-1 edit rates are indicated with dots for samples' corresponding mRNA concentration (ug / mL) used for electroporation. Non-linear regressions, using hyperbola interpolations, were derived for groups of experiments and are identified with the curved lines. Example recommendations of mRNA concentrations (ug / mL) are identified with vertical lines at 10 ug / mL (green) and 12.5 ug / mL (orange dotted line). This discovery identifies that the selection of mRNA concentration, irrespective of the number of cells, can be used to optimize KO efficiency for on-target editing.

[0298] Figs.2A-2F show PD-1 off-target signals for Candidates 3, 1, 19, 9, 17, and 4, respectively. Observed edit rates for select PD-1 candidate off-targets are indicated with dots for samples' corresponding mRNA concentration (ug / mL) used for electroporation. Non-linear regressions, using hyperbola interpolations, were derived for groups of experiments and are identified with the curved lines. Example recommendations of mRNA concentrations (ug / mL) are identified with vertical lines at 10 ug / mL (green) and 12.5 ug / mL (orange dotted line). The discovery identifies that selection of mRNA concentration, irrespective of the number of cells, can be used to minimize off-target editing.

[0299] Fig.3 shows TIGIT on-target hyperbolafit options. Observed edit rates for TIGIT are indicated with dots for samples' corresponding mRNA concentration (ug / mL) used for electroporation. Non-linear regressions, using hyperbola interpolations, were derived for groups of experiments and are identified with the curved lines. Example recommendations of mRNA concentrations (ug / mL) are identified with vertical lines at 40 ug / mL (green) and 50 ug / mL (blue dotted line). This discovery identifies that the selection of mRNA concentration, irrespective of the number of cells, can be used to optimize KO efficiency for on-target editing. 101 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO

[0300] Figs.4A-4B show TIGIT off-target signals for Candidates 1 and 2, respectively. Observed edit rates for select TIGIT candidate off-targets are indicated with dots for samples' corresponding mRNA concentration (ug / mL) used for electroporation. Non-linear regressions, using hyperbola interpolations, were derived for groups of experiments and are identified with the curved lines. Example recommendations of mRNA concentrations (ug / mL) are identified with vertical lines at 40 ug / mL (green) and 50 ug / mL (blue dotted line). The discovery identifies that selection of mRNA concentration, irrespective of the number of cells, can be used to minimize off-target editing.

[0301] The examples set forth above are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the embodiments of the compositions, systems and methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above-described modes for carrying out the invention that are obvious to persons of skill in the art are intended to be within the scope of the following claims. All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains.

[0302] All headings and section designations are used for clarity and reference purposes only and are not to be considered limiting in any way. For example, those of skill in the art will appreciate the usefulness of combining various aspects from different headings and sections as appropriate according to the spirit and scope of the invention described herein.

[0303] All references cited herein are hereby incorporated by reference herein in their entireties and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0304] Many modifications and variations of this application can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments and examples described herein are offered by way of example only, and the application is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which the claims are entitled. 102 DB1 / 150591471.1

Claims

Attorney Docket No.: 116983-5129-WO WHAT IS CLAIMED IS:

1. A method for gene-editing a population of cells using a programmable nuclease, the method comprising introducing an RNA molecule associated with the programmable nuclease into the population of cells in a volume, wherein the RNA molecule is present at afixed concentration not dependent on the number of cells in the volume, to generate a population of gene-edited cells.

2. The method of claim 1, wherein the programmable nuclease is selected from the group consisting of a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease (ZFN), a CRISPR-associated (Cas) nuclease, and a meganuclease (MN).

3. The method of claim 1 or 2, wherein the RNA molecule is an mRNA molecule encoding the programmable nuclease.

4. The method of claim 3, wherein thefixed concentration of the mRNA molecule is from about 1 µg / mL to about 100 µg / mL.

5. The method of claim 3, wherein thefixed concentration of the mRNA molecule is from about 10 µg / mL to about 50 µg / mL.

6. The method of claim 3, wherein thefixed concentration of the mRNA molecule is about 10 µg / mL.

7. The method of claim 3, wherein thefixed concentration of the mRNA molecule is about 12.5 µg / mL.

8. The method of claim 3, wherein thefixed concentration of the mRNA molecule is about 20 µg / mL.

9. The method of claim 3, wherein thefixed concentration of the mRNA molecule is about 30 µg / mL.

10. The method of claim 3, wherein thefixed concentration of the mRNA molecule is about 40 µg / mL.

11. The method of claim 3, wherein thefixed concentration of the mRNA molecule is about 50 µg / mL.

12. The method of claim 1 or 2, wherein the RNA molecule is a gRNA molecule.

13. The method of any one of claims 1-12, wherein the volume is about 1 mL to about 100 mL. 103 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO 14. The method of any one of claims 1-13, wherein the volume comprises about 105to about 109cells.

15. The method of any one of claims 1-14, wherein the RNA molecule is introduced via a transfection technique selected from the group consisting of electroporation, nanoparticle mediated transfection, magnetic bead-based transfection, and calcium phosphate precipitation.

16. The method of any one of claims 1-15, wherein the RNA molecule is introduced via electroporation.

17. The method of claim 16, wherein the volume comprises an electroporation buffer.

18. The method of claim 17, wherein the electroporation buffer is CTS™ Xenon™ Genome Editing Buffer.

19. The method of any one of claims 16-18, wherein the electroporation is conducted using the Xenon™ electroporator with the settings 2300 V, 2 ms pulse width, 3 pulses.

20. The method of any one of claims 1-19, further comprising activating the population of cells for 1 day or 2 days before introducing the RNA molecule.

21. The method of claim 20, wherein the activating step comprises contacting the population of cells with TransAct.

22. The method of claim 20 or 21, wherein the activating step comprises contacting the population of cells with TransAct at a ratio of 1:

100.

23. The method of any one of claims 1-22, further comprising resting the population of cells for 1 day or 2 days after introducing the RNA molecule.

24. The method of any one of claims 1-23, wherein the programmable nuclease cuts a DNA sequence in a target gene.

25. The method of claim 24, wherein the target gene is selected from the group consisting of an immune checkpoint gene, a TCR gene, a PCSK9 gene, a hATTR gene, aSOD1 gene, a TAU gene, a LRRK2 gene, a SMN2 gene, a TTR gene, a Factor XI gene, an APOL1 gene, and a CCR5 gene.

26. The method of claim 25, 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, 104 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11, and BCOR.

27. The method of claim 25, wherein the immune checkpoint gene is selected from the group consisting of PD-1, TIGIT, CTLA-4, LAG-3, and CISH.

28. The method of any one of claims 24-27, wherein the expression of the target gene is reduced.

29. The method of any one of claims 24-27, wherein the expression of the target gene is reduced by at least 50%.

30. The method of any one of claims 24-27, wherein the expression of the target gene is reduced by at least 80%.

31. The method of any one of claims 1-30, wherein the cells are T cells.

32. The method of claim 31, wherein the T cells are tumor infiltrating lymphocytes (TILs).

33. The method of claim 31, wherein the T cells express a chimeric antigen receptor (CAR).

34. The method of claim 33, wherein the CAR binds to an antigen chosen from: CD19, CD20, CD22, BCMA, mesothelin, EGFRvIII, GD2, Tn antigen, sTn antigen, Tn-O-Glycopeptides, sTn-O- Glycopeptides, PSMA, CD97, TAG72, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, leguman, GD3, CD171, IL-11Ra, PSCA, MAD-CT-1, MAD-CT-2, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, folate receptor alpha, ERBBs (e.g., ERBB2), Her2 / neu, MUC1, EGFR, NCAM, Ephrin B2, CAIX, LMP2, sLe, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, FAP, Legumain, HPV E6 or E7, ML-IAP, CLDN6, TSHR, GPRC5D, ALK, Polysialic acid, Fos-related antigen, neutrophil elastase, TRP-2, CYP1B1, sperm protein 17, beta human chorionic gonadotropin, AFP, thyroglobulin, PLAC1, globoH, RAGE1, MN-CA IX, human telomerase reverse transcriptase, intestinal carboxyl esterase, mut hsp 70-2, NA-17, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, NY-ESO-1, GPR20, Ly6k, OR51E2, TARP, GFRα4, or a peptide of any of these antigens presented on MHC.

35. A therapeutic population of gene-edited cells produced by the method of any one of claims 1- 34.

36. The therapeutic population of gene-edited cells of claim 35, wherein the gene-edited cells have reduced expression of a target gene.

37. The therapeutic population of gene-edited cells of claim 36, wherein the target gene is selected from the group consisting of an immune checkpoint gene, a TCR gene, a PCSK9 gene, a hATTR gene, aSOD1 gene, a TAU gene, a LRRK2 gene, a SMN2 gene, a TTR gene, a Factor XI gene, an APOL1 gene, and a CCR5 gene. 105 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO 38. The therapeutic population of gene-edited cells of claim 37, 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.

39. The therapeutic population of gene-edited cells of any one of claims 36-38, wherein the expression of the target gene is reduced by at least 50%.

40. The therapeutic population of gene-edited cells of any one of claims 36-38, wherein the expression of the target gene is reduced by at least 60%.

41. The therapeutic population of gene-edited cells of any one of claims 36-38, wherein the expression of the target gene is reduced by at least 80%.

42. The therapeutic population of gene-edited cells of any one of claims 35-41, wherein the cells are T cells.

43. The therapeutic population of gene-edited cells of claim 42, wherein the T cells are tumor infiltrating lymphocytes (TILs).

44. The therapeutic population of gene-edited cells of claim 42, wherein the T cells express a chimeric antigen receptor (CAR).

45. The therapeutic population of gene-edited cells of claim 44, wherein the CAR binds to an antigen chosen from: CD19, CD20, CD22, BCMA, mesothelin, EGFRvIII, GD2, Tn antigen, sTn antigen, Tn-O-Glycopeptides, sTn-O-Glycopeptides, PSMA, CD97, TAG72, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, leguman, GD3, CD171, IL-11Ra, PSCA, MAD-CT-1, MAD-CT-2, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, folate receptor alpha, ERBBs (e.g., ERBB2), Her2 / neu, MUC1, EGFR, NCAM, Ephrin B2, CAIX, LMP2, sLe, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, FAP, Legumain, HPV E6 or E7, ML-IAP, CLDN6, TSHR, GPRC5D, ALK, Polysialic acid, Fos-related antigen, neutrophil elastase, TRP-2, CYP1B1, sperm protein 17, beta human chorionic gonadotropin, AFP, thyroglobulin, PLAC1, globoH, RAGE1, MN-CA IX, human telomerase reverse transcriptase, intestinal carboxyl esterase, mut hsp 70-2, NA-17, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, NY-ESO-1, GPR20, Ly6k, OR51E2, TARP, GFRα4, or a peptide of any of these antigens presented on MHC. 106 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO 46. A method for gene-editing a population of TILs using a programmable nuclease, comprising: (a) performing afirst expansion of the population of TILs in a cell culture medium comprising IL- 2, wherein thefirst expansion is performed for about 3-14 days; and (b) performing a second expansion of the population of TILs in a cell culture medium comprising IL-2, OKT-3, and antigen presenting cells (APCs), wherein the second expansion is performed for about 7-14 days, wherein the method further comprises introducing afirst RNA molecule associated with the programmable nuclease into the population of TILs in a volume, wherein the RNA molecule is present at afixed concentration not dependent on the number of TILs in the volume, and wherein thefirst RNA molecule is introduced before step (a), after step (a) and before step (b), or after step (b).

47. The method of claim 46, further comprising introducing a second RNA molecule associated with a second programmable nuclease into the population of TILs.

48. The method of claim 46 or 47, wherein the introducing thefirst and / or second RNA molecule is performed after thefirst expansion and before the second expansion.

49. The method of any one of claims 46-48, further comprising resting the population of TILs for 1 day or 2 days after introducing thefirst RNA molecule.

50. The method of any one of claims 46-49, further comprising activating the population of TILs for 1 day or 2 days before introducing thefirst RNA molecule.

51. The method of claim 50, wherein the activating step comprises contacting the population of TILs with TransAct.

52. The method of claim 50 or 51, wherein the activating step comprises contacting the population of TILs with TransAct at a ratio of 1:

100.

53. The method of any one of claims 46-52, wherein thefirst RNA molecule is afirst mRNA molecule encoding thefirst programmable nuclease.

54. The method of any one of claims 47-53, wherein the second RNA molecule is a second mRNA molecule encoding the second programmable nuclease.

55. The method of any one of claims 46-54, wherein thefirst and / or second programmable nuclease is selected from the group consisting of a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease (ZFN), a CRISPR-associated (Cas) nuclease, and a meganuclease 107 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO (MN).

56. The method of any one of claims 53-55, wherein thefixed concentration of thefirst and / or second mRNA molecule is from about 1 µg / mL to about 100 µg / mL.

57. The method of any one of claims 53-55, wherein thefixed concentration of thefirst and / or second mRNA molecule is from about 10 µg / mL to about 50 µg / mL.

58. The method of any one of claims 53-55, wherein thefixed concentration of thefirst and / or second mRNA molecule is about 10 µg / mL.

59. The method of any one of claims 53-55, wherein thefixed concentration of thefirst and / or second mRNA molecule is about 12.5 µg / mL.

60. The method of any one of claims 53-55, wherein thefixed concentration of thefirst and / or second mRNA molecule is about 20 µg / mL.

61. The method of any one of claims 53-55, wherein thefixed concentration of thefirst and / or second mRNA molecule is about 30 µg / mL.

62. The method of any one of claims 53-55, wherein thefixed concentration of thefirst and / or second mRNA molecule is about 40 µg / mL.

63. The method of any one of claims 53-55, wherein thefixed concentration of thefirst and / or second mRNA molecule is about 50 µg / mL.

64. The method of any one of claims 46-63, wherein thefirst programmable nuclease cuts a DNA sequence in afirst target gene.

65. The method of any one of claims 47-64, wherein the second programmable nuclease cuts a DNA sequence in a second target gene.

66. The method of claim 64 or 65, wherein thefirst and / or second target gene is selected from the group consisting of an immune checkpoint gene, a TCR gene, a PCSK9 gene, a hATTR gene, aSOD1 gene, a TAU gene, a LRRK2 gene, a SMN2 gene, a TTR gene, a Factor XI gene, an APOL1 gene, and a CCR5 gene.

67. The method of claim 66, 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, 108 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, TOX, SOCS1, ANKRD11, and BCOR.

68. The method of claim 66, wherein the immune checkpoint gene is selected from the group consisting of PD-1, TIGIT, CTLA-4, LAG-3, and CISH.

69. The method of claim 66, wherein thefirst target gene is PD-1 and the second target gene is CTLA-4.

70. The method of claim 66, wherein thefirst target gene is PD-1 and the second target gene is TIGIT.

71. The method of claim 66, wherein thefirst target gene is PD-1 and the second target gene is LAG-3.

72. The method of claim 66, wherein thefirst target gene is PD-1 and the second target gene is CISH.

73. The method of any one of claims 46-72, wherein thefirst and / or second RNA molecule is a gRNA molecule.

74. The method of any one of claims 46-73, wherein thefirst and / or second RNA molecule is introduced via a transfection technique selected from the group consisting of electroporation, nanoparticle mediated transfection, magnetic bead-based transfection, and calcium phosphate precipitation.

75. The method of any one of claims 46-73, wherein thefirst and / or second RNA molecule is introduced via electroporation.

76. The method of claim 75, wherein the volume comprises an electroporation buffer.

77. The method of claim 76, wherein the electroporation buffer is CTS™ Xenon™ Genome Editing Buffer.

78. The method of claim 76 or 77, wherein the electroporation is conducted using the Xenon™ electroporator with the settings 2300 V, 2 ms pulse width, 3 pulses.

79. The method of any one of claims 46-78, wherein the volume is about 1 mL to about 100 mL.

80. The method of any one of claims 46-79, wherein the volume comprises about 105to about 109cells.

81. The method of any of claims 46-80, wherein thefirst expansion is performed for about 3-11 days. 109 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO 82. The method of any of claims 46-81, wherein the second expansion is performed for about 7-11 days.

83. The method of any of claims 46-82, wherein the IL-2 is present at an initial concentration of between 1000 IU / mL and 6000 IU / mL in the cell culture medium in thefirst expansion.

84. The method of any of claims 46-83, wherein in the second expansion step, the IL-2 is present at an initial concentration of between 1000 IU / mL and 6000 IU / mL and the OKT-3 antibody is present at an initial concentration of about 30 ng / mL.

85. A method for gene-editing a population of TILs using a programmable nuclease, comprising: (a) culturing afirst population of TILs in afirst cell culture medium comprising IL-2 for about 5-7 days to produce a second population of TILs; (b) activating the second population of TILs for about 2-4 days, to produce a third population of TILs; (c) introducing an RNA molecule associated with the programmable nuclease into the third population of TILs in a volume, wherein the RNA molecule is present at afixed concentration not dependent on the number of TILs in the volume, to produce a fourth population of TILs, wherein the programmable nuclease cuts the DNA sequence of a target gene; and (d) culturing the fourth population of TILs in a second cell culture medium comprising antigen presenting cells (APCs), OKT-3, and IL-2 for about 7-11 days, to produce sixth population of TILs having reduced expression of the target gene.

86. The method of claim 85, wherein the activating step comprises contacting the population of TILs with TransAct.

87. The method of claim 85 or 86, wherein the activating step comprises contacting the population of TILs with TransAct at a ratio of 1:

100.

88. The method of any one of claims 85-87, wherein the programmable nuclease is selected from the group consisting of a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease (ZFN), a CRISPR-associated (Cas) nuclease, and a meganuclease (MN).

89. The method of any one of claims 85-88, wherein thefixed concentration of the mRNA molecule is from about 1 µg / mL to about 100 µg / mL.

90. The method of any one of claims 85-88, wherein thefixed concentration of the mRNA molecule is from about 10 µg / mL to about 50 µg / mL.

91. The method of any one of claims 85-88, wherein thefixed concentration of the mRNA molecule 110 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO is about 10 µg / mL.

92. The method of any one of claims 85-88, wherein thefixed concentration of the mRNA molecule is about 12.5 µg / mL.

93. The method of any one of claims 85-88, wherein thefixed concentration of the mRNA molecule is about 20 µg / mL.

94. The method of any one of claims 85-88, wherein thefixed concentration of the mRNA molecule is about 30 µg / mL.

95. The method of any one of claims 85-88, wherein thefixed concentration of the mRNA molecule is about 40 µg / mL.

96. The method of any one of claims 85-88, wherein thefixed concentration of the mRNA molecule is about 50 µg / mL.

97. The method of any one of claims 85-96, wherein the programmable nuclease cuts a DNA sequence in a target gene.

98. The method of claim 97, wherein the target gene is selected from the group consisting of an immune checkpoint gene, a TCR gene, a PCSK9 gene, a hATTR gene, aSOD1 gene, a TAU gene, a LRRK2 gene, a SMN2 gene, a TTR gene, a Factor XI gene, an APOL1 gene, and a CCR5 gene.

99. The method of claim 98, 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.

100. The method of claim 98, wherein the immune checkpoint gene is selected from the group consisting of PD-1, TIGIT, CTLA-4, LAG-3, and CISH.

101. A method for gene-editing a population of TILs using two programmable nucleases, comprising: (a) culturing afirst population of TILs in afirst cell culture medium comprising IL-2 for about 5-7 days to produce a second population of TILs; 111 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO (b) activating the second population of TILs for about 2-4 days, to produce a third population of TILs; (c) introducing afirst RNA molecule associated with afirst programmable nuclease into the third population of TILs in a volume, wherein thefirst RNA molecule is present at afixed concentration not dependent on the number of TILs in the volume, to produce a fourth population of TILs, wherein thefirst programmable nuclease cuts the DNA sequence of afirst target gene; (d) resting the fourth population of TILs in thefirst cell culture medium comprising IL-2 for about 2 to 3 days; (e) introducing a second RNA molecule associated with a second programmable nuclease into the fourth population of TILs in a volume, wherein the second RNA molecule is present at a fixed concentration not dependent on the number of TILs in the volume, to produce afifth population of TILs, wherein the second programmable nuclease cuts the DNA sequence of a second target gene; and (f) culturing thefifth population of TILs in a second cell culture medium comprising antigen presenting cells (APCs), OKT-3, and IL-2 for about 7-11 days, to produce sixth population of TILs having reduced expression of thefirst target gene and the second target gene.

102. The method of claim 101, wherein the activating step comprises contacting the population of TILs with TransAct.

103. The method of claim 101 or 102, wherein the activating step comprises contacting the population of TILs with TransAct at a ratio of 1:

100.

104. The method of any one of claims 101-103, wherein thefirst RNA molecule is afirst mRNA molecule encoding thefirst programmable nuclease.

105. The method of any one of claims 101-104, wherein the second RNA molecule is a second mRNA molecule encoding the second programmable nuclease.

106. The method of any one of claims 101-105, wherein thefirst and / or second programmable nuclease is selected from the group consisting of a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease (ZFN), a CRISPR-associated (Cas) nuclease, and a meganuclease (MN).

107. The method of any one of claims 101-106, wherein thefixed concentration of thefirst and / or 112 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO second mRNA molecule is from about 1 µg / mL to about 100 µg / mL.

108. The method of any one of claims 101-106, wherein thefixed concentration of thefirst and / or second mRNA molecule is from about 10 µg / mL to about 50 µg / mL.

109. The method of any one of claims 101-106, wherein thefixed concentration of thefirst and / or second mRNA molecule is about 10 µg / mL.

110. The method of any one of claims 101-106, wherein thefixed concentration of thefirst and / or second mRNA molecule is about 12.5 µg / mL.

111. The method of any one of claims 101-106, wherein thefixed concentration of thefirst and / or second mRNA molecule is about 20 µg / mL.

112. The method of any one of claims 101-106, wherein thefixed concentration of thefirst and / or second mRNA molecule is about 30 µg / mL.

113. The method of any one of claims 101-106, wherein thefixed concentration of thefirst and / or second mRNA molecule is about 40 µg / mL.

114. The method of any one of claims 101-106, wherein thefixed concentration of thefirst and / or second mRNA molecule is about 50 µg / mL.

115. The method of any one of claims 101-114, wherein thefirst programmable nuclease cuts a DNA sequence in afirst target gene.

116. The method of any one of claims 101-115, wherein the second programmable nuclease cuts a DNA sequence in a second target gene.

117. The method of claim 115 or 116, wherein thefirst and / or second target gene is selected from the group consisting of an immune checkpoint gene, a TCR gene, a PCSK9 gene, a hATTR gene, aSOD1 gene, a TAU gene, a LRRK2 gene, a SMN2 gene, a TTR gene, a Factor XI gene, an APOL1 gene, and a CCR5 gene.

118. The method of claim 117, 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. 113 DB1 / 150591471.1Attorney Docket No.: 116983-5129-WO 119. The method of claim 117, wherein the immune checkpoint gene is selected from the group consisting of PD-1, TIGIT, CTLA-4, LAG-3, and CISH.

120. The method of claim 117, wherein thefirst target gene is PD-1 and the second target gene is CTLA-4.

121. The method of claim 117, wherein thefirst target gene is PD-1 and the second target gene is TIGIT.

122. The method of claim 117, wherein thefirst target gene is PD-1 and the second target gene is LAG-3.

123. The method of claim 117, wherein thefirst target gene is PD-1 and the second target gene is CISH. 114 DB1 / 150591471.1

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