A method for generating enhanced tumor-infiltrating lymphocytes by microfluidic delivery

Abstract

The present application provides TILs containing factors that enhance the activity and / or proliferative capacity of TILs, methods for producing such TILs, and methods for using such modified TILs to enhance immune responses.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 278,447, filed November 11, 2021, which is incorporated by reference in its entirety herein.

[0002] Reference to sequence listings filed electronically via EFSWEB The contents of the sequence listing electronically filed in this application (4821_079PC04_Seqlisting_ST26.xml, size: 20,165 bytes, creation date: November 9, 2022) are incorporated by reference in their entirety into this specification.

[0003] The present disclosure relates generally to TILs that contain factors that enhance the activity and / or proliferative capacity of TILs, methods of producing such TILs, and methods of using such modified TILs to enhance immune responses. [Background technology]

[0004] The development of tumor infiltrating lymphocyte (TIL) therapy is fraught with challenges. Although TIL therapy has shown remarkable activity against solid tumors in patients, current TIL compositions require lymphodepletion of patients and maintenance of high doses of IL-2 following cell infusion to support clinical activity. Ex vivo engineering of TIL products with mRNA can improve efficacy, potentially expanding the number of patients who can receive treatment and allowing for repeated dosing or combination therapy with other therapies.

[0005] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety. In particular, patent publications WO2016070136, US20180142198, WO2017008063, US20180201889, WO2019178005, WO2019178006, and WO2020176789 are incorporated by reference in their entirety. Summary of the Invention

[0006] Provided herein is a method for increasing expression of granzyme B in tumor-infiltrating lymphocytes (TILs), the method comprising: (a) modifying TILs to increase expression of (i) a costimulatory molecule, (ii) a cytokine, or (ii) both a costimulatory molecule and a cytokine; and (b) culturing the TILs in the absence of exogenous cytokines, wherein after the modification and culturing, the TILs exhibit higher expression of granzyme B upon activation than corresponding unmodified TILs (reference TILs).

[0007] The present disclosure further provides a method for increasing expression of granzyme B in tumor-infiltrating lymphocytes (TILs), the method comprising culturing TILs in the absence of exogenous cytokines, wherein the TILs have been modified to increase expression of (i) a costimulatory molecule, (ii) a cytokine, or (iii) both a costimulatory molecule and a cytokine, and following the culture, the TILs exhibit greater proliferation upon activation than corresponding unmodified TILs (reference TILs).

[0008] In some embodiments, expression of Granzyme B is at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, or at least about 10-fold higher than expression by a reference TIL.

[0009] In some embodiments, the culturing is for at least about 1 day, at least about 2 days, or at least about 3 days. In some embodiments, the costimulatory molecule is CD86. In some embodiments, the cytokine comprises membrane-bound IL-2, membrane-bound IL-12, or both.

[0010] In some embodiments, the modification comprises perturbing the TILs by passing a cell suspension comprising the TILs through a cell deformation constriction such that a nucleic acid encoding a costimulatory molecule and / or a nucleic acid encoding a cytokine enters the TILs through the perturbation when contacted with the TILs, in some embodiments, the modification further comprises contacting the TILs with a nucleic acid encoding a costimulatory molecule and / or a nucleic acid encoding a cytokine.

[0011] In some embodiments, the TILs are modified by passing a cell suspension containing the added TILs through a cell deformation constriction, thereby perturbing the added TILs such that a nucleic acid encoding a costimulatory molecule and / or a nucleic acid encoding a cytokine enters the TIL when contacted with the added TILs, such that after entry of the nucleic acid encoding a costimulatory molecule and / or the nucleic acid encoding a cytokine, the added TILs exhibit increased expression of the costimulatory molecule and / or the cytokine and become modified TILs.

[0012] In some embodiments, the method further comprises contacting the cell suspension with a nucleic acid encoding a costimulatory molecule and / or a nucleic acid encoding a cytokine. In some embodiments, the cell suspension is contacted with the nucleic acid encoding a costimulatory molecule and / or the nucleic acid encoding a cytokine prior to passing through the cell deformation constriction. In some embodiments, the cell suspension is contacted with the nucleic acid encoding a costimulatory molecule and / or the nucleic acid encoding a cytokine while passing through the cell deformation constriction. In some embodiments, the cell suspension is contacted with the nucleic acid encoding a costimulatory molecule and / or the nucleic acid encoding a cytokine while passing through the cell deformation constriction.

[0013] In some aspects, the nucleic acid encoding a costimulatory molecule and / or the nucleic acid encoding a cytokine is an mRNA.

[0014] In some embodiments, the constriction for cell deformation has a width of about 10% to about 99% of the average diameter of the TILs. In some embodiments, the constriction for cell deformation has a width of about 10% to about 99% of the average diameter of the introduced TILs. In some embodiments, the width of the constriction is about 3.5 μm to about 4.2 μm, or about 3.5 μm to about 4.8 μm, or about 3.5 μm to about 6 μm, or about 5 μm to about 12 μm, or about 12 μm to about 15 μm, or about 6 μm to about 12 μm, or about 8 μm to about 11 μm, or about 9 μm to about 11 μm. In some embodiments, the width of the constriction is about 3 μm to about 5 μm. In some embodiments, the width of the constriction is about 4 μm.

[0015] In some aspects, the present invention provides methods of modulating the activity and / or proliferation of tumor infiltrating lymphocytes (TILs), by modifying the TILs to increase expression of one or more costimulatory molecules and / or one or more cytokines. In some aspects, the present invention provides methods of modulating the activity and / or proliferation of tumor infiltrating lymphocytes (TILs), by modifying the TILs to increase expression of one or more costimulatory molecules. In some embodiments, the costimulatory molecule is B7-H2 (ICOSL), B7-1 (CD80), B7-2 (CD86), CD70, LIGHT, HVEM, CD40, 4-1BBL, OX40L, TL1A, GITRL, CD30L, TIM4, SLAM, CD48, CD58, CD155, or CD112. In some embodiments, the costimulatory molecule is CD86. In some aspects, the invention provides methods of modulating the phenotype and / or proliferative capacity of TILs, in which the TILs are modified to increase expression of one or more cytokines.

[0016] In some embodiments of the methods described herein, the TILs are modified to contain a membrane-bound chimeric cytokine. In some embodiments, the membrane-bound chimeric cytokine is a fusion protein comprising a cytokine and a transmembrane domain. In some embodiments, the cytokine is linked to the transmembrane domain by a peptide linker. In some embodiments, the peptide linker is (G4S)3 (SEQ ID NO:3) or (EAAAK)3 (SEQ ID NO:4). In some embodiments, the cytokine is a type I cytokine. In some embodiments, the cytokine is IL-15, IL-12, IL-2, IFNα, IFNβ, or IL-21, or a functional variant thereof. In some embodiments, the cytokine is IL-2 or a functional variant thereof and / or IL-12 or a functional variant thereof. In some embodiments, the membrane-bound chimeric cytokine comprises the amino acid sequence of SEQ ID NO:7-10.

[0017] In some embodiments of the invention, modified TILs have increased expression of one or more cytokines and / or one or more costimulatory molecules, and are prepared by a method comprising: a) passing a cell suspension comprising the input TILs through a cell deformation constriction to perturb the input TILs sufficiently to allow passage of one or more nucleic acids encoding one or more cytokines and / or one or more nucleic acids encoding one or more costimulatory molecules to form perturbed input TILs, where the diameter of the constriction is a function of the diameter of the input TILs in suspension, and b) incubating the input perturbed TILs with one or more nucleic acids encoding one or more cytokines and / or one or more nucleic acids encoding one or more costimulatory molecules to allow the one or more nucleic acids encoding one or more cytokines and / or one or more nucleic acids encoding one or more costimulatory molecules to enter the input perturbed TILs and express the nucleic acids, thereby generating modified TILs comprising one or more cytokines and / or one or more costimulatory molecules. In some embodiments of the invention, modified TILs have increased expression of one or more cytokines and / or one or more costimulatory molecules, and are prepared by a method comprising: a) passing a cell suspension comprising the input TILs through a cell deformation constriction to perturb the input TILs sufficiently to allow passage of one or more nucleic acids encoding one or more cytokines and / or one or more costimulatory molecules to form perturbed input TILs, where the diameter of the constriction is a function of the diameter of the input TILs in suspension, and b) incubating the perturbed input TILs with one or more nucleic acids encoding one or more cytokines and / or one or more costimulatory molecules to enter the input perturbed TILs and express the nucleic acids, thereby generating modified TILs comprising one or more cytokines and / or one or more costimulatory molecules.In some embodiments, the method comprises: (a) incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine before, during, and / or after passing the cell suspension through a constriction for cell deformation; (b) incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine and a costimulatory molecule before, during, and / or after passing the cell suspension through a constriction for cell deformation; (c) incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine and a nucleic acid encoding a costimulatory molecule before, during, and / or after passing the cell suspension through a constriction for cell deformation; or (d) incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine and a nucleic acid encoding one or more costimulatory molecules before, during, and / or after passing the cell suspension through a constriction for cell deformation. In some embodiments, the method comprises (a) incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine prior to passing the cell suspension through the cell deformation constriction, (b) incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine and a costimulatory molecule prior to passing the cell suspension through the cell deformation constriction, (c) incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine and a nucleic acid encoding a costimulatory molecule prior to passing the cell suspension through the cell deformation constriction, or (d) incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine and a nucleic acid encoding one or more costimulatory molecules prior to passing the cell suspension through the cell deformation constriction. In some embodiments, one or more of the nucleic acids is mRNA.

[0018] In some embodiments of the invention, the modified TILs have increased expression of one or more of T-bet, EOMES, TCF1, and CD62L over corresponding unmodified TILs. In some embodiments, the modified TILs have increased expression of one or more of T-bet, EOMES, TCF1, and CD62L over corresponding unmodified TILs when cultured in the absence of exogenous cytokines. In some embodiments, the expression of one or more of T-bet, EOMES, TCF1, and CD62L in the modified TILs is increased by about 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 3-fold, 4-fold, 5-fold, 8-fold, or more than 10-fold over corresponding unmodified TILs.

[0019] In some embodiments of the present invention, the modified TILs have increased expression of granzyme B compared to the corresponding unmodified TILs. In some embodiments, the modified TILs have increased expression of granzyme B compared to the corresponding unmodified TILs when cultured in the absence of exogenous cytokines. In some embodiments, the modified TILs have increased expression of granzyme B compared to the corresponding unmodified TILs when co-cultured with tumor cells in the absence of exogenous cytokines. In some embodiments, the expression of granzyme B in the modified TILs is increased by about 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 3-fold, 4-fold, 5-fold, 8-fold, or 10-fold or more compared to the corresponding unmodified TILs.

[0020] In some embodiments, the modified TILs exhibit greater proliferation than their unmodified counterparts. In some embodiments, the modified TILs exhibit greater proliferation when cultured in media that does not contain exogenous cytokines than their unmodified counterparts. In some embodiments, the exogenous cytokine is IL-2 and / or IL-12, and optionally, the exogenous cytokine is IL-2. In some embodiments, the modified TILs exhibit greater proliferation when co-cultured with tumor cells in media that does not contain exogenous cytokines than their unmodified counterparts. In some embodiments, the tumor is an autologous tumor.

[0021] In some aspects, the invention provides compositions comprising modified TILs that have been modified to increase expression of one or more costimulatory molecules and / or one or more cytokines. In some aspects, the invention provides compositions comprising modified TILs, where the TILs have been modified to increase expression of one or more costimulatory molecules. In some embodiments, the costimulatory molecule is B7-H2 (ICOSL), B7-1 (CD80), B7-2 (CD86), CD70, LIGHT, HVEM, CD40, 4-1BBL, OX40L, TL1A, GITRL, CD30L, TIM4, SLAM, CD48, CD58, CD155, or CD112. In some embodiments, the costimulatory molecule is CD86. In some embodiments, the TILs have been modified to increase expression of one or more cytokines. In some aspects, the invention provides compositions comprising modified TILs that have been modified to include a membrane-bound chimeric cytokine.

[0022] In some embodiments of the compositions described herein, the membrane-bound chimeric cytokine is a fusion protein comprising a cytokine and a transmembrane domain. In some embodiments, the cytokine is linked to the transmembrane domain by a peptide linker. In some embodiments, the peptide linker is (G4S)3 (SEQ ID NO:3) or (EAAAK)3 (SEQ ID NO:4). In some embodiments, the cytokine is a type I cytokine. In some embodiments, the cytokine is IL-15, IL-12, IL-2, IFNα, IFNβ, or IL-21, or a functional variant thereof. In some embodiments, the cytokine is IL-2 or a functional variant thereof, and / or IL-12 or a functional variant thereof. In some embodiments, the membrane-bound chimeric cytokine comprises the amino acid sequence of SEQ ID NO:7-10.

[0023] In some embodiments of the compositions described herein, the modified TILs have increased expression of one or more cytokines and / or one or more costimulatory molecules, and the modified TILs have been prepared by a method comprising: a) passing a cell suspension comprising the input TILs through a cell deformation constriction, thereby perturbing the input TILs sufficiently to allow passage of one or more nucleic acids encoding one or more cytokines and / or one or more nucleic acids encoding one or more costimulatory molecules to form a perturbed input TIL, where the diameter of the constriction is a function of the diameter of the input TILs in suspension, and b) incubating the input perturbed TILs with one or more nucleic acids encoding one or more cytokines and / or one or more nucleic acids encoding one or more costimulatory molecules to allow the one or more nucleic acids encoding one or more cytokines and / or one or more nucleic acids encoding one or more costimulatory molecules to enter the input perturbed TILs and express the nucleic acids, thereby generating a modified TIL comprising one or more cytokines and / or one or more costimulatory molecules. In some embodiments, the TILs have increased expression of one or more cytokines and / or one or more costimulatory molecules, and the TILs have been prepared by a method comprising: a) passing a cell suspension comprising the input TILs through a cell deformation constriction, thereby perturbing the input TILs sufficiently to allow passage of one or more nucleic acids encoding one or more cytokines and / or one or more costimulatory molecules to form perturbed input TILs, where the diameter of the constriction is a function of the diameter of the input TILs in suspension, and b) incubating the perturbed input TILs with one or more nucleic acids encoding one or more cytokines and / or one or more costimulatory molecules to enter the input perturbed TILs and express the nucleic acids, thereby generating modified TILs comprising one or more cytokines and / or one or more costimulatory molecules.In some embodiments, a method of preparing modified TILs comprises: (a) incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine before, during, and / or after passing a cell suspension through a constriction for cell deformation; (b) incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine and a costimulatory molecule before, during, and / or after passing a cell suspension through a constriction for cell deformation; (c) incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine and a nucleic acid encoding a costimulatory molecule before, during, and / or after passing a cell suspension through a constriction for cell deformation; or (d) incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine and a nucleic acid encoding one or more costimulatory molecules before, during, and / or after passing a cell suspension through a constriction for cell deformation. In some embodiments, a method of preparing modified TILs comprises (a) incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine prior to passing a cell suspension through a constriction for cell deformation, (b) incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine and a costimulatory molecule prior to passing a cell suspension through a constriction for cell deformation, (c) incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine and a nucleic acid encoding a costimulatory molecule prior to passing a cell suspension through a constriction for cell deformation, or (d) incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine and a nucleic acid encoding one or more costimulatory molecules prior to passing a cell suspension through a constriction for cell deformation. In some embodiments, one or more of the nucleic acids is mRNA.

[0024] In some embodiments of the compositions described herein, the modified TILs have increased expression of one or more of T-bet, EOMES, TCF1, and CD62L over corresponding unmodified TILs. In some embodiments, the modified TILs have increased expression of one or more of T-bet, EOMES, TCF1, and CD62L over corresponding unmodified TILs when cultured in the absence of exogenous cytokines. In some embodiments, the expression of one or more of T-bet, EOMES, TCF1, and CD62L in the modified TILs is increased by about 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 3-fold, 4-fold, 5-fold, 8-fold, or more than 10-fold over corresponding unmodified TILs.

[0025] In some embodiments of the present invention, the modified TILs have increased expression of granzyme B compared to the corresponding unmodified TILs. In some embodiments, the modified TILs have increased expression of granzyme B compared to the corresponding unmodified TILs when cultured in the absence of exogenous cytokines. In some embodiments, the modified TILs have increased expression of granzyme B compared to the corresponding unmodified TILs when co-cultured with tumor cells in the absence of exogenous cytokines. In some embodiments, the expression of granzyme B in the modified TILs is increased by about 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 3-fold, 4-fold, 5-fold, 8-fold, or 10-fold or more compared to the corresponding unmodified TILs.

[0026] In some embodiments, the modified TILs of the composition exhibit greater proliferation than their unmodified counterparts. In some embodiments, the modified TILs exhibit greater proliferation when cultured in media that does not contain exogenous cytokines than their unmodified counterparts. In some embodiments, the exogenous cytokine is IL-2 and / or IL-12, and optionally, the exogenous cytokine is IL-2. In some embodiments, the modified TILs exhibit greater proliferation when co-cultured with tumor cells in media that does not contain exogenous cytokines than their unmodified counterparts. In some embodiments, the tumor is an autologous tumor.

[0027] In some aspects, the invention provides methods of modulating an immune response in an individual, the methods comprising administering to the individual modified TILs, the modified TILs being prepared according to the methods described herein. In some aspects, the invention provides methods of modulating an immune response in an individual, the methods comprising administering a composition described herein.

[0028] In some aspects, the present invention provides a method of treating cancer, infectious disease, or virus-related disease in an individual, comprising administering modified TILs to the individual, the modified TILs being prepared according to the methods described herein. In some embodiments, the present invention provides a method of treating cancer, infectious disease, or virus-related disease in an individual, comprising administering a composition described herein. In some embodiments, the method comprises administering the modified TILs multiple times, or administering the composition multiple times. In some embodiments, the modified TILs are administered intravenously or intratumorally. In some embodiments, the individual is a human. In some embodiments, the modified TILs are administered before, simultaneously with, or after administration of another therapeutic agent.

[0029] In some aspects, the present invention provides a pharmaceutical composition for stimulating an immune response in an individual, the composition comprising an effective amount of a composition described herein. In some embodiments, the present invention provides a pharmaceutical composition for use as a medicament, the pharmaceutical composition comprising an effective amount of a composition described herein. In some embodiments, the present invention provides a pharmaceutical composition for treating cancer, an infectious disease, or a virus-related disease in an individual, the pharmaceutical composition comprising an effective amount of a composition described herein. In some embodiments, the composition comprises modified TILs administered before, simultaneously with, or after administration of another therapeutic agent.

[0030] In some embodiments of the delivery through a constriction described herein, the width of the constriction is about 10% to about 99% of the average diameter of the loaded TILs. In some embodiments, the width of the constriction is about 3.5 μm to about 4.2 μm, or about 3.5 μm to about 4.8 μm, or about 3.5 μm to about 6 μm, or about 5 μm to about 12 μm, or about 12 μm to about 15 μm, or about 6 μm to about 12 μm, or about 8 μm to about 11 μm, or about 9 μm to about 11 μm. In some embodiments, the width of the constriction is about 3 μm to about 5 μm. In some embodiments, the width of the constriction is about 4 μm. In some embodiments, the cell suspension containing a plurality of loaded TILs is passed through a plurality of constrictions, where the plurality of constrictions are arranged in series and / or parallel.

[0031] In some aspects, the present invention provides a kit for use in any of the methods described herein.In some aspects, the present invention provides a kit that includes the composition described herein.In some embodiments, the kit further includes one or more of a buffer, a diluent, a filter, a needle, a syringe, or a package insert that includes instructions for administering the composition to an individual.

[0032] In some aspects, the present invention provides a method for producing TILs comprising a membrane-bound chimeric cytokine, the method comprising introducing a nucleic acid encoding a membrane-bound chimeric cytokine into TILs. In some embodiments, the TILs comprising a membrane-bound chimeric cytokine are prepared by: a) passing a cell suspension comprising the input TILs through a cell deformation constriction to perturb the input TILs sufficiently large to allow passage of the nucleic acid encoding the membrane-bound chimeric cytokine, forming a perturbed input TILs, where the diameter of the constriction is a function of the diameter of the input TILs in suspension; and b) incubating the perturbed input TILs with a nucleic acid encoding a membrane-bound chimeric cytokine, allowing the nucleic acid to enter the perturbed input TILs and express the nucleic acid encoding the membrane-bound chimeric cytokine, thereby generating a TIL comprising a membrane-bound chimeric cytokine. In some embodiments, the method comprises incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine before, during, and / or after passing the cell suspension through the cell deformation constriction. In some embodiments, the method includes incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine prior to passing the cell suspension through a cell deformation constriction. In some embodiments, the nucleic acid encoding a membrane-bound chimeric cytokine is an mRNA encoding a membrane-bound chimeric cytokine. In some embodiments, the width of the constriction is about 10% to about 99% of the average diameter of the input TILs. In some embodiments, the width of the constriction is about 5 μm to about 12 μm, or about 6 μm to about 12 μm, or about 8 μm to about 11 μm, or about 9 μm to about 11 μm, or about 12 μm to about 15 μm. In some embodiments, the width of the constriction is about 10 μm. In some aspects, the width of the constriction is about 8 μm. In some embodiments, the cell suspension including a plurality of input TILs is passed through a plurality of constrictions, where the plurality of constrictions are arranged in series and / or parallel. In some aspects, the invention provides the use of a pharmaceutical composition in the manufacture of a medicament for stimulating an immune response in an individual, the pharmaceutical composition comprising an effective amount of a composition described herein.In some aspects, the present invention provides the use of a pharmaceutical composition in the manufacture of a medicament for treating cancer, an infectious disease, or a virus-related disease in an individual, the pharmaceutical composition comprising an effective amount of a composition described herein. In some embodiments, the pharmaceutical composition is formulated for multiple administration. In some embodiments, the pharmaceutical composition is administered intravenously or intratumorally. In some embodiments, the individual is a human. In some embodiments, the pharmaceutical composition is formulated to be administered before, simultaneously with, or after administration of another therapeutic agent. [Brief description of the drawings]

[0033] [Figure 1A] FIG. 1 shows the viability of tumor-infiltrating lymphocytes expressing membrane-bound IL-2 ("mbIL-2"), membrane-bound IL-12 ("mbIL-12"), or both mbIL-2 and mbIL-12 resulting from constriction-mediated mRNA delivery at various time points after delivery. No contact ("NC", e.g., cells were not treated with constriction-mediated delivery), empty compression (e.g., constriction-mediated delivery was performed without input material), and GFP resulting from constriction-mediated delivery of mRNA were used as controls in these experiments. [Figure 1B] 1 shows GFP expression in tumor-infiltrating lymphocytes expressing membrane-bound IL-2 ("mbIL-2"), membrane-bound IL-12 ("mbIL-12"), or both mbIL-2 and mbIL-12 resulting from mRNA delivery through a constriction at various time points after delivery. No contact ("NC"), empty compression (e.g., constriction delivery was performed without input material), and GFP resulting from mRNA delivery through a constriction were used as controls in these experiments. [Figure 1C]FIG. 1 shows mbIL-2 expression in tumor-infiltrating lymphocytes expressing membrane-bound IL-2 ("mbIL-2"), membrane-bound IL-12 ("mbIL-12"), or both mbIL-2 and mbIL-12 resulting from constriction-mediated mRNA delivery at various time points after delivery. No contact ("NC"), empty compression (e.g., constriction-mediated delivery was performed without input material), and GFP resulting from constriction-mediated delivery of mRNA were used as controls in these experiments. [Figure 1D] FIG. 1 shows mbIL-12 expression in tumor-infiltrating lymphocytes expressing membrane-bound IL-2 ("mbIL-2"), membrane-bound IL-12 ("mbIL-12"), or both mbIL-2 and mbIL-12 resulting from mRNA delivery through a constriction at various time points after delivery. No contact ("NC"), empty compression (e.g., constriction delivery was performed without input material), and GFP resulting from mRNA delivery through a constriction were used as controls in these experiments. [Figure 2A] FIG. 1 shows the mean fluorescence intensity ("MFI") for GFP expression in tumor-infiltrating lymphocytes expressing membrane-bound IL-2 ("mbIL-2"), membrane-bound IL-12 ("mbIL-12"), or both mbIL-2 and mbIL-12 resulting from mRNA delivery through a constriction at various time points after delivery. No contact ("NC"), empty constriction (e.g., constriction delivery was performed without input material), and GFP resulting from mRNA delivery through a constriction were used as controls in these experiments. [Figure 2B] FIG. 1 shows the mean fluorescence intensity ("MFI") for mbIL-2 expression in tumor-infiltrating lymphocytes expressing membrane-bound IL-2 ("mbIL-2"), membrane-bound IL-12 ("mbIL-12"), or both mbIL-2 and mbIL-12 resulting from mRNA delivery through a constriction at various time points after delivery. No contact ("NC"), empty constriction (e.g., constriction delivery was performed without input material), and GFP resulting from mRNA delivery through a constriction were used as controls in these experiments. [Figure 2C] FIG. 1 shows the mean fluorescence intensity ("MFI") for membrane-bound IL-2 ("mbIL-2"), membrane-bound IL-12 ("mbIL-12"), or mbIL-2 and mbIL-12 expression in tumor-infiltrating lymphocytes expressing either membrane-bound IL-2 or membrane-bound IL-12 at various time points after delivery resulting from mRNA delivery through a constriction. No contact ("NC"), empty constriction (e.g., constriction delivery was performed without input material), and GFP resulting from mRNA delivery through a constriction were used as controls in these experiments. [Diagram 3] A-D show the viability of tumor-infiltrating lymphocytes expressing membrane-bound IL-2 ("mbIL-2"), membrane-bound IL-12 ("mbIL-12"), or both mbIL-2 and mbIL-12, following mRNA delivery through a constriction and subsequent culture in media containing different concentrations of IL-2 (3000 IU / mL, 300 IU / mL, 30 IU / mL, or 0 IU / mL). No contact ("NC") and empty compression (e.g., constriction delivery without input material) were used as controls in these experiments. [Figure 4] A-D show proliferation of tumor-infiltrating lymphocytes expressing membrane-bound IL-2 ("mbIL-2"), membrane-bound IL-12 ("mbIL-12"), or both mbIL-2 and mbIL-12 following mRNA delivery through a constriction and subsequent culture in media containing different concentrations of IL-2 (3000 IU / mL, 300 IU / mL, 30 IU / mL, or 0 IU / mL). No contact ("NC") and empty compression (e.g., constriction delivery without input material) were used as controls in these experiments. [Diagram 5]A-D show CD39+ expression of tumor-infiltrating lymphocytes expressing membrane-bound IL-2 ("mbIL-2"), membrane-bound IL-12 ("mbIL-12"), or both mbIL-2 and mbIL-12, resulting from mRNA delivery through a constriction and then cultured in medium containing different concentrations of IL-2 (3000 IU / mL, 300 IU / mL, 30 IU / mL, or 0 IU / mL). No contact ("NC") and empty compression (e.g., constriction delivery performed without input material) were used as controls in these experiments. [Figure 6] A-D show CD39+CD69+ expression of tumor-infiltrating lymphocytes expressing membrane-bound IL-2 ("mbIL-2"), membrane-bound IL-12 ("mbIL-12"), or both mbIL-2 and mbIL-12, resulting from mRNA delivery through a constriction and then cultured in medium containing different concentrations of IL-2 (3000 IU / mL, 300 IU / mL, 30 IU / mL, or 0 IU / mL). No contact ("NC") and empty compression (e.g., constriction delivery was performed without input material) were used as controls in these experiments. [Figure 7] A-D show CD62L+ expression of tumor-infiltrating lymphocytes expressing membrane-bound IL-2 ("mbIL-2"), membrane-bound IL-12 ("mbIL-12"), or both mbIL-2 and mbIL-12, following mRNA delivery through a constriction and subsequent culture in media containing different concentrations of IL-2 (3000 IU / mL, 300 IU / mL, 30 IU / mL, or 0 IU / mL). No contact ("NC") and empty compression (e.g., constriction delivery without input material) were used as controls in these experiments. [Figure 8A] FIG. 1 shows CD69+ expression of tumor-infiltrating lymphocytes expressing membrane-bound IL-2 ("mbIL-2"), membrane-bound IL-12 ("mbIL-12"), or both mbIL-2 and mbIL-12, following mRNA delivery through a constriction and subsequent culture in medium containing different concentrations of IL-2 (3000 IU / mL, 300 IU / mL, 30 IU / mL, or 0 IU / mL). [Figure 8B]FIG. 1 shows CD69+ expression of tumor-infiltrating lymphocytes expressing membrane-bound IL-2 ("mbIL-2"), membrane-bound IL-12 ("mbIL-12"), or both mbIL-2 and mbIL-12, following mRNA delivery through a constriction and subsequent culture in medium containing different concentrations of IL-2 (3000 IU / mL, 300 IU / mL, 30 IU / mL, or 0 IU / mL). [Figure 8C] FIG. 1 shows CD69+ expression of tumor-infiltrating lymphocytes expressing membrane-bound IL-2 ("mbIL-2"), membrane-bound IL-12 ("mbIL-12"), or both mbIL-2 and mbIL-12, following mRNA delivery through a constriction and subsequent culture in medium containing different concentrations of IL-2 (3000 IU / mL, 300 IU / mL, 30 IU / mL, or 0 IU / mL). [Figure 8D] FIG. 1 shows CD69+ expression of tumor-infiltrating lymphocytes expressing membrane-bound IL-2 ("mbIL-2"), membrane-bound IL-12 ("mbIL-12"), or both mbIL-2 and mbIL-12, following mRNA delivery through a constriction and subsequent culture in medium containing different concentrations of IL-2 (3000 IU / mL, 300 IU / mL, 30 IU / mL, or 0 IU / mL). [Figure 8E] FIG. 1 shows CD69+ expression of tumor-infiltrating lymphocytes expressing membrane-bound IL-2 ("mbIL-2"), membrane-bound IL-12 ("mbIL-12"), or both mbIL-2 and mbIL-12, following mRNA delivery through a constriction and subsequent culture in medium containing different concentrations of IL-2 (3000 IU / mL, 300 IU / mL, 30 IU / mL, or 0 IU / mL). [Figure 8F] FIG. 1 shows CD69+ expression of tumor-infiltrating lymphocytes expressing membrane-bound IL-2 ("mbIL-2"), membrane-bound IL-12 ("mbIL-12"), or both mbIL-2 and mbIL-12, following mRNA delivery through a constriction and subsequent culture in medium containing different concentrations of IL-2 (3000 IU / mL, 300 IU / mL, 30 IU / mL, or 0 IU / mL). [Figure 8G]FIG. 1 shows CD69+ expression of tumor-infiltrating lymphocytes expressing membrane-bound IL-2 ("mbIL-2"), membrane-bound IL-12 ("mbIL-12"), or both mbIL-2 and mbIL-12, following mRNA delivery through a constriction and subsequent culture in medium containing different concentrations of IL-2 (3000 IU / mL, 300 IU / mL, 30 IU / mL, or 0 IU / mL). [Figure 8H] FIG. 1 shows CD69+ expression of tumor-infiltrating lymphocytes expressing membrane-bound IL-2 ("mbIL-2"), membrane-bound IL-12 ("mbIL-12"), or both mbIL-2 and mbIL-12, following mRNA delivery through a constriction and subsequent culture in media containing different concentrations of IL-2 (3000 IU / mL, 300 IU / mL, 30 IU / mL, or 0 IU / mL). No contact ("NC") and empty compression (e.g., constriction delivery without input material) were used as controls in these experiments. [Figure 9] A-D show expression of mbIL-2 in tumor-infiltrating lymphocytes expressing membrane-bound IL-2 ("mbIL-2"), membrane-bound IL-12 ("mbIL-12"), or both mbIL-2 and mbIL-12, following mRNA delivery through a constriction and subsequent culture in media containing different concentrations of IL-2 (3000 IU / mL, 300 IU / mL, 30 IU / mL, or 0 IU / mL). No contact ("NC") and empty compression (e.g., constriction delivery without input material) were used as controls in these experiments. [Figure 10] A-D show expression of mbIL-12 in tumor-infiltrating lymphocytes expressing membrane-bound IL-2 ("mbIL-2"), membrane-bound IL-12 ("mbIL-12"), or both mbIL-2 and mbIL-12, following mRNA delivery through a constriction and subsequent culture in medium containing different concentrations of IL-2 (3000 IU / mL, 300 IU / mL, 30 IU / mL, or 0 IU / mL). No contact ("NC") and empty compression (e.g., constriction delivery without input material) were used as controls in these experiments. [Figure 11]A-D show expression of both mbIL-2 and mbIL-12 in tumor-infiltrating lymphocytes expressing membrane-bound IL-2 ("mbIL-2"), membrane-bound IL-12 ("mbIL-12"), or both mbIL-2 and mbIL-12, resulting from mRNA delivery through a constriction and then cultured in medium containing different concentrations of IL-2 (3000 IU / mL, 300 IU / mL, 30 IU / mL, or 0 IU / mL). No contact ("NC") and empty compression (e.g., constriction delivery performed without input material) were used as controls in these experiments. [Figure 12] A-D show T-bet expression in tumor-infiltrating lymphocytes expressing membrane-bound IL-2 ("mbIL-2"), membrane-bound IL-12 ("mbIL-12"), or both mbIL-2 and mbIL-12, following mRNA delivery through a constriction and subsequent culture in media containing different concentrations of IL-2 (3000 IU / mL, 300 IU / mL, 30 IU / mL, or 0 IU / mL). No contact ("NC") and empty compression (e.g., delivery through a constriction without input material) were used as controls in these experiments. [Figure 13] A-D show Eomes+ and TCF-1+ expression in tumor-infiltrating lymphocytes expressing membrane-bound IL-2 ("mbIL-2"), membrane-bound IL-12 ("mbIL-12"), or both mbIL-2 and mbIL-12, following mRNA delivery through a constriction and subsequent culture in medium containing different concentrations of IL-2 (3000 IU / mL, 300 IU / mL, 30 IU / mL, or 0 IU / mL). No contact ("NC") and empty compression (e.g., constriction delivery performed without input material) were used as controls in these experiments. [Figure 14]14A-B show the viability of tumor infiltrating lymphocytes expressing both membrane-bound IL-2 ("mbIL-2") and membrane-bound IL-12 ("mbIL-12") following constriction delivery of 0.1 mg / mL, 0.25 mg / mL, or 0.5 mg / mL of mRNA, and the viability of tumor infiltrating lymphocytes expressing CD86 and mbIL-2, CD86 and mb-IL12, and CD86 and both mbIL-2 and mbIL-12 following constriction delivery of 0.25 mg / mL of mRNA. Tumor infiltrating lymphocytes were cultured in medium containing either 3000 IU / mL (FIG. 14A) or 0 IU / mL (FIG. 14B) of IL-2 following mRNA delivery. No contact ("NC") and empty compression (e.g., constriction delivery without input material) were used as controls in these experiments. [Figure 15] 15A-B show the proliferation of tumor infiltrating lymphocytes expressing both membrane-bound IL-2 ("mbIL-2") and membrane-bound IL-12 ("mbIL-12") following constriction delivery of 0.1 mg / mL, 0.25 mg / mL, or 0.5 mg / mL of mRNA, and the proliferation of tumor infiltrating lymphocytes expressing CD86 and mbIL-2, CD86 and mb-IL12, and CD86 and both mbIL-2 and mbIL-12 following constriction delivery of 0.25 mg / mL of mRNA. Tumor infiltrating lymphocytes were cultured in medium containing either 3000 IU / mL (FIG. 15A) or 0 IU / mL (FIG. 15B) of IL-2 following mRNA delivery. No contact ("NC") and empty compression (e.g., constriction delivery without input material) were used as controls in these experiments. [Figure 16]16A-B show CD39+ expression of tumor infiltrating lymphocytes expressing both membrane-bound IL-2 ("mbIL-2") and membrane-bound IL-12 ("mbIL-12") resulting from constriction delivery of 0.1 mg / mL, 0.25 mg / mL, or 0.5 mg / mL mRNA, and CD39+ expression of tumor infiltrating lymphocytes expressing CD86 and mbIL-2, CD86 and mb-IL12, and CD86 and both mbIL-2 and mbIL-12 resulting from constriction delivery of 0.25 mg / mL mRNA. Tumor infiltrating lymphocytes were cultured in media containing either 3000 IU / mL (FIG. 16A) or 0 IU / mL (FIG. 16B) IL-2 after mRNA delivery. No contact ("NC") and empty compression (e.g., constriction delivery was performed without input material) were used as controls in these experiments. [Figure 17] 17A-B show CD62L+ expression of tumor infiltrating lymphocytes expressing both membrane-bound IL-2 ("mbIL-2") and membrane-bound IL-12 ("mbIL-12") resulting from constriction delivery of 0.1 mg / mL, 0.25 mg / mL, or 0.5 mg / mL mRNA, and CD86 and mbIL-2, CD86 and mb-IL12, and CD86 and both mbIL-2 and mbIL-12 resulting from constriction delivery of 0.25 mg / mL mRNA. Tumor infiltrating lymphocytes were cultured in medium containing either 3000 IU / mL (FIG. 17A) or 0 IU / mL (FIG. 17B) IL-2 after mRNA delivery. In these experiments, no contact ("NC") and empty compression (eg, delivery through the stricture was performed without any input material) were used as controls. [Figure 18]18A-B show CD86+ expression of tumor infiltrating lymphocytes expressing both membrane-bound IL-2 ("mbIL-2") and membrane-bound IL-12 ("mbIL-12") resulting from constriction delivery of 0.1 mg / mL, 0.25 mg / mL, or 0.5 mg / mL mRNA, and CD86+ expression of tumor infiltrating lymphocytes expressing CD86 and mbIL-2, CD86 and mb-IL12, and CD86 and both mbIL-2 and mbIL-12 resulting from constriction delivery of 0.25 mg / mL mRNA. Tumor infiltrating lymphocytes were cultured in media containing either 3000 IU / mL (FIG. 18A) or 0 IU / mL (FIG. 18B) IL-2 after mRNA delivery. No contact ("NC") and empty compression (e.g., constriction delivery was performed without input material) were used as controls in these experiments. [Figure 19A] FIG. 1 shows mbIL-2 expression in tumor infiltrating lymphocytes expressing both membrane-bound IL-2 ("mbIL-2") and membrane-bound IL-12 ("mbIL-12") resulting from constriction delivery of 0.1 mg / mL, 0.25 mg / mL, or 0.5 mg / mL mRNA, and mbIL-2 expression in tumor infiltrating lymphocytes expressing CD86 and mbIL-2, CD86 and mb-IL12, and CD86 and both mbIL-2 and mbIL-12 resulting from constriction delivery of 0.25 mg / mL mRNA. Tumor infiltrating lymphocytes were cultured in medium containing 3000 IU / mL IL-2 after mRNA delivery. No contact ("NC") and empty compression (e.g., constriction delivery without input material) were used as controls in these experiments. [Figure 19B]FIG. 1 shows mbIL-2 expression in tumor infiltrating lymphocytes expressing both membrane-bound IL-2 ("mbIL-2") and membrane-bound IL-12 ("mbIL-12") resulting from constriction delivery of 0.1 mg / mL, 0.25 mg / mL, or 0.5 mg / mL mRNA, and mbIL-2 expression in tumor infiltrating lymphocytes expressing CD86 and mbIL-2, CD86 and mb-IL12, and CD86 and both mbIL-2 and mbIL-12 resulting from constriction delivery of 0.25 mg / mL mRNA. Tumor infiltrating lymphocytes were cultured in medium containing 0 IU / mL IL-2 after mRNA delivery. No contact ("NC") and empty compression (e.g., constriction delivery without input material) were used as controls in these experiments. [Figure 19C] FIG. 1 shows the mean fluorescence intensity ("MFI") of mbIL-2 expression in tumor infiltrating lymphocytes expressing both membrane-bound IL-2 ("mbIL-2") and membrane-bound IL-12 ("mbIL-12") resulting from constriction delivery of 0.1 mg / mL, 0.25 mg / mL, or 0.5 mg / mL of mRNA, and the mean fluorescence intensity ("MFI") of mbIL-2 expression in tumor infiltrating lymphocytes expressing CD86 and mbIL-2, CD86 and mb-IL12, and CD86 and both mbIL-2 and mbIL-12 resulting from constriction delivery of 0.25 mg / mL of mRNA. Tumor infiltrating lymphocytes were cultured in medium containing 3000 IU / mL of IL-2 after mRNA delivery. Non-contact ("NC") and empty compression (e.g., constriction delivery without input material) were used as controls in these experiments. [Figure 19D]FIG. 1 shows the mean fluorescence intensity ("MFI") of mbIL-2 expression in tumor infiltrating lymphocytes expressing both membrane-bound IL-2 ("mbIL-2") and membrane-bound IL-12 ("mbIL-12") resulting from constriction delivery of 0.1 mg / mL, 0.25 mg / mL, or 0.5 mg / mL of mRNA, and the mean fluorescence intensity ("MFI") of mbIL-2 expression in tumor infiltrating lymphocytes expressing CD86 and mbIL-2, CD86 and mb-IL12, and CD86 and both mbIL-2 and mbIL-12 resulting from constriction delivery of 0.25 mg / mL of mRNA. Tumor infiltrating lymphocytes were cultured in medium containing 0 IU / mL IL-2 after mRNA delivery. No contact ("NC") and empty compression (e.g., constriction delivery without input material) were used as controls in these experiments. [Figure 20A] FIG. 1 shows mbIL-12 expression in tumor infiltrating lymphocytes expressing both membrane-bound IL-12 ("mbIL-12") and membrane-bound IL-2 ("mbIL-2") resulting from constriction delivery of 0.1 mg / mL, 0.25 mg / mL, or 0.5 mg / mL mRNA, and mbIL-12 expression in tumor infiltrating lymphocytes expressing CD86 and mbIL-2, CD86 and mb-IL12, and CD86 and both mbIL-2 and mbIL-12 resulting from constriction delivery of 0.25 mg / mL mRNA. Tumor infiltrating lymphocytes were cultured in medium containing 3000 IU / mL IL-2 after mRNA delivery. No contact ("NC") and empty compression (e.g., constriction delivery without input material) were used as controls in these experiments. [Figure 20B]FIG. 1 shows mbIL-12 expression in tumor infiltrating lymphocytes expressing both membrane-bound IL-12 ("mbIL-12") and membrane-bound IL-2 ("mbIL-2") resulting from constriction delivery of 0.1 mg / mL, 0.25 mg / mL, or 0.5 mg / mL of mRNA, and mbIL-12 expression in tumor infiltrating lymphocytes expressing CD86 and mbIL-2, CD86 and mb-IL12, and CD86 and both mbIL-2 and mbIL-12 resulting from constriction delivery of 0.25 mg / mL of mRNA. Tumor infiltrating lymphocytes were cultured in medium containing either 0 IU / mL of IL-2 after mRNA delivery. No contact ("NC") and empty compression (e.g., constriction delivery without input material) were used as controls in these experiments. [Figure 20C] FIG. 1 shows the mean fluorescence intensity ("MFI") of mbIL-12 expression in tumor infiltrating lymphocytes expressing both membrane-bound IL-12 ("mbIL-12") and membrane-bound IL-2 ("mbIL-2") resulting from constriction delivery of 0.1 mg / mL, 0.25 mg / mL, or 0.5 mg / mL of mRNA, and the mean fluorescence intensity ("MFI") of mbIL-12 expression in tumor infiltrating lymphocytes expressing CD86 and mbIL-2, CD86 and mb-IL12, and CD86 and both mbIL-2 and mbIL-12 resulting from constriction delivery of 0.25 mg / mL of mRNA. Tumor infiltrating lymphocytes were cultured in medium containing 3000 IU / mL of IL-2 after mRNA delivery. Non-contact ("NC") and empty compression (e.g., constriction delivery without input material) were used as controls in these experiments. [Figure 20D]FIG. 1 shows the mean fluorescence intensity ("MFI") of mbIL-12 expression in tumor infiltrating lymphocytes expressing both membrane-bound IL-12 ("mbIL-12") and membrane-bound IL-2 ("mbIL-2") resulting from constriction delivery of 0.1 mg / mL, 0.25 mg / mL, or 0.5 mg / mL of mRNA, and the mean fluorescence intensity ("MFI") of mbIL-12 expression in tumor infiltrating lymphocytes expressing CD86 and mbIL-2, CD86 and mb-IL12, and CD86 and both mbIL-2 and mbIL-12 resulting from constriction delivery of 0.25 mg / mL of mRNA. Tumor infiltrating lymphocytes were cultured in medium containing 0 IU / mL IL-2 after mRNA delivery. No contact ("NC") and empty compression (e.g., constriction delivery without input material) were used as controls in these experiments. [Figure 21] 21A-B show T-bet+ expression in tumor infiltrating lymphocytes expressing both membrane-bound IL-2 ("mbIL-2") and membrane-bound IL-12 ("mbIL-12") resulting from constriction delivery of 0.1 mg / mL, 0.25 mg / mL, or 0.5 mg / mL of mRNA, and T-bet expression in tumor infiltrating lymphocytes expressing CD86 and mbIL-2, CD86 and mb-IL12, and CD86 and both mbIL-2 and mbIL-12 resulting from constriction delivery of 0.25 mg / mL of mRNA. Tumor infiltrating lymphocytes were cultured in medium containing either 3000 IU / mL (FIG. 21A) or 0 IU / mL (FIG. 21B) of IL-2 after mRNA delivery. In these experiments, no contact ("NC") and empty compression (eg, delivery through the stricture was performed without any input material) were used as controls. [Figure 22]22A-B show Eomes+ expression of tumor infiltrating lymphocytes expressing both membrane-bound IL-2 ("mbIL-2") and membrane-bound IL-12 ("mbIL-12") resulting from constriction delivery of 0.1 mg / mL, 0.25 mg / mL, or 0.5 mg / mL of mRNA, and Eomes+ expression of tumor infiltrating lymphocytes expressing CD86 and mbIL-2, CD86 and mb-IL12, and CD86 and both mbIL-2 and mbIL-12 resulting from constriction delivery of 0.25 mg / mL of mRNA. Tumor infiltrating lymphocytes were cultured in medium containing either 3000 IU / mL (FIG. 22A) or 0 IU / mL (FIG. 22B) of IL-2 after mRNA delivery. No contact ("NC") and empty compression (e.g., constriction delivery was performed without input material) were used as controls in these experiments. [Diagram 23] 23A-B show Eomes+ and TCF-1 expression in tumor infiltrating lymphocytes expressing both membrane-bound IL-2 ("mbIL-2") and membrane-bound IL-12 ("mbIL-12") resulting from constriction delivery of 0.1 mg / mL, 0.25 mg / mL, or 0.5 mg / mL mRNA, and Eomes+ and TCF-1 expression in tumor infiltrating lymphocytes expressing CD86 and mbIL-2, CD86 and mb-IL12, and CD86 and both mbIL-2 and mbIL-12 resulting from constriction delivery of 0.25 mg / mL mRNA. Tumor infiltrating lymphocytes were cultured in medium containing either 3000 IU / mL (FIG. 23A) or 0 IU / mL (FIG. 23B) IL-2 after mRNA delivery. In these experiments, no contact ("NC") and empty compression (eg, delivery through the stricture was performed without any input material) were used as controls. [Figure 24A] FIG. 1 shows the percentage of viable cells over time in human tumor infiltrating lymphocytes (TILs) transfected with the indicated total amounts of mbIL-2 and mbIL-12 mRNA or TILs compressed without any input material (control TILs). [Figure 24B]FIG. 1 shows the percentage of cells expressing mbIL-2 and mbIL-12, respectively, over time in TILs transduced by compression with the total amount of mbIL-2 and mbIL-12 mRNA indicated or in TILs compressed without any input material (control TILs). [Figure 24C] FIG. 1 shows the percentage of cells expressing mbIL-2 and mbIL-12, respectively, over time in TILs transduced by compression with the total amount of mbIL-2 and mbIL-12 mRNA indicated or in TILs compressed without any input material (control TILs). [Figure 24D] FIG. 1 shows representative expression of mbIL-2 and mbIL-12 at 1 day after compression in TILs transduced with the indicated total amounts of mbIL-2 and mbIL-12 mRNA or in TILs treated with compression without any input material (control TILs). [Figure 24E] FIG. 1 shows representative expression of mbIL-2 and mbIL-12 at 1 day after compression in TILs transduced with the indicated total amounts of mbIL-2 and mbIL-12 mRNA or in TILs treated with compression without any input material (control TILs). [Diagram 25] (A and B) Number of viable CD8+ T cells and percentage of Ki67 positive cells measured by flow cytometry in TILs from three human melanoma patients that were pressed with no input material (control TILs), pressed with mbIL-2 mRNA, or pressed with mbIL-2 and mbIL-12 mRNA and co-cultured with autologous tumor cells for 3 days in the absence of exogenous cytokines; and (B) Number of viable CD8+ T cells and percentage of Ki67 positive cells measured by flow cytometry in TILs pressed with no input material and then co-cultured with autologous tumor cells for 3 days in the presence of exogenous IL-2 and IL-12 (control TILs+rhIL-2+12). [Figure 26]Figure 1 shows the percentage of granzyme B (GZMB) positive cells measured by flow cytometry in TILs from three human melanoma patients that were pressed with no input material (control TILs), pressed with mbIL-2 mRNA, or pressed with mbIL-2 and mbIL-12 mRNA and co-cultured with autologous tumor cells for 3 days in the absence of exogenous cytokines, and the percentage of granzyme B (GZMB) positive cells measured by flow cytometry in TILs that were pressed with no input material and then co-cultured with autologous tumor cells for 3 days in the presence of exogenous IL-2 and IL-12 (control TILs+rhIL-2+12). [Figure 27A] Fluorescence images showing the amount of tumor killed by TILs from two human melanoma patients that were pressed with no input material (control TILs), pressed with mbIL-2 mRNA, or pressed with mbIL-2 and mbIL-12 mRNA and co-cultured with autologous tumor cells for 24 hours in the absence of exogenous cytokines, and by TILs that were pressed with no input material and then co-cultured with autologous tumor cells for 24 hours in the presence of exogenous IL-2 and IL-12 (control TILs+rhIL-2+12). [Figure 27B] Fluorescence images showing the amount of tumor killed by TILs from two human melanoma patients that were pressed with no input material (control TILs), pressed with mbIL-2 mRNA, or pressed with mbIL-2 and mbIL-12 mRNA and co-cultured with autologous tumor cells for 24 hours in the absence of exogenous cytokines, and by TILs that were pressed with no input material and then co-cultured with autologous tumor cells for 24 hours in the presence of exogenous IL-2 and IL-12 (control TILs+rhIL-2+12). [Figure 27C]Scatter plot showing the amount of tumor killed by TILs from two human melanoma patients that were compressed with no input material (control TILs), compressed with mbIL-2 mRNA, or compressed with mbIL-2 and mbIL-12 mRNA and co-cultured with autologous tumor cells for 24 hours in the absence of exogenous cytokines, and the amount of tumor killed by TILs that were compressed with no input material and then co-cultured with autologous tumor cells for 24 hours in the presence of exogenous IL-2 and IL-12 (control TILs+rhIL-2+12). [Figure 27D] Scatter plot showing the amount of tumor killed by TILs from two human melanoma patients that were compressed with no input material (control TILs), compressed with mbIL-2 mRNA, or compressed with mbIL-2 and mbIL-12 mRNA and co-cultured with autologous tumor cells for 24 hours in the absence of exogenous cytokines, and the amount of tumor killed by TILs that were compressed with no input material and then co-cultured with autologous tumor cells for 24 hours in the presence of exogenous IL-2 and IL-12 (control TILs+rhIL-2+12). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] In some aspects, methods are provided for modulating the activity and / or proliferative capacity of tumor infiltrating lymphocytes (TILs), comprising modifying the TILs to increase expression of one or more costimulatory molecules and / or one or more cytokines. In some embodiments, there is provided a method of modulating the activity and / or proliferative capacity of tumor infiltrating lymphocytes (TILs), the method comprising: a) passing a cell suspension comprising input TILs through a cell deformation constriction to perturb the input TILs sufficiently to allow passage of one or more nucleic acids encoding one or more cytokines and / or one or more nucleic acids encoding one or more costimulatory molecules to form perturbed input TILs, wherein the diameter of the constriction is a function of the diameter of the input TILs in suspension; and b) incubating the perturbed input TILs with one or more nucleic acids encoding one or more cytokines and / or one or more nucleic acids encoding one or more costimulatory molecules to allow the one or more nucleic acids encoding one or more cytokines and / or one or more nucleic acids encoding one or more costimulatory molecules to enter the perturbed input TILs and express the nucleic acids, thereby generating TILs comprising the one or more cytokines and / or one or more costimulatory molecules. In some aspects, provided herein are methods of modulating the activity and / or proliferative capacity of TILs, the methods comprising intracellular delivery to the TILs of one or more nucleic acids encoding one or more cytokines. In some aspects, provided herein are methods of modulating the activity and / or proliferative capacity of TILs, the methods comprising intracellular delivery to the TILs of one or more nucleic acids encoding one or more costimulatory molecules. In some aspects, provided herein are methods of modulating the activity and / or proliferative capacity of TILs, the methods comprising intracellular delivery to the TILs of one or more nucleic acids encoding (i) one or more cytokines, and (ii) one or more costimulatory molecules.

[0035] Thus, in some embodiments, provided herein are methods of modulating the activity and / or proliferation capacity of TILs, the methods comprising perturbing the TILs by passing a cell suspension comprising TILs through a cell deformation constriction such that (i) one or more nucleic acids encoding one or more cytokines, (ii) one or more nucleic acids encoding one or more costimulatory molecules, or (iii) both (i) and (ii) enter the TILs through the perturbation section when contacted with the TILs. In some embodiments, such methods may further comprise contacting the TILs with (i) one or more nucleic acids encoding one or more cytokines, (ii) one or more nucleic acids encoding one or more costimulatory molecules, or (iii) both (i) and (ii). As further described herein, in some embodiments, the one or more cytokines and / or one or more costimulatory molecules can modulate the activity and / or proliferation capacity of TILs.

[0036] In some aspects, compositions are provided herein that include modified TILs, in which the TILs have been modified to increase expression of one or more costimulatory molecules and / or one or more cytokines. Thus, in some aspects, compositions are provided herein that include modified TILs, in which the modified TILs exhibit higher expression of one or more costimulatory molecules than a reference TIL (e.g., a corresponding TIL that has not been modified as described herein). In some aspects, compositions are provided herein that include modified TILs, in which the modified TILs exhibit higher expression of one or more cytokines than a reference TIL (e.g., a corresponding TIL that has not been modified as described herein). In some aspects, compositions are provided herein that include modified TILs exhibit higher expression of both one or more cytokines and one or more costimulatory molecules than a reference TIL (e.g., a corresponding TIL that has not been modified as described herein).

[0037] In some embodiments, a composition is provided comprising modified TILs, the modified TILs being prepared by a method comprising: a) passing a cell suspension comprising the input TILs through a cell deformation constriction, thereby perturbing the input TILs sufficiently to allow passage of one or more nucleic acids encoding one or more cytokines and / or one or more nucleic acids encoding one or more costimulatory molecules to form perturbed input TILs, where the diameter of the constriction is a function of the diameter of the input TILs in suspension; and b) incubating the perturbed input TILs with one or more nucleic acids encoding one or more cytokines and / or one or more nucleic acids encoding one or more costimulatory molecules to allow the one or more nucleic acids encoding one or more cytokines and / or one or more nucleic acids encoding one or more costimulatory molecules to enter the perturbed input TILs and express the nucleic acids, thereby generating modified TILs comprising one or more cytokines and / or one or more costimulatory molecules. Thus, in some embodiments, the modified TILs described herein have passed through a cell deformation constriction, which deforms the TIL to cause a perturbation in the TIL, such that one or more nucleic acids encoding one or more cytokines enter the TIL through the perturbation when contacted with the TIL. In some embodiments, the modified TILs described herein have passed through a cell deformation constriction, which deforms the TIL to cause a perturbation in the TIL, such that one or more nucleic acids encoding one or more costimulatory molecules enter the TIL through the perturbation when contacted with the TIL. In some embodiments, the modified TILs described herein have passed through a cell deformation constriction, which deforms the TIL to cause a perturbation in the TIL, such that both (i) one or more nucleic acids encoding one or more cytokines and (ii) one or more nucleic acids encoding one or more costimulatory molecules enter the TIL through the perturbation when contacted with the TIL.

[0038] In some aspects, a method for producing a modified TIL comprising a membrane-bound chimeric cytokine is provided, the method comprising introducing a nucleic acid encoding a membrane-bound chimeric cytokine into the TIL. In some embodiments, a method for producing a modified TIL comprising a membrane-bound chimeric cytokine is provided, the method comprising: a) passing a cell suspension comprising the input TIL through a cell deformation constriction, thereby perturbing the input TIL sufficiently large to allow passage of the nucleic acid encoding the membrane-bound chimeric cytokine, to form a perturbed input TIL, the diameter of the constriction being a function of the diameter of the input TIL in suspension; and b) incubating the perturbed input TIL with a nucleic acid encoding a membrane-bound chimeric cytokine, allowing the nucleic acid to enter the perturbed input TIL and express the nucleic acid encoding the membrane-bound chimeric cytokine, thereby generating a modified TIL comprising a membrane-bound chimeric cytokine.

[0039] The techniques and procedures described or referenced herein are generally conventional and well understood and commonly used by those skilled in the art. Such techniques and procedures include, for example, the widely used methods described in Molecular Cloning: A Laboratory Manual (Sambrook et al., 2004). thed.,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,2012)、Current Protocols in Molecular Biology(F.M.Ausubel,et al.eds.,2003)、the series Methods in Enzymology(Academic Press,Inc.)、PCR 2:A Practical Approach(M.J.MacPherson,B.D.Hames and G.R.Taylor eds.,1995)、Antibodies,A Laboratory Manual(Harlow and Lane,eds.,1988)、Culture of Animal Cells:A Manual of Basic Technique and Specialized Applications(R.I.Freshney,6 thed.,J.Wiley and Sons,2010)、Oligonucleotide Synthesis(M.J.Gait,ed.,1984)、Methods in Molecular Biology,Humana Press、Cell Biology:A Laboratory Notebook(J.E.Cellis,ed.,Academic Press,1998)、Introduction to Cell and Tissue Culture(J.P.Mather and P.E.Roberts,Plenum Press,1998)、Cell and Tissue Culture:Laboratory Procedures(A.Doyle,J.B.Griffiths,and D.G.Newell,eds.,J.Wiley and Sons,1993-8)、Handbook of Experimental Immunology(D.M.Weir and C.C.Blackwell,eds.,1996)、Gene Transfer Vectors for Mammalian Cells(J.M.Miller and M.P.Calos,eds.,1987)、PCR:The Polymerase Chain Reaction,(Mullis et al.,eds.,1994)、Current Protocols in Immunology(J.E.Coligan et al.,eds.,1991)、Short Protocols in Molecular Biology(Ausubel et al.,eds.,J.Wiley and Sons,2002)、Immunobiology(C.A.Janeway et al.,2004)、Antibodies(P.Finch,1997)、Antibodies:A Practical Approach(D.Catty.,ed.,IRL Press,1988-1989)、Monoclonal Antibodies:A Practical Approach(P.Shepherd and C.Dean,eds., Oxford University Press, 2000), Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999), The Antibodies (M. Zanetti and JDCapra, eds., Harwood Academic Publishers, 1995), and Cancer: Principles and Practice of Oncology (VTDeVita et al., eds., JBLippincott Company, 2011). .

[0040] definition For purposes of interpreting this specification, the following definitions shall apply and whenever appropriate, terms used in the singular shall also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth below shall control.

[0041] As used herein, the singular forms "a," "an," and "the" include plural referents unless otherwise indicated.

[0042] It is understood that some aspects and embodiments of the invention described herein include "comprising," "consisting," and "consisting essentially of."

[0043] The term "about" as used herein refers to a normal error range for the respective value that is readily apparent to one of ordinary skill in the art. Reference herein to a value or parameter with "about" includes (and describes) an embodiment relating to the value or parameter itself.

[0044] As used herein, "treatment" is an approach to obtain beneficial or desired clinical results. As used herein, "treatment" encompasses the administration or application of a therapeutic agent to a disease in a mammal, including a human. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, any one or more of the following: alleviation of one or more symptoms, reduction in the extent of the disease, prevention or delay of the spread of the disease (e.g., metastasis, e.g., to the lungs or lymph nodes), prevention or delay of the recurrence of the disease, delay or slowing of the progression of the disease, improvement of the disease state, inhibition of the disease or the progression of the disease, inhibition or slowing of the disease or its progression, prevention of the onset of the disease, and remission (whether partial or total). Relief of the pathological consequences of a proliferative disease is also encompassed by "treatment". The method of the present invention contemplates one or more of these aspects of treatment.

[0045] The term "prophylactic treatment" as used herein refers to treatment when an individual is known or suspected to have a disease or be at risk for a disease, but has no or minimal symptoms of the disease. An individual receiving prophylactic treatment may be treated before symptoms appear. In some embodiments, an individual may be treated when they have a precancerous lesion.

[0046] As used herein, "combination therapy" refers to administering a first agent in combination with another agent. "In combination" refers to administering one treatment modality in addition to another treatment modality, for example, administering the TIL composition described herein in addition to administering the immunoconjugate described herein to the same individual. Thus, "in combination" refers to administering one treatment modality to an individual before, during, or after administering another treatment modality.

[0047] As used herein, the term "co-administration" means that the first and second therapeutic agents in a combination therapy are administered within about 15 minutes, e.g., within about 10 minutes, 5 minutes, or 1 minute of each other. When the first and second therapeutic agents are administered simultaneously, the first and second therapeutic agents can be included in the same composition (e.g., a composition that includes both the first and second therapeutic agents) or can be included in separate compositions (e.g., the first therapeutic agent is included in one composition and the second therapeutic agent is included in another composition).

[0048] As used herein, the term "sequential administration" refers to administration of a first therapeutic agent and a second therapeutic agent in a combination therapy, separated by a time interval of about 15 minutes or more, such as about 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, or more. Either the first therapeutic agent or the second therapeutic agent may be administered first. When the first therapeutic agent and the second therapeutic agent are contained in separate compositions, they may be contained in the same or different packages or kits.

[0049] As used herein, the term "co-administration" means that the administration of a first therapeutic agent and a second therapeutic agent overlap with each other in combination therapy.

[0050] The term "treatment" with respect to cancer includes any or all of killing cancer cells, inhibiting the growth of cancer cells, inhibiting the replication of cancer cells, reducing the overall tumor burden, and ameliorating one or more symptoms associated with the disease.

[0051] The term "pore" as used herein refers to an opening, including but not limited to a hole, crevice, cavity, opening, break, gap, or perforation in a material. In some instances (where indicated), the term refers to a pore in a surface of the present disclosure. In other instances (where indicated), the pore may refer to a hole in a cell membrane.

[0052] The term "membrane" as used herein refers to a selective barrier or sheet having pores. The term includes flexible sheet-like structures that function as a boundary or lining. In some instances, the term refers to a surface or filter that contains pores. This term is distinct from the term "cell membrane."

[0053] As used herein, the term "filter" refers to a porous article that allows selective passage through the pores. In some instances, the term refers to a surface or membrane that contains pores.

[0054] The term "exogenous" when used in reference to a factor such as an antigen or adjuvant in relation to a cell refers to the fact that a factor is extracellular or that a factor is delivered into the cell from outside the cell, and that the factor may or may not already be present in the cell, and that the cell may or may not produce the exogenous factor after it is delivered.

[0055] The term "heterogeneous" as used herein refers to something that is mixed or not uniform in structure or composition. In some instances, the term refers to pores having a variety of sizes, shapes, or distributions within a particular surface.

[0056] The term "homogeneous" as used herein refers to something being constant or uniform throughout in structure or composition. In some instances, the term refers to pores having a consistent size, shape, or distribution within a particular surface.

[0057] As used herein, the term "homologous" refers to a molecule being derived from the same organism. In some instances, the term refers to a nucleic acid or protein that is normally present or expressed in a particular organism.

[0058] The term "heterologous" in reference to nucleic acid sequences, such as coding sequences and control sequences, refers to sequences that are not normally associated with each other and / or sequences that are not normally associated with a particular cell. Thus, a "heterologous" region of a nucleic acid construct or vector is a segment of nucleic acid that is present within or joined to another nucleic acid molecule that does not naturally occur together. For example, a heterologous region of a nucleic acid construct may include a coding sequence, but the coding sequence is flanked by sequences that do not naturally occur together with the coding sequence. Another example of a heterologous coding sequence is a construct in which the coding sequence itself is not naturally occurring (e.g., a synthetic sequence with codons different from the native gene). Similarly, a cell transformed with a construct that is not normally occurring in the cell is considered heterologous for the purposes of the present invention. Allelic variations or naturally occurring mutational events do not give rise to heterologous DNA as used herein.

[0059] The term "heterologous" in reference to an amino acid sequence, such as a peptide or polypeptide sequence, refers to sequences that are not normally associated with each other and / or sequences that are not normally associated with a particular cell. Thus, a "heterologous" region of a peptide sequence is a segment of amino acids that is within or attached to another amino acid molecule that does not naturally occur together. For example, a heterologous region of a peptide construct may include an amino acid sequence of a peptide, but the amino acid sequence of the peptide is flanked by sequences that do not naturally occur together with the amino acid sequence of the peptide. Another example of a heterologous peptide sequence is a construct in which the peptide sequence itself does not occur in nature (e.g., a synthetic sequence with amino acids different from those encoded by a natural gene). Similarly, a cell transformed with a vector that expresses an amino acid construct that does not normally occur in the cell is considered heterologous for the purposes of the present invention. Allelic variations or naturally occurring mutational events do not give rise to a heterologous peptide as used herein.

[0060] The term "inhibition" as used herein may refer to the act of preventing, reducing, eliminating, or otherwise antagonizing the presence or action of a particular target. Inhibition may refer to partial or complete inhibition. For example, inhibiting an immune response may refer to any act that leads to blocking, reducing, eliminating, or otherwise antagonizing an immune response. In another example, inhibiting expression of a nucleic acid includes, but is not limited to, reducing transcription of the nucleic acid, reducing mRNA abundance (e.g., silencing mRNA transcription), degrading mRNA, inhibiting mRNA translation, and the like. In another example, inhibition may refer to the act of slowing or stopping proliferation, for example, slowing or preventing proliferation of tumor cells.

[0061] The term "suppression" as used herein may refer to the act of decreasing, reducing, hindering, limiting, diminishing, or otherwise reducing the presence or action of a particular target. Suppression may refer to partial or complete suppression. For example, suppressing an immune response may refer to any act that leads to decreasing, reducing, hindering, limiting, diminishing, or otherwise reducing an immune response. In other examples, suppressing the expression of a nucleic acid includes, but is not limited to, reducing the transcription of the nucleic acid, reducing mRNA abundance (e.g., silencing mRNA transcription), degrading mRNA, inhibiting mRNA translation, and the like.

[0062] The term "enhancement" as used herein may refer to the act of improving, promoting, enhancing, or otherwise increasing the presence or action of a particular target. For example, enhancing an immune response may refer to any act that leads to improving, promoting, enhancing, or otherwise increasing an immune response. In one example, enhancing an immune response may refer to using an antigen and / or an adjuvant to improve, promote, enhance, or otherwise increase an immune response. In another example, enhancing expression of a nucleic acid may include, but is not limited to, increased transcription of the nucleic acid, increased mRNA abundance (e.g., increased mRNA transcription), decreased mRNA degradation, increased mRNA translation, and the like. The term used herein to describe the modified TILs of the present disclosure may, in some embodiments, refer to an improvement or increase in one or more properties of the modified TILs over the corresponding unmodified TILs. Non-limiting examples of such properties are provided throughout the present disclosure.

[0063] The term "modulation" as used herein may refer to the act of changing, altering, differing, or otherwise modifying the presence or action of a particular target. For example, modulating an immune response may refer to any act that leads to changing, altering, differing, or otherwise modifying an immune response. In some examples, "modulation" refers to enhancing the presence or action of a particular target. In some examples, "modulation" refers to suppressing the presence or action of a particular target. In other examples, modulating the expression of a nucleic acid includes, but is not limited to, altering the transcription of the nucleic acid, altering mRNA abundance (e.g., increasing mRNA transcription), corresponding changes in mRNA degradation, altering mRNA translation, and the like.

[0064] The term "induction" as used herein may refer to the act of causing, promoting, stimulating, establishing, or otherwise resulting in a result. For example, inducing an immune response may refer to any act that leads to causing, promoting, stimulating, establishing, or otherwise resulting in a desired immune response. In other examples, inducing expression of a nucleic acid may include, but is not limited to, initiating transcription of the nucleic acid, initiating mRNA translation, and the like.

[0065] The term "polynucleotide" or "nucleic acid" as used herein refers to a polymer of any length of nucleotides, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other naturally occurring, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of a polynucleotide can be composed of sugar and phosphate groups (as typically found in RNA or DNA), or it can be composed of modified or substituted sugar or phosphate groups. Alternatively, the backbone of a polynucleotide can be composed of polymers of synthetic subunits such as phosphoramidates and phosphorothioates, and thus may be oligodeoxynucleoside phosphoramidates (P-NH2), mixed phosphorothioate-phosphodiester oligomers, or mixed phosphoramidate-phosphodiester oligomers. Additionally, double-stranded polynucleotides can be obtained from single-stranded polynucleotide products of chemical synthesis by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo with DNA polymerase using an appropriate primer.

[0066] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues can contain naturally occurring or non-naturally occurring amino acid residues, including, but not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. This definition includes both full-length proteins and fragments thereof. These terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present invention, "polypeptide" refers to a protein that contains modifications (e.g., deletions, additions, and substitutions, generally conservative in nature) to the native sequence, so long as the protein maintains the desired activity. These modifications may be deliberate, such as by site-directed mutagenesis, or may be accidental, such as by mutations of the host that produces the protein or errors during PCR amplification.

[0067] As used herein, "pharmacologically acceptable" or "pharmacologically compatible" means that a material is not biologically or otherwise undesirable, e.g., that the material can be incorporated into a pharmaceutical composition administered to a patient without causing significant undesirable biological effects or interacting in a deleterious manner with other components of the composition in which it is included. Pharmaceutically acceptable carriers or excipients preferably have met the required standards of toxicological and manufacturing testing and / or are described in the Inactive Ingredients Guide prepared by the U.S. Food and Drug Administration.

[0068] As used herein, a "microfluidic system" refers to a system that processes small volumes of fluid (e.g., m\L, nL, pL, fL) for individual processing of small volumes of liquid. Certain implementations described herein include multiplexing, automation, and high-throughput screening. Fluids (e.g., buffers, solutions, solutions containing payloads, or cell suspensions) can be moved, mixed, separated, or otherwise processed. In certain embodiments described herein, a microfluidic system is used to mechanically constrict cells suspended in a buffer to induce perturbations (e.g., pores) in the cells and allow the payload or compound to enter the cytosol of the cells.

[0069] For any of the structural and functional properties described herein, methods for determining such properties are known in the art.

[0070] Methods for enhancing activity and / or proliferation of tumor-infiltrating lymphocytes - Patents.com T cell activation initiates an intracellular signaling cascade that ultimately leads to proliferation, effector function, or death, depending on the strength of the TCR and associated signals. To prevent premature or excessive activation, T cells require two independent signals for full activation. Signal 1 is an antigen-specific signal delivered by the binding of the TCR to an antigenic peptide in complex with MHC. Signal 2 is mediated by cytokines or by the engagement of costimulatory molecules such as B7.1 (CD80) and B7.2 (CD86) on antigen-presenting cells (APCs). Signal 3 is mediated by inflammatory cytokines such as IL-2, IL-12, and IFN-α.

[0071] The term "tumor infiltrating lymphocytes (TILs)" as used herein may include any or all of the lymphocyte cell populations that have invaded tumor tissue. TILs have been reported in many solid tumors, including breast cancer, and have emerged as important biomarkers in predicting treatment efficacy and outcomes. In breast cancer, TILs appear to be composed primarily of cytotoxic T cells (CD8+) and helper T cells (CD4+), with a smaller proportion of B-cells and NK cells (Pruneri et al., Breast. 2018 Feb; 37: 207-214; Whitford et al., Eur J Cancer, 1992; 28(2-3): 350-6). High numbers of TILs correlate with the presence of tertiary lymphoid structures in tumors, which also house follicular helper T cells (Tfh) responsible for lymphopoiesis (Gu-Trantien et al., J Clin Invest, 2013 Jul; 123(7): 2873-92).

[0072] TILs are useful for therapy due to their high specificity for tumor antigens. For therapeutic purposes, TILs can be isolated from tumor tissue by one or more methods, including but not limited to enzymatic digestion. After isolation from the tumor, a subset of TILs can be optionally isolated to increase the purity of the subpopulation. TILs can then be expanded in one or more stages (Dudley et al., J Immunother. 2003; 26(4): 332-342; Jin et al., J Immunother. 2012 Apr; 35(3): 283-292). In some examples, TILs can be expanded from thousands of cells to billions of cells. Expanded TILs can be injected into an individual to treat one or more tumors. In some embodiments, TILs are autologous to the individual. In some embodiments, TILs are allogeneic to the individual.

[0073] In some embodiments, TILs may be isolated from carcinomas by mechanical separation, enzymatic separation, and / or density gradient centrifugation (Baldan et al., Br J Cancer 2015 Apr 28;112(9):1510-8; Tan and Lei, Methods Mol Biol. 2019;1960:93-99).

[0074] Methods for enhancing activity and / or proliferation of tumor-infiltrating lymphocytes - Patents.com In some aspects, methods are provided for modulating the activity and / or proliferation of tumor infiltrating lymphocytes (TILs), comprising modifying the TILs to increase expression of one or more mediators of signal 2 and / or signal 3.

[0075] In some aspects, methods are provided for modulating the activity and / or proliferation of tumor infiltrating lymphocytes (TILs), comprising modifying the TILs to increase expression of one or more costimulatory molecules and / or one or more cytokines. In some aspects, methods are provided for modulating the activity and / or proliferation of TILs, comprising modifying the TILs to increase expression of one or more costimulatory molecules. In some embodiments, the costimulatory molecule is B7-H2 (ICOSL), B7-1 (CD80), B7-2 (CD86), CD70, LIGHT, HVEM, CD40, 4-1BBL, OX40L, TL1A, GITRL, CD30L, TIM4, SLAM, CD48, CD58, CD155, or CD112. In some embodiments, the costimulatory molecule is CD86. In some embodiments, the TILs are modified to increase expression of one or more of B7-H2 (ICOSL), B7-1 (CD80), B7-2 (CD86), CD70, LIGHT, HVEM, CD40, 4-1BBL, OX40L, TL1A, GITRL, CD30L, TIM4, SLAM, CD48, CD58, CD155, or CD112. In some embodiments, the TILs are modified to increase expression of CD86.

[0076] In some embodiments, a method for modulating the activity and / or proliferation capacity of TILs is provided, the method comprising expressing a nucleic acid encoding a costimulatory molecule in the TILs. In some embodiments, the method comprises expressing one or more nucleic acids encoding one or more of B7-H2 (ICOSL), B7-1 (CD80), B7-2 (CD86), CD70, LIGHT, HVEM, CD40, 4-1BBL, OX40L, TL1A, GITRL, CD30L, TIM4, SLAM, CD48, CD58, CD155, or CD112. In some embodiments, the method comprises expressing one or more nucleic acids encoding CD86. In some embodiments, the nucleic acid encoding the costimulatory molecule is an mRNA. In some embodiments, the nucleic acid encoding CD86 is an mRNA. Thus, in some aspects, modifying the TILs to increase expression of one or more costimulatory molecules comprises intracellularly delivering one or more nucleic acids (e.g., mRNA) encoding one or more cytokines to the TILs.

[0077] In some embodiments, methods are provided for modulating the activity and / or proliferative capacity of tumor infiltrating lymphocytes (TILs), comprising modifying the TILs to increase expression of one or more costimulatory molecules and / or one or more cytokines. In some embodiments, the methods comprise modifying the TILs to increase expression of one or more costimulatory molecules relative to a reference TIL (e.g., a corresponding TIL that has not been modified as described herein). In some embodiments, the methods comprise modifying the TILs to increase expression of one or more cytokines relative to a reference TIL (e.g., a corresponding TIL that has not been modified as described herein). In some embodiments, the methods comprise modifying the TILs to increase expression of both one or more cytokines and one or more costimulatory molecules relative to a reference TIL (e.g., a corresponding TIL that has not been modified as described herein).

[0078] In some aspects, methods are provided for modulating the activity and / or proliferation capacity of TILs, in which the TILs are modified to increase expression of one or more cytokines. In some embodiments, the cytokine is a type I cytokine. In some embodiments, the cytokine is a type 2 cytokine. In some embodiments, the cytokine comprises one or more of IL-2, IL-15, IL-10, IL-12, IFN-α, or IL-21, or functional variants thereof. In some embodiments, the cytokine is IL-2 or a functional variant thereof and / or IL-12 or a functional variant thereof. As used herein, the term "functional variant" refers to a variant of a polypeptide or protein (e.g., a cytokine) that has substantial or significant sequence identity with the polypeptide or protein and retains at least one biological activity of the polypeptide or protein. Functional variants of a polypeptide or protein can be prepared by means known in the art in view of the present disclosure. Functional variants can include one or more modifications to the amino acid sequence of the polypeptide or protein. In some embodiments, the modification alters one or more physicochemical properties of the polypeptide or protein, for example, by improving the thermostability of the polypeptide or protein, altering the substrate specificity, altering the pH optimum, reducing immunogenicity, etc. In some embodiments, the modification alters one or more of the biological activities of the polypeptide or protein, so long as it does not destroy or eliminate all of the biological activities of the polypeptide or protein.

[0079] In some aspects, a method of modulating the activity and / or proliferation capacity of TILs is provided, the method comprising expressing a nucleic acid encoding a membrane-bound chimeric cytokine in TILs. In some embodiments, the method comprises expressing one or more nucleic acids encoding one or more of the membrane-bound chimeric cytokines in TILs, where the cytokines are IL-2, IL-15, IL-10, IL-12, IFN-α, or IL-21. In some embodiments, the method comprises expressing one or more nucleic acids encoding a membrane-bound chimeric cytokine in TILs, where the cytokines are IL-2 and / or IL-12. In some embodiments, the nucleic acid encoding the membrane-bound chimeric cytokine is an mRNA encoding the membrane-bound chimeric cytokine. In some embodiments, the one or more mRNA encoding the membrane-bound chimeric cytokine is an mRNA encoding IL-2 and / or IL-12.

[0080] In some embodiments, the TILs are modified to increase expression of one or more cytokines. In some embodiments, the cytokine is IL-10, IL-15, IL-12, IL-2, IFN-α, IFN-γ, IL-21, or a functional variant thereof. In some embodiments, the cytokine is IFN-α2 or a functional variant thereof. In some embodiments, the cytokine is a mutant cytokine (e.g., a modified cytokine), such as a membrane-bound chimeric cytokine. In some embodiments, the TILs are modified to increase expression of one or more membrane-bound chimeric cytokines (e.g., membrane-bound IL-10, IL-15, IL-12, IL-2, IFN-α, IFN-γ, IL-21).

[0081] In some embodiments, the cytokine is modified and the modified cytokine is a fusion protein comprising a cytokine and a transmembrane domain. In some embodiments, the cytokine is linked to the transmembrane domain by a peptide linker. In some embodiments, the transmembrane domain is a transferrin receptor protein 1 (TFRC) or a tumor necrosis factor (e.g., FasL) transmembrane domain. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12. In some embodiments, the peptide linker is a G4S linker or an EAAAK linker. In embodiments, the G4S linker comprises any one of 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of the G4S sequence. In some embodiments, the EAAAK linker comprises any one of 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of the EAAK sequence. In some embodiments, the peptide linker is (G4S)3 (SEQ ID NO: 3) or (EAAAK)3 (SEQ ID NO: 4). In some embodiments, the membrane-bound chimeric cytokine comprises an amino acid sequence of SEQ ID NO: 7-10. In some embodiments, the TIL is modified to comprise a nucleic acid that results in increased expression and / or secretion of one or more cytokines. In some embodiments, the nucleic acid encoding the membrane-bound chimeric cytokine comprises a nucleotide sequence of SEQ ID NO: 1 or 2. In some embodiments, the cytokine is a signal 3 effector that stimulates T cell activation. In some embodiments, the membrane-bound chimeric cytokine has a longer half-life of the cytokine in an individual than a non-membrane-bound cytokine. In some embodiments, the half-life of the membrane-bound chimeric cytokine is increased by any one of about 10%, 25%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 200-fold, or 500-fold or more than the non-membrane-bound cytokine.In some embodiments, the membrane-bound cytokine in the engineered TILs has a spatial association between the cytokine and an antigen presented by an antigen-presenting cell that is about any one of 1, 2, 3, 4, 6, 8, 12, 16, 20, 24, 28, 32, 36, 48, 72, 96 hours or more longer than a TIL comprising a non-membrane-bound cytokine. In some embodiments, the TIL comprising a membrane-bound cytokine exhibits a local cytokine concentration that is about any one of 10%, 25%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 200-fold, or 500-fold or more higher than a corresponding TIL comprising a non-membrane-bound cytokine. In some embodiments, the membrane-bound cytokine is a membrane-bound chemokine.

[0082] In some aspects, a composition is provided that enhances the activity of TILs, the composition comprising a membrane-bound chimeric cytokine in the TILs. In some embodiments, the cytokine is modified, and the modified cytokine is a fusion protein comprising a cytokine and a transmembrane domain. In some embodiments, the cytokine is linked to the transmembrane domain by a peptide linker. In some embodiments, the transmembrane domain is a transferrin receptor protein 1 (TFRC) or a tumor necrosis factor (e.g., FasL) transmembrane domain. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12. In some embodiments, the peptide linker is a G4S linker or an EAAAK linker. In some embodiments, the G4S linker comprises any one of 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of the G4S sequence. In some embodiments, the EAAAK linker comprises any one of 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of the EAAAK sequence. In some embodiments, the peptide linker is (G4S)3 (SEQ ID NO:3) or (EAAAK)3 (SEQ ID NO:4). In some embodiments, the membrane-bound chimeric cytokine comprises an amino acid sequence of any one of SEQ ID NOs:7-10. In some embodiments, the TIL comprises a nucleic acid that results in increased expression and / or secretion of one or more cytokines. In some embodiments, the nucleic acid encoding the membrane-bound chimeric cytokine comprises a nucleotide sequence of SEQ ID NO:1 or 2. In some embodiments, the TIL comprises an mRNA that results in increased expression and / or secretion of one or more cytokines. In some embodiments, the cytokine is a signal 3 effector that stimulates activation of T cells. In some embodiments, the membrane-bound chimeric cytokine provides a longer half-life of the cytokine in an individual than a non-membrane-bound cytokine. In some embodiments, the half-life of the membrane-bound chimeric cytokine is increased by any one of about 10%, 25%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 200-fold, or 500-fold or more over the non-membrane-bound cytokine.In some embodiments, the membrane-bound cytokine provides a spatial association between the antigen and cytokine presented by the membrane-bound chimeric cytokine-introduced TILs that is about 1, 2, 3, 4, 6, 8, 12, 16, 20, 24, 28, 32, 36, 48, 72, 96 hours or more longer than a corresponding TIL that includes a non-membrane-bound cytokine. In some embodiments, the membrane-bound cytokine-introduced TILs provide a local cytokine concentration that is about 10%, 25%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 200-fold, or 500-fold or more higher than a corresponding TIL that includes a non-membrane-bound cytokine. In some embodiments, the membrane-bound cytokine is a membrane-bound chemokine.

[0083] In some embodiments, there is provided a method of modulating the activity and / or proliferative capacity of tumor infiltrating lymphocytes (TILs), the method comprising: a) passing a cell suspension comprising the input TILs through a cell deformation constriction, thereby perturbing the input TILs sufficiently to allow passage of one or more nucleic acids encoding one or more cytokines and / or one or more nucleic acids encoding one or more costimulatory molecules to form perturbed input TILs, the diameter of the constriction being a function of the diameter of the input TILs in suspension; and b) incubating the perturbed input TILs with one or more nucleic acids encoding one or more cytokines and / or one or more nucleic acids encoding one or more costimulatory molecules to allow the one or more nucleic acids encoding one or more cytokines and / or one or more nucleic acids encoding one or more costimulatory molecules to enter the perturbed input TILs and express the nucleic acids, thereby generating modified TILs comprising one or more cytokines and / or one or more costimulatory molecules. In some embodiments, the nucleic acids encoding the cytokines and / or the nucleic acids encoding the costimulatory molecules are mRNA. In some embodiments where the cytokine is a membrane-bound cytokine, the nucleic acid encoding the membrane-bound chimeric cytokine is an mRNA encoding the membrane-bound chimeric cytokine.

[0084] In some embodiments, there is provided a method of modulating the activity and / or proliferation capacity of tumor infiltrating lymphocytes (TILs), the method comprising: a) passing a cell suspension comprising the input TILs through a cell deformation constriction, thereby perturbing the input TILs sufficiently large to allow passage of one or more nucleic acids encoding one or more cytokines and / or one or more costimulatory molecules to form a perturbed input TILs, the diameter of the constriction being a function of the diameter of the input TILs in suspension; and b) incubating the input perturbed TILs with one or more nucleic acids encoding one or more cytokines and / or one or more costimulatory molecules, allowing the input perturbed TILs to enter and express the nucleic acids, thereby generating a modified TIL comprising one or more cytokines and / or one or more costimulatory molecules. In some embodiments, the nucleic acid encoding one or more cytokines is an mRNA. In some embodiments where the cytokine is a membrane-bound cytokine, the nucleic acid encoding a membrane-bound chimeric cytokine is an mRNA encoding a membrane-bound chimeric cytokine.

[0085] In some embodiments, a method of modulating the activity and / or proliferation of tumor infiltrating lymphocytes (TILs) is provided, the method comprising incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine before, during, and / or after passing a cell suspension through a constriction for cell deformation. In some embodiments, the method comprises incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine and a costimulatory molecule before, during, and / or after passing a cell suspension through a constriction for cell deformation. In some embodiments, the method comprises incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine and a nucleic acid encoding a costimulatory molecule before, during, and / or after passing a cell suspension through a constriction for cell deformation. In some embodiments, the method comprises incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine and a nucleic acid encoding one or more costimulatory molecules before, during, and / or after passing a cell suspension through a constriction for cell deformation. In some embodiments, the nucleic acid encoding the membrane-bound chimeric cytokine is an mRNA encoding the membrane-bound chimeric cytokine. As used herein, the term "incubating" encompasses bringing together at least a first component (e.g., a nucleic acid encoding a cytokine) and a second component (e.g., a TIL) such that the first and second components are in physical proximity and therefore capable of interacting (i.e., contacting).

[0086] In some embodiments, a method of modulating the activity and / or proliferation of tumor infiltrating lymphocytes (TILs) is provided, the method comprising incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine prior to passing a cell suspension through a cell deformation constriction. In some embodiments, the method comprises incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine and a costimulatory molecule prior to passing the cell suspension through a cell deformation constriction. In some embodiments, the method comprises incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine and a nucleic acid encoding a costimulatory molecule prior to passing the cell suspension through a cell deformation constriction. In some embodiments, the method comprises incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine and a nucleic acid encoding one or more costimulatory molecules prior to passing the cell suspension through a cell deformation constriction. In some embodiments, the nucleic acid encoding a membrane-bound chimeric cytokine is an mRNA encoding a membrane-bound chimeric cytokine.

[0087] In some embodiments according to any one of the methods described herein, the TIL is a mammalian TIL.In some embodiments, the TIL is a monkey, mouse, dog, cat, horse, rat, sheep, goat, pig or rabbit TIL.In some embodiments, the TIL is a primate TIL.In some embodiments, the TIL is a human TIL.

[0088] Compositions of modified TILs with enhanced activity and / or proliferation In some embodiments, compositions are provided that include modified TILs with enhanced activity and / or proliferative capacity.

[0089] In some aspects, a composition is provided comprising modified TILs, wherein the TILs have been modified to increase expression of one or more signal 2 mediators and / or signal 3 mediators.

[0090] In some aspects, compositions are provided that include modified TILs, in which the TILs are modified to have increased expression of one or more costimulatory molecules and / or one or more cytokines. In some aspects, compositions are provided that include modified TILs, in which the TILs are modified to have increased expression of one or more costimulatory molecules. In some embodiments, the costimulatory molecule is B7-H2 (ICOSL), B7-1 (CD80), B7-2 (CD86), CD70, LIGHT, HVEM, CD40, 4-1BBL, OX40L, TL1A, GITRL, CD30L, TIM4, SLAM, CD48, CD58, CD155, or CD112. In some embodiments, the costimulatory molecule is CD86. In some embodiments, the TILs are modified to increase expression of one or more of B7-H2 (ICOSL), B7-1 (CD80), B7-2 (CD86), CD70, LIGHT, HVEM, CD40, 4-1BBL, OX40L, TL1A, GITRL, CD30L, TIM4, SLAM, CD48, CD58, CD155, or CD112. In some embodiments, the TILs are modified to increase expression of CD86.

[0091] In some embodiments, a composition is provided that includes modified TIL, wherein the TIL is modified to express a nucleic acid encoding a costimulatory molecule in the TIL. In some embodiments, the TIL is modified to express one or more nucleic acids encoding one or more of B7-H2 (ICOSL), B7-1 (CD80), B7-2 (CD86), CD70, LIGHT, HVEM, CD40, 4-1BBL, OX40L, TL1A, GITRL, CD30L, TIM4, SLAM, CD48, CD58, CD155, or CD112. In some embodiments, the TIL is modified to express one or more nucleic acids encoding CD86. In some embodiments, one or more of the nucleic acids is mRNA.

[0092] In some aspects, compositions are provided comprising modified TILs, wherein the TILs are modified to have increased expression of one or more costimulatory molecules and / or one or more cytokines. In some aspects, compositions are provided comprising modified TILs, wherein the TILs are modified to have increased expression of one or more cytokines. In some embodiments, the cytokine is a type I cytokine. In some embodiments, the cytokine is a type 2 cytokine. In some embodiments, the cytokine comprises one or more of IL-2, IL-15, IL-10, IL-12, IFN-α, or IL-21, or functional variants thereof. In some embodiments, the cytokine is IL-2 or a functional variant thereof and / or IL-12 or a functional variant thereof.

[0093] In some aspects, compositions are provided that include modified TILs, in which the TILs are modified to express a nucleic acid encoding a membrane-bound chimeric cytokine in the TILs. In some embodiments, the TILs are modified to express one or more nucleic acids encoding one or more membrane-bound chimeric cytokines in the TILs, where the cytokines are IL-2, IL-15, IL-10, IL-12, IFN-α, or IL-21. In some embodiments, the TILs are modified to express one or more nucleic acids encoding one or more membrane-bound chimeric cytokines in the TILs, where the cytokines are IL-2 and / or IL-12. In some embodiments, one or more of the nucleic acids is mRNA.

[0094] In some embodiments, the TILs are modified to increase expression of one or more cytokines. In some embodiments, the cytokine is IL-10, IL-15, IL-12, IL-2, IFN-α, IFN-γ, IL-21, or a functional variant thereof. In some embodiments, the cytokine is IFN-α2 or a functional variant thereof. In some embodiments, the cytokine is a mutant cytokine (such as a modified cytokine), such as a membrane-bound chimeric cytokine. In some embodiments, the TILs are modified to increase expression of one or more membrane-bound chimeric cytokines (such as membrane-bound IL-10, IL-15, IL-12, IL-2, IFN-α, IFN-γ, IL-21, etc.).

[0095] In some embodiments, the cytokine is modified and the modified cytokine is a fusion protein comprising a cytokine and a transmembrane domain. In some embodiments, the cytokine is linked to the transmembrane domain by a peptide linker. In some embodiments, the transmembrane domain is a transferrin receptor protein 1 (TFRC) or a tumor necrosis factor (e.g., FasL) transmembrane domain. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO:11 or SEQ ID NO:12. In some embodiments, the peptide linker is a G4S linker or an EAAAK linker. In embodiments, the G4S linker comprises any one of 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of the G4S sequence. In some embodiments, the EAAAK linker comprises any one of 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of the EAAAK sequence. In some embodiments, the peptide linker is (G4S)3 (SEQ ID NO:3) or (EAAAK)3 (SEQ ID NO:4). In some embodiments, the membrane-bound chimeric cytokine comprises an amino acid sequence of SEQ ID NO: 7-10. In some embodiments, the TIL is modified to comprise a nucleic acid that results in increased expression and / or secretion of one or more cytokines. In some embodiments, the nucleic acid encoding the membrane-bound chimeric cytokine comprises a nucleotide sequence of SEQ ID NO: 1 or 2. In some embodiments, the TIL is modified to comprise an mRNA that results in increased expression and / or secretion of one or more cytokines. In some embodiments, the cytokine is a signal 3 effector that stimulates T cell activation. In some embodiments, the membrane-bound chimeric cytokine has a longer half-life of the cytokine in an individual than a non-membrane-bound cytokine. In some embodiments, the half-life of the membrane-bound chimeric cytokine is increased by any one of about 10%, 25%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 200-fold, or 500-fold or more than the non-membrane-bound cytokine.In some embodiments, the membrane-bound cytokine in the engineered TILs has a spatial association between the cytokine and an antigen presented by an antigen-presenting cell that is about any one of 1, 2, 3, 4, 6, 8, 12, 16, 20, 24, 28, 32, 36, 48, 72, 96 hours or more longer than a TIL comprising a non-membrane-bound cytokine. In some embodiments, the TIL comprising a membrane-bound cytokine exhibits a local cytokine concentration that is about any one of 10%, 25%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 200-fold, or 500-fold or more higher than a corresponding TIL comprising a non-membrane-bound cytokine. In some embodiments, the membrane-bound cytokine is a membrane-bound chemokine.

[0096] In some aspects, a composition is provided that enhances the activity of TILs, the composition comprising a membrane-bound chimeric cytokine in the TILs. In some embodiments, the cytokine is modified, and the modified cytokine is a fusion protein comprising a cytokine and a transmembrane domain. In some embodiments, the cytokine is linked to the transmembrane domain by a peptide linker. In some embodiments, the transmembrane domain is a transferrin receptor protein 1 (TFRC) or a tumor necrosis factor (e.g., FasL) transmembrane domain. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12. In some embodiments, the peptide linker is a G4S linker or an EAAAK linker. In some embodiments, the G4S linker comprises any one of 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of the G4S sequence. In some embodiments, the EAAAK linker comprises any one of 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of the EAAAK sequence. In some embodiments, the peptide linker is (G4S)3 (SEQ ID NO:3) or (EAAAK)3 (SEQ ID NO:4). In some embodiments, the membrane-bound chimeric cytokine comprises any one of the amino acid sequences of SEQ ID NOs:7-10. In some embodiments, the TIL comprises a nucleic acid that results in increased expression and / or secretion of one or more cytokines. In some embodiments, the nucleic acid encoding the membrane-bound chimeric cytokine comprises the nucleotide sequence of SEQ ID NO:1 or 2. In some embodiments, the cytokine is a signal 3 effector that stimulates T cell activation. In some embodiments, the membrane-bound chimeric cytokine has a longer half-life of the cytokine in an individual than a non-membrane-bound cytokine. In some embodiments, the half-life of the membrane-bound chimeric cytokine is increased by any one of about 10%, 25%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 200-fold, or 500-fold or more than the non-membrane-bound cytokine.In some embodiments, the membrane-bound cytokine provides a spatial association between the antigen and cytokine presented by the membrane-bound chimeric cytokine-introduced TILs that is about 1, 2, 3, 4, 6, 8, 12, 16, 20, 24, 28, 32, 36, 48, 72, 96 hours or more longer than a corresponding TIL that includes a non-membrane-bound cytokine. In some embodiments, the membrane-bound cytokine-introduced TILs provide a local cytokine concentration that is about 10%, 25%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 200-fold, or 500-fold or more higher than a corresponding TIL that includes a non-membrane-bound cytokine. In some embodiments, the membrane-bound cytokine is a membrane-bound chemokine.

[0097] In some embodiments, a composition is provided that includes modified TILs, in which the TILs have been modified by a method that includes: a) passing a cell suspension that includes the input TILs through a cell deformation constriction, thereby creating a perturbation in the input TILs that is large enough to allow passage of one or more nucleic acids encoding one or more cytokines and / or one or more nucleic acids encoding one or more costimulatory molecules to form a perturbed input TILs, where the diameter of the constriction is a function of the diameter of the input TILs in suspension, and b) incubating the perturbed input TILs with one or more nucleic acids encoding one or more cytokines and / or one or more nucleic acids encoding one or more costimulatory molecules to allow the one or more nucleic acids encoding one or more cytokines and / or one or more nucleic acids encoding one or more costimulatory molecules to enter the perturbed input TILs and express the nucleic acids, thereby generating modified TILs that include one or more cytokines and / or one or more costimulatory molecules. In some embodiments, the nucleic acids encoding the cytokines and / or the nucleic acids encoding the costimulatory molecules are mRNA. In some embodiments where the cytokine is a membrane-bound cytokine, the nucleic acid encoding the membrane-bound chimeric cytokine is an mRNA encoding the membrane-bound chimeric cytokine.

[0098] In some embodiments, a composition is provided that includes modified TILs, in which the TILs have been modified by a method that includes: a) passing a cell suspension that includes the input TILs through a cell deformation constriction, thereby perturbing the input TILs sufficiently to allow passage of one or more nucleic acids encoding one or more cytokines and / or one or more costimulatory molecules to form a perturbed input TIL, where the diameter of the constriction is a function of the diameter of the input TILs in suspension; and b) incubating the perturbed input TILs with one or more nucleic acids encoding one or more cytokines and / or one or more costimulatory molecules to enter the input perturbed TILs and express the nucleic acids, thereby generating modified TILs that include one or more cytokines and / or one or more costimulatory molecules. In some embodiments, the nucleic acid encoding one or more cytokines is an mRNA. In some embodiments where the cytokine is a membrane-bound cytokine, the nucleic acid encoding a membrane-bound chimeric cytokine is an mRNA encoding a membrane-bound chimeric cytokine.

[0099] In some embodiments, a composition is provided that includes modified TILs, in which the TILs have been modified by a method that includes incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine before, during, and / or after passing a cell suspension through a constriction for cell deformation. In some embodiments, the TILs have been modified by a method that includes incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine and a costimulatory molecule before, during, and / or after passing a cell suspension through a constriction for cell deformation. In some embodiments, the TILs have been modified by a method that includes incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine and a nucleic acid encoding a costimulatory molecule before, during, and / or after passing a cell suspension through a constriction for cell deformation. In some embodiments, the TILs have been modified by a method that includes incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine and a nucleic acid encoding one or more costimulatory molecules before, during, and / or after passing a cell suspension through a constriction for cell deformation. In some embodiments, the nucleic acid encoding a membrane-bound chimeric cytokine is an mRNA encoding a membrane-bound chimeric cytokine.

[0100] In some embodiments, a composition is provided that includes modified TILs, in which the TILs are modified in a manner that includes incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine before passing a cell suspension through a constriction for cell deformation. In some embodiments, the TILs are modified in a manner that includes incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine and a costimulatory molecule before passing a cell suspension through a constriction for cell deformation. In some embodiments, the TILs are modified in a manner that includes incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine and a nucleic acid encoding a costimulatory molecule before passing a cell suspension through a constriction for cell deformation. In some embodiments, the TILs are modified in a manner that includes incubating the TILs with a nucleic acid encoding a membrane-bound chimeric cytokine and a nucleic acid encoding one or more costimulatory molecules before passing a cell suspension through a constriction for cell deformation. In some embodiments, the nucleic acid encoding a membrane-bound chimeric cytokine is an mRNA encoding a membrane-bound chimeric cytokine.

[0101] In some embodiments according to any one of the compositions described herein, the TIL is a mammalian TIL.In some embodiments, the TIL is a monkey, mouse, dog, cat, horse, rat, sheep, goat, pig or rabbit TIL.In some embodiments, the TIL is a primate TIL.In some embodiments, the TIL is a human TIL.

[0102] Methods of treatment, compositions for use as medicaments, and use of pharmaceutical compositions in the manufacture of medicaments In some embodiments, a method of modulating an immune response in an individual is provided, the method comprising administering modified TILs exhibiting increased expression of one or more cytokines and / or one or more costimulatory molecules. For example, in some embodiments, the present disclosure provides a method of modulating an immune response in an individual in need of modulating an immune response, the method comprising administering to a subject modified TILs exhibiting increased expression of one or more cytokines. In some embodiments, the present disclosure provides a method of modulating an immune response in an individual in need of modulating an immune response, the method comprising administering to a subject modified TILs exhibiting increased expression of one or more costimulatory molecules. In some embodiments, the present disclosure provides a method of modulating an immune response in an individual in need of modulating an immune response, the method comprising administering to a subject modified TILs exhibiting increased expression of both (i) one or more cytokines and (ii) one or more costimulatory molecules. In some embodiments, the modified TILs are prepared according to any one of the methods described herein. In some embodiments, a method of modulating an immune response in an individual is provided, the method comprising administering to a subject any one of the compositions comprising modified TILs described herein.

[0103] In some embodiments, a method of treating cancer, infectious disease, or virus-related disease in an individual is provided, comprising administering modified TILs prepared according to any one of the methods described herein. In some embodiments, a method of treating cancer, infectious disease, or virus-related disease in an individual is provided, comprising administering any one of the compositions comprising modified TILs described herein. Thus, some embodiments of the present disclosure relate to a method of treating cancer in a subject in need of cancer treatment, comprising administering to the subject any of the modified TILs described herein. In some embodiments, a method of treating an infectious disease in a subject in need of treatment, comprising administering to the subject any of the modified TILs described herein. In some embodiments, a method of treating a virus-related disease in a subject in need of treatment, comprising administering to the subject any of the modified TILs described herein. Non-limiting examples of cancer, infectious disease, and virus-related disease that can be treated by the present disclosure are described elsewhere herein.

[0104] In some embodiments, the method includes administering the modified TILs multiple times or administering the composition comprising the modified TILs multiple times. As is evident from the present disclosure, the modified TILs (or the composition comprising the modified TILs) can be administered to the subject by any suitable route of administration. Non-limiting examples of such routes of administration include intratumoral, oral, intrapulmonary, intranasal, parenteral (intravenous, intraarterial, intramuscular, intraperitoneal, or subcutaneous), intrarectal, intralymphatic, intrathecal, periocular, or topical. In some embodiments, the modified TILs or the composition comprising the modified TILs is administered intravenously or intratumorally. In some embodiments, the individual is a mammal. In some embodiments, the individual is a monkey, mouse, dog, cat, horse, rat, sheep, goat, pig, or rabbit. In some embodiments, the individual is a primate. In some embodiments, the individual is a human. In some embodiments, the modified TILs or the composition comprising the modified TILs is administered before, simultaneously with, or after administration of another therapeutic agent. In some embodiments, the other therapeutic agent is a checkpoint therapy.

[0105] In some embodiments, a pharmaceutical composition is provided for stimulating an immune response in an individual, the composition comprising an effective amount of any one of the compositions comprising modified TILs described herein. In some embodiments, a pharmaceutical composition is provided for use as a medicament, the pharmaceutical composition comprising an effective amount of any one of the compositions comprising modified TILs described herein.

[0106] In some embodiments, a pharmaceutical composition for treating cancer, an infectious disease, or a viral-related disease in an individual is provided, the pharmaceutical composition comprising an effective amount of any one of the compositions comprising modified TILs described herein.

[0107] In some aspects, there is provided a use of a pharmaceutical composition in the manufacture of a medicament for stimulating an immune response in an individual, the pharmaceutical composition comprising an effective amount of any one of the compositions comprising modified TILs described herein.

[0108] In some embodiments, there is provided a use of a pharmaceutical composition in the manufacture of a medicament for treating cancer, an infectious disease, or a viral-related disease in an individual, the pharmaceutical composition comprising an effective amount of any one of the compositions comprising modified TILs described herein.

[0109] In some embodiments, a composition is provided for use as a medicament, the composition comprises an effective amount of modified TILs comprising membrane-bound chimeric cytokines. In some embodiments, a composition is provided for treating cancer, infectious disease, or virus-related disease in an individual with a composition, the composition comprises an effective amount of TILs comprising membrane-bound chimeric cytokines. In some embodiments, a method is provided for treating cancer, infectious disease, or virus-related disease in an individual, the method comprises administering to the individual a composition comprising an effective amount of TILs comprising membrane-bound chimeric cytokines.

[0110] In some embodiments, there is provided the use of a composition comprising an effective amount of a TIL comprising a membrane-bound chimeric cytokine in the manufacture of a medicament for stimulating an immune response in an individual and / or for treating cancer, an infectious disease, or a viral-related disease in an individual.

[0111] In some embodiments according to any one of the pharmaceutical compositions, compositions for use, or uses described herein, the composition is formulated for multiple administration. In some embodiments, the composition is administered intravenously or intratumorally. In some embodiments, the individual is a mammal. In some embodiments, the individual is a monkey, mouse, dog, cat, horse, rat, sheep, goat, pig, or rabbit. In some embodiments, the individual is a primate. In some embodiments, the individual is a human. In some embodiments, the composition is formulated to be administered before, simultaneously with, or after administration of another therapeutic agent. In some embodiments, the other therapeutic agent is a checkpoint therapeutic agent.

[0112] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described herein, the TILs are modified by a method comprising: a) passing a cell suspension comprising the input TILs through a cell deformation constriction, thereby creating a perturbation in the input TILs sufficient to allow passage of one or more nucleic acids encoding one or more cytokines and / or one or more nucleic acids encoding one or more costimulatory molecules to form a perturbed input TILs, wherein the diameter of the constriction is a function of the diameter of the input TILs in suspension; and b) incubating the perturbed input TILs with one or more nucleic acids encoding one or more cytokines and / or one or more nucleic acids encoding one or more costimulatory molecules to allow the one or more nucleic acids encoding one or more cytokines and / or one or more nucleic acids encoding one or more costimulatory molecules to enter the perturbed input TILs and express the nucleic acids, thereby generating modified TILs comprising one or more cytokines and / or one or more costimulatory molecules. In some embodiments, the nucleic acids encoding the cytokines and / or the nucleic acids encoding the costimulatory molecules are mRNA. In some embodiments where the cytokine is a membrane-bound cytokine, the nucleic acid encoding the membrane-bound chimeric cytokine is an mRNA encoding the membrane-bound chimeric cytokine.

[0113] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described herein, the TILs are modified by a method comprising: a) passing a cell suspension comprising the input TILs through a cell deformation constriction, thereby perturbing the input TILs sufficiently large to allow passage of one or more nucleic acids encoding one or more cytokines and / or one or more costimulatory molecules to form a perturbed input TILs, wherein the diameter of the constriction is a function of the diameter of the input TILs in suspension; and b) incubating the perturbed input TILs with one or more nucleic acids encoding one or more cytokines and / or one or more costimulatory molecules to enter the input perturbed TILs and express the nucleic acids, thereby generating modified TILs comprising one or more cytokines and / or one or more costimulatory molecules. In some embodiments, the nucleic acid encoding one or more cytokines is an mRNA. In some embodiments where the cytokine is a membrane-bound cytokine, the nucleic acid encoding a membrane-bound chimeric cytokine is an mRNA encoding a membrane-bound chimeric cytokine.

[0114] Regulation of TIL activity and proliferation In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described herein, the modified TILs exhibit greater in vivo persistence than corresponding unmodified TILs, hi some embodiments, modified TILs that express membrane-bound IL-2 and / or IL-12 exhibit greater in vivo persistence than corresponding TILs that do not express membrane-bound IL-2 and / or IL-12.

[0115] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described herein, the modified TILs exhibit a longer in vivo circulation time than corresponding unmodified TILs, hi some embodiments, modified TILs that express membrane-bound IL-2 and / or IL-12 exhibit a longer in vivo circulation time than corresponding TILs that do not express membrane-bound IL-2 and / or IL-12.

[0116] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described herein, the modified TILs exhibit greater in vivo persistence in the absence of exogenous cytokines than corresponding unmodified TILs, hi some embodiments, modified TILs that express membrane-bound IL-2 and / or IL-12 exhibit greater in vivo persistence in the absence of exogenous cytokines than corresponding TILs that do not express membrane-bound IL-2 and / or IL-12.

[0117] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described herein, the modified TILs exhibit a longer in vivo circulation time in the absence of exogenous cytokines than corresponding unmodified TILs, hi some embodiments, modified TILs that express membrane-bound IL-2 and / or IL-12 exhibit a longer in vivo circulation time in the absence of exogenous cytokines than corresponding TILs that do not express membrane-bound IL-2 and / or IL-12.

[0118] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described herein, the modified TILs exhibit greater in vivo persistence when cultured in the absence of exogenous cytokines for any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days than corresponding unmodified TILs. In some embodiments, the modified TILs that express membrane-bound IL-2 and / or IL-12 exhibit greater in vivo persistence when cultured in the absence of exogenous cytokines for any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days than corresponding TILs that do not express membrane-bound IL-2 and / or IL-12.

[0119] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described herein, the modified TILs exhibit a longer in vivo circulation time than corresponding unmodified TILs when cultured for any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days in the absence of exogenous cytokines. In some embodiments, the modified TILs that express membrane-bound IL-2 and / or IL-12 exhibit a longer in vivo circulation time than corresponding TILs that do not express membrane-bound IL-2 and / or IL-12 when cultured for any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days in the absence of exogenous cytokines.

[0120] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described herein, the modified TILs have increased expression of one or more markers of central memory T cells (e.g., but not limited to, CD62L) relative to corresponding unmodified TILs. As used herein, "central memory T cells" or "T CM The term "membrane-associated T cells" refers to memory T cells that express at least CD45RO, CCR7, CD62L, and / or CD127. Thus, in some aspects, the described methods of modifying TILs are useful for increasing expression of one or more central memory T cell markers in TILs. In some embodiments, modified TILs that express membrane-bound IL-2 and / or IL-12 have increased expression of one or more markers of central memory T cells over corresponding TILs that do not express membrane-bound IL-2 and / or IL-12.

[0121] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described herein, the modified TILs have increased expression of one or more markers of central memory T cells when cultured in the absence of exogenous cytokines, compared to corresponding unmodified TILs. In some embodiments, the modified TILs have increased expression of one or more markers of central memory T cells when cultured in the absence of exogenous cytokines for any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, compared to corresponding unmodified TILs. In some embodiments, the modified TILs expressing membrane-bound IL-2 and / or IL-12 have increased expression of one or more markers of central memory T cells when cultured in the absence of exogenous cytokines, compared to corresponding TILs that do not express membrane-bound IL-2 and / or IL-12. In some embodiments, the exogenous cytokine comprises exogenous IL-2.

[0122] In some embodiments according to any one of the above-mentioned methods, pharmaceutical compositions, compositions for use, or uses, expression of one or more markers of central memory T cells in the modified TILs is increased by any one of about 10%, 25%, 50%, 75%, 100%, 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 3-fold, 4-fold, 5-fold, 8-fold, or more than 10-fold compared to the corresponding unmodified TILs.

[0123] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described herein, the modified TILs have increased expression of one or more markers of T cell self-renewal (e.g., but not limited to, TCF1) relative to corresponding unmodified TILs. In some embodiments, modified TILs that express membrane-bound IL-2 and / or IL-12 have increased expression of one or more markers of T cell self-renewal relative to corresponding TILs that do not express membrane-bound IL-2 and / or IL-12.

[0124] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described herein, the modified TILs have increased expression of one or more markers of T cell self-renewal when cultured in the absence of exogenous cytokines, compared to corresponding unmodified TILs. In some embodiments, the modified TILs have increased expression of one or more markers of T cell self-renewal when cultured in the absence of exogenous cytokines for any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, compared to corresponding unmodified TILs. In some embodiments, the modified TILs expressing membrane-bound IL-2 and / or IL-12 have increased expression of one or more markers of T cell self-renewal when cultured in the absence of exogenous cytokines, compared to corresponding TILs that do not express membrane-bound IL-2 and / or IL-12. In some embodiments, the exogenous cytokine comprises exogenous IL-2.

[0125] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described above, expression of one or more markers of T cell self-renewal in the modified TILs is increased by any one of about 10%, 25%, 50%, 75%, 100%, 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 3-fold, 4-fold, 5-fold, 8-fold, or more than 10-fold over the corresponding unmodified TILs.

[0126] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described herein, the modified TILs have reduced expression of one or more markers of T cell exhaustion (e.g., but not limited to, CD39 or CD69) than corresponding unmodified TILs. In some embodiments, modified TILs that express membrane-bound IL-2 and / or IL-12 have reduced expression of one or more markers of T cell auto-exhaustion than corresponding TILs that do not express membrane-bound IL-2 and / or IL-12. Non-limiting examples of exhaustion markers include PD-1, CD39, TIM-3, TIGIT, and / or LAG-3.

[0127] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described herein, the modified TILs have reduced expression of one or more markers of T cell exhaustion when cultured in the absence of exogenous cytokines, compared to corresponding unmodified TILs. In some embodiments, the modified TILs have reduced expression of one or more markers of T cell exhaustion when cultured in the absence of exogenous cytokines for any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, compared to corresponding unmodified TILs. In some embodiments, the modified TILs expressing membrane-bound IL-2 and / or IL-12 have reduced expression of one or more markers of T cell exhaustion when cultured in the absence of exogenous cytokines, compared to corresponding TILs that do not express membrane-bound IL-2 and / or IL-12. In some embodiments, the exogenous cytokine comprises exogenous IL-2.

[0128] In some embodiments according to any one of the above-mentioned methods, pharmaceutical compositions, compositions for use, or uses, expression of one or more markers of T cell exhaustion in the modified TILs is reduced by any one of about 10%, 25%, 50%, 75%, 100%, 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 3-fold, 4-fold, 5-fold, 8-fold, or more than 10-fold compared to the corresponding unmodified TILs.

[0129] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described herein, the modified TILs have increased expression of one or more of T-bet, EOMES, TCF1, CD62L relative to corresponding unmodified TILs. In some embodiments, modified TILs that express membrane-bound IL-2 and / or IL-12 have increased expression of one or more of T-bet, EOMES, TCF1, CD62L relative to corresponding TILs that do not express membrane-bound IL-2 and / or IL-12.

[0130] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described herein, the modified TILs have increased expression of one or more of T-bet, EOMES, TCF1, CD62L when cultured in the absence of exogenous cytokines, relative to corresponding unmodified TILs. In some embodiments, the modified TILs have increased expression of one or more of T-bet, EOMES, TCF1, CD62L when cultured in the absence of exogenous cytokines for any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, relative to corresponding unmodified TILs. In some embodiments, modified TILs that express membrane-bound IL-2 and / or IL-12 have increased expression of one or more of T-bet, EOMES, TCF1, CD62L when cultured in the absence of exogenous cytokines, relative to corresponding TILs that do not express membrane-bound IL-2 and / or IL-12. In some embodiments, the exogenous cytokine comprises exogenous IL-2.

[0131] In some embodiments according to any one of the above-mentioned methods, pharmaceutical compositions, compositions for use, or uses, expression of one or more of T-bet, EOMES, TCF1, CD62L in the modified TILs is increased by any one of about 10%, 25%, 50%, 75%, 100%, 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 3-fold, 4-fold, 5-fold, 8-fold, or more than 10-fold in the modified TILs compared to the corresponding unmodified TILs.

[0132] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described herein, the modified TILs have reduced expression of one or more of T-bet, EOMES, CD39, and CD69 relative to corresponding unmodified TILs. In some embodiments, modified TILs that express membrane-bound IL-2 and / or IL-12 have reduced expression of one or more of T-bet, EOMES, CD39, and CD69 relative to corresponding TILs that do not express membrane-bound IL-2 and / or IL-12.

[0133] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described herein, the modified TILs have reduced expression of one or more of T-bet, EOMES, CD39, and CD69 when cultured in the absence of exogenous cytokines, compared to corresponding unmodified TILs. In some embodiments, the modified TILs have reduced expression of one or more of T-bet, EOMES, CD39, and CD69 when cultured in the absence of exogenous cytokines for any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, compared to corresponding unmodified TILs. In some embodiments, the modified TILs that express membrane-bound IL-2 and / or IL-12 have reduced expression of one or more of T-bet, EOMES, CD39, and CD69 when cultured in the absence of exogenous cytokines, compared to corresponding TILs that do not express membrane-bound IL-2 and / or IL-12. In some embodiments, the exogenous cytokine comprises exogenous IL-2.

[0134] In some embodiments according to any one of the above-mentioned methods, pharmaceutical compositions, compositions for use, or uses, expression of one or more of T-bet, EOMES, CD39, and CD69 in the modified TILs is reduced by any one of about 10%, 25%, 50%, 75%, 100%, 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 3-fold, 4-fold, 5-fold, 8-fold, or less than 10-fold in the modified TILs compared to the corresponding unmodified TILs.

[0135] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described herein, the modified TILs exhibit greater proliferation than corresponding unmodified TILs, hi some embodiments, modified TILs that express membrane-bound IL-2 and / or IL-12 exhibit greater proliferation than corresponding TILs that do not express membrane-bound IL-2 and / or IL-12.

[0136] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described herein, the modified TILs exhibit greater proliferation than corresponding unmodified TILs when cultured in medium that does not contain an exogenous cytokine (e.g., but not limited to, exogenous IL-2). In some embodiments, the modified TILs exhibit greater proliferation than corresponding unmodified TILs when cultured in medium that does not contain an exogenous cytokine (e.g., but not limited to, exogenous IL-2) for any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In some embodiments, the modified TILs expressing membrane-bound IL-2 and / or IL-12 exhibit greater proliferation than corresponding TILs that do not express membrane-bound IL-2 and / or IL-12 when cultured in medium that does not contain an exogenous cytokine (e.g., but not limited to, exogenous IL-2). In some embodiments, the exogenous cytokine comprises exogenous IL-2.

[0137] In some embodiments according to any one of the above-mentioned methods, pharmaceutical compositions, compositions for use, or uses, the proliferation rate of the modified TILs is increased by any one of about 10%, 25%, 50%, 75%, 100%, 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 3-fold, 4-fold, 5-fold, 8-fold, or more than 10-fold compared to the corresponding unmodified TILs.

[0138] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described herein, the modified TILs exhibit greater proliferation when co-cultured with tumor cells than corresponding unmodified TILs, in some embodiments, modified TILs that express membrane-bound IL-2 and / or IL-12 exhibit greater proliferation when co-cultured with tumor cells than corresponding TILs that do not express membrane-bound IL-2 and / or IL-12.

[0139] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described herein, the modified TILs exhibit greater proliferation when co-cultured with tumor cells in the absence of exogenous cytokines (e.g., but not limited to, exogenous IL-2 and / or IL-12) than corresponding unmodified TILs. In some embodiments, the modified TILs exhibit greater proliferation when co-cultured with tumor cells in the absence of exogenous cytokines (e.g., but not limited to, exogenous IL-2 and / or IL-12) for any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days than corresponding unmodified TILs. In some embodiments, the modified TILs expressing membrane-bound IL-2 and / or IL-12 exhibit greater proliferation when co-cultured with tumor cells in the absence of exogenous cytokines (e.g., but not limited to, exogenous IL-2 and / or IL-12) than corresponding TILs that do not express membrane-bound IL-2 and / or IL-12. In some embodiments, the exogenous cytokine comprises exogenous IL-2.

[0140] In some embodiments according to any one of the above-mentioned methods, pharmaceutical compositions, compositions for use, or uses, the proliferation rate of the modified TILs is increased by any one of about 10%, 25%, 50%, 75%, 100%, 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 3-fold, 4-fold, 5-fold, 8-fold, 10-fold, 20-fold, 50-fold, or 100-fold or more than the corresponding unmodified TILs when co-cultured with tumor cells in the absence of exogenous cytokines.

[0141] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described herein, the modified TILs exhibit higher expression of granzyme B than corresponding unmodified TILs. In some embodiments, modified TILs that express membrane-bound IL-2 and / or IL-12 exhibit higher expression of granzyme B than corresponding TILs that do not express membrane-bound IL-2 and / or IL-12.

[0142] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described herein, the modified TILs exhibit higher expression of granzyme B when cultured in the absence of exogenous cytokines than corresponding unmodified TILs. In some embodiments, modified TILs that express membrane-bound IL-2 and / or IL-12 exhibit higher expression of granzyme B when cultured in the absence of exogenous cytokines than corresponding TILs that do not express membrane-bound IL-2 and / or IL-12.

[0143] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described herein, the modified TILs exhibit higher expression of granzyme B than corresponding unmodified TILs when co-cultured with tumor cells in the absence of exogenous cytokines. In some embodiments, the modified TILs expressing membrane-bound IL-2 and / or IL-12 exhibit higher expression of granzyme B than corresponding TILs that do not express membrane-bound IL-2 and / or IL-12 when co-cultured with tumor cells in the absence of exogenous cytokines. In some embodiments, the tumor cells are autologous tumor cells.

[0144] In some embodiments according to any one of the above-mentioned methods, pharmaceutical compositions, compositions for use, or uses, expression of Granzyme B in the modified TILs is increased by any one of about 10%, 25%, 50%, 75%, 100%, 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 3-fold, 4-fold, 5-fold, 8-fold, 10-fold, 20-fold, 50-fold, or 100-fold or more compared to the corresponding unmodified TILs.

[0145] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described herein, the percentage of cells expressing granzyme B in the modified TILs is higher than the percentage in the corresponding unmodified TILs. In some embodiments, the percentage of cells expressing granzyme B in the modified TILs that express membrane-bound IL-2 and / or IL-12 is higher than the percentage in the corresponding TILs that do not express membrane-bound IL-2 and / or IL-12.

[0146] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described herein, the percentage of cells expressing granzyme B in the modified TILs is higher than the percentage in the corresponding unmodified TILs when cultured in the absence of exogenous cytokines. In some embodiments, the percentage of cells expressing granzyme B in the modified TILs that express membrane-bound IL-2 and / or IL-12 is higher than the percentage in the corresponding TILs that do not express membrane-bound IL-2 and / or IL-12 when cultured in the absence of exogenous cytokines.

[0147] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described herein, the percentage of cells expressing granzyme B in the modified TILs is higher than the percentage in the corresponding unmodified TILs when co-cultured with tumor cells in the absence of exogenous cytokines. In some embodiments, the percentage of cells expressing granzyme B in the modified TILs expressing membrane-bound IL-2 and / or IL-12 is higher than the percentage in the corresponding TILs that do not express membrane-bound IL-2 and / or IL-12 when co-cultured with tumor cells in the absence of exogenous cytokines. In some embodiments, the tumor cells are autologous tumor cells.

[0148] In some embodiments according to any one of the above-mentioned methods, pharmaceutical compositions, compositions for use, or uses, the percentage of cells expressing Granzyme B in the modified TILs is any one of about 10%, 25%, 50%, 75%, 100%, 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 3-fold, 4-fold, 5-fold, 8-fold, 10-fold, 20-fold, 50-fold, or more than 100-fold higher than the percentage in the corresponding unmodified TILs.

[0149] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described herein, the modified TILs kill co-cultured tumor cells more than corresponding unmodified TILs, hi some embodiments, modified TILs that express membrane-bound IL-2 and / or IL-12 kill co-cultured tumor cells more than corresponding TILs that do not express membrane-bound IL-2 and / or IL-12.

[0150] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described herein, the modified TILs kill co-cultured tumor cells in the absence of exogenous cytokines to a greater extent than corresponding unmodified TILs, in some embodiments, modified TILs that express membrane-bound IL-2 and / or IL-12 kill co-cultured tumor cells in the absence of exogenous cytokines to a greater extent than corresponding TILs that do not express membrane-bound IL-2 and / or IL-12.

[0151] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described above, enhanced tumor cell killing in co-culture by the modified TILs is increased by any one of about 10%, 25%, 50%, 75%, 100%, 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 3-fold, 4-fold, 5-fold, 8-fold, 10-fold, 20-fold, 50-fold, or 100-fold or more than that by the corresponding unmodified TILs. In some embodiments, the tumor cells are autologous tumor cells.

[0152] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described herein, killing of tumor cells co-cultured in the absence of exogenous cytokines by the modified TILs is equivalent to killing of tumor cells co-cultured in the presence of one or more exogenous cytokines by corresponding unmodified TILs. In some embodiments, the exogenous cytokines comprise exogenous IL-2. In some embodiments, the exogenous cytokines comprise exogenous IL-12. In some embodiments, the exogenous cytokines comprise exogenous IL-2 and / or exogenous IL-12.

[0153] In some embodiments according to any one of the methods, pharmaceutical compositions, compositions for use, or uses described above, the killing of co-cultured tumor cells promoted by the modified TILs differs by no more than any one of 1%, 2%, 5%, 8%, 10%, 25%, 50%, 75%, 100%, 1.2-fold, 1.5-fold, 1.8-fold, or 2-fold from the killing of tumor cells promoted by corresponding unmodified TILs in the presence of one or more exogenous cytokines. In some embodiments, the exogenous cytokines comprise exogenous IL-2 and / or exogenous IL-12. In some embodiments, the tumor cells are autologous tumor cells.

[0154] In some embodiments according to the methods, pharmaceutical compositions, compositions for use, or uses described herein, TILs comprising membrane-bound chimeric IL-2 and / or membrane-bound chimeric IL-12 are prepared by: a) passing a cell suspension comprising input TILs through a cell deformation constriction, thereby perturbing the input TILs sufficiently large to allow passage of the nucleic acid encoding membrane-bound chimeric IL-2 and / or membrane-bound chimeric IL-12 to form perturbed input TILs, where the diameter of the constriction is a function of the diameter of the input TILs in suspension; and b) incubating the perturbed input TILs with nucleic acid encoding membrane-bound chimeric IL-2 and / or membrane-bound chimeric IL-12, allowing the nucleic acid to enter the perturbed input TILs and express the nucleic acid encoding membrane-bound chimeric IL-2 and / or membrane-bound chimeric IL-12, thereby generating TILs comprising membrane-bound chimeric IL-2 and / or membrane-bound chimeric IL-12.

[0155] Stenosis delivery As described and illustrated herein, the method of modifying TILs provided herein includes passing TILs (e.g., a cell suspension containing TILs) through a cell deformation constriction, which perturbs the TILs such that one or more nucleic acids encoding cytokines and / or one or more nucleic acids encoding costimulatory molecules enter the TILs through the perturbation when contacted with the TILs. As used herein, a "perturbation" refers to any opening in a cell membrane (e.g., the cell membrane of a TIL) that is not present under normal steady-state conditions (e.g., conditions in which no deformation force is applied to the cell). A perturbation includes a hole, a cleft, a cavity, an opening, a pore, a break, a gap, a perforation, or a combination thereof. As described herein, when a cell passes through a constriction (such as that disclosed herein), the cell is subjected to various forces (including, but not limited to, mechanical deformation forces and / or shear forces) due to the compressive physical environment, which creates a perturbation in the cell membrane.

[0156] In some embodiments, the width of the constriction is about 10% to about 99% of the average diameter of the introduced TILs. In some embodiments, the width of the constriction is any one of about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 20% to about 60%, about 40% to about 60%, about 30% to about 45%, about 50% to about 99%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 60% to about 90%, about 60% to about 80%, or about 60% to about 70% of the average diameter of the introduced TILs. In some embodiments, the width of the narrowed portion is about 5 μm to about 12 μm, about 6 μm to about 12 μm, about 8 μm to about 11 μm, about 9 μm to about 11 μm, about 9.5 μm to about 10.5 μm, about 8 μm to about 15 μm, about 10 μm to about 15 μm, or about 12 μm to about 15 μm. In some embodiments, the width of the narrowed portion is about 10 μm. In some embodiments, the width of the narrowed portion is about 3 μm to about 6 μm. In some embodiments, the width of the narrowed portion is about 4.2 μm to about 6 μm. In some embodiments, the width of the narrowed portion is about 4.2 μm to about 4.8 μm. In some embodiments, the width of the narrowed portion is about 3 μm to about 5 μm. In some embodiments, the width of the narrowed portion is about 3 μm to about 3.5 μm. In some embodiments, the width of the narrowed portion is about 3.5 μm to about 4 μm. In some embodiments, the width of the constriction is about 4 μm to about 4.5 μm. In some embodiments, the width of the constriction is about 3.2 μm to about 3.8 μm. In some embodiments, the width of the constriction is about 3.8 μm to about 4.3 μm. In some embodiments, the width of the constriction is less than or equal to any one of about 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm, 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5 μm, 8 μm, 8.5 μm, 9 μm, 9.5 μm, 10 μm, 10.5 μm, 11 μm, 11.5 μm, 12 μm, 12.5 μm, 13 μm, 13.5 μm, 14 μm, 14.5 μm, or 15 μm.In some embodiments, the width of the constriction is less than or equal to about any one of 3.0 μm, 3.1 μm, 3.2 μm, 3.3 μm, 3.4 μm, 3.5 μm, 3.6 μm, 3.7 μm, 3.8 μm, 3.9 μm, 4.0 μm, 4.1 μm, 4.2 μm, 4.3 μm, 4.4 μm, 4.5 μm, 4.6 μm, 4.7 μm, 4.8 μm, 4.9 μm, or 5.0 μm. In some embodiments, the width of the constriction is about 3.5 μm. In some embodiments, the width of the constriction is about 4 μm. In some embodiments, the input cell suspension comprising TILs is passed through multiple constrictions, where multiple constrictions are arranged in series and / or parallel.

[0157] Constrictions used to generate compositions of TILs that contain costimulatory molecules and / or cytokines In some embodiments, the invention provides compositions of TILs comprising costimulatory molecules or cytokines, hi some embodiments, nucleic acids encoding costimulatory molecules and / or cytokines are delivered intracellularly to TILs.

[0158] In some embodiments, the nucleic acid is introduced into the TIL by passing the cell through a constriction, which generates a temporary hole in the membrane of the cell, thereby allowing the nucleic acid to enter the cell. Examples of using a constriction to deliver a compound into a cell are shown in WO2013 / 059343, WO2015 / 023982, WO2016 / 070136, WO2017041050, WO2017008063, WO2017 / 192785, WO2017 / 192786, WO2019 / 178005, WO2019 / 178006, WO2020 / 072833, WO2020154696, and WO2020176789.

[0159] In some embodiments, nucleic acids are delivered into TILs to generate TILs of the invention by passing a cell suspension containing TILs through a constriction, which deforms the cells and perturbs them, thereby allowing the nucleic acid to enter the cells and be expressed. In some embodiments, the constriction is included within a microfluidic channel. In some embodiments, multiple constrictions can be arranged in parallel and / or in series within the microfluidic channel.

[0160] In some embodiments, the constriction in the microfluidic channel has an inlet, a center, and an outlet. In some embodiments, the length, depth, and width of the constriction in the microfluidic channel can vary. In some embodiments, the width of the constriction in the microfluidic channel is a function of the diameter of the TIL. Methods for determining the diameter of the TIL are known in the art, for example, high content imaging, cell counting devices, or flow cytometry.

[0161] In some embodiments where the constriction is used to deliver a nucleic acid encoding a costimulatory molecule or cytokine to a TIL, the width of the constriction is about 2 μm to about 15 μm. In some embodiments, the width of the constriction is about 3 μm to about 10 μm. In some embodiments, the width of the constriction is about 3 μm to about 6 μm. In some embodiments, the width of the constriction is about 4.2 μm to about 6 μm. In some embodiments, the width of the constriction is about 4.2 μm to about 4.8 μm. In some embodiments, the width of the constriction is about 3 μm to about 5 μm. In some embodiments, the width of the constriction is about 3 μm to about 3.5 μm. In some embodiments, the width of the constriction is about 3.5 μm to about 4 μm. In some embodiments, the width of the constriction is about 4 μm to about 4.5 μm. In some embodiments, the width of the constriction is about 3.2 μm to about 3.8 μm. In some embodiments, the width of the constriction is about 3.8 μm to about 4.3 μm. In some embodiments, the width of the constriction is less than or equal to about any one of 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm, 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5 μm, 8 μm, 8.5 μm, 9 μm, 9.5 μm, 10 μm, 10.5 μm, 11 μm, 11.5 μm, 12 μm, 12.5 μm, 13 μm, 13.5 μm, 14 μm, 14.5 μm, or 15 μm. In some embodiments, the width of the constriction is about 3.0 μm, 3.1 μm, 3.2 μm, 3.3 μm, 3.4 μm, 3.5 μm, 3.6 μm, 3.7 μm, 3.8 μm, 3.9 μm, 4.0 μm, 4.1 μm, 4.2 μm, 4.3 μm, 4.4 μm, 4.5 μm, 4.6 μm, 4.7 μm, 4.8 μm, 4.9 μm, or 5.0 μm or less. In some embodiments, the width of the constriction is about 3.5 μm. In some embodiments, the width of the constriction is about 4 μm.

[0162] In some embodiments where a constriction is used to deliver a nucleic acid encoding a costimulatory molecule or cytokine to a TIL, the width of the constriction is about 3 μm to about 20 μm. In some embodiments, the width of the constriction is about 5 μm to about 15 μm. In some embodiments, the width of the constriction is about 8 μm to about 12 μm. In some embodiments, the width of the constriction is about 9 μm to about 11 μm. In some embodiments, the width of the constriction is about 9.5 μm to about 10.5 μm. In some embodiments, the width of the constriction is about 7 μm to about 9 μm. In some embodiments, the width of the constriction is about 8 μm to about 10 μm. In some embodiments, the width of the constriction is about 9 μm to about 11 μm. In some embodiments, the width of the constriction is about 10 μm to about 12 μm. In some embodiments, the width of the constriction is about 11 μm to about 13 μm. In some embodiments, the width of the constriction is about 5 μm to about 12 μm, about 6 μm to about 12 μm, about 8 μm to about 11 μm, about 9 μm to about 11 μm, about 9.5 μm to about 10.5 μm, about 8 μm to about 15 μm, about 10 μm to about 15 μm, or about 12 μm to about 15 μm. In some embodiments, the width of the constriction is about 9.7 μm to about 10.3 μm. In some embodiments, the width of the constriction is less than or equal to about any one of 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm, 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5 μm, 8 μm, 8.5 μm, 9 μm, 9.5 μm, 10 μm, 10.5 μm, 11 μm, 11.5 μm, 12 μm, 12.5 μm, 13 μm, 13.5 μm, 14 μm, 14.5 μm, 15 μm, 15.5 μm, 16 μm, 16.5 μm, 17 μm, 17.5 μm, or 18 μm. In some embodiments, the width of the constriction is less than or equal to about 9.0 μm, 9.1 μm, 9.2 μm, 9.3 μm, 9.4 μm, 9.5 μm, 9.6 μm, 9.7 μm, 9.8 μm, 9.9 μm, 10.0 μm, 10.1 μm, 10.2 μm, 10.3 μm, 10.4 μm, 10.5 μm, 10.6 μm, 10.7 μm, 10.8 μm, 10.9 μm, or 11.0 μm. In some embodiments, the width of the constriction is about 10.0 μm.

[0163] In some embodiments of the present invention, the composition comprises a plurality of TILs. In some embodiments, the width of the constriction is about 10% to about 99% of the average diameter of the subpopulation of TILs having the smallest diameter in the TIL population. In some embodiments, the width of the constriction is any one of about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 20% to about 60%, about 40% to about 60%, about 30% to about 45%, about 50% to about 99%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 60% to about 90%, about 60% to about 80%, or about 60% to about 70% of the average diameter of the subpopulation of TILs having the smallest diameter in the TIL population. In some embodiments, the width of the constriction is any one of about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 99% of the average diameter of the subpopulation of TILs having the smallest diameter in the TIL population. In some embodiments, the width of the constriction is any one of about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the average diameter of the subpopulation of TILs having the smallest diameter in the TIL population.

[0164] In some embodiments of the present invention, the composition comprises a plurality of TILs. In some embodiments, the width of the constriction is about 10% to about 99% of the average diameter of the subpopulation of TILs having the largest diameter in the TIL population. In some embodiments, the width of the constriction is any one of about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 20% to about 60%, about 40% to about 60%, about 30% to about 45%, about 15% to about 30%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 20% to about 30%, about 30% to about 70%, or about 30% to about 60% of the average diameter of the subpopulation of TILs having the largest diameter in the TIL population. In some embodiments, the width of the constriction is any one of about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 99% of the average diameter of the subpopulation of TILs having the largest diameter in the TIL population. In some embodiments, the width of the constriction is any one of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the average diameter of the subpopulation of TILs having the largest diameter in the TIL population.

[0165] Several parameters may affect the delivery of compounds to TILs to stimulate an immune response by the methods described herein. In some embodiments, the cell suspension is contacted with the compound before, during, or after passing through the constriction. The TILs may be passed through the constriction while suspended in a solution containing the compound to be delivered, or the compound may be added to the cell suspension after the TILs have passed through the constriction. In some embodiments, the compound to be delivered is coated on the constriction.

[0166] Examples of parameters that may affect the delivery of compounds to TILs include, but are not limited to, the dimensions of the constriction, the inlet angle of the constriction, the surface characteristics of the constriction (e.g., roughness, chemical modification, hydrophilicity, hydrophobicity, etc.), the operating flow rate (e.g., cell passage time through the constriction), the cell concentration, the concentration of the compound in the cell suspension, the buffer in the cell suspension, and the time for the TILs to recover or repair after passing through the constriction. These may affect the passage of the delivered compound to the TILs. Other parameters that affect the delivery of compounds to the TILs include the velocity of the TILs in the constriction, the shear rate in the constriction, the viscosity of the cell suspension, the velocity component perpendicular to the flow rate, and the time in the constriction. Additionally, multiple chips configuring serial and / or parallel flow paths may affect the delivery to the TILs. Multiple chips arranged in parallel may help to increase throughput. Such parameters can be designed to control the delivery of compounds. In some embodiments, the cell concentration is between about 10 and at least about 10 12 In some embodiments, the concentration of the delivery compound may range from about 10 ng / mL to about 1 g / mL, or any concentration or concentration range therebetween. In some embodiments, the concentration of the delivery compound may range from about 1 pM to at least about 2 M, or any concentration or concentration range therebetween.

[0167] In some embodiments, the concentration of the costimulatory molecule incubated with the TIL is about 0.01 μM to about 10 mM. For example, in some embodiments, the concentration of the costimulatory molecule incubated with the TIL is less than about 0.01 μM, less than about 0.1 μM, less than about 1 μM, less than about 10 μM, less than about 100 μM, less than about 1 mM, or less than about 10 mM. In some embodiments, the concentration of the costimulatory molecule incubated with the TIL is greater than about 10 mM. In some embodiments, the concentration of the costimulatory molecule incubated with the TIL is about 0.01 μM to about 0.1 μM, about 0.1 μM to about 1 μM, about 1 μM to about 10 μM, about 10 μM to about 100 μM, about 100 μM to about 1 mM, or 1 mM to about 10 mM. In some embodiments, the concentration of the costimulatory molecule incubated with the TIL is about 0.1 μM to about 1 mM. In some embodiments, the concentration of the costimulatory molecule incubated with the TILs is about 0.1 μM to about 10 μM. In some embodiments, the concentration of the costimulatory molecule incubated with the TILs is 1 μM.

[0168] In some embodiments, the concentration of nucleic acid encoding a cytokine and / or costimulatory molecule incubated with the TIL is about 1 nM to about 1 mM. In some embodiments, the concentration of nucleic acid encoding a cytokine and / or costimulatory molecule incubated with the TIL is less than about 0.1 nM, less than about 1 nM, less than about 0.01 μM, less than about 0.1 μM, less than about 1 μM, less than about 10 μM, less than about 100 μM, less than about 1 mM, or less than about 10 mM. In some embodiments, the concentration of nucleic acid encoding a cytokine and / or costimulatory molecule incubated with the TIL is greater than about 10 mM. In some embodiments, the concentration of the nucleic acid encoding the cytokine and / or costimulatory molecule incubated with the TIL is about 0.1 nM to about 1 nM, about 1 nM to about 10 nM, about 10 nM to about 100 nM, about 0.1 μM to about 1 μM, about 1 μM to about 10 μM, about 10 μM to about 100 μM, about 100 μM to about 1 mM, or 1 mM to about 10 mM. In some embodiments, the concentration of the nucleic acid encoding the cytokine and / or costimulatory molecule incubated with the TIL is about 10 nM to about 100 nM. In some embodiments, the concentration of the nucleic acid encoding the cytokine and / or costimulatory molecule incubated with the TIL is about 1 nM to about 10 nM. In some embodiments, the concentration of the nucleic acid encoding the cytokine and / or costimulatory molecule incubated with the TIL is about 50 nM. In some embodiments, the nucleic acid is an mRNA.

[0169] In some embodiments, the TILs comprise nucleic acids encoding cytokines and / or costimulatory molecules at a concentration of about 1 nM to about 1 mM. In some embodiments, the TILs comprise nucleic acids encoding cytokines and / or costimulatory molecules at a concentration of less than about 0.1 nM, less than about 1 nM, less than about 0.01 μM, less than about 0.1 μM, less than about 1 μM, less than about 10 μM, less than about 100 μM, less than about 1 mM, or less than about 10 mM. In some embodiments, the TILs comprise nucleic acids encoding cytokines and / or costimulatory molecules at a concentration of greater than about 10 mM. In some embodiments, the TIL comprises a nucleic acid encoding a cytokine and / or a costimulatory molecule at a concentration of about 0.1 nM to about 1 nM, about 1 nM to about 10 nM, about 10 nM to about 100 nM, about 0.1 μM to about 1 μM, about 1 μM to about 10 μM, about 10 μM to about 100 μM, about 100 μM to about 1 mM, or 1 mM to about 10 mM. In some embodiments, the TIL comprises a nucleic acid encoding a cytokine and / or a costimulatory molecule at a concentration of about 10 nM to about 100 nM. In some embodiments, the TIL comprises a nucleic acid encoding a cytokine and / or a costimulatory molecule at a concentration of about 1 nM to about 10 nM. In some embodiments, the TIL comprises a nucleic acid encoding a cytokine and / or a costimulatory molecule at a concentration of about 50 nM. In some embodiments, the nucleic acid is mRNA.

[0170] Systems and Kits In some aspects, the invention provides a system comprising one or more of a constriction, a TIL suspension, a nucleic acid encoding a cytokine and / or a costimulatory molecule for use in the methods disclosed herein. The system can include any of the embodiments described in the methods above. Such embodiments include a microfluidic channel providing a constriction for cell deformation or a surface with pores providing a constriction for cell deformation, a cell suspension, a cell perturbation, delivery parameters, compounds, and / or applications, etc. In some embodiments, the constriction for cell deformation is of a suitable size for delivery to the TIL. In some embodiments, delivery parameters such as operating flow rate, cell and compound concentration, velocity of cells within the constriction, and composition of the cell suspension (e.g., osmolality, salt concentration, serum content, cell concentration, pH, etc.) are optimized to enhance activity and / or proliferation capacity of the TIL.

[0171] Further provided are kits or articles of manufacture for use in treating an individual with cancer or an infectious disease. In some embodiments, the kits include TILs that contain intracellular nucleic acids encoding cytokines and / or costimulatory molecules. In some embodiments, the kits include one or more of a constriction, a TIL suspension, and a nucleic acid encoding a cytokine and / or costimulatory molecule for use in generating modified TILs for use in treating an individual with cancer or an infectious disease. In some embodiments, the kits include the compositions described herein (e.g., a microfluidic channel or surface comprising pores, a cell suspension, and / or a compound) in suitable packaging. Suitable packaging materials are known in the art and include, for example, vials (such as sealed vials), containers, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar bags or sealed plastic bags), and the like. These articles may be further sterilized and / or sealed.

[0172] The invention further provides kits containing components of the methods described herein. The kits may further include instructions for carrying out the methods to treat an individual in need of treatment and / or instructions for introducing cytokines and / or costimulatory molecules into TILs. The kits described herein may further include other materials, such as other buffers, diluents, filters, needles, syringes, and inserts containing instructions for carrying out any of the methods described herein (e.g., instructions for treating an individual in need of treatment or instructions for modifying TILs to contain cytokines and / or costimulatory molecules intracellularly).

[0173] Exemplary embodiments Embodiment 1. A method of modulating the activity and / or proliferative capacity of tumor infiltrating lymphocytes (TILs), comprising modifying said TILs to increase expression of one or more costimulatory molecules and / or one or more cytokines.

[0174] Embodiment 2. A method of modulating the activity and / or proliferative capacity of tumor infiltrating lymphocytes (TILs), comprising modifying said TILs to increase expression of one or more costimulatory molecules.

[0175] Embodiment 3. The method of embodiment 1 or 2, wherein said costimulatory molecule is B7-H2 (ICOSL), B7-1 (CD80), B7-2 (CD86), CD70, LIGHT, HVEM, CD40, 4-1BBL, OX40L, TL1A, GITRL, CD30L, TIM4, SLAM, CD48, CD58, CD155, or CD112.

[0176] Embodiment 4 The method of embodiment 1 or 2, wherein said costimulatory molecule is CD86.

[0177] Embodiment 5. A method of modulating the phenotype and / or proliferative capacity of TILs, wherein the TILs are modified to have increased expression of one or more cytokines.

[0178] Embodiment 6. The method of any one of embodiments 1 and 3-5, wherein the TIL is modified to comprise a membrane-bound chimeric cytokine.

[0179] Embodiment 7. The method of embodiment 6, wherein said membrane-bound chimeric cytokine is a fusion protein comprising a cytokine and a transmembrane domain.

[0180] Embodiment 8 The method of embodiment 7, wherein said cytokine is linked to said transmembrane domain by a peptide linker.

[0181] Embodiment 9. The method of embodiment 8, wherein the peptide linker is (G4S)3 (SEQ ID NO:3) or (EAAAK)3 (SEQ ID NO:4).

[0182] Embodiment 10. The method of any one of embodiments 1 and 3-9, wherein the cytokine is a type I cytokine.

[0183] Embodiment 11. The method of any one of embodiments 1 and 3-10, wherein the cytokine is IL-15, IL-12, IL-2, IFNα, IFNβ, or IL-21 or a functional variant thereof.

[0184] Embodiment 12. The method of embodiment 11, wherein said cytokine is IL-2 or a functional variant thereof and / or IL-12 or a functional variant thereof.

[0185] Embodiment 13. The method of any one of embodiments 7 to 12, wherein the membrane-bound chimeric cytokine comprises an amino acid sequence of SEQ ID NO: 7 to 10.

[0186] Embodiment 14. The modified TILs have increased expression of one or more cytokines and / or one or more costimulatory molecules, and the TILs are a) passing a cell suspension containing input TILs through a cell deformation constriction to perturb the input TILs sufficiently to allow passage of one or more nucleic acids encoding one or more cytokines and / or one or more nucleic acids encoding one or more costimulatory molecules to form perturbed input TILs, wherein the diameter of the constriction is a function of the diameter of the input TILs in the suspension; b) incubating the perturbed input TILs with the one or more nucleic acids encoding one or more cytokines and / or the one or more nucleic acids encoding one or more costimulatory molecules, and allowing the one or more nucleic acids encoding one or more cytokines and / or the one or more nucleic acids encoding one or more costimulatory molecules to enter the perturbed input TILs and express the nucleic acids, thereby generating the modified TILs comprising the one or more cytokines and / or the one or more costimulatory molecules.

[0187] Embodiment 15. The modified TILs have increased expression of one or more cytokines and / or one or more costimulatory molecules, and the TILs are a) passing a cell suspension containing input TILs through a cell deformation constriction to perturb the input TILs sufficiently to allow passage of one or more nucleic acids encoding the one or more cytokines and / or the one or more costimulatory molecules to form perturbed input TILs, wherein the diameter of the constriction is a function of the diameter of the input TILs in the suspension; and b) incubating the perturbed input TILs with the one or more nucleic acids encoding the one or more cytokines and / or the one or more costimulatory molecules to allow the perturbed input TILs to enter and express the nucleic acids, thereby generating the modified TILs comprising the one or more cytokines and / or the one or more costimulatory molecules.

[0188] Embodiment 16. (a) incubating the TILs with the nucleic acid encoding the membrane-bound chimeric cytokine before, during, and / or after passing the cell suspension through the cell deformation constriction; (b) incubating the TILs with the nucleic acid encoding the membrane-bound chimeric cytokine and the costimulatory molecule before, during, and / or after passing the cell suspension through the cell deformation constriction; (c) incubating the TILs with the nucleic acid encoding the membrane-bound chimeric cytokine and the nucleic acid encoding the costimulatory molecule before, during, and / or after passing the cell suspension through the cell deformation constriction; or (d) incubating the TILs with the nucleic acid encoding the membrane-bound chimeric cytokine and the nucleic acid encoding the one or more costimulatory molecules before, during, and / or after passing the cell suspension through the cell deformation constriction.

[0189] EMBODIMENT 17. (a) incubating the TILs with the nucleic acid encoding the membrane-bound chimeric cytokine prior to passing the cell suspension through the cell deformation constriction; (b) incubating the TILs with the nucleic acid encoding the membrane-bound chimeric cytokine and the costimulatory molecule prior to passing the cell suspension through the cell deformation constriction; (c) incubating the TILs with the nucleic acid encoding the membrane-bound chimeric cytokine and the nucleic acid encoding the costimulatory molecule prior to passing the cell suspension through the cell deformation constriction; or (d) incubating the TILs with the nucleic acid encoding the membrane-bound chimeric cytokine and the nucleic acid encoding the one or more costimulatory molecules prior to passing the cell suspension through the cell deformation constriction.

[0190] Embodiment 18 The method of any one of embodiments 14 to 17, wherein one or more of the nucleic acids is mRNA.

[0191] Embodiment 19. The method of any one of embodiments 1 to 18, wherein the modified TILs have increased expression of one or more of T-bet, EOMES, TCF1, and CD62L compared to corresponding unmodified TILs.

[0192] Embodiment 20. The method of any one of embodiments 1 to 18, wherein the modified TILs have increased expression of one or more of T-bet, EOMES, TCF1, and CD62L compared to corresponding unmodified TILs when cultured in the absence of exogenous cytokines.

[0193] Embodiment 21. The method of embodiment 19 or 20, wherein expression of one or more of T-bet, EOMES, TCF1, and CD62L in the modified TILs is increased by more than about 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 3-fold, 4-fold, 5-fold, 8-fold, or 10-fold compared to corresponding unmodified TILs.

[0194] Embodiment 21A. The method of any one of embodiments 1-21, wherein the modified TILs have increased expression of Granzyme B compared to corresponding unmodified TILs.

[0195] Embodiment 21B. The method of any one of embodiments 1 to 21, wherein the modified TILs have increased expression of granzyme B compared to corresponding unmodified TILs when cultured in the absence of exogenous cytokines.

[0196] Embodiment 21C. The method of any one of embodiments 1-21, wherein the modified TILs have increased expression of granzyme B compared to corresponding unmodified TILs when co-cultured with tumor cells in the absence of exogenous cytokines.

[0197] Embodiment 21D. The method of embodiment 21A, 21B or 21C, wherein expression of Granzyme B in the modified TILs is increased by about 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 3-fold, 4-fold, 5-fold, 8-fold, or 10-fold or more compared to the corresponding unmodified TILs.

[0198] Embodiment 22 The method of any one of embodiments 1 to 21, wherein the modified TILs exhibit greater proliferation than corresponding unmodified TILs.

[0199] Embodiment 23. The method of any one of embodiments 1 to 21, wherein the modified TILs exhibit greater proliferation than corresponding unmodified TILs when cultured in medium that does not contain exogenous cytokines.

[0200] Embodiment 23A. The method of any one of embodiments 1-21, wherein the modified TILs exhibit greater proliferation than corresponding unmodified TILs when co-cultured with tumor cells in the absence of exogenous cytokines.

[0201] Embodiment 24 The method of any one of embodiments 20-23A, wherein the exogenous cytokine is IL-2 and / or IL-12, optionally wherein the exogenous cytokine is IL-2.

[0202] Embodiment 25. A composition comprising modified TILs, wherein the TILs have been modified to increase expression of one or more costimulatory molecules and / or one or more cytokines.

[0203] Embodiment 26. A composition comprising modified TILs, wherein the TILs have been modified to have increased expression of one or more costimulatory molecules.

[0204] Embodiment 27. The composition of embodiment 25 or 26, wherein said costimulatory molecule is B7-H2 (ICOSL), B7-1 (CD80), B7-2 (CD86), CD70, LIGHT, HVEM, CD40, 4-1BBL, OX40L, TL1A, GITRL, CD30L, TIM4, SLAM, CD48, CD58, CD155, or CD112.

[0205] Embodiment 28 The composition of embodiment 27, wherein the costimulatory molecule is CD86.

[0206] Embodiment 29. A composition comprising modified TILs, wherein the TILs have been modified to increase expression of one or more cytokines.

[0207] Embodiment 30. The composition of any one of embodiments 25 and 27-29, wherein the TILs are modified to contain a membrane-bound chimeric cytokine.

[0208] Embodiment 31. The composition of embodiment 30, wherein the membrane-bound chimeric cytokine is a fusion protein comprising a cytokine and a transmembrane domain.

[0209] Embodiment 32 The composition of embodiment 31, wherein the cytokine is linked to the transmembrane domain by a peptide linker.

[0210] Embodiment 33. The composition of embodiment 32, wherein the peptide linker is (G4S)3 (SEQ ID NO:3) or (EAAAK)3 (SEQ ID NO:4).

[0211] Embodiment 34. The composition of any one of embodiments 25 and 27-33, wherein the cytokine is a type I cytokine.

[0212] Embodiment 35. The composition of any one of embodiments 25 and 27-34, wherein the cytokine is IL-15, IL-12, IL-2, IFNα, IFNβ, or IL-21 or a functional variant thereof.

[0213] Embodiment 36 The method of embodiment 35, wherein the cytokine is IL-2 or a functional variant thereof and / or IL-12 or a functional variant thereof.

[0214] Embodiment 37. The composition of any one of embodiments 30 to 36, wherein the membrane-bound chimeric cytokine comprises an amino acid sequence of SEQ ID NO: 7 to 10.

[0215] Embodiment 38. The modified TILs have increased expression of one or more cytokines and / or one or more costimulatory molecules, and the modified TILs are a) passing a cell suspension containing input TILs through a cell deformation constriction to perturb the input TILs sufficiently to allow passage of one or more nucleic acids encoding one or more cytokines and / or one or more nucleic acids encoding one or more costimulatory molecules to form perturbed input TILs, wherein the diameter of the constriction is a function of the diameter of the input TILs in the suspension; b) incubating the perturbed input TILs with the one or more nucleic acids encoding one or more cytokines and / or the one or more nucleic acids encoding one or more costimulatory molecules, and allowing the one or more nucleic acids encoding one or more cytokines and / or the one or more nucleic acids encoding one or more costimulatory molecules to enter the perturbed input TILs and express the nucleic acids, thereby generating the modified TILs comprising the one or more cytokines and / or the one or more costimulatory molecules.

[0216]

[0036] Embodiment 39. The TILs have increased expression of one or more cytokines and / or one or more costimulatory molecules, and the TILs are a) passing a cell suspension containing input TILs through a cell deformation constriction to perturb the input TILs sufficiently to allow passage of the one or more nucleic acids encoding one or more cytokines and / or one or more costimulatory molecules to form perturbed input TILs, wherein the diameter of the constriction is a function of the diameter of the input TILs in the suspension; and b) incubating the perturbed input TILs with the one or more nucleic acids encoding one or more cytokines and / or one or more costimulatory molecules to enter the perturbed input TILs and express the nucleic acids, thereby generating the modified TILs comprising the one or more cytokines and / or the one or more costimulatory molecules.

[0217] Embodiment 40. The method for preparing the modified TILs comprises: (a) incubating the TILs with the nucleic acid encoding the membrane-bound chimeric cytokine before, during, and / or after passing the cell suspension through the cell deformation constriction; (b) incubating the TILs with the nucleic acid encoding the membrane-bound chimeric cytokine and the costimulatory molecule before, during, and / or after passing the cell suspension through the cell deformation constriction; (c) incubating the TILs with the nucleic acid encoding the membrane-bound chimeric cytokine and the nucleic acid encoding the costimulatory molecule before, during, and / or after passing the cell suspension through the cell deformation constriction; or (d) incubating the TILs with the nucleic acid encoding the membrane-bound chimeric cytokine and the nucleic acid encoding the one or more costimulatory molecules before, during, and / or after passing the cell suspension through the cell deformation constriction.

[0218] Embodiment 41. The method for preparing the modified TILs comprises: (a) incubating the TILs with the nucleic acid encoding the membrane-bound chimeric cytokine prior to passing the cell suspension through the cell deformation constriction; (b) incubating the TILs with the nucleic acid encoding the membrane-bound chimeric cytokine and the costimulatory molecule prior to passing the cell suspension through the cell deformation constriction; (c) incubating the TILs with the nucleic acid encoding the membrane-bound chimeric cytokine and the nucleic acid encoding the costimulatory molecule prior to passing the cell suspension through the cell deformation constriction; or (d) incubating the TILs with the nucleic acid encoding the membrane-bound chimeric cytokine and the nucleic acid encoding the one or more costimulatory molecules prior to passing the cell suspension through the cell deformation constriction.

[0219] Embodiment 42 The composition of any one of embodiments 38 to 41, wherein one or more of the nucleic acids is mRNA.

[0220] Embodiment 43. The composition of any one of embodiments 25 to 42, wherein the modified TILs have increased expression of one or more of T-bet, EOMES, TCF1, and CD62L compared to corresponding unmodified TILs.

[0221] Embodiment 44. The composition of any one of embodiments 25 to 43, wherein the modified TILs have increased expression of one or more of T-bet, EOMES, TCF1, and CD62L compared to corresponding unmodified TILs when cultured in the absence of exogenous cytokines.

[0222] Embodiment 45. The composition of embodiment 43 or 44, wherein expression of one or more of T-bet, EOMES, TCF1, and CD62L in the modified TILs is increased by more than about 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 3-fold, 4-fold, 5-fold, 8-fold, or 10-fold compared to the corresponding unmodified TILs.

[0223] Embodiment 45A. The composition of any one of embodiments 25-45, wherein the modified TILs have increased expression of Granzyme B compared to corresponding unmodified TILs.

[0224] Embodiment 45B. The composition of any one of embodiments 25 to 45, wherein the modified TILs have increased expression of granzyme B compared to corresponding unmodified TILs when cultured in the absence of exogenous cytokines.

[0225] Embodiment 45C. The composition of any one of embodiments 25 to 45, wherein the modified TILs have increased expression of granzyme B compared to corresponding unmodified TILs when co-cultured with tumor cells in the absence of exogenous cytokines.

[0226] Embodiment 45D. The composition of embodiment 45A or 45B or 45C, wherein expression of granzyme B in the modified TILs is increased by about 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 3-fold, 4-fold, 5-fold, 8-fold, or 10-fold or more compared to the corresponding unmodified TILs.

[0227] Embodiment 46 The composition of any one of embodiments 25 to 45, wherein the modified TILs exhibit greater proliferation than corresponding unmodified TILs.

[0228] Embodiment 47. The composition of any one of embodiments 25 to 45, wherein the modified TILs exhibit greater proliferation than corresponding unmodified TILs when cultured in a medium that does not contain exogenous cytokines.

[0229] Embodiment 47A. The composition of any one of embodiments 25 to 45, wherein the modified TILs exhibit greater proliferation than corresponding unmodified TILs when co-cultured with tumor cells in the absence of exogenous cytokines.

[0230] Embodiment 48. The composition of any one of embodiments 44 to 47A, wherein the exogenous cytokine is IL-2 and / or IL-12, optionally wherein the exogenous cytokine is IL-2.

[0231] Embodiment 49. A method for modulating an immune response in an individual, comprising administering to the individual the modified TILs, wherein the modified TILs have been prepared according to the method of any one of embodiments 1 to 24.

[0232] Embodiment 50. A method of modulating an immune response in an individual comprising administering the composition of any one of embodiments 25 to 48.

[0233] Embodiment 51. A method for treating cancer, an infectious disease, or a virus-related disease in an individual, comprising administering to the individual the modified TILs, wherein the modified TILs have been prepared according to any one of the methods of embodiments 1 to 24.

[0234] Embodiment 52. A method of treating cancer, an infectious disease, or a virus-related disease in an individual comprising administering the composition of any one of embodiments 25 to 48.

[0235] Embodiment 53. The method of any one of embodiments 49 to 52, comprising administering the modified TILs multiple times or administering the composition multiple times.

[0236] Embodiment 54 The method of any one of embodiments 49 to 53, wherein the modified TILs are administered intravenously or intratumorally.

[0237] Embodiment 55 The method of any one of embodiments 49-54, wherein the individual is a human.

[0238] Embodiment 56 The method of any one of embodiments 49-55, wherein the modified TILs are administered prior to, concurrently with, or following administration of another therapeutic agent.

[0239] Embodiment 57. A pharmaceutical composition for stimulating an immune response in an individual, comprising an effective amount of the composition of any one of embodiments 25 to 48.

[0240] Embodiment 58. A pharmaceutical composition for use as a medicament, comprising an effective amount of the composition of any one of embodiments 25 to 48.

[0241] Embodiment 59. A pharmaceutical composition for treating cancer, an infectious disease, or a virus-related disease in an individual, comprising an effective amount of the composition of any one of embodiments 25 to 48.

[0242] Embodiment 60. The pharmaceutical composition of embodiment 58 or 59, wherein said composition comprising modified TILs is administered prior to, simultaneously with, or after administration of another therapeutic agent.

[0243] Embodiment 61. The method of any one of embodiments 1-24 and 49-56, or the composition of any one of embodiments 25-48 and 57-60, wherein the width of the constriction is about 10% to about 99% of the average diameter of the introduced TILs.

[0244] Embodiment 62. The method of any one of embodiments 1 to 24 and 49 to 56, or the composition of any one of embodiments 25 to 48, wherein the width of the constriction is from about 3.5 μm to about 4.2 μm, or from about 3.5 μm to about 4.8 μm, or from about 3.5 μm to about 6 μm, or from about 5 μm to about 12 μm, or from about 12 μm to about 15 μm, or from about 6 μm to about 12 μm, or from about 8 μm to about 11 μm, or from about 9 μm to about 11 μm.

[0245] Embodiment 63. The method of any one of embodiments 1-24 and 49-56, or the composition of any one of embodiments 25-48 and 57-60, wherein the width of the constriction is from about 3 μm to about 5 μm.

[0246] Embodiment 64. The method of any one of embodiments 1-24 and 49-56, or the composition of any one of embodiments 25-48 and 57-60, wherein the width of the constriction is about 4 μm.

[0247] Embodiment 65. The method of any one of embodiments 1 to 24 and 49 to 56, or the composition of any one of embodiments 25 to 48 and 57 to 60, wherein the cell suspension containing a plurality of input TILs is passed through a plurality of constrictions, wherein the plurality of constrictions are arranged in series and / or parallel.

[0248] Embodiment 66. A kit for use in any one of the methods of embodiments 1 to 24, 49 to 56, and 61 to 65.

[0249] Embodiment 67. A kit comprising the composition of any one of embodiments 25 to 48 and 57 to 65.

[0250] Embodiment 68 The kit of embodiment 66 or 67, further comprising one or more of a buffer, a diluent, a filter, a needle, a syringe, or a package insert containing instructions for administering said composition to an individual.

[0251] Embodiment 69. A method for producing a TIL comprising a membrane-bound chimeric cytokine, the method comprising introducing a nucleic acid encoding said membrane-bound chimeric cytokine into a TIL.

[0252] Embodiment 70. The TILs comprising the membrane-bound chimeric cytokine are a) passing a cell suspension containing input TILs through a cell deformation constriction to perturb the input TILs sufficiently to allow passage of a nucleic acid encoding said membrane-bound chimeric cytokine to form perturbed input TILs, wherein the diameter of the constriction is a function of the diameter of the input TILs in the suspension; The method of embodiment 69, wherein the TILs are prepared by: b) incubating the perturbed input TILs with the nucleic acid encoding the membrane-bound chimeric cytokine, thereby allowing the nucleic acid to enter the perturbed input TILs and express the nucleic acid encoding the membrane-bound chimeric cytokine, thereby generating TILs comprising the membrane-bound chimeric cytokine.

[0253] Embodiment 71. The method of embodiment 70, comprising incubating the TILs with the nucleic acid encoding the membrane-bound chimeric cytokine before, during, and / or after passing the cell suspension through the cell deformation constriction.

[0254] Embodiment 72. The method of embodiment 70, comprising incubating the TILs with the nucleic acid encoding the membrane-bound chimeric cytokine prior to passing the cell suspension through the cell deformation constriction.

[0255] Embodiment 73. The method of any one of embodiments 69 to 72, wherein the nucleic acid encoding the membrane-bound chimeric cytokine is an mRNA encoding the membrane-bound chimeric cytokine.

[0256] Embodiment 74. The method of any one of embodiments 70 to 73, wherein the width of the constriction is about 10% to about 99% of the average diameter of the introduced TILs.

[0257] Embodiment 75. The method of any one of embodiments 70 to 74, wherein the width of the constriction is from about 5 μm to about 12 μm, or from about 6 μm to about 12 μm, or from about 8 μm to about 11 μm, or from about 9 μm to about 11 μm, or from about 12 μm to about 15 μm.

[0258] Embodiment 76. The method of any one of embodiments 70 to 75, wherein the width of the constriction is about 10 μm.

[0259] Embodiment 77. The method of any one of embodiments 70 to 76, wherein the width of the constriction is about 8 μm.

[0260] Embodiment 78. The method of any one of embodiments 70 to 77, wherein the cell suspension containing the plurality of input TILs is passed through a plurality of constrictions, wherein the plurality of constrictions are arranged in series and / or in parallel.

[0261] Embodiment 79. Use of a pharmaceutical composition in the manufacture of a medicament for stimulating an immune response in an individual, wherein the pharmaceutical composition comprises an effective amount of the composition of any one of embodiments 25 to 48.

[0262] Embodiment 80. Use of a pharmaceutical composition in the manufacture of a medicament for treating cancer, an infectious disease, or a viral-related disease in an individual, wherein the pharmaceutical composition comprises an effective amount of the composition of any one of embodiments 25 to 48.

[0263] Embodiment 81. The use of embodiment 79 or 80, wherein the pharmaceutical composition is formulated for multiple administration.

[0264] Embodiment 82. The use of any one of embodiments 79 to 81, wherein the pharmaceutical composition is administered intravenously or intratumorally.

[0265] Embodiment 83 The use of any one of embodiments 79 to 82, wherein the individual is a human.

[0266] Embodiment 84. The use of any one of embodiments 79 to 83, wherein the pharmaceutical composition is formulated to be administered prior to, concurrently with, or following administration of another therapeutic agent. EXAMPLES

[0267] As will be appreciated by those skilled in the art, several embodiments are possible within the scope and spirit of the present invention. The present invention will now be described in more detail with reference to the following non-limiting examples. The following examples further illustrate the present invention but, of course, should not be construed as in any way limiting the scope of the invention.

[0268] Example 1: Tumor infiltrating lymphocytes (TILs) express membrane-bound cytokines for 48 hours following constriction-mediated delivery of mRNA encoding the membrane-bound cytokine This example shows that following constriction-mediated delivery of membrane-bound IL-2 (e.g., mbIL-2) and / or membrane-bound IL-12 (e.g., mbIL-12) mRNA, mbIL-2 and / or mbIL-12 are expressed in tumor-infiltrating lymphocytes (TILs) for 48 hours.

[0269] method TILs were thawed and cultured in medium containing high concentration IL-2 (3000 IU / mL) for 24 hours. After culture, mRNA encoding mbIL-2 (0.25 mg / mL), mbIL-12 (0.25 mg / mL), or mbIL-2 and mbIL-12 (0.25 mg / mL each, 0.5 mg / mL total) was delivered to the TILs by constriction delivery ("squeezing"). Specifically, in the presence of the above mRNAs, TILs were squeezed at 30 psi using an ST-10-040-70 tip (10 um long, 4 um wide, 70 um deep). As a control for downstream analysis, constriction delivery was performed similarly without input material and with GFP mRNA. TILs containing the squeezed mRNA were resuspended in culture medium containing high concentration IL-2 (3000 IU / mL). Fluorescence-activated cell sorting (FACS) was used to measure the expression of membrane-bound cytokines at 4, 24, and 48 hours after resuspension. In particular, cell viability, the percentage of cells expressing GFP, mbIL-2, and / or mbIL-12, and the mean fluorescence intensity (e.g., MFI) of GFP expression, mbIL-2 expression, and mbIL-12 expression were assessed (Figures 1A-1D and Figures 2A-2C). Table 1 outlines the FACS protocol used to evaluate TILs. [Table 1]

[0270] result As shown in Figure 1A, the viability was consistently 80-90% at all measurement time points after compression (4 h, day 1, and day 2). The viability of TILs increases over time in high IL-2 culture medium. This is evident by comparing the viability of TILs after 4 h and 2 days.

[0271] As shown by the GFP mRNA positive control, mRNA was effectively delivered to and expressed by TILs using the constriction delivery method (Figure 1B and Figure 2A). When mRNA was delivered by the constriction delivery method, TILs successfully expressed mbIL-2 and mbIL-12, and also successfully expressed both mbIL-2 and mbIL-12 simultaneously, as revealed by both the percentage of cytokine-expressing cells and MFI expression (see, e.g., Figures 1C-1D and Figures 2B-2C).

[0272] Example 2: TILs express membrane-bound cytokines over a 7-day period when mRNA encoding the membrane-bound cytokine is delivered via constriction and cultured with various IL-2 concentrations In this example, we demonstrate that delivery of mRNA encoding membrane-bound IL-2 (e.g., mbIL-2) and membrane-bound IL-12 (e.g., mbIL-12) through a constriction results in expression of mbIL-2 or mbIL-12 over a period of 7 days when cultured in the presence of various IL-2 concentrations.

[0273] method TILs were thawed and cultured in medium containing high concentration IL-2 (3000 IU / mL) for 24 hours. After culture, mRNA encoding mbIL-2 (0.25 mg / mL), mbIL-12 (0.25 mg / mL), or both mbIL-2 and mbIL-12 (0.25 mg / mL) was delivered to the TILs by constriction delivery. In the presence of the mRNA, the TILs were compressed at 30 psi using an ST-10-040-70 tip (10 um long, 4 um wide, 70 um deep). TILs containing the compressed mRNA were resuspended in culture medium containing IL-2 at concentrations of 3000 IU / mL, 300 IU / mL, 30 IU / mL, and 0 IU / mL and cultured for a total of 7 days. Cytokine expression was measured 1, 3, and 7 days after resuspension using fluorescence-activated cell sorting (FACS). In particular, cell viability, proliferation, percentage of cells expressing cytokines, and mean fluorescence intensity (e.g., MFI) of expression of mbIL-2 and mbIL-12 were assessed. Viability of TILs after mRNA compression delivery and incubation in the presence of IL-2 is shown in Figures 3A-3D. Proliferation of viable TILs after mRNA compression delivery and incubation in the presence of IL-2 is shown in Figures 4A-4D. Expression of CD39 in TILs after mRNA compression delivery and incubation in the presence of IL-2 is shown in Figures 5A-5D. Co-expression of CD39 and CD69 in TILs after mRNA compression delivery and incubation in the presence of IL-2 is shown in Figures 6A-6D. Expression of CD62L in TILs after mRNA compression delivery and incubation in the presence of IL-2 is shown in Figures 7A-7D. Expression of CD69 in TILs after mRNA compression delivery and incubation in the presence of IL-2 is shown in Figures 8A-8H. Figures 9A-9D, 10A-10D, and 11A-11D show the percentage of TILs expressing mbIL-2, or mbIL-12, or both mbIL-2 and mbIL-12, respectively. The percentage of TILs expressing T-bet after delivery by mRNA compression and incubation in the presence of IL-2 is shown in Figures 12A-12D. The percentage of TILs expressing Eomes and TCF-1 after delivery by mRNA compression and incubation in the presence of IL-2 is shown in Figures 13A-13D.Each assay was performed for each concentration of IL-2 in the culture medium.

[0274] Tables 2 and 3 outline the FACS protocols used to assess the surface and nuclear components of TILs. [Table 2] [Table 3]

[0275] result As shown in Figures 3A-3C, viability was consistently greater than 85% at all time points for all cells tested in 3000 IU / mL, 300 IU / mL, and 30 IU / mL IL-2 culture medium. Viability of TILs in medium without IL-2 remained greater than 85% by day 3 (Figure 3D). Proliferation was increased for all TILs in medium with 3000 IU / mL, 300 IU / mL, and 30 IU / mL IL-2 (Figures 4A-4C), and proliferation of TILs cultured in medium without IL-2 was similar to that of corresponding TILs cultured in 3000 IU / mL IL-2 medium by day 3.

[0276] When mRNA was delivered by compression, TILs successfully expressed mbIL-2, mbIL-12, and both mbIL-2 and mbIL-12 simultaneously, as evidenced by the percentage of cells expressing each cytokine (see, e.g., Figures 9A-9D, 10A-10D, and 11A-11D). As shown in Figures 9A-9D, mbIL-2 expression decreased at each IL-2 media concentration over the two days following compression delivery. mbIL-2 expression was highest on day 1 and negligible by day 3 following mRNA delivery (Figures 9A-9D). mbIL-12 expression similarly decreased at each IL-2 media concentration over the two days following compression delivery during incubation following mRNA compression delivery (Figures 10A-10D). As shown in Figures 10A-10D, expression of mbIL-12 was highest on day 1 after compression delivery of mRNA and gradually decreased over time. Similarly, co-expression of mb-IL2 and mbIL-12 decreased at each IL-2 medium concentration during incubation after compression delivery of the respective mRNAs and at 2 days after compression delivery (Figures 11A-11D).

[0277] The phenotype of TILs was further evaluated after mRNA was delivered by compression and then incubated with IL-2. As shown in Figures 5A-5D, CD39 expression was similar across all TILs regardless of IL-2 culture condition. CD62L (central memory T cells) expression was highest on day 1 when a combination of mbIL-2 and mbIL-12 mRNA was delivered followed by incubation with IL-2 in culture medium (Figures 7A-7D). CD69 expression was similar on days 1 and 7 in all TILs cultured in IL-2 medium, but decreased by day 3 (Figures 8A-8H). As shown in Figures 12A-12D, TILs expressing mbIL-2, mbIL-12, or both mbIL-2 and mbIL-12 showed increased expression of T-bet with increasing concentrations of IL-2 in the culture medium. TILs showed higher expression of Eomes and TCF-1 on day 3 after mRNA delivery by compression (Figures 13A-13D).

[0278] Example 3: TILs express membrane-bound cytokines over a 3-day period when mRNA encoding mbIL-2, mbIL-12, and CD86 is delivered via constriction and cultured at various IL-2 concentrations In this example, we show that TILs delivered by compression with mRNA encoding mbIL-2 and mbIL-12 or CD86 express mbIL-2 and mbIL-12 and demonstrate their effect on the phenotype of the TILs.

[0279] method TILs were thawed and cultured for 24 hours in medium containing high concentration IL-2 (3000 IU / mL).After culture, mRNAs encoding mbIL-2 and mbIL-12 (0.1mg / mL), mbIL-2 and mbIL-12 (0.25mg / mL), mbIL-2 and mbIL-12 (0.5mg / mL), CD86 and mbIL-2 (0.25mg / mL), CD86 and mbIL-12 (0.25mg / mL), or CD86, mbIL-2, and mbIL-12 (0.25mg / mL each) were delivered to the TILs by compression at 30 psi using a ST-10-040-70 tip (length 10um, width 4um, depth 70um). TILs containing mRNA delivered through the constriction were resuspended in culture medium with or without IL-2 at 3000 IU / mL and cultured for 3 days. Fluorescence-activated cell sorting (FACS) was used to measure cytokine expression 1 and 3 days after resuspension. Viability of TILs after mRNA compression delivery and incubation in the presence of IL-2 is shown in Figures 14A and 14B. Proliferation of viable TILs after mRNA compression delivery and incubation in the presence of IL-2 is shown in Figures 15A and 15B. Expression of CD39 on TILs after mRNA compression delivery and incubation in the presence of IL-2 is shown in Figures 16A and 16B. Expression of CD62L on TILs after mRNA compression delivery and incubation in the presence of IL-2 is shown in Figures 17A and 17B. Figures 18A and 18B show the mean fluorescence intensity of CD86. The percentage of TILs expressing mbIL-2 after delivery by mRNA compression and incubation in the presence of IL-2 is shown in Figures 19A-19D. The percentage of TILs expressing mbIL-12 after delivery by mRNA compression and incubation in the presence of IL-2 is shown in Figures 20A-20D. The percentage of TILs expressing T-bet after delivery by mRNA compression and incubation in the presence of IL-2 is shown in Figures 21A and 21B. The percentage of TILs expressing Eomes after delivery by mRNA compression and incubation in the presence of IL-2 is shown in Figures 22A and 22B.The percentage of TILs expressing Eomes+TCF-1 after mRNA compression delivery and incubation in the presence of IL-2 is shown in Figures 23A and 23B.

[0280] Tables 4 and 5 outline the FACS protocols used to assess the surface and nuclear components of TILs. [Table 4] [Table 5]

[0281] result As shown in Figures 14A-14B, for all cells tested at 3000 IU / mL, viability was consistently greater than about 85% in all replicates (e.g., days 1 and 3). Viability of TILs in medium without IL-2 remained greater than about 85% by day 3 (Figure 14B). TILs containing mRNA encoding mbIL-2 and mbIL-12 or CD86 show high viability in 0 IU / mL IL-2 medium. TILs delivered by compression with mRNA encoding mbIL-2 and mbIL-12 and / or mRNA for CD86 showed increased proliferation (Figures 15A-15B).

[0282] When mRNA was delivered by compression, TILs successfully expressed mbIL-2, mbIL-12, and DC86, as evidenced by both the percentage of cells expressing the cytokines and MFI expression (see, e.g., Figures 19A-19D and 20A-20D). As shown in Figures 19A-19D and 20A-20D, a higher concentration of mRNA (0.5 mg / mL) encoding mbIL-2 and / or mbIL-12 treated with compression resulted in a similar percentage of cells expressing each cytokine compared to 0.25 mg / mL mRNA encoding mbIL-2 and / or mbIL-12 treated with compression, and showed an overall increase in expression of mbIL-2 and / or mbIL-12, respectively, over a 3-day period.

[0283] The phenotype of the TILs transduced with mRNA by compression treatment was further evaluated. As shown in Figures 16A-16B, CD39 was similar for all TILs regardless of IL-2 culture conditions. Expression of CD62L (central memory T cells) was enhanced in the presence of mbIL-12 mRNA (Figures 17A-17B). CD86 MFI was similarly enhanced by the presence of mbIL-12 mRNA in TILs (Figures 18A-18B). High IL-2 concentrations in the culture medium in mRNA-transduced TILs enhanced the expression of Eomes+ and TCF-1+ (Figures 23A-23B).

[0284] Example 4: Tumor infiltrating lymphocytes (TILs) express membrane-bound cytokines for up to 72 hours following constriction-mediated delivery of mRNA encoding the membrane-bound cytokine, without the addition of exogenous rhIL2 In this example, we demonstrate that following constriction-mediated delivery of membrane-bound IL-2 (e.g., mbIL-2) and membrane-bound IL-12 (e.g., mbIL-12) mRNA, mbIL-2 and mbIL-12 are expressed in TILs for 72 hours.

[0285] method TILs were thawed and cultured for 24 hours in medium containing a concentration of IL-2 (3000 IU / mL). After culture, in two groups, (1) mRNAs encoding mbIL-2 and mbIL-12 (total 0.25 mg / mL) and (2) mRNAs encoding mbIL-2 and mbIL-12 (total 0.50 mg / mL) were delivered to the TILs by constriction delivery ("squeezing"), respectively. In the presence of the above mRNAs, the TILs were squeezed at 30 psi using an ST-10-04-70 tip (length 10 μm, width 4 μm, depth 70 μm). As a control for downstream analysis (control TILs), constriction delivery was performed in the same way, but without input material. TILs containing the mRNAs delivered by squeezing were resuspended in culture medium without exogenous rhIL-2 and cultured for 3 days. Viability and expression of mbIL-2 and mbIL-12 were quantified by flow cytometry (Figures 24A-24E).

[0286] result As shown in Figure 24A, the viability of modified TILs containing mbIL-2 and mbIL-12 mRNA delivered by compression was consistently high (approximately 80%) at all measurement time points after compression (days 0, 1, 2, and 3).

[0287] As shown in Figures 24B-E, mRNAs encoding mbIL-2 and mbIL-12 were efficiently delivered simultaneously to TILs by the constriction delivery method and subsequently expressed.

[0288] Example 5: TILs transfected by compression with mRNA encoding membrane-bound cytokines proliferate in co-culture with tumors As shown in this example, proliferation of TILs containing mbIL-2 and mbIL-12 RNA delivered by compression was analyzed on day 3, the number of viable CD8+ T cells in each sample was measured in coculture with tumor cells, Ki67 expression was analyzed by flow cytometry, and granzyme B expression was analyzed by flow cytometry.

[0289] method TILs were isolated from tumor tissues of three melanoma patients. TILs were thawed and cultured overnight in medium without rhIL-2. After culture, in two groups, (1) mRNA encoding mbIL-2 and (2) mRNA encoding mbIL-2 and mbIL-12 were delivered to TILs by constriction delivery ("squeezing"), respectively. In the presence of the above mRNAs, TILs were squeezed at 30 psi using an ST-10-04-70 tip (length 10 μm, width 4 μm, depth 70 μm). As a control for downstream analysis (control TILs), constriction delivery was performed in the same way, but without input material. The squeezed TILs were then cultured for 3 days in the presence of autologous tumor cells without exogenous cytokines. As an additional control, TILs squeezed without input material (control TILs) were cultured with autologous tumor cells in medium containing 3000 IU / mL rhIL-2 and 10 ng / mL rhIL-12.

[0290] result As shown in Figure 25A, TILs compressed with mbIL2 mRNA alone or with mbIL-2 and mbIL-12 mRNA and cultured in the presence of autologous tumor cells without exogenous IL-2 or IL-12 showed higher cell proliferation as measured by the number of CD8+ T cells in each donor sample than TILs compressed with no input material (control TILs) for all three donor samples. As shown in Figure 25B, TILs compressed with mbIL2 mRNA alone or with mbIL-2 and mbIL-12 mRNA and cultured in the presence of autologous tumor cells without exogenous IL-2 or IL-12 showed higher Ki67 expression and a higher percentage of Ki67 positive cells in each sample than TILs compressed with no input material (control TILs) for all three donor samples. As shown in Figure 26, TILs were analyzed for granzyme B expression by flow cytometry 20 hours after co-culture with autologous tumor cells, and similarly, TILs pressed with (1) mbIL2 mRNA only or (2) mbIL-2 and mbIL-12 mRNA and co-cultured with autologous tumor cells without exogenous IL-2 or IL-12 showed higher expression of granzyme B than control TILs.

[0291] Example 6: TILs transfected by compression with mRNA encoding a membrane-bound cytokine kill tumors when co-cultured with autologous tumor cells As shown in this example, in comparison to control TILs co-cultured with tumor cells in the presence of exogenous rhIL-2 and rhIL-12, (1) TILs pressed with mbIL2 mRNA or (2) with mbIL-2 and mbIL-12 mRNA and co-cultured with tumor cells in the absence of exogenous cytokines kill tumors.

[0292] method TILs were thawed and cultured with autologous tumor cell lines in a 2:1 ratio in cytokine-free medium for 1-2 days. Autologous tumor cell lines were stained with Cytolight Red and imaged using an IncuCyte fluorescent microscope for 24 hours. After culture, in two groups, (1) mRNA encoding mbIL-2 and (2) mRNA encoding mbIL-2 and mbIL-12, respectively, were delivered to TILs by constriction delivery ("squeezing"). In the presence of the above mRNAs, TILs were squeezed at 30 psi using an ST-10-04-70 tip (length 10 μm, width 4 μm, depth 70 μm). As a control for downstream analysis (control TILs), constriction delivery was performed in the same way, but without input material. The squeezed TILs were then co-cultured with autologous tumor cells in the absence of exogenous cytokines. As an additional control, TILs compressed without input material (control TILs) were cultured with autologous tumor cells in medium containing 3000 IU / mL rhIL-2 and 10 ng / mL rhIL-12. A red mask was applied to IncuCyte images and used to calculate percent tumor confluence.

[0293] result As shown in Figures 27A-D, TILs compressed with (1) mbIL-2 mRNA or (2) mbIL-2 and mbIL-12 mRNA in co-culture in the absence of exogenous cytokines exhibited tumor killing capacity equivalent to that of control TILs co-cultured with tumor cells supported by exogenous rhIL-2 and rhIL-12. Furthermore, TILs compressed with (1) mbIL-2 mRNA or (2) mbIL-2 and mbIL-12 mRNA in co-culture in the absence of exogenous cytokines exhibited tumor killing capacity significantly higher than that of control TILs co-cultured in the absence of exogenous cytokines. [Table 6] JPEG2024545582000007.jpg231159JPEG2024545582000008.jpg238159JPEG2024545582000009.jpg155159

Claims

1. A method for increasing the expression of granzyme B in tumor-infiltrating lymphocytes (TILs), comprising: (a) modifying the TILs to increase the expression of (i) a costimulatory molecule, (ii) a cytokine, or (iii) both a costimulatory molecule and a cytokine; and (b) culturing the TILs in the absence of exogenous cytokines, wherein after the modification and culturing, the TILs exhibit higher expression of granzyme B upon activation than corresponding unmodified TILs (reference TILs).

2. A method for increasing the expression of granzyme B in tumor-infiltrating lymphocytes (TILs), comprising culturing the TILs in the absence of exogenous cytokines, wherein the TILs have been modified to increase expression of (i) costimulatory molecules, (ii) cytokines, or (iii) both costimulatory molecules and cytokines, and after the culturing, the TILs exhibit greater proliferation upon activation than corresponding unmodified TILs (reference TILs).

3. The method of claim 1 or 2, wherein the expression of Granzyme B is increased by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, or at least about 10-fold compared to the reference TIL.

4. 3. The method of claim 1 or 2, wherein the culturing is carried out for at least about 1 day, at least about 2 days, or at least about 3 days.

5. The method of claim 1 or 2, wherein the costimulatory molecule is CD86.

6. The method of claim 1 or 2, wherein the cytokine comprises membrane-bound IL-2, membrane-bound IL-12, or both.

7. The method of claim 1, wherein the modification comprises perturbing the TIL by passing a cell suspension containing the TIL through a cell deformation constriction, such that when the nucleic acid encoding the costimulatory molecule and / or the nucleic acid encoding the cytokine is contacted with the TIL, it passes through the perturbation section and enters the TIL.

8. The method of claim 7, wherein the modifying further comprises contacting the TIL with the nucleic acid encoding the costimulatory molecule and / or the nucleic acid encoding the cytokine.

9. 3. The method of claim 2, wherein the TILs are modified by perturbing the input TILs by passing a cell suspension containing the input TILs through a cell deformation constriction such that the nucleic acid encoding the costimulatory molecule and / or the nucleic acid encoding the cytokine enters the input TILs upon contact with the TILs, and after the nucleic acid encoding the costimulatory molecule and / or the nucleic acid encoding the cytokine, the input TILs exhibit increased expression of the costimulatory molecule and / or the cytokine, becoming modified TILs.

10. 10. The method of claim 9, further comprising contacting the cell suspension with the nucleic acid encoding the costimulatory molecule and / or the nucleic acid encoding the cytokine.

11. 11. The method of claim 10, wherein the cell suspension is contacted with the nucleic acid encoding the costimulatory molecule and / or the nucleic acid encoding the cytokine before passing the cell suspension through the cell deformation constriction.

12. 12. The method of claim 10 or 11, wherein the cell suspension is contacted with the nucleic acid encoding the costimulatory molecule and / or the nucleic acid encoding the cytokine while passing the cell suspension through the cell deformation constriction.

13. The method of claim 1 or 2, wherein the nucleic acid encoding the costimulatory molecule and / or the nucleic acid encoding the cytokine is mRNA.

14. The method of any one of claims 7 to 9, wherein the cell deformation constriction has a width of about 10% to about 99% of the average diameter of the TIL.

15. The method of claim 14, wherein the cell deformation constriction has a width of about 10% to about 99% of the average diameter of the introduced TILs.

16. 16. The method of claim 15, wherein the width of the constriction is from about 3.5 μm to about 4.2 μm, or from about 3.5 μm to about 4.8 μm, or from about 3.5 μm to about 6 μm, or from about 5 μm to about 12 μm, or from about 12 μm to about 15 μm, or from about 6 μm to about 12 μm, or from about 8 μm to about 11 μm, or from about 9 μm to about 11 μm.

17. The method of claim 15, wherein the width of the constriction is from about 3 μm to about 5 μm.

18. 18. The method of claim 17, wherein the width of the constriction is about 4 μm.

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