Method for activating and proliferating tumor-infiltrating lymphocyte

Abstract

To provide a method for activating and proliferating tumor-infiltrating lymphocytes.SOLUTION: Provided is a method for activating and proliferating tumor-infiltrating lymphocytes (TIL) by a single-step process. A method for activating and proliferating TIL without using a feeder cell is also provided. In addition to a population of isolated and proliferated TIL in which a central memory T cell phenotype is enriched, a composition of the population of proliferated TIL is also provided. Provided is a method for activating and proliferating TIL which uses a more rationalized approach, that is, a single-step approach, an approach requiring a shorter proliferation period, an approach of using soluble irritant reagent, an approach more suitable for clinical production, and an approach using no feeder cell or the like. In addition to a population of isolated and proliferated TIL with abundant central memory T cell phenotype, a composition of the population of proliferated TIL is also provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 940,035, filed November 25, 2019, and U.S. Provisional Patent Application No. 63 / 081,539, filed September 22, 2020, each of which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to methods for activating and expanding lymphocyte populations, particularly tumor-infiltrating lymphocytes. [Background technology]

[0003] Adoptive transfer of tumor-infiltrating lymphocytes (TILs) is a powerful approach for the treatment of large, intractable cancers, especially in patients with poor prognosis. Successful immunotherapy requires a large number of TILs, necessitating a robust and reliable expansion process. Typically, a multistep process involving IL-2-based TIL expansion (pre-REP) followed by a rapid expansion protocol (REP) has become the preferred method for TIL expansion due to its ability to generate therapeutically effective numbers of TILs. However, the process remains limited by its time-consuming nature. After the pre-REP stage, which can last up to six weeks, REP can result in a 1,000-fold expansion of TILs over a 14-day period. REP is a challenging process that requires large amounts of anti-CD3 antibody (OKT3) and IL-2, as well as a large excess (e.g., 200-fold) of feeder cells to activate the TILs.

[0004] One of the most challenging aspects of currently available REP-based TIL expansion methods is the need to obtain and use feeder cells. In these REP-based methods, TIL activation depends on the presence of feeder cells. Feeder cell populations are typically collected from 3–5 allogeneic donors, making the process of collecting, exposing, and maintaining the feeder cell population expensive and difficult to control. Therefore, despite the cost and difficulty of using feeder cells, they are considered essential for the TIL activation and expansion process.

[0005] Therefore, there is a need for more streamlined methods for expanding TILs. Summary of the Invention [Means for solving the problem]

[0006] Methods for activating and expanding TILs are provided that use more streamlined approaches, i.e., single-step approaches, approaches requiring shorter expansion periods, approaches using soluble stimulating reagents, approaches more suitable for clinical manufacturing, and approaches that do not use feeder cells, etc. In addition to isolated expanded TIL populations enriched for central memory T cell phenotype, compositions of expanded TIL populations are also provided.

[0007] As disclosed herein, it has been surprisingly discovered that TILs can be activated and expanded in the absence of feeder cells using a combination of a T cell receptor (TCR) agonist (e.g., a CD3 agonist) and a CD28 agonist. The TCR agonist and CD28 agonist can be antibodies linked or conjugated to each other or linked to a nanomatrix. Surprisingly, the feeder-free TIL activation and expansion process described herein can result in a 150,000-fold expansion of TILs and enrichment for central memory T cell phenotypes. Surprisingly, the feeder-free TIL activation and expansion process described herein can also result in a 4,000- to 100,000-fold expansion by day 14 of the one-step process. This robust expansion was also observed in samples from multiple donors that were not expanded under pre-REP conditions. Thus, the process described herein is capable of generating expanded TILs in situations where the current standard of practice, a two-step TIL expansion method, fails.

[0008] As disclosed herein, it has also been surprisingly found that TILs can be activated and expanded using a one-step process that obviates the need for separate pre-REP and REP stages.

[0009] In one aspect, the present invention relates to a method for expanding a population of TILs in a dissociated tumor sample, the method comprising culturing the dissociated tumor sample in a medium in which the TILs are contacted with a TCR agonist, a CD28 agonist, and a T cell stimulating cytokine.

[0010] In some embodiments, the medium is supplemented with a T cell stimulating cytokine at intervals selected from the group consisting of 1 day, 2 days, 3 days, 4 days, 5 days, and 6 days.

[0011] In some embodiments, the final concentration of the T cell stimulating cytokine is 10 U / ml to 7,000 U / ml. In some embodiments, the T cell stimulating cytokine is IL-2. In some embodiments, the medium is changed at an interval selected from the group consisting of 1 day, 2 days, 3 days, 4 days, 5 days, and 6 days.

[0012] In some embodiments, the composition of the medium is maintained, hi some embodiments, 30% to 99% of the medium is changed at intervals selected from the group consisting of 1 day, 2 days, 3 days, 4 days, 5 days, and 6 days.

[0013] In some embodiments, the methods herein can retrieve a TIL sample from a previously failed pre-REP expansion. In some embodiments, the tumor sample is from a subject who previously submitted a tumor sample for TIL expansion, wherein the previous TIL expansion included a pre-REP stage, and the number of TILs isolated from the pre-REP stage was less than 1000 TILs. In some embodiments, the tumor sample is from a subject who previously submitted a tumor sample for TIL expansion, wherein the previous TIL expansion included a pre-REP stage, and the number of TILs isolated from the pre-REP stage was less than 5-fold expansion.

[0014] In some embodiments, the dissociated tumor sample is between 0.5 and 4 mm in size. 3 In some embodiments, the dissociated tumor sample comprises digested tumor fragments.

[0015] In some embodiments, the medium comprises feeder cells. In some embodiments, the feeder cells are peripheral blood mononuclear cells or antigen-presenting cells. In some embodiments, the feeder cells express a TCR agonist, a CD28 agonist, and / or a 4-1BB agonist. In some embodiments, the TCR agonist, a CD28 agonist, and / or the 4-1BB agonist is expressed on the surface of the feeder cells. In some embodiments, the feeder cells are genetically modified to express the TCR agonist, the CD28 agonist, and / or the 4-1BB ligand. In some embodiments, the TCR agonist is a CD3 agonist. In some embodiments, the CD3 agonist is OKT3. In some embodiments, the CD28 agonist is CD86. In some embodiments, the feeder cells are antigen-presenting cells. In some embodiments, the antigen-presenting cells comprise K562 cells. In some embodiments, the feeder cells express the TCR agonist and / or 4-1BB agonist. In some embodiments, the 4-1BB agonist is a 4-1BB ligand. In some embodiments, the feeder cells are genetically modified to express a T cell stimulating cytokine. In some embodiments, the T cell stimulating cytokine is IL-2.

[0016] In some embodiments, the medium does not contain feeder cells. In some embodiments, the CD28 agonist is dissolved in the medium.

[0017] In some embodiments, the TCR agonist is a CD3 agonist.

[0018] In some embodiments, the TCR agonist and / or the CD28 agonist are linked to a nanomatrix comprising a colloidal suspension of a matrix of polymer chains, each nanomatrix having a maximum dimension of 1-500 nm in length. In some embodiments, the TCR agonist and the CD28 agonist are linked to the same polymer chain. In some embodiments, the TCR agonist and the CD28 agonist are linked to different polymer chains. In some embodiments, the TCR agonist is linked to the nanomatrix at 25 μg per mg of nanomatrix.

[0019] In some embodiments, the TCR agonist comprises a soluble monospecific complex comprising two linked anti-CD3 antibodies. In some embodiments, the CD28 agonist comprises a soluble monospecific complex comprising two linked anti-CD28 antibodies. In some embodiments, the medium comprises a CD2 agonist. In some embodiments, the CD2 agonist comprises a soluble monospecific complex comprising two linked anti-CD2 antibodies.

[0020] In another aspect, the present invention relates to a method for expanding a population of TILs, comprising contacting the population of TILs with a nanomatrix comprising a colloidal suspension of a matrix of polymer chains, the matrix being bound to a CD3 agonist and a CD28 agonist, the nanomatrix providing an activation signal to the population of TILs, thereby activating and inducing the proliferation of the population of TILs, each matrix having a maximum dimension of 1 to 500 nm in length, and the method does not involve the use of feeder cells during the expansion of the population of TILs.

[0021] In some embodiments, the population of TILs in contact with the nanomatrix further comprises tumor cells.

[0022] In some embodiments, the population of TILs is isolated from a subject and contacted with the nanomatrix, without further proliferation processes of the population of TILs prior to contacting the population of TILs with the nanomatrix.

[0023] In some embodiments, the CD3 agonist and the CD28 agonist are attached to the same polymer chain. In some embodiments, the CD3 agonist and the CD28 agonist are attached to different polymer chains. In some embodiments, the CD3 agonist is attached to the nanomatrix at 25 μg per mg of nanomatrix.

[0024] In some embodiments, the nanomatrix further comprises magnetic, paramagnetic, or superparamagnetic nanocrystals embedded between or within the matrix of polymer chains.

[0025] In some embodiments, the matrix of polymer chains comprises a polymer of dextran.

[0026] In some embodiments, the polymer chains are colloidal polymer chains.

[0027] In some embodiments, the ratio of the volume of the nanomatrix to the volume of the TIL is 1:5 or greater. In some embodiments, the ratio of the volume of the nanomatrix to the volume of the TIL is 1:500 or greater. In some embodiments, the ratio of the volume of the nanomatrix to the volume of the TIL is 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:200, 1:300, 1:400, or 1:500.

[0028] In some embodiments, the CD28 agonist is bound to the nanomatrix at 25 μg per mg of nanomatrix. In some embodiments, the agonist is a recombinant agonist. In some embodiments, the agonist is an antibody. In some embodiments, the agonist is a humanized antibody. In some embodiments, the CD3 agonist is OKT3 or UCHT1.

[0029] In some embodiments, the methods herein can salvage a TIL sample from a previously failed pre-REP expansion. In some embodiments, the TILs to be expanded are derived from a subject who previously submitted a sample of TILs for expansion, the previous expansion of TILs including a pre-REP stage, where the number of TILs isolated from the pre-REP stage was less than 1000 TILs. In some embodiments, the TILs to be expanded are derived from a subject who previously submitted a sample of TILs for expansion, the previous expansion of TILs including a pre-REP stage, where the number of TILs isolated from the pre-REP stage was less than 5-fold expansion.

[0030] In another aspect, the invention relates to a method of expanding a population of TILs, comprising contacting the population of TILs with a composition comprising first, second, and third soluble monospecific complexes, each soluble monospecific complex comprising two antibodies or fragments thereof linked together, each antibody or fragment thereof of each soluble monospecific complex specifically binding to the same antigen on the population of TILs, the first soluble monospecific complex comprising an anti-CD3 antibody, the second soluble monospecific complex comprising an anti-CD28 antibody, and the third soluble monospecific complex comprising an anti-CD2 antibody, and the method does not involve the use of feeder cells during the expansion of the population of TILs.

[0031] In some embodiments, the population of TILs contacted with the composition further comprises tumor cells.

[0032] In some embodiments, the population of TILs is isolated from a subject and contacted with the composition, without further proliferation processes of the population of TILs prior to contacting the population of TILs with the composition.

[0033] In some embodiments, the soluble monospecific complex has a concentration of 0.2 to 25 μl / ml.

[0034] In some embodiments, the soluble monospecific complexes are tetrameric antibody complexes (TACs). In some embodiments, each TAC comprises two antibodies from a first animal species linked by two antibody molecules from a second species that specifically bind to the Fc portion of the antibodies from the first animal species.

[0035] In some embodiments, the anti-CD3 antibody is an OKT3 antibody or a UCHT1 antibody. In some embodiments, the method further comprises contacting the population of TILs with the cytokine IL-2. In some embodiments, the TILs are contacted with the cytokine IL-2 at intervals selected from the group consisting of 1 day, 2 days, 3 days, 4 days, 5 days, and 6 days. In some embodiments, the final concentration of the cytokine IL-2 is 100 U / ml to 7,000 U / ml.

[0036] In some embodiments, the methods herein can salvage a TIL sample from a previously failed pre-REP expansion. In some embodiments, the TILs to be expanded are from a subject who previously submitted a sample of TILs for expansion, the previous expansion of TILs including a pre-REP stage, where the number of TILs isolated from the pre-REP stage was less than 1000 TILs. In some embodiments, the sample to be expanded is from a subject who previously submitted a sample of TILs for expansion, the previous expansion of TILs including a pre-REP stage, where the number of TILs isolated from the pre-REP stage was less than 5-fold expansion.

[0037] In some embodiments, the TILs are expanded for up to 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days. In some embodiments, the TILs are expanded for 9-25 days, 9-21 days, or 9-14 days.

[0038] In some embodiments, the TILs are expanded 500-500,000 fold. In some embodiments, the population of TILs is expanded from an initial population of 100-100,000 TILs. In some embodiments, the population of TILs is expanded at least 1,500-fold at day 14 of expansion. In some embodiments, the population of TILs is expanded up to 100,000-fold at day 14 of expansion. In some embodiments, the population of TILs is expanded at least 15,000-fold at day 21 of expansion. In some embodiments, the population of TILs is expanded up to 500,000-fold at day 21 of expansion.

[0039] In some embodiments, members of the population of TILs are genetically modified.

[0040] In some embodiments, the members of the TIL population are modified with a gene regulatory system. In some embodiments, the members of the TIL population are modified using RNA interference. In some embodiments, the members of the TIL population are modified using transcription activator-like effector nucleases (TALENs). In some embodiments, the members of the TIL population are modified using zinc finger nucleases. In one embodiment, the members of the TIL population are modified using RNA-guided nucleases. In some embodiments, the members of the TIL population are modified using a Cas enzyme and at least one guide RNA. In some embodiments, the Cas enzyme is Cas9.

[0041] In some embodiments, members of the population of TILs are selected from the group consisting of ANKRD11, BCL2L11, BCL3, BCOR, CALM2, CBLB, CHIC2, CTLA4, DHODH, E2F8, EGR2, FLI1, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, LAG3, MAP4K, NFKBIA, NR4A3, NRP1, PBRM1, PCBP1, PDCD1, PELI1, P In some embodiments, members of the TIL population are modified in one or more genes selected from the group consisting of IK3CD, PPP2R2D, PTPN1, PTPN2, PTPN22, PTPN6, RBM39, RC3H1, SEMA7A, SERPINA3, SETD5, SH2B3, SH2D1A, SMAD2, SOCS1, TANK, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TNIP1, TRAF6, UMPS, WDR6, and ZC3H12A. In some embodiments, members of the TIL population are modified in one or more genes selected from the group consisting of SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA. In some embodiments, the modification in the one or more genes is an insertion, deletion, or mutation of one or more nucleic acids. In some embodiments, the modification in the one or more genes reduces or inhibits expression of the gene and / or the function of the protein encoded by the gene. In some embodiments, members of the TIL population are epigenetically modified, hi some embodiments, the epigenetic modification is a histone modification.

[0042] In some embodiments, members of the population of TILs are selected from the group consisting of ANKRD11, BCL2L11, BCL3, BCOR, CALM2, CBLB, CHIC2, CTLA4, DHODH, E2F8, EGR2, FLI1, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, LAG3, MAP4K, NFKBIA, NR4A3, NRP1, PBRM1, PCBP1, PDCD1, PELI1, P The gene is modified in one or more genes selected from the group consisting of IK3CD, PPP2R2D, PTPN1, PTPN2, PTPN22, PTPN6, RBM39, RC3H1, SEMA7A, SERPINA3, SETD5, SH2B3, SH2D1A, SMAD2, SOCS1, TANK, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TNIP1, TRAF6, UMPS, WDR6, and ZC3H12A. The modification in the one or more genes is methylation of one or more nucleic acids. In some embodiments, the modification in the one or more genes is methylation of one or more nucleic acids. In some embodiments, the modification in the one or more genes reduces or inhibits expression of the gene and / or the function of the protein encoded by the gene.

[0043] In some embodiments, members of the TIL population are altered in the SOCS1 gene, which in some embodiments reduces or inhibits expression of the gene and / or the function of the protein encoded by the gene.

[0044] In some embodiments, members of the TIL population are altered in multiple genes. In some embodiments, members of the TIL population are altered in two or more genes selected from the group consisting of SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA. In some embodiments, the two or more genes are ANKRD11, BCL2L11, BCL3, BCOR, CALM2, CBLB, CHIC2, CTLA4, DHODH, E2F8, EGR2, FLI1, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, LAG3, MAP4K, NFKBIA, NR4A3, NRP1, PBRM1, PCBP1, PDCD1 , PELI1, PIK3CD, PPP2R2D, PTPN1, PTPN2, PTPN22, PTPN6, RBM39, RC3H1, SEMA7A, SERPINA3, SETD5, SH2B3, SH2D1A, SMAD2, SOCS1, TANK, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TNIP1, TRAF6, UMPS, WDR6, and ZC3H12A. In some embodiments, the members of the TIL population are modified in the SOCS1 gene and one or more additional genes. In some embodiments, the members of the TIL population are modified in the SOCS1 gene and one or more additional genes selected from the group consisting of ZC3H12A, PTPN2, CBLB, RC3H1, or NFKBIA. In certain embodiments, the members of the TIL population are modified in the SOCS1 and ZC3H12A genes. In some embodiments, members of the TIL population are modified in the SOCS1 and PTPN2 genes. In some embodiments, modification of the SOCS1 and PTPN2 genes reduces or inhibits expression of the genes and / or the function of the proteins encoded by the genes. In some embodiments, members of the TIL population are modified in the SOCS1 and ZC3H12A genes. In some embodiments, modification of the SOCS1 and ZC3H12A genes reduces or inhibits expression of the genes and / or the function of the proteins encoded by the genes.In some embodiments, members of the TIL population are modified in the SOCS1 and CBLB genes. In some embodiments, modifying the SOCS1 and CBLB genes reduces or inhibits expression of the genes and / or function of the proteins encoded by the genes. In some embodiments, members of the TIL population are modified in the SOCS1 and RC3H1 genes. In some embodiments, modifying the SOCS1 and RC3H1 genes reduces or inhibits expression of the genes and / or function of the proteins encoded by the genes. In some embodiments, members of the TIL population are modified in the SOCS1 and NFKBIA genes. In some embodiments, modifying the SOCS1 and NFKBIA genes reduces or inhibits expression of the genes and / or function of the proteins encoded by the genes.

[0045] In some embodiments, the population of TILs is expanded to produce a population of expanded TILs, wherein at least 10% of the expanded population have a central memory T cell phenotype. In some embodiments, the population of TILs is expanded to produce a population of expanded TILs, wherein at least 15% of the expanded population have a central memory T cell phenotype. In some embodiments, the population of TILs is expanded to produce a population of expanded TILs, wherein 5-50% of the expanded population have a central memory T cell phenotype at 14 days of expansion. In some embodiments, the population of TILs is expanded to produce a population of expanded TILs, wherein 10-25% of the expanded population have a central memory T cell phenotype at 14 days of expansion.

[0046] In another aspect, the invention relates to a composition comprising a population of expanded TILs produced by any of the methods disclosed herein.

[0047] The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0048] [Figure 1] 1 is a schematic diagram showing the workflow of TIL production using feeder cells. [Figure 2] FIG. 1 is a schematic diagram showing four methods for producing TILs: Method 1 (one-step REP using feeder cells but without a pre-REP step ("REP-like")), Method 2 (using anti-CD3 and anti-CD28 antibody-conjugated Dynabeads ("Dynabeads")), Method 3 (using anti-CD3, anti-CD2, and anti-CD28 antibody-based tetrameric antibody complexes (TACs) ("Stemcell")), and Method 4 (using anti-CD3 and anti-CD28 antibody-conjugated nanomatrix ("Transact"). [Figure 3] A shows a plot depicting the fold expansion of TILs on day 14. B shows a plot depicting the fold expansion of TILs on day 21. In each figure panel, each dot represents an independent melanoma donor. Data generated using Method 1 (REP-like), Method 2 (Dynabeads, 1.5 or 0.5 x 106 beads / well), Method 3 (Stemcell), Method 4 (Transact), and control (IL-2 alone) are shown. [Figure 4] A series of FACS analyses showing the gating strategy for cell counting are shown. [Figure 5] A shows a plot depicting the percentage of viable cells that are CD3+ T cells in culture on days 0, 9, and 14. B shows a plot depicting the percentage of T cells with a central memory phenotype (Tcm, defined as CCR7+CD45RO+) on day 14. Data generated using Method 1 (REP-like), Method 3 (Stemcell), Method 4 (Transact), and a control (IL-2 alone) are shown. [Figure 6] 1 shows a series of FACS analyses showing the percentage of central memory T cell phenotypes at day 14 from three independent donors. [Figure 7] A bar graph showing fold expansion at day 14 using FACS counting bead analysis of TIL pan-T cell proliferation from three independent donors is shown. [Figure 8] 1 shows a bar graph depicting fold expansion using FACS counting bead analysis of the proliferation of matched donor-derived TIL pan-T cells at day 14. Data generated using Method 1 (REP-like), Method 2 (Dynabeads, 1.5 or 0.5 x 10 beads / well), Method 3 (Stemcell), Method 4 (Transact), and control (IL-2 alone) are shown. [Figure 9] 1 shows a bar graph depicting extrapolated fold expansion at day 21 using FACS counting bead analysis of TIL pan-T cell proliferation from three independent donors. Data generated using Method 1 (REP-like), Method 3 (Stemcell), and Method 4 (Transact) are shown. [Figure 10] A plot showing fold expansion over time at day 14 relative to day 0 of culture as a function of seeding density is shown. [Figure 11] Bar graphs showing the fold induction of expression of IFNγ (A), IL-2 (B), IL-6 (C), and TNFα (D) in TIL donor samples produced using Method 1 (REP-like), Method 3 (Stemcell), and Method 4 (Transact). [Figure 12] Shown are the percent CD45 editing and viability of TILs 8 days after electroporation, which were expanded using method 1 (REP-like), method 3 (Stemcell), or method 4 (Transact). [Figure 13] 1 shows a bar graph showing the fold expansion for soluble tetramer and aAPC on day 10 or 11 using Method 1 (REP-like or "REP"), Method 3 (Stemcell or "Stem"), Method 4 (Transact or "Trans"), Method 5 (aAPC-OKT3 or "OKT3"), and Method 6 (aAPC-OKT3-CD86 or "OKT3+CD86"). [Figure 14] Bar graphs showing fold expansion at day 18 (D3399) or day 23 (D6752 and D6755) for soluble tetramer and aAPC-edited TILs are shown. [Figure 15]1 shows a bar graph depicting the central memory phenotype for edited TILs at day 18 (D3399) or day 23 (D6752 and D6755). [Figure 16] A table of editing frequencies on day 18 (D3399) or day 23 (D6752 and D6755) is shown. [Figure 17] Bar graphs showing extrapolated cell counts of tumor fragments of TILs on days 14 or 20 are shown. [Figure 18] Bar graphs showing central memory phenotype (%) on days 14 or 20 are shown. [Figure 19] A table of editing frequencies on days 14 and 20 is shown. [Figure 20] A table of editing frequencies on days 14 and 20 is shown. [Figure 21] 1 shows a bar graph depicting the viability of TILs from different donors prepared from tumor fragments and digests. [Figure 22] 1 shows a bar graph depicting cell counts for TILs from different donors prepared from tumor fragments and digests. DETAILED DESCRIPTION OF THE INVENTION

[0049] The traditional activation and expansion process of TILs requires multiple steps, including at least separate pre-REP and REP stages, as well as the use of feeder cells. Both requirements make this traditional process time-consuming and expensive. Patients requiring immunotherapy using adoptive transfer of TILs often have a very poor prognosis, and having a population of expanded and differentiated TILs available for therapy more quickly can be a matter of life and death.

[0050] The need for a generally slow pre-REP step followed by a generally fast REP step to achieve sufficient TIL activation and expansion fold for therapeutic use is both time-consuming and expensive. In certain applications, the conventional pre-REP step can last 2-6 weeks, with an additional 1-3 weeks of REP. Therefore, it is necessary to eliminate the pre-REP step and streamline the TIL production process into a single step, with or without the use of feeder cells.

[0051] Reliance on feeder cells is particularly challenging for at least several reasons. First, obtaining a viable feeder cell population is extremely difficult because the cells are harvested from 3-5 allogeneic donors. This heterogeneous source of feeder cells precludes standardization of their use, as each population of donor cells must be individually characterized for its ability to expand TILs. Similarly, the inherent variability of feeder cells makes the expansion of TILs using feeder cells less reproducible and less predictable. Second, when feeder cells are used, TILs cannot be manipulated in the presence of feeder cells, so they can only be manipulated or genetically modified before or after the REP phase, not during it. Third, TIL production methods that include a REP step cannot shorten the REP because populations of TILs expanded until the feeder cells die cannot be used. Fourth, the use of feeder cells to stimulate TILs precludes the ability to wash off and / or remove the stimulatory agent. Therefore, in some cases, the pre-REP and REP stages of the conventional process cannot produce the desired number of TILs. For at least the above four reasons, it is necessary to eliminate dependency on feeder cells, which is what has been achieved by the present invention and is disclosed herein. Eliminating feeder cells allows for a high degree of control over the TIL proliferation process. For example, the TIL proliferation process can be stopped once the required number of TILs has been obtained.

[0052] To provide improved, faster, and simpler methods for producing TILs, the present disclosure provides methods for activating and expanding TILs using more streamlined approaches, i.e., single-step approaches, approaches requiring shorter expansion periods, approaches using soluble stimulating reagents, and approaches that do not use feeder cells, etc. In addition to isolated expanded TIL populations enriched for central memory T cell phenotype, compositions of expanded TIL populations are also provided.

[0053] In some aspects, the present disclosure relates to a method for activating and expanding TILs in a single-step process without the use of feeder cells, wherein activation occurs via contact with a CD3 and CD28 agonist. In certain embodiments, the CD3 and CD28 agonist are bound to a nanomatrix of polymer chains. In certain embodiments, the CD3 and CD28 agonist are antibodies or fragments thereof linked or conjugated to each other. In certain embodiments, the expanded TILs have a higher percentage of cells with a central memory T cell phenotype than TILs isolated using feeder cell-based methods. In certain embodiments, the method further comprises activating the TILs with at least one 4-1BB agonist. In some embodiments, the 4-1BB agonist is a 4-1BB ligand.

[0054] In some aspects, the present disclosure relates to methods for activating and expanding TILs in a one-step process that eliminates the need for a pre-REP stage and separate rapid expansion protocols ("REP") and pre-REP.

[0055] In general, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein is well known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods known in the art and as described in the various general and more specific references cited and discussed throughout the specification, unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications as commonly performed in the art or as described herein. The nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein, as well as the laboratory methods and techniques thereof, are well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0056] Unless otherwise defined herein, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. In the case of potential ambiguity, the definitions provided herein take precedence over any dictionary or extrinsic definitions. Unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include the singular. Unless otherwise specified, the use of "or" means "and / or." The use of the term "including," as well as other forms such as "includes" and "included," is not limiting.

[0057] As used herein, the terms "about" and "approximately" refer to values ​​that are within 5% of a given value or range.

[0058] As used herein, the term "tumor-infiltrating lymphocytes" or "TILs" refers to a population of lymphocytes that have left the bloodstream of a subject and migrated into a tumor. TILs include CD8 + Cytotoxic T cells, Th1 and Th17 CD4 +These include, but are not limited to, T cells, and natural killer (NK) cells. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from a patient tissue sample as described herein (sometimes referred to as "freshly harvested"); "secondary TILs" are any TIL cell population that has been expanded or proliferated as discussed herein, including, but not limited to, bulk TILs and expanded TILs ("REP TILs" or "post-REP TILs"). In some embodiments, primary TILs comprise tumor-reactive T cells obtained from a patient's peripheral blood. TIL cell populations may include genetically modified TILs. "TILs" also refers to a population of lymphocytes that have left a subject's bloodstream, migrated into a tumor, and then left again to re-enter the bloodstream.

[0059] As used herein, the phrase "population of cells" or "population of TILs" refers to a plurality of cells or TILs that share a common characteristic. Generally, a population is generally on the order of 1 x 10 6 ~1×10 10 The range is 1 x 10 cells, with different populations of TILs containing different numbers. For example, initial growth of primary TILs in the presence of IL-2 results in approximately 1 x 10 cells. 7 Bulk TIL populations of cells can be obtained. Rep expansion typically involves 1.5 x 10 cells for injection. 9 ~1.5×10 10 This is done to provide a population of cells.

[0060] As used herein, the phrase "expanding a population of TILs" is synonymous with "proliferating a population of TILs" and refers to increasing the number of cells within a population of TILs.

[0061] As used herein, the term "expansion process" refers to a process in which the number of cells in a population of TILs increases. A process in which TILs are simply isolated or enriched without substantially increasing the number of TILs is not an expansion process.

[0062] As used herein, the term "matrix" or "flexible matrix" refers to a discrete, isolable, three-dimensional lattice-type structure, the backbone of which may be flexible or flexible and may be composed of materials such as polymers and ceramics. Because of its three-dimensional structure, the matrix may have a minimum dimension and a maximum dimension, e.g., length. Flexible matrices may be collagen, purified proteins, purified peptides, polysaccharides, glycosaminoglycans, or extracellular matrix compositions. Polysaccharides may include, for example, cellulose ethers, starches, gum arabic, agarose, dextran, chitosan, hyaluronic acid, pectin, xanthan, guar gum, or alginate. Other polymers may include polyesters, polyethers, polyacrylates, polyacrylamides, polyamines, polyethyleneimines, polyquaternium polymers, polyphosphazenes, polyvinyl alcohols, polyvinyl acetates, polyvinylpyrrolidones, block copolymers, or polyurethanes. The flexible matrix may include a polymer of dextran. "Matrices" refers to a collection of matrices.

[0063] As used herein, the phrase "maximum dimension" in the context of a matrix refers to the longest length of the matrix.

[0064] As used herein, the term "agonist" refers to a chemical, molecule, macromolecule, complex of molecules, or complex of macromolecules that binds to a target either on the surface of a cell or in a soluble form. In certain embodiments, when an agonist binds to a target on the surface of a cell, the agonist activates the target, resulting in a biological response. Agonists include hormones, neurotransmitters, antibodies, and fragments of antibodies.

[0065] As used herein, the term "nanomatrix" refers to a colloidal suspension of multiple matrices of polymer chains. A nanomatrix is ​​a multiphase material with dimensions less than 500 nm or a structure with nanoscale repeat distances between the different phases that make up the material. Polymers may include polyethylene, polypropylene, polystyrene, polysaccharides, dextran, and other polymers composed of many repeating subunits. Nanomatrices may also have additional functional compounds embedded therein, such as magnetic, paramagnetic, or superparamagnetic nanocrystals. Additionally, functional moieties, such as ligands or agonists, may be covalently bonded or attached to the polymer chains for specific applications.

[0066] As used herein, the term "dextran" refers to a complex branched glucan, i.e., a polysaccharide obtained by condensation of glucose. Dextran chains vary in length from 3 to 2000 kilodaltons. The polymer backbone consists of α-1,6 glycosidic bonds between glucose monomers, branching from α-1,3 bonds.

[0067] As used herein, the phrase "nanomatrix-bound agonist" refers to an agonist that is covalently attached within the nanomatrix to the polymer chains that comprise the matrix.

[0068] As used herein, the term "colloidal suspension" refers to a mixture in which one substance, e.g., a matrix, is suspended throughout another substance, e.g., a liquid. A colloidal suspension thus has a dispersed phase, i.e., the suspended substance, and a continuous phase, i.e., the suspending medium, e.g., a liquid.

[0069] As used herein, the phrase "contacting a population of TILs with a nanomatrix" refers to bringing the TILs and nanomatrix together so that the TILs can associate with functional moieties, e.g., ligands or agonists, bound to the nanomatrix or functional compounds, e.g., nanocrystals, embedded in the nanomatrix via ionic bonds, hydrogen bonds, or other types of physical or chemical interactions.

[0070] As used herein, the term "subject" refers to a human having a tumor into which a population of lymphocytes has migrated from the human's bloodstream and converted into TILs, and the human may be a patient in need of immunotherapy involving an expanded population of the patient's own TILs.

[0071] As used herein, the term "CD3" refers to the CD3 (cluster of differentiation 3) T cell coreceptor, which promotes the activation of both cytotoxic T cells (CD8+ naive T cells) and T helper cells (CD4+ naive T cells). CD3 is a protein complex composed of six distinct polypeptide chains: two CD3 zeta chains, two CD3 epsilon chains, one CD3e gamma chain, and one CD3 delta chain. These chains associate with the alpha and beta chains (or gamma and delta chains) of the T cell receptor (TCR) to generate activation signals in T lymphocytes. The alpha and beta chains (or gamma and delta chains) of the TCR and the CD3 molecule together constitute the TCR complex. The human CD3E gene is identified by National Center for Biotechnology Information (NCBI) gene ID 916. An exemplary nucleic acid sequence for the human CD3E gene is NCBI Reference Sequence: NG_007383.1. An exemplary amino acid sequence of a human CD3E polypeptide is provided as SEQ ID NO:876. [Table 1]

[0072] In Table 1, predicted leader sequences for proteins that have a leader sequence are underlined.

[0073] As used herein, the term "CD28" refers to cluster of differentiation 28, a type of protein expressed on T cells that provides a costimulatory signal necessary for T cell activation and survival. In addition to the T cell receptor (TCR), stimulation of T cells through CD28 can provide a potent signal for the production of various cytokines, e.g., interleukins. CD28 is a receptor for CD80 and CD86 proteins. Upon activation by toll-like receptor ligands, CD80 expression is upregulated on antigen-presenting cells (APCs). The human CD28 gene is identified by NCBI Gene ID 940. An exemplary nucleotide sequence for the human CD28 gene is NCBI Reference Sequence: NG_029618.1. An exemplary amino acid sequence of a human CD28 polypeptide is provided as SEQ ID NO: 877.

[0074] As used herein, the term "CD2" refers to cluster of differentiation 2, which is a cell adhesion molecule found on the surface of T cells and natural killer (NK) cells. CD2 interacts with other adhesion molecules and acts as a costimulatory molecule on T cells and NK cells. The human CD2 gene is identified by NCBI gene ID 914. An exemplary nucleic acid sequence for the human CD2 gene is NCBI Reference Sequence: NG_050908.1. An exemplary amino acid sequence of a human CD2 polypeptide is provided as SEQ ID NO: 878.

[0075] As used herein, the term "4-1BB" refers to CD137, which is a T cell costimulatory molecule. An exemplary nucleotide sequence for the human 4-1BB gene is NCBI Reference Sequence: NC_000001.11. An exemplary amino acid sequence of human 4-1BB is NCBI Reference Sequence: NP_001552.2 (SEQ ID NO: 880).

[0076] As used herein, the term "4-1BB ligand" refers to a type 2 transmembrane glycoprotein that is expressed in activated T lymphocytes and binds to 4-1BB. An exemplary nucleotide sequence for human 4-1BB ligand is NCBI Reference Sequence: NC_000019.10. An exemplary amino acid sequence for human 4-1BB ligand is NCBI Reference Sequence: AAA53134.1 (SEQ ID NO: 881).

[0077] As used herein, the term "fragment" in reference to an agonist or antibody refers to a fragment of an agonist or antibody that retains the ability to specifically bind to an antigen. Examples of antibody fragments include: (i) a Fab fragment, i.e., a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, i.e., a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single antibody arm; (v) a dAb fragment comprising a single variable domain; and (vi) an isolated complementarity-determining region (CDR). Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant techniques by a synthetic linker that allows the VL and VH domains to combine into a single protein chain to form a monovalent molecule (known as a single-chain Fv (ScFv)). Such short-chain antibodies are also intended to be encompassed by the term "antigen-binding protein" of an antibody. Other forms of short-chain antibodies, such as diabodies, are also encompassed. Additionally, short-chain antibodies include "linear antibodies," which comprise a pair of tandem Fv segments (VH-CH1-VH-CH1), which, together with complementary light chain polypeptides, form a pair of antigen-binding regions.

[0078] The term "antibody" generally refers to an immunoglobulin (Ig) molecule composed of four polypeptide chains, i.e., two heavy (H) chains and two light (L) chains, or a functional fragment, mutant, variant, or derivative thereof that retains the epitope binding properties of an Ig molecule. Such fragment, mutant, variant, or derivative antibody types are known in the art. In full-length antibody embodiments, each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain variable region (domain) is also designated as VDH in this disclosure. The CH is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The CL is composed of a single CL domain. The light chain variable region (domain) is also designated as VDL in this disclosure. The VH and VL are further divided into regions of hypervariability called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Generally, each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Immunoglobulin molecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0079] As used herein, the phrase "selective binding" or "selectively binds" refers to the binding of an agonist to an epitope on a given antigen. Typically, the agonist binds selectively to an epitope on a given antigen with a high affinity (K D ) about 10 -5 Less than m, e.g., about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 K below M or even lower D Combine with.

[0080] As used herein, "K DThe term "agonist-antigen interaction" refers to the dissociation equilibrium constant of a particular agonist-antigen interaction. Typically, the agonists described herein have a dissociation equilibrium constant (K) for a target, as measured, for example, using surface plasmon resonance (SPR) technology on a Biacore instrument, with the agonist as the ligand and the target as the analyte. D ) about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 K below M or even lower D and has a K that is at least 10 times lower, e.g., at least 100 times lower, e.g., at least 1000 times lower, e.g., at least 10,000 times lower, e.g., at least 100,000 times lower, e.g., at least 100,000 times lower, than the affinity for binding to a nonspecific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein). D The amount of affinity reduction is the K of the agonist. D Because it depends on the K D is very low (ie, the agonist is highly specific), the amount by which affinity for the antigen is reduced below affinity for a nonspecific antigen may be at least 10,000-fold.

[0081] As used herein, "k d ”(seconds -1 The term k ) refers to the dissociation rate constant of a particular agonist-antigen interaction. off Also called value.

[0082] As used herein, "k a " (M -1 × seconds -1 The term ) refers to the association rate constant of a particular agonist-antigen interaction.

[0083] As used herein, the term "KD" (M) refers to the dissociation equilibrium constant of a particular agonist-antigen interaction.

[0084] As used herein, "KA " (M -1 The term k ) refers to the association equilibrium constant of a particular agonist-antigen interaction. a k d It is obtained by dividing by

[0085] As used herein, the phrase "activation signal" refers to one or more non-endogenous stimuli that activate T cells. In the endogenous process, T cells are activated when peptide antigens are presented by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Upon activation, the T cells rapidly divide and secrete cytokines that regulate or support the immune response. The endogenous T cell activation process includes at least (a) activation of the TCR complex, which includes CD3, and (b) costimulation of CD28 or 4-1BB by proteins on the surface of APCs. It is known in the art that endogenous T cell activation can be stimulated by stimulation of T cells with CD3, CD28, or 4-1BB agonists (e.g., antibodies). Thus, CD3, CD28, and / or 4-1BB can together provide an activation signal to T cells.

[0086] As used herein, the phrase "activating a population of TILs to induce their proliferation" refers to the process of subjecting a population of TILs to activation signals so that the TILs can increase in number or proliferate and begin producing cytokines (activated TILs) to enhance the immune response.

[0087] As used herein, the term "nanocrystal" refers to a material molecule having at least one dimension less than 100 nm, based on quantum dots, and composed of atoms in either a monocrystalline or polycrystalline arrangement. The size of nanocrystals distinguishes them from larger crystals.

[0088] As used herein, the phrase "magnetic, paramagnetic, or superparamagnetic nanocrystals" refers to nanocrystals that can be manipulated using a magnetic field. Such nanocrystals generally consist of at least one component that is a magnetic material, such as iron, nickel, or cobalt.

[0089] As used herein, the terms "tumor cells" or "cancer cells" refer to cells that divide uncontrollably, forming solid tumors or filling the blood with abnormal cells. Healthy cells stop dividing when they no longer require additional daughter cells, but tumor or cancer cells continue to produce copies. They can also spread from one part of the body to another in a process known as metastasis. Tumor cells can be isolated from many types of cancer, including bladder cancer, breast cancer, cervical cancer, colon and rectal cancer, endometrial cancer, kidney cancer, lip and oral cancer, liver cancer, melanoma, mesothelioma, lung cancer, non-small cell lung cancer, head and neck cancer, neuroblastoma, glioblastoma multiforme, non-melanoma skin cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, small cell lung cancer, and thyroid cancer. Tumor cells can be isolated from primary tumors and metastases.

[0090] As used herein, the term "tumor sample" refers to tumor cells isolated from a subject. In certain embodiments, a tumor sample is at least a portion of a solid tumor isolated in whole or in part from a subject or patient with a tumor. Tumor samples can be isolated from many cancer types, including bladder cancer, breast cancer, cervical cancer, colon and rectal cancer, endometrial cancer, kidney cancer, lip and oral cancer, liver cancer, melanoma, mesothelioma, lung cancer, non-small cell lung cancer, head and neck cancer, neuroblastoma, glioblastoma multiforme, non-melanoma skin cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, small cell lung cancer, and thyroid cancer. Tumor samples can be isolated from primary tumors and metastases.

[0091] As used herein, the term "dissociated tumor sample" refers to a tumor sample that has been fragmented into "tumor fragments." The fragmentation can be physical fragmentation, mechanical fragmentation, ultrasonic fragmentation, enzymatic fragmentation, or any combination thereof. The fragmentation can be performed mechanically, and optionally, the tumor fragments can then be enzymatically digested to a single-cell suspension. Mechanical dissociation methods can involve chopping and slicing the tumor into smaller tumor fragments, while enzymatic dissociation methods can involve treating the tumor fragments with specific enzymes, such as proteases.

[0092] In some embodiments, the methods herein can retrieve TIL samples from previously failed pre-REP expansions. In certain embodiments, the tumor sample is isolated from a subject whose sample was previously subjected to a TIL expansion technique. In some embodiments, the previous TIL expansion technique consisted of pre-REP expansion. In some embodiments, the pre-REP expansion comprises administering IL-2 to a dissociated tumor sample from the subject. In some embodiments, IL-2 is the only immunomodulator administered to the tumor sample or TILs expanded from the tumor sample in the pre-REP expansion. In some embodiments, the previous TIL expansion technique has failed. In some embodiments, a TIL expansion technique has failed if it does not expand an adequate number of TILs. In some embodiments, the appropriate number of TILs is greater than 1,000, 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000 TILs. In some embodiments, a TIL expansion technique is unsuccessful if it does not induce an appropriate TIL expansion fold. In some embodiments, an appropriate TIL expansion fold is greater than 50, 100, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000-fold expansion. In some embodiments, the previous TIL expansion technique and the TIL expansion method disclosed herein use portions of the same tumor sample. In some embodiments, two different samples are isolated from the same subject. In some embodiments, the methods described herein can provide greater numbers or fold expansion of TILs than previous expansion techniques, hi some embodiments, the methods described herein can provide clinically useful numbers of TILs that previous expansion techniques were unable to provide.

[0093] As used herein, the phrase "T cell receptor agonist" or "TCR agonist" refers to an agonist of the T cell receptor complex. Suitable TCR agonists include, but are not limited to, CD3 agonists (e.g., anti-CD3 antibodies).

[0094] As used herein, the term "culture medium" refers to a liquid or gel designed to support the survival, growth, and / or proliferation of cells in an artificial environment. Culture media generally contain a set of defined components. Such components may include an energy source, growth factors, hormones, stimulants, activators, sugars, salts, vitamins, and / or amino acids, and / or combinations thereof.

[0095] As used herein, the expression "the composition of the medium is maintained" refers to a medium that contains a set of defined components, such as specific stimulants and activators, in which the identity of the components remains constant, but the concentration of one or more of the components may change.In certain embodiments, the concentration of one or more components in the medium changes over time while cells are cultured in the medium.However, when the medium is changed, the new medium has the same components every time it is changed.

[0096] As used herein, the term "feeder cells" refers to cells used to provide extracellular secretions that promote the growth of another cell type. In certain embodiments, the feeder cells referred to herein are peripheral blood mononuclear cells (PBMCs) or antigen-presenting cells (APCs).

[0097] As used herein, the term "recombinant agonist" refers to an agonist protein encoded by a recombinant gene cloned into a system that supports gene expression and mRNA translation. The recombinant gene is designed to be under the control of a well-characterized promoter and to express the target agonist protein in a selected host cell to achieve high levels of protein expression. Genetic modification by recombinant DNA technology can result in the expression of mutant proteins or the expression of large amounts of protein.

[0098] As used herein, the expression "colloidal polymer chains" refers to polymer chains that can form colloidal suspensions when covalently linked to one another or linked by other physical or chemical interactions.

[0099] As used herein, the phrase "specifically binds" refers to a protein complex, e.g., an agonist, antagonist, antibody, or soluble monospecific complex, that interacts with a particular antigen with high specificity compared to other antigens with which the protein complex has a lower association affinity. The specific binding interaction may be mediated by ionic bonds, hydrogen bonds, or other types of chemical or physical associations. In certain embodiments, a protein complex specifically binds to a particular antigen when it recognizes its target antigen in a complex mixture of proteins and / or macromolecules. Two or more agonists, antagonists, antibodies, or soluble monospecific complexes "bind to the same epitope" if the agonists cross-compete (one prevents the binding or modulatory action of the other).

[0100] As used herein, the expression "central memory T cell phenotype" refers to T cells that are CD45RO+ and CCR7 (CCR7 hi ) and CD62L (CD62 hi) refers to a subset of T cells that express the CD4 receptor. The surface phenotype of central memory T cells also includes TCR, CD3, and CD127 (IL-7R). Central memory T cells primarily secrete IL-2 and CD40L as effector molecules after TCR triggering. Central memory T cells predominate in the CD4 compartment in the blood and, in humans, are relatively abundant in lymph nodes and tonsils.

[0101] As used herein, the term "anti-CD3 antibody" refers to an antibody or variant thereof, e.g., a monoclonal antibody, including human, humanized, chimeric, or murine antibodies directed against the CD3 receptor in the T cell antigen receptor of mature T cells. Anti-CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include the UCHT1 clone, also known as T3 and CD3c. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0102] As used herein, the phrase "anti-CD28 antibody" refers to an antibody or variant thereof, e.g., a monoclonal antibody, including a human, humanized, chimeric, or murine antibody directed against the CD28 receptor in the T cell antigen receptor of mature T cells.

[0103] As used herein, the term "anti-4-1BB antibody" refers to an antibody or variant thereof, such as a monoclonal antibody, including human, humanized, chimeric, or murine antibodies against 4-1BB. In some embodiments, an anti-4-1BB antibody can be used as a 4-1BB ligand.

[0104] As used herein, the phrase "anti-CD2 antibody" refers to an antibody or variant thereof, e.g., a monoclonal antibody, including human, humanized, chimeric, or murine antibodies directed against the CD2 receptor in the T cell antigen receptor of mature T cells.

[0105] As used herein, the term "OKT-3" (also referred to herein as "OKT3") refers to an anti-CD3 antibody manufactured by Miltenyi Biotech, Inc., San Diego, Calif., USA, and its biosimilars or variants (e.g., humanized, chimeric, or affinity matured variants). A hybridoma capable of producing OKT-3 is available at the American Type Culture Collection and has been assigned ATCC accession number CRL 8001. A hybridoma capable of producing OKT-3 is available at the European Collection of Authenticated Cell Cultures (ECACC) and has been assigned catalog number 86022706.

[0106] As used herein, the term "UCHT1" refers to the anti-CD3 antibody described in Beverley and Callard (1981) Eur. J. Immunol. 11:329-334, and biosimilars or variants thereof (e.g., humanized, chimeric, or affinity matured variants). An exemplary hybridoma capable of producing UCHT1 is available from Creative Diagnostics, Shirley, NY, USA, and has been assigned catalog number CSC-H3068.

[0107] As used herein, the term "tetrameric antibody complex" or "TAC" refers to a protein complex comprising two antibodies acting as first and second agonists linked by one or two linker antibodies that connect the antibodies acting as the first and second agonists. The linker antibody may bind to the constant regions of the agonist antibodies, and the constant regions may be of different isotypes. Bispecific antibodies, with one binding region per isotype, may also be used. Support for these complexes can be found in U.S. Pat. No. 4,868,109, which is incorporated herein by reference in its entirety. In other embodiments, the antibodies, or antigen-binding fragments thereof, acting as the first and second ligands may be covalently or noncovalently linked by one or more linker molecules. Non-limiting examples of such linker molecules include avidin and streptavidin, which may be used to conjugate biotinylated antibodies, e.g., antibodies with a biotin moiety in the Fc region. In further embodiments, the tetrameric antibody complex may be used as a mixture of complexes. This includes the use of multiple conjugates in a mixture of conjugates, where the conjugates in the total mixture may be contacted with three or more different ligands.

[0108] As used herein, the term "RNA-guided nuclease" refers to a nucleic acid / protein complex based on the naturally occurring type II CRISPR-Cas system, which is a programmable endonuclease that can be used to perform targeted genome editing. RNA-guided nucleases consist of two components: a short, approximately 100-nucleotide guide RNA (gRNA) that base-pairs with the target genomic DNA sequence using 20 variable nucleotides at its 5' end, and a nuclease, such as Cas9 endonuclease, that cleaves the target DNA.

[0109] As used herein, the term "Cas9" refers to CRISPR-associated protein 9, a protein that plays a key role in the immunological defense against certain bacterial DNA viruses and is frequently used in genetic engineering applications. Cas9 is an RNA-guided DNA endonuclease enzyme associated with the CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system in Streptococcus pyogenes. Cas9 can interrogate sections of DNA by matching sites complementary to a guide RNA (gRNA). If the DNA substrate is complementary to the gRNA, Cas9 cleaves the DNA. Because the targeting specificity of Cas9 comes from gRNA:DNA complementarity and not from modifications to the protein itself (such as TALENs and zinc fingers), it is easy to engineer Cas9 to target new DNA. Forms of Cas9 that bind but do not cleave cognate DNA can be used to target transcriptional activators or repressors to specific DNA sequences to control transcriptional activation and repression. Native Cas9 requires a guide RNA, which is composed of two distinct RNAs related to CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA). Cas9 targeting has been simplified by engineering chimeric single-stranded guide RNAs.

[0110] As used herein, the term "inactive Cas9" or "dCas9" refers to Cas9 endonuclease Dead, a mutant form of Cas9 in which endonuclease activity has been eliminated by a point mutation in its endonuclease domain. Like its unmutated form, dCas9 is used in CRISPR systems with a gRNA to target specific genes or nucleotides complementary to the gRNA, equipped with a PAM sequence that allows Cas9 to bind. Cas9 typically contains two endonuclease domains, called the RuvC and HNH domains. The point mutations D10A and H840A alter two residues critical for endonuclease activity, ultimately resulting in its inactivation. Although dCas9 lacks endonuclease activity, its binding to guide RNAs and targeted DNA strands is still possible because such binding is governed by other domains. When the gRNA positions dCas9 so that transcription factors and RNA polymerase cannot access the DNA, this alone is often sufficient to attenuate, if not completely block, transcription of the target gene. However, because dCas9 typically has modifiable regions at the N- and C-termini of the protein that can be used to bind transcriptional activators, this ability to bind DNA can also be harnessed for activation.

[0111] As used herein, the term "cytokine" refers to a broad category of small proteins (approximately 5-20 kDa in size) important in cell signaling. Cytokines are peptides that cannot cross the lipid bilayer of cells into the cytoplasm. Cytokines have been shown to participate in autocrine, paracrine, and endocrine signaling as immunomodulators. While the terms overlap to some extent, cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors, and generally do not include hormones or growth factors. Cytokines are produced by a wide range of cells, including immune cells such as macrophages, B lymphocytes, T lymphocytes, and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells. Cytokines generally act through binding to cell surface receptors and are particularly important in the immune response because they are involved in regulating the maturation, growth, and responsiveness of specific cell populations.

[0112] As used herein, the phrase "T cell stimulating cytokine" refers to a cytokine that stimulates and / or activates T cell lymphocytes. In some embodiments, the T cell stimulating cytokine is IL-2.

[0113] As used herein, the term "IL-2" (also referred to herein as "IL2") refers to the cytokine and T-cell growth factor known as interleukin-2, and includes all forms of IL-2, i.e., human and mammalian forms, forms with conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-2 is described, for example, in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated herein by reference in their entireties. The term IL-2 encompasses human, recombinant IL-2, such as aldesleukin (PROLEUKIN, commercially available from multiple sources at 22 million IU per single-use vial) and recombinant IL-2 marketed by CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-209-b), as well as other commercially available equivalents from other suppliers. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a non-glycosylated human recombinant IL-2 with a molecular weight of approximately 15 kDa. The term IL-2 also encompasses pegylated forms of IL-2, including the pegylated IL-2 prodrug NKTR-214 available from Nektar Therapeutics, South San Francisco, Calif., USA. NKTR-214 and pegylated IL-2 suitable for use in the present invention are described in U.S. Patent Application Publication No. US2014 / 0328791A1 and International Patent Application Publication No. WO2012 / 065086A1, the disclosures of which are incorporated herein by reference in their entireties. Other types of conjugated IL-2 suitable for use in the present invention are described in U.S. Patent Nos. 4,766,106, 5,206,344, 5,089,261, and 4,902,502, the disclosures of which are incorporated herein by reference in their entireties. Formulations of IL-2 suitable for use in the present invention are described in U.S. Patent No. 6,706,289.The disclosure of which is incorporated herein by reference in its entirety. The human IL2 gene is identified by NCBI gene ID 3558. An exemplary nucleotide sequence for the human IL2 gene is NCBI reference sequence NG_016779.1. An exemplary amino acid sequence of a human IL-2 polypeptide is provided as SEQ ID NO: 879.

[0114] As used herein, the phrase "soluble monospecific complex" refers to a complex comprising two binding proteins that are directly or indirectly linked to each other and bind to the same antigen, wherein the two binding proteins are soluble and not immobilized on a surface, particle, or bead.

[0115] Furthermore, in accordance with the present disclosure, conventional molecular biology, microbiology, and recombinant DNA techniques may be employed that are within the skill of one in the art. Such techniques are fully explained in the literature, see, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition, 1999. Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (herein referred to as "Sambrook et al., 1989"), DNA Cloning: A Practical Approach, Volumes I and II (DNGlover ed.1985), Oligonucleotide Synthesis (MJGait ed.1984), Nucleic Acid Hybridization [BD Hames & SJHiggins eds.(1985)], Transcription And Translation[BDHames & SJHiggins, eds.(1984)], Animal Cell Culture[RIFreshney, ed.(1986)], Immobilized Cells And Enzymes[IRL Press, (1986)], B.Perbal, A Practical Guide To Molecular Cloning(1984), FMAusubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994), each of which is incorporated herein by reference in its entirety.

[0116] Unless otherwise specified, the sequence identity / similarity values ​​provided herein refer to values ​​obtained using the BLAST 2.0 suite of programs, using default parameters (Altschul, et al., (1997) Nucleic Acids Res. 25:3389-402, incorporated herein by reference in its entirety).

[0117] As used herein, "nucleic acid target sequence" and "nucleic acid binding sequence" are used interchangeably and refer to a sequence that binds to and / or targets a nucleic acid.

[0118] As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences includes reference to residues of the two sequences that are identical when aligned for maximum correspondence over a specified comparison window. When the percentage of sequence identity is used in the context of proteins, it is recognized that non-identical residue positions often differ by conservative amino acid substitutions, in which an amino acid residue is replaced with another amino acid residue with similar chemical properties (e.g., charge or hydrophobicity), thereby not changing the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity can be adjusted upward to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Means for making this adjustment are well known to those skilled in the art. Typically, this involves scoring conservative substitutions as partial rather than complete mismatches, thereby increasing the percentage sequence identity. Thus, for example, if identical amino acids are given a score of 1 and non-conservative substitutions are given a score of zero, conservative substitutions are given a score between zero and 1. Scoring of conservative substitutions is calculated, for example, according to the algorithm of Meyers and Miller, (1988) Computer Applic. Biol. Sci. 4:11-17, as implemented in the program PC / GENE (Intelligenetics, Mountain View, Calif., USA). Each of these references is incorporated herein by reference in its entirety.

[0119] As used herein, "sequence identity percentage" refers to the value determined by comparing two optimally aligned sequences over a comparison window, and the portion of the polynucleotide sequence in the comparison window may have additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) due to the optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage.

[0120] The terms "substantial identity" or "substantially identical" in the context of polynucleotide sequences mean that the polynucleotide comprises a sequence having 50-100% sequence identity, preferably at least 50% sequence identity, preferably at least 60% sequence identity, preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and most preferably at least 95%, when compared to a reference sequence using one of the alignment programs described using standard parameters. Those skilled in the art will recognize that these values ​​may be appropriately adjusted to determine the identity of corresponding proteins encoded by two nucleotide sequences, taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. For these purposes, substantial amino acid sequence identity typically means 55-100%, preferably at least 55%, preferably at least 60%, more preferably at least 70%, 80%, 90%, and most preferably at least 95% sequence identity.

[0121] I. Tumor-infiltrating lymphocytes (TILs) Tumor-infiltrating lymphocytes, or TILs, are a population of cells originally obtained as leukocytes that have left the subject's bloodstream and migrated into the tumor. TILs include CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 +These include, but are not limited to, T cells, and natural killer (NK) cells. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from a patient tissue sample as described herein (sometimes referred to as "freshly harvested"), and "secondary TILs" are any TIL cell population that has been expanded or proliferated as discussed herein.

[0122] TILs are generally defined biochemically using cell surface markers or functionally by their ability to infiltrate tumors and effect therapy. TILs are generally classified as expressing one or more of the following biomarkers: CD4, CD8, TCRαβ, TCRγδ, CD27, CD28, CD56, CCR7, CD45RA, CD45RO, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors after reintroduction into patients. TILs can be further characterized by efficacy. For example, TILs can be considered effective if, for example, interferon gamma (IFNγ) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL after TCR stimulation.

[0123] Adoptive cell therapy using TILs cultured ex vivo by conventional TIL manufacturing processes involves at least two stages: at least one rapid expansion protocol (REP) stage and a preceding pre-REP stage. Adoptive cell therapy has been shown to be successful in melanoma patients after host immunosuppression. Current infusion acceptance parameters depend on TIL composition readings (e.g., CD28, CD8, or CD4 positivity) as well as the numerical fold expansion and viability of the REP product.

[0124] Experimental findings indicate that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes by removing regulatory T cells and competing elements of the immune system ("cytokine sinks") plays an important role in enhancing therapeutic efficacy. Accordingly, some embodiments of the present invention may employ a lymphodepletion step (sometimes referred to as "immunosuppressive conditioning") in patients prior to the introduction of the TILs of the present invention. In some embodiments, no lymphodepletion step is employed.

[0125] A. TIL proliferation As generally described herein, TILs are generally harvested from a patient sample and manipulated to expand their numbers before transplantation into the patient. In some embodiments, the TILs may be genetically engineered as discussed below. Generally, TILs are first harvested from a patient tumor sample ("primary TILs") and then expanded into larger populations for further manipulation as described herein, optionally cryopreserved and restimulated, and optionally evaluated for phenotypic and metabolic parameters indicative of TIL health.

[0126] Patient tumor samples can be obtained using methods known in the art, typically via surgical resection, needle biopsy, or other means to obtain a sample containing a mixture of tumor and TIL cells. Generally, the tumor sample can be derived from any solid tumor, including primary tumors, invasive tumors, or metastases. The solid tumor can be of any cancer type, including, but not limited to, bladder cancer, breast cancer, cervical cancer, colon and rectal cancer, endometrial cancer, kidney cancer, lip and oral cancer, liver cancer, melanoma, mesothelioma, lung cancer, non-small cell lung cancer, head and neck cancer, neuroblastoma, glioblastoma multiforme, non-melanoma skin cancer, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, small cell lung cancer, and thyroid cancer. In some embodiments, useful TILs can be obtained from malignant melanoma tumors, which have been reported to have particularly high levels of TILs. TILs can be obtained using primary melanoma tumors or their metastases.

[0127] In some embodiments, a solid tumor is an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can be benign or malignant. Solid tumor cancer refers to a malignant, neoplastic, or cancerous solid tumor. Solid tumor cancers include, but are not limited to, lymphoma, sarcoma, breast (including triple-negative breast cancer), pancreas, prostate, colon, rectum, bladder, lung (including non-small cell lung cancer (NSCLC)), brain, kidney, stomach, and skin (including, but not limited to, squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, the cancer is selected from cervical cancer, head and neck cancer (including, for example, head and neck squamous cell carcinoma (HNSCC)), neuroblastoma, glioblastoma, glioblastoma multiforme, liver cancer, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple-negative breast cancer, and non-small cell lung cancer. The tissue architecture of solid tumors comprises interdependent tissue compartments, including the parenchyma (cancer cells) and supporting stromal cells in which the cancer cells are dispersed and which may provide a supportive microenvironment.

[0128] Once harvested, the tumor sample is typically separated into approximately 1 to 8 mm sections using sharp scraping. 3 , or about 0.5 to about 4 mm 3 fragmented into small pieces of about 2-3 mm 3are particularly useful. The TILs are cultured from these fragments using an enzymatic tumor digest. Such tumor digests can be generated by incubation in an enzymatic medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamic acid, 10 mcg / mL gentamicin, 30 units / mL DNase, and 1.0 mg / mL collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests can be generated by placing the tumor in the enzymatic medium, mechanically dissociating the tumor for approximately 1 minute, followed by incubation for 30 minutes at 37°C in 5% CO2, and then repeating cycles of mechanical dissociation and incubation under the above conditions until only small tissue fragments are contained. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using FICOLL branched hydrophilic polysaccharides can be performed to remove these cells. Other methods known in the art may also be used, such as those described in U.S. Patent Application Publication No. 2012 / 0244133A1, the disclosure of which is incorporated herein by reference in its entirety. Any of the above methods may be used in any of the embodiments described herein for methods of expanding TILs or methods of treating cancer.

[0129] Generally, the harvested cell suspension is referred to as a "primary cell population" or a "freshly harvested" cell population. In some embodiments, fragmentation comprises physical fragmentation, such as exfoliation and digestion. In some embodiments, the fragmentation is physical fragmentation. In some embodiments, the fragmentation is by exfoliation. In some embodiments, the fragmentation is by digestion. In some embodiments, TILs can be initially cultured from enzymatic tumor digests and tumor fragments harvested from patients.

[0130] In some embodiments, if the tumor is a solid tumor, after a tumor sample is collected, the tumor is subjected to physical fragmentation. In some embodiments, the fragmentation occurs before lyophilization. In some embodiments, the fragmentation occurs after lyophilization. In some embodiments, the fragmentation occurs after the tumor is collected, without lyophilization. In some embodiments, the tumor is fragmented and 10, 20, 30, 40 or more fragments or pieces are placed in each container for first growth. In some embodiments, the tumor is fragmented and 30 or 40 fragments or pieces are placed in each container for first growth. In some embodiments, the tumor is fragmented and 40 fragments or pieces are placed in each container for first growth. In some embodiments, the plurality of fragments comprises about 4-50 fragments, each fragment having a volume of about 27 mm. 3 In some embodiments, the plurality of fragments comprises about 30 to about 60 fragments, and the total volume is about 1300 m 3 ~about 1500mm 3 In some embodiments, the plurality of pieces comprises about 50 pieces and has a total volume of about 1350 mm 3 In some embodiments, the plurality of fragments comprises about 50 fragments, with a total mass of about 1 gram to about 1.5 grams. In some embodiments, the plurality of fragments comprises about 4 fragments.

[0131] In some embodiments, the TILs are obtained from tumor fragments. In some embodiments, the tumor fragments are obtained by sharp abrasion. In some embodiments, the tumor fragments are about 1 mm 3 ~10mm 3 In some embodiments, the tumor fragment is about 1 mm 3 ~8mm 3 In some embodiments, the tumor fragment is about 0.5 mm 3 ~4mm 3 In some embodiments, the tumor fragment is about 1 mm 3 In some embodiments, the tumor fragment is about 2 mm 3 In some embodiments, the tumor fragment is about 3 mm3 In some embodiments, the tumor fragment is about 4 mm 3 In some embodiments, the tumor fragment is about 5 mm 3 In some embodiments, the tumor fragment is about 6 mm 3 In some embodiments, the tumor fragment is about 7 mm 3 In some embodiments, the tumor fragment is about 8 mm 3 In some embodiments, the tumor fragment is about 9 mm 3 In some embodiments, the tumor fragment is about 10 mm 3 is.

[0132] In some embodiments, the TILs are obtained from tumor digests. In some embodiments, tumor digests are generated by incubation in an enzyme medium, such as, but not limited to, RPMI 1640, 2 mM GlutaMAX, 10 mg / mL gentamicin, 30 U / mL DNase, and 1.0 mg / mL collagenase, followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, Calif.). After placing the tumor in the enzyme medium, the tumor may be mechanically dissociated for approximately 1 minute. The solution may then be incubated at 37°C in 5% CO2 for 30 minutes, after which the tumor may be mechanically dissociated again for approximately 1 minute. After another 30-minute incubation at 37°C in 5% CO2, the tumor may be mechanically dissociated a third time for approximately 1 minute. In some embodiments, after the third mechanical disruption, if large tissue fragments are present, the sample may be subjected to one or two additional mechanical dissociations, with or without an additional 30 minute incubation in 5% CO at 37° C. In some embodiments, if the cell suspension contains a large number of red blood cells or dead cells at the end of the final incubation, density gradient separation using FICOLL may be performed to remove these cells.

[0133] In some embodiments, the cells may optionally be frozen or cryopreserved after collection of the sample, and may be cryopreserved before entering the proliferation phase.

[0134] 1. Summary of conventional TIL expansion methods In addition to the use of feeder cells, conventional methods for activating and expanding TILs employ a multi-step process that includes at least a rapid expansion protocol (REP) step preceded by a separate pre-REP step.

[0135] a. First expansion step in conventional multi-step TIL production: pre-REP A conventional multi-step TIL production process begins with pre-REP or first expansion. Generally, pre-REP begins with a fragmented and / or enzymatically digested tumor sample, to which IL-2 is added to induce cytokine-induced slow TIL growth within the tumor sample. Generally, IL-2 is the only cytokine, or immunomodulator, added to the pre-REP. The pre-REP or first expansion stage can take anywhere from two weeks to several months. Pre-REP may begin with harvesting new TILs, which may offer additional therapeutic benefit compared to older TILs (i.e., TILs that have undergone more replication prior to administration to a subject / patient) because they can increase their replication cycle after administration to a subject / patient.

[0136] In some embodiments, during pre-REP, tumor tissue or cells derived from tumor tissue are grown in standard synthetic medium (including, but not limited to, RPMI) and treated with reagents such as irradiated feeder cells and anti-CD3 antibodies to achieve a desired effect, such as increasing the number of TILs and / or enriching the population for cells containing desired cell surface markers or other structural, biochemical, or functional characteristics. Pre-REP may use laboratory-grade reagents, which facilitates the incorporation of alternative strategies for improving TIL production (with the understanding that laboratory-grade reagents will be diluted in the subsequent REP stage). Thus, in some embodiments, a TLR agonist and / or peptide or peptidomimetic of the present disclosure may be included in the medium for the pre-REP stage. The pre-REP culture may, in some embodiments, include IL-2.

[0137] In some cases, after detachment or digestion of tumor fragments, the resulting cells are cultured in medium containing IL-2 under conditions that favor the growth of TILs over tumor and other cells. Tumor digests are incubated in 2 mL wells in medium containing inactivated human AB serum and 6000 IU / mL IL-2. In some instances, 300–6000 IU / mL IL-2 is added. During pre-REP, this primary cell population is cultured for several days to several months to produce a bulk TIL population, generally approximately 1 × 10 8 of bulk TIL cells are obtained.

[0138] In some cases, during the pre-REP or first expansion stage, TIL cultures grow to small (approximately 2 mm 3 ) Start with explants of tumor fragments or 1 x 10 cells of a single-cell suspension of enzymatically digested tumor tissue. 6 Viable cells are initiated by plating in 2 ml of complete medium (RPMI 1640-based medium supplemented with 10% human serum) containing 6000 IU / ml of IL-2. The cultures are grown at a cell density of 5 x 10 5 ~2×10 6TIL cells are typically maintained at 5x10 cells / ml for 2-4 weeks until millions of TIL cells are available. Multiple independent cultures are screened by cytokine secretion assay for recognition of autologous tumor cells (if possible) and HLA-A2+ tumor cell lines. Two to six independent TIL cultures showing the highest cytokine secretion are then further cultured at a cell count of 5x10 cells / ml. 7 Cells are grown in complete medium containing 6000 IU / ml IL-2 until they reach more than 100 cells (this number is usually reached 3-6 weeks after tumor resection).

[0139] In some cases, the primary propagation of the pre-REP is carried out in a closed bioreactor, such as a G-REX-10 or G-REX-100.

[0140] If genetically modified TILs are used therapeutically, the first TIL population (also called bulk TIL population) can be genetically modified prior to a second expansion at the REP stage.

[0141] In conventional processes incorporating the pre-REP stage, the boundary between pre-REP and REP occurs after TILs undergo proliferation in the presence of IL-2 to reach the appropriate cell number required to initiate REP, or after a predetermined period of pre-REP. In various embodiments, pre-REP occurs when the number of TILs obtained reaches 1×10 or more, depending on the manufacturing protocol used. 6 , 10×10 6 , 4×10 6 or 40 x 10 6 In another embodiment, the pre-REP can be completed when the culture period reaches 3-14 days or up to 9-14 days after fragmentation. The TILs can then be directly cryopreserved for further use or transferred to the REP.

[0142] In some cases, the TILs obtained from the pre-REP or first expansion step are stored until a phenotype for selection is determined. In some cases, the TILs obtained from the first expansion are not stored and proceed directly to a second expansion or REP step. In some cases, the TILs obtained from the pre-REP step are not cryopreserved after the first expansion and before the second expansion or REP step.

[0143] b. Second and Subsequent Expansion Steps in Conventional Multi-Step TIL Production: REP In traditional multi-step TIL production, the TIL cell population is optionally expanded in number after harvest and an initial bulking process, i.e., pre-REP. This further expansion is referred to as secondary expansion, which may include an expansion process commonly referred to in the art as a rapid expansion protocol (REP). The secondary expansion, or REP, is generally carried out in a gas-permeable container using a medium containing several components, including feeder cells, a cytokine source, and an anti-CD3 antibody. Optionally, the secondary expansion, or REP, can be carried out using any TIL flask or container known to those skilled in the art and can continue for 7-14 days or longer.

[0144] In some cases, the second expansion or REP can be carried out in a gas-permeable container using methods known in the art. For example, TILs can be rapidly expanded using non-specific T cell receptor stimulation in the presence of interleukin-2 (IL-2). The non-specific T cell receptor stimulation can include, for example, an anti-CD3 antibody, such as about 30 ng / ml OKT3, a mouse monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, Calif.), or UCHT-1 (commercially available from BioLegend, San Diego, Calif., USA). During the second expansion, TILs can be expanded in vitro to further stimulate the TILs by incorporating one or more cancer antigens, including antigenic portions, e.g., epitopes, thereof, such as human leukocyte antigen A2 (HLA-A2)-binding peptides, e.g., 0.3 μM MART-1:26-35(27L) or gpl00:209-217(210M), optionally expressed from a vector, optionally in the presence of a T cell growth factor, e.g., 300 IU / mL IL-2. Other suitable antigens may include, for example, NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic portions thereof. TILs can also be rapidly expanded by restimulation with the same cancer antigen(s) pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, the TILs can be further restimulated with, for example, irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, the restimulation is performed as part of the second expansion. In some embodiments, the second expansion is performed in the presence of irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0145] Optionally, the secondary expansion or REP can be performed in a supplemented cell medium containing IL-2, OKT-3, and antigen-presenting feeder cells. Optionally, the antigen-presenting feeder cells (APCs) are PBMCs (peripheral blood mononuclear cells). Optionally, the ratio of TILs to PBMCs and / or antigen-presenting cells in the rapid expansion and / or secondary expansion is 1-25 and 1-500, respectively. Optionally, REP and / or the secondary expansion are performed in a flask containing the bulk TILs mixed with a 100-fold or 200-fold excess of inactivated feeder cells, 30 mg / mL OKT3 anti-CD3 antibody, and 3000 IU / mL IL-2 in 150 ml of medium. Medium changes (typically 1 / 2 or 1 / 3 medium changes via respiration with fresh medium) are performed until the cells are transferred to another growth chamber. Alternative growth chambers include G-REX flasks and gas-permeable containers.

[0146] In some cases, the second expansion or REP is performed, which further includes a step of selecting TILs for superior tumor reactivity. Any selection method known in the art can be used. For example, TILs can be selected for superior tumor reactivity using the method described in U.S. Patent Application Publication No. 2016 / 0010058A1, the disclosure of which is incorporated herein by reference in its entirety. Optionally, a cell viability assay can be performed after the second expansion (including the expansion referred to as REP expansion) using a standard assay known in the art. For example, a trypan blue dye exclusion assay can be performed on a sample of the bulk TILs, which selectively labels dead cells and allows for viability assessment. In some cases, TIL samples can be counted using a Cellometer K2 automated cell counter (Nexcelom Bioscience, Lawrence, Mass.) to determine viability.

[0147] Optionally, a further propagation step can be performed in addition to the second propagation.

[0148] c. feeder cells In many cases, the feeder cells used in conventional multi-step feeder cell-based TIL expansion methods are peripheral blood mononuclear cells (PBMCs) obtained from a standard whole blood unit derived from a healthy blood donor. The PBMCs are obtained using standard methods, such as FICOLL-Paque gradient separation. Generally, the allogeneic PBMCs are inactivated by either irradiation or heat treatment and used in the REP procedure. In some cases, PBMCs are considered replication-incompetent and acceptable for use in the TIL expansion procedure if the total viable cell number at day 14 is lower than the initial viable cell number transferred to culture on day 0 of the REP and / or day 0 of the secondary expansion (i.e., the start day of the secondary expansion).

[0149] Optionally, PBMCs are considered replication incompetent and acceptable for use in the TIL expansion procedures described herein if the total number of viable cells cultured in the presence of OKT3 and IL-2 does not increase on days 7 and 14 from the initial number of viable cells placed into culture on day 0 of the REP and / or day 0 of the secondary expansion (i.e., the start day of the secondary expansion). Optionally, the PBMCs are cultured in the presence of 30 ng / ml of OKT3 antibody and 3000 IU / ml of IL-2.

[0150] In some cases, the second propagation or REP procedure is performed using about 2.5 x 10 9 12.5 x 10 feeder cells 6 TIL~100×10 6 The ratio of TILs is required.

[0151] After the second expansion step or REP, cells can be harvested. In some embodiments, the TILs are harvested after one, two, three, four or more expansion steps. TILs can be harvested by any suitable aseptic method, including, for example, centrifugation. Methods for harvesting TILs are well known in the art, and any such known method can be used with this process.

[0152] 2. A novel one-step method for the expansion and activation of TILs The conventional multi-step feeder cell-dependent proliferation and activation process of TILs requires multiple steps and feeder cells, both of which make the conventional process time-consuming and expensive. Patients requiring immunotherapy using adoptive transfer of tumor-infiltrating lymphocytes (TILs) often have extremely poor prognosis, and rapid treatment with a population of expanded and differentiated TILs can be a matter of life and death.

[0153] For example, because patients who need such treatment are often in a moribund state, the period of growth is difficult, and delays may lead to death before the treatment is administered.The ability to shorten the period required for TIL growth by the method described herein provides significant advantages compared with the traditional long-term process.In addition, the TILs genetically engineered to increase effector function described herein are advantageous because of their ability to grow more rapidly, thus shortening the time for growth and their ability to kill tumor cells more effectively.

[0154] Furthermore, reliance on feeder cells poses challenges for at least several reasons. First, obtaining a viable feeder cell population is extremely difficult because the cells are harvested from 3 to 5 allogeneic donors. This heterogeneous source of feeder cells precludes standardization of their use, as each population of donor cells must be individually characterized for its ability to expand TILs. Similarly, the inherent variability in feeder cells makes the expansion of TILs using feeder cells less reproducible and less predictable. Second, when feeder cells are used, TILs cannot be manipulated in the presence of feeder cells, so they can only be manipulated or genetically modified before or after the REP phase, not during it. Third, TIL production methods involving rapid expansion protocols (REPs) cannot shorten REP because populations of TILs expanded until the feeder cells die cannot be used. Fourth, the use of feeder cells to stimulate TILs precludes the ability to wash off and / or remove the stimulatory agent. Therefore, in some cases, the pre-REP and REP stages of the conventional process are unable to produce the desired number of TILs. For at least the above three reasons, in some embodiments, it is necessary to eliminate dependency on feeder cells, which is what has been achieved in the present invention and is disclosed herein. Eliminating feeder cells allows for a high degree of control over the TIL proliferation process. For example, the TIL proliferation process can be stopped once the required number of TILs has been obtained.

[0155] In one aspect of the methods disclosed herein, the pre-REP step of conventional TIL expansion protocols is completely omitted. Surprisingly, this single expansion step allows for large numbers of TILs to be obtained in 21 days or less, without the use of a pre-REP step, i.e., a single-step TIL activation and expansion process. In some embodiments, TILs are expanded on feeder cells using a single-step REP-like process. In some embodiments, TILs are expanded in a single-step process using particles such as Dynabeads. In some embodiments, TILs are expanded in a single-step process using tetrameric antibody complexes (TACs), such as those manufactured by Stemcell. In some embodiments, TILs are expanded in a single-step process using nanomatrices, such as those manufactured by Miltenyi Biotec (Transact). In some embodiments, TILs are engineered or genetically modified during the single-step TIL expansion process.

[0156] In some embodiments, the TILs are derived from a previous failure using conventional pre-REP as described above. In certain embodiments, a pre-REP failure refers to the failure of TILs isolated from a human subject to 4x10 blasts at 23 days using the pre-REP protocol. 7 In another embodiment, a failure of pre-REP refers to the inability to expand TILs isolated from a human subject to more than 100-fold their original number. In another embodiment, a failure of pre-REP refers to the inability to expand TILs isolated from a human subject to more than 1 x 10 cells using the pre-REP protocol. 6 or 1×10 7 In certain embodiments, the methods provided herein are capable of reversing pre-REP failure, i.e., growing cells from samples that have experienced pre-REP failure.

[0157] In one aspect of the methods disclosed herein, a method of expanding a population of TILs in a dissociated tumor sample comprises culturing the dissociated tumor sample in a medium in which the TILs are contacted with a T cell receptor (TCR) agonist, a CD28 agonist, and / or a T cell stimulating cytokine. In some embodiments, the TILs are contacted with a 4-1BB agonist.

[0158] In some embodiments, the dissociated tumor sample is between 0.5 and 4 mm in size. 3 In some embodiments, the tumor fragments are between 0.5 and 1 mm in size. 3 In some embodiments, the tumor fragments are 0.5 to 1.5 mm in size. 3 In some embodiments, the tumor fragments are 1.5 to 2 mm in size. 3 In some embodiments, the tumor fragment is 2 to 2.5 mm in size. 3 In some embodiments, the tumor fragment is 2.5 to 3 mm in size. 3 In some embodiments, the tumor fragment is 3 to 3.5 mm in size. 3 In some embodiments, the tumor fragment is 3.5 to 4 mm in size. 3 In some embodiments, the dissociated tumor sample comprises digested tumor fragments.

[0159] In some embodiments, the medium is supplemented with T cell stimulatory cytokines at intervals of 1-2 days, 2-3 days, 3-4 days, 4-5 days, or 5-6 days. In some embodiments, the interval is 1 day. In some embodiments, the interval is 2 days. In some embodiments, the interval is 3 days. In some embodiments, the interval is 4 days. In some embodiments, the interval is 5 days. In some embodiments, the interval is 6 days.

[0160] In some embodiments, the final concentration of the T cell stimulatory cytokine is between 10 U / ml and 7,000 U / ml. In some embodiments, the final concentration of the T cell stimulatory cytokine is between 10 U / ml and 200 U / ml. In some embodiments, the final concentration of the T cell stimulatory cytokine is between 200 U / ml and 300 U / ml. In some embodiments, the final concentration of the T cell stimulatory cytokine is between 300 U / ml and 400 U / ml. In some embodiments, the final concentration of the T cell stimulatory cytokine is between 400 U / ml and 500 U / ml. In some embodiments, the final concentration of the T cell stimulatory cytokine is between 500 U / ml and 600 U / ml. In some embodiments, the final concentration of the T cell stimulatory cytokine is between 600 U / ml and 700 U / ml. In some embodiments, the final concentration of the T cell stimulatory cytokine is between 700 U / ml and 800 U / ml. In some embodiments, the final concentration of the T cell stimulatory cytokine is between 800 U / ml and 900 U / ml. In some embodiments, the final concentration of the T cell stimulatory cytokine is 900 U / ml to 1000 U / ml. In some embodiments, the final concentration of the T cell stimulatory cytokine is 1000 U / ml to 1500 U / ml. In some embodiments, the final concentration of the T cell stimulatory cytokine is 1500 U / ml to 2000 U / ml. In some embodiments, the final concentration of the T cell stimulatory cytokine is 2000 U / ml to 2500 U / ml. In some embodiments, the final concentration of the T cell stimulatory cytokine is 2500 U / ml to 3000 U / ml. In some embodiments, the final concentration of the T cell stimulatory cytokine is 3000 U / ml to 3500 U / ml. In some embodiments, the final concentration of the T cell stimulatory cytokine is 3500 U / ml to 4000 U / ml. In some embodiments, the final concentration of the T cell stimulatory cytokine is 4000 U / ml to 4500 U / ml. In some embodiments, the final concentration of the T cell stimulating cytokine is 4,500 U / ml to 5,000 U / ml, hi some embodiments, the final concentration of the T cell stimulating cytokine is 5,000 U / ml to 5,500 U / ml.In some embodiments, the final concentration of the T cell stimulating cytokine is 5,500 U / ml to 6,000 U / ml. In some embodiments, the final concentration of the T cell stimulating cytokine is 6,000 U / ml to 6,500 U / ml. In some embodiments, the final concentration of the T cell stimulating cytokine is 6,500 U / ml to 7,000 U / ml.

[0161] The T cell stimulating cytokine can be any cytokine effective in stimulating T cells. In some embodiments, the T cell stimulating cytokine is IL-2. In some embodiments, the methods disclosed herein further comprise contacting the dissociated tumor sample and / or population of TILs with the cytokine IL-2. In some embodiments, the TILs are contacted with the cytokine IL-2 every other day. In some embodiments, the TILs are contacted with the cytokine IL-2 at intervals of 2, 3, 4, 5, or 6 days. In some embodiments, the TILs are contacted with the cytokine IL-2 at intervals of 2 days. In some embodiments, the TILs are contacted with the cytokine IL-2 at intervals of 3 days. In some embodiments, the TILs are contacted with the cytokine IL-2 at intervals of 4 days. In some embodiments, the TILs are contacted with the cytokine IL-2 at intervals of 5 days. In some embodiments, the TILs are contacted with the cytokine IL-2 at intervals of 6 days.

[0162] In some embodiments, the final concentration of the cytokine IL-2 is 100 U / ml to 7,000 U / ml. In some embodiments, the final concentration of the cytokine IL-2 is 10 U / ml to 200 U / ml. In some embodiments, the final concentration of the cytokine IL-2 is 200 U / ml to 300 U / ml. In some embodiments, the final concentration of the cytokine IL-2 is 300 U / ml to 400 U / ml. In some embodiments, the final concentration of the cytokine IL-2 is 400 U / ml to 500 U / ml. In some embodiments, the final concentration of the cytokine IL-2 is 500 U / ml to 600 U / ml. In some embodiments, the final concentration of the cytokine IL-2 is 600 U / ml to 700 U / ml. In some embodiments, the final concentration of the cytokine IL-2 is 700 U / ml to 800 U / ml. In some embodiments, the final concentration of the cytokine IL-2 is 800 U / ml to 900 U / ml. In some embodiments, the final concentration of the cytokine IL-2 is 900 U / ml to 1000 U / ml. In some embodiments, the final concentration of the cytokine IL-2 is 1000 U / ml to 1500 U / ml. In some embodiments, the final concentration of the cytokine IL-2 is 1500 U / ml to 2000 U / ml. In some embodiments, the final concentration of the cytokine IL-2 is 2000 U / ml to 2500 U / ml. In some embodiments, the final concentration of the cytokine IL-2 is 2500 U / ml to 3000 U / ml. In some embodiments, the final concentration of the cytokine IL-2 is 3000 U / ml to 3500 U / ml. In some embodiments, the final concentration of the cytokine IL-2 is 3500 U / ml to 4000 U / ml. In some embodiments, the final concentration of the cytokine IL-2 is 4000 U / ml to 4500 U / ml. In some embodiments, the final concentration of the cytokine IL-2 is 4,500 U / ml to 5,000 U / ml, hi some embodiments, the final concentration of the cytokine IL-2 is 5,000 U / ml to 5,500 U / ml.In some embodiments, the final concentration of the cytokine IL-2 is 5,500 U / ml to 6,000 U / ml. In some embodiments, the final concentration of the cytokine IL-2 is 6,000 U / ml to 6,500 U / ml. In some embodiments, the final concentration of the cytokine IL-2 is 6,500 U / ml to 7,000 U / ml.

[0163] In some embodiments, the composition of the medium is maintained. In some embodiments, 30% to 99% of the medium is changed at intervals of 1 to 2 days, 2 to 3 days, 3 to 4 days, 4 to 5 days, or 5 to 6 days. In some embodiments, the interval is 1 day. In some embodiments, the interval is 2 days. In some embodiments, the interval is 3 days. In some embodiments, the interval is 4 days. In some embodiments, the interval is 5 days. In some embodiments, the interval is 6 days.

[0164] a. Feeder cells In some embodiments, the medium comprises feeder cells. In some embodiments, the feeder cells are peripheral blood mononuclear cells (PBMCs). In some embodiments, the feeder cells are antigen-presenting cells (APCs). In some embodiments, the feeder cells express a T cell receptor (TCR) agonist, a CD28 agonist, and / or a 4-1BB agonist. In some embodiments, the feeder cells express a 41BB agonist as described in Bartkowiak and Curran, Front Oncol, 5:117 (2015), which is incorporated herein by reference in its entirety. In some embodiments, the 4-1BB agonist is a 4-1BB ligand. In some embodiments, the T cell receptor (TCR) agonist, a CD28 agonist, and / or a 4-1BB agonist is expressed on the surface of the feeder cells. In some embodiments, the TCR agonist is a CD3 agonist. In some embodiments, the CD3 agonist is OKT3 or UCHT. In some embodiments, the CD28 agonist is CD80 or CD86. In some embodiments, the CD28 agonist is CD86. In some embodiments, the CD28 agonist is dissolved in the medium. In some embodiments, the feeder cells are APCs. In some embodiments, the APCs are K562 cells. In some embodiments, the APCs are modified to express the above proteins.

[0165] In some embodiments, the TILs are genetically modified to reduce expression of another gene. Further disclosure regarding these genetic modifications is provided below.

[0166] In some embodiments, K562 cells are engineered to express OKT3 "aAPC-OKT3." In some embodiments, the engineered cells are irradiated (e.g., with 15,000 rad) before use. In some embodiments, K562 cells are engineered to express OKT3 and CD86 "aAPC-OKT3-CD86." In some embodiments, the engineered cells are irradiated (e.g., with 15,000 rad) before use. In some embodiments, pre-REP-failed TILs are expanded with soluble activators or artificial antigen-presenting cells (aAPCs).

[0167] In some embodiments, the feeder cells are genetically modified to express the T cell stimulating cytokine. In some embodiments, the T cell stimulating cytokine that the feeder cells are genetically modified to express is IL-2. In some embodiments, the medium does not contain feeder cells.

[0168] In some embodiments, the TILs are expanded using T cell stimulating cytokines and feeder cells. In some embodiments, the TILs are expanded using T cell stimulating cytokines and without feeder cells.

[0169] b. Nanomatrix Nanomatrices of 1 μm or less than 500 nm, having a flexible matrix and stimulatory agent(s) or agonists bound thereto, can stimulate T cells. In certain embodiments, the sub-500 nm matrix, in contrast to beads or microspheres of the same size, lacks a solid surface (leading to a flexible, mobile phase). The nanomatrix resembles a mesh or net made of a flexible polymeric material. In certain embodiments, the polymeric material is dextran. In certain embodiments, the nanomatrix is ​​a plastic that provides access to the cell membrane of target cells, e.g., T cells. Thus, the nanomatrix binds to the respective targets (e.g., receptors) on the cell surface with the agonist bound to the flexible matrix, allowing optimal interaction with the binding partner due to the flexibility of the matrix. To a certain extent, the shape of the nanomatrix conforms to the target cell surface, thereby enlarging the contact surface between the nanomatrix and the target cell. The size of the matrix, between 1 and 500 nm, is too small to cause cell perturbations. That is, the nanomatrix is ​​biologically inert with respect to altering cell function. When beads or microspheres with a size of 1 μm or larger are used, such perturbations caused by direct cell / bead contact are problematic. Furthermore, preferentially, the nanomatrix is ​​biodegradable and non-toxic to cells due to its composition consisting of biodegradable polymeric materials, such as dextran polymers. As a result, the nanomatrix is ​​a completely biologically inert, yet biodegradable entity with respect to altering cell function. Therefore, there is no need to remove the nanomatrix after contact with T cells for stimulation and proliferation. Subsequent analysis, experiments, and / or clinical applications of these cells will not be affected by the presence of the nanomatrix in the activated T cell composition.

[0170] Furthermore, being soluble or colloidal, the unbound nanomatrix can be easily diluted by repeated washing steps after the T cell stimulation process to an effective concentration below the activation threshold of T cells.

[0171] The flexible matrix of the nanomatrix is ​​bound to one or more stimulatory agonists that provide activation signals(s) to T cells, thereby activating and inducing proliferation of the T cells. The agonists are molecules that can bind to cell surface structures and induce polyclonal stimulation of the cells. One example of an agent that can be bound to the flexible matrix of the nanomatrix is ​​a combination of an anti-CD3 monoclonal antibody (mAb) and a costimulatory protein, such as anti-CD28 mAb.

[0172] The nanomatrix's ability to pass through a sterile filter allows it to be added to closed cell culture systems that are equipped with or can be equipped with a sterile filter, such as cell culture bags (Miltenyi Biotec, Baxter, CellGenics), G-Rex devices (Wilson Wolf manufacturing), WAVE Bioreactors (GE Healthcare), Quantum Cell Expansion System (Terumo BCT), or CliniMACS® Prodigy (Miltenyi Biotec, see Apel et al. 2013, Chemie Ingenieur Technik 85:103-110, incorporated herein by reference in its entirety). The nanomatrix can be added to a closed cell culture system using a syringe to push the nanomatrix through the filter or a pump to pull the nanomatrix from a bag or vial (connected to a vented vial adapter) through the filter.

[0173] The contacting can be carried out in any container capable of holding cells, for example, in vitro, preferably in a sterile environment. Such a container can be, for example, a culture flask, a culture bag, a bioreactor, or any device that can be used to grow cells (e.g., the sample processing system of WO2009072003, i.e., the CliniMACS® Prodigy system, which is incorporated herein by reference in its entirety).

[0174] The nanomatrix used in the present invention can be a nanomatrix in which at least one first drug and one second drug are bound to the same flexible matrix. Contacting this type of matrix with T cells activates the T cells and induces their proliferation. The ratio of the first and second drugs bound to the same flexible matrix can be in the range of 100:1 to 1:100, preferentially 10:1 to 1:10, and most preferentially 2:1 to 1:2.

[0175] Furthermore, the nanomatrix of the present invention can be a nanomatrix in which at least one first agonist and one second agonist are bound to separate flexible matrices. Contacting a mixture of these nanomatrices with T cells activates the T cells and induces their proliferation. The ratio and / or concentration of the first agent-bound flexible matrix and the second agent-bound flexible matrix can be varied to achieve optimal stimulation results depending on the type of T cells and / or agents used. This facilitates optimization of activation conditions for specific T cell subsets by titrating different concentrations and ratios of the first agent-bound flexible matrix and the second agent-bound flexible matrix.

[0176] Nanomatrices can be prepared by a variety of methods known in the art, including solvent evaporation, phase separation, spray drying, or solvent extraction at low temperatures. The process selected should be simple, reproducible, and scalable. The resulting nanomatrix should be flowable and non-agglomerated to produce a uniform, injectable suspension. The nanomatrix should be sterile. This can be ensured, for example, by filtration, a terminal sterilization step, and / or through an aseptic process. The preparation of the nanomatrix is ​​described in Example 1.

[0177] As used herein, the terms "matrix of mobile polymer chains" and "mobile matrix" are interchangeable. The term "mobile" refers to a common and well-described characteristic of nanoparticles of organic biopolymers, such as dextran or others (see Bertholon et al., Langmuir, 2006, pp. 45485-5490, incorporated herein by reference in its entirety). Because these polymers contain mobile (mobile), preferentially highly mobile (mobile), chains, the matrix is ​​characterized by the absence of a solid surface as a binding point for the stimulant, e.g., an antibody, in stark contrast to currently used beads or microspheres, which typically have a rigid, inflexible surface. As a result, the nanomatrix, comprising a matrix of mobile polymer chains, is flexible and can adjust to the shape of the cell surface. Furthermore, the result is a nanomatrix in which the majority (i.e., more than 50%), preferentially more than 80% and more preferentially more than 90% and most preferentially more than 99% of the total volume of the nanomatrix in aqueous solution is made up of mobile polymer chains.

[0178] The contact between the nanomatrix conjugated with one or more stimulating agents and the cells to be stimulated benefits from the fact that the nanomatrix does not have a fixed, rigid or hard surface, allowing the nanomatrix to access the cell surface.In certain embodiments, the nanomatrix is ​​made of hydrophilic polymer chains, which allows the chains to be hydrated in aqueous solution and thus achieves maximum mobility.The flexible matrix is ​​the only component or at least the main component of the nanomatrix, regardless of the drug to be bound.

[0179] Agonists can be bound or coupled to the flexible matrix by various methods known in the art. The binding can be covalent or non-covalent, electrostatic or hydrophobic, and can be achieved by various binding means, including, for example, chemical, mechanical, enzymatic, or other means that allow a drug to stimulate the cells. For example, an antibody against a cell surface structure can be first bound to the matrix, or avidin or streptavidin can be bound to the matrix for binding to a biotinylated drug. The antibody against the cell surface structure can be bound to the matrix directly or indirectly, for example, via an anti-isotype antibody. Another example is the use of protein A or protein G, or other nonspecific antibody-binding molecules bound to the matrix to bind antibodies. Alternatively, the drug can be bound to the matrix by chemical means, for example, cross-linking to the matrix.

[0180] As used herein, the term "biologically inert" refers to the property of a nanomatrix that is non-toxic to living cells and, due to its small size, does not induce strong changes in cellular function through physical interaction with the cell surface, except for the triggering function of the specific ligand / receptor of the bound ligand or antibody. The nanomatrix may also be biodegradable, e.g., degraded by enzymatic activity or removed by phagocytic cells. The biodegradable material may be derived from a natural or synthetic material that degrades in biological fluids, such as cell culture medium and blood. The degradation may occur using or without enzymatic methods. The biodegradable material degrades within days, weeks, or months, depending on the environmental conditions to which it is exposed. The biodegradable material should be non-toxic and non-antigenic to living cells and humans. The degradation products should produce non-toxic by-products. An important aspect in the context of biological inertness is the fact that the nanomatrix does not induce strong changes in the structure, function, activity state, or viability of the labeled cells, i.e., does not cause perturbation of the cells and does not interfere with subsequent experiments and therapeutic applications of stimulated cells. Mechanical or chemical stimulation of the cells is reduced due to the nanomatrix's extremely small size, i.e., in the nanoscale range, and its properties of having a flexible matrix that hugs the cell surface rather than changing the shape of the cell surface or exerting strong shear forces on the cells, e.g., leading to membrane rupture.

[0181] In some embodiments, the TCR agonist and / or the CD28 agonist is linked to a nanomatrix comprising a colloidal suspension of a matrix of polymer chains, each nanomatrix having a length of 1-500 nm along its largest dimension. In some embodiments, the nanomatrix has a length of 1-50 nm along its largest dimension. In some embodiments, the nanomatrix has a length of 50-100 nm along its largest dimension. In some embodiments, the nanomatrix has a length of 100-150 nm along its largest dimension. In some embodiments, the nanomatrix has a length of 150-200 nm along its largest dimension. In some embodiments, the nanomatrix has a length of 200-250 nm along its largest dimension. In some embodiments, the nanomatrix has a length of 250-300 nm along its largest dimension. In some embodiments, the nanomatrix has a length of 300-350 nm along its largest dimension. In some embodiments, the nanomatrix has a length of 350-400 nm along its largest dimension. In some embodiments, the nanomatrix has a length of 400-450 nm in its largest dimension, hi some embodiments, the nanomatrix has a length of 450-500 nm in its largest dimension.

[0182] In some embodiments, the TCR agonist and the CD28 agonist are conjugated to the same polymer chain. In some embodiments, the TCR agonist and the CD28 agonist are conjugated to different polymer chains. In some embodiments, the TCR agonist, or fragment thereof, is conjugated to the nanomatrix at 25 μg per mg of nanomatrix. In some embodiments, the TCR agonist, or fragment thereof, is conjugated to the nanomatrix at about 5 μg to about 10 μg per mg of nanomatrix. In some embodiments, the TCR agonist, or fragment thereof, is conjugated to the nanomatrix at about 10 μg to about 15 μg per mg of nanomatrix. In some embodiments, the TCR agonist, or fragment thereof, is conjugated to the nanomatrix at about 15 μg to about 20 μg per mg of nanomatrix. In some embodiments, the TCR agonist, or fragment thereof, is conjugated to the nanomatrix at about 20 μg to about 25 μg per mg of nanomatrix. In some embodiments, the TCR agonist, or fragment thereof, is bound to the nanomatrix at about 25 μg to about 30 μg per mg of nanomatrix. In some embodiments, the TCR agonist, or fragment thereof, is bound to the nanomatrix at about 30 μg to about 35 μg per mg of nanomatrix. In some embodiments, the TCR agonist, or fragment thereof, is bound to the nanomatrix at about 35 μg to about 40 μg per mg of nanomatrix. In some embodiments, the TCR agonist, or fragment thereof, is bound to the nanomatrix at about 40 μg to about 45 μg per mg of nanomatrix. In some embodiments, the TCR agonist, or fragment thereof, is bound to the nanomatrix at about 45 μg to about 50 μg per mg of nanomatrix. In some embodiments, the TCR agonist is a CD3 agonist.

[0183] In some embodiments, the CD28 agonist or fragment thereof is bound to the nanomatrix at 25 μg per mg of nanomatrix. In some embodiments, the CD28 agonist or fragment thereof is bound to the nanomatrix at about 5 μg to about 10 μg per mg of nanomatrix. In some embodiments, the CD28 agonist or fragment thereof is bound to the nanomatrix at about 10 μg to about 15 μg per mg of nanomatrix. In some embodiments, the CD28 agonist or fragment thereof is bound to the nanomatrix at about 15 μg to about 20 μg per mg of nanomatrix. In some embodiments, the CD28 agonist or fragment thereof is bound to the nanomatrix at about 20 μg to about 25 μg per mg of nanomatrix. In some embodiments, the CD28 agonist or fragment thereof is bound to the nanomatrix at about 25 μg to about 30 μg per mg of nanomatrix. In some embodiments, the CD28 agonist or fragment thereof is bound to the nanomatrix at about 30 μg to about 35 μg per mg of nanomatrix. In some embodiments, the CD28 agonist or fragment thereof is bound to the nanomatrix at about 35 μg to about 40 μg per mg of nanomatrix. In some embodiments, the CD28 agonist or fragment thereof is bound to the nanomatrix at about 40 μg to about 45 μg per mg of nanomatrix. In some embodiments, the CD28 agonist or fragment thereof is bound to the nanomatrix at about 45 μg to about 50 μg per mg of nanomatrix.

[0184] In some embodiments, the nanomatrix further comprises magnetic, paramagnetic, or superparamagnetic nanocrystals embedded between or within the matrix of polymer chains. In some embodiments, the matrix of polymer chains comprises a dextran polymer. In some embodiments, the polymer chains are colloidal polymer chains.

[0185] In some embodiments, the ratio of the volume of nanomatrix to the volume of TILs in the dissociated tumor sample is 1:5 or greater. In some embodiments, the ratio of the volume of nanomatrix to the volume of TILs is 1:10 or greater. In some embodiments, the ratio of the volume of nanomatrix to the volume of TILs is 1:25 or greater. In some embodiments, the ratio of the volume of nanomatrix to the volume of TILs is 1:50 or greater. In some embodiments, the ratio of the volume of nanomatrix to the volume of TILs is 1:100 or greater. In some embodiments, the ratio of the volume of nanomatrix to the volume of TILs is 1:200 or greater. In some embodiments, the ratio of the volume of nanomatrix to the volume of TILs is 1:300 or greater. In some embodiments, the ratio of the volume of nanomatrix to the volume of TILs is 1:400 or greater. In some embodiments, the ratio of the volume of nanomatrix to the volume of TILs is 1:500 or greater. In some embodiments, the ratio of the volume of nanomatrix to the volume of TILs is 1:600 ​​or greater. In some embodiments, the ratio of the volume of the nanomatrix to the volume of the TIL is 1:700 or greater. In some embodiments, the ratio of the volume of the nanomatrix to the volume of the TIL is 1:800 or greater. In some embodiments, the ratio of the volume of the nanomatrix to the volume of the TIL is 1:900 or greater. In some embodiments, the ratio of the volume of the nanomatrix to the volume of the TIL is 1:1,000 or greater.

[0186] In some embodiments, the ratio of matrix to TIL numbers in the dissociated tumor sample is 1:500 or greater. In some embodiments, the ratio of matrix to TIL numbers is 1:500 to 1:750. In some embodiments, the ratio of matrix to TIL numbers is 1:750 to 1:1,000. In some embodiments, the ratio of matrix to TIL numbers is 1:1,000 to 1:1,250. In some embodiments, the ratio of matrix to TIL numbers is 1:1,250 to 1:1,500. In some embodiments, the ratio of matrix to TIL numbers is 1:1,500 to 1:1,750. In some embodiments, the ratio of matrix to TIL numbers is 1:1,750 to 1:2,000. In some embodiments, the ratio of matrix to TIL numbers is 1:2,000 to 1:2,250. In some embodiments, the ratio of the number of matrix to TILs is 1:2,250 to 1:2,500. In some embodiments, the ratio of the number of matrix to TILs is 1:2,500 to 1:2,750. In some embodiments, the ratio of the number of matrix to TILs is 1:2,750 to 1:3,000. In some embodiments, the ratio of the number of matrix to TILs is 1:3,000 to 1:3,500. In some embodiments, the ratio of the number of matrix to TILs is 1:3,500 to 1:4,000. In some embodiments, the ratio of the number of matrix to TILs is 1:4,000 to 1:5,000.

[0187] In some embodiments, the agonist is a recombinant agonist. In some embodiments, the agonist is an antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the CD3 agonist is an OKT3 antibody or a UCHT1 antibody.

[0188] In another aspect of the methods disclosed herein, a method for expanding a population of TILs includes contacting a population of TILs with a nanomatrix comprising a colloidal suspension of a matrix of polymer chains, the matrix being bound to a CD3 agonist and a CD28 agonist, the nanomatrix providing an activation signal to the population of TILs, thereby activating and inducing proliferation of the population of TILs, each matrix having a maximum dimension of 1 to 500 nm in length, and the method does not include the use of feeder cells during the expansion of the population of TILs.

[0189] In some embodiments, the population of TILs contacted with the nanomatrix further comprises tumor cells, hi some embodiments, the population of TILs is isolated from a subject and contacted with the nanomatrix, and does not include any further proliferation processes of the population of TILs prior to contacting the population of TILs with the nanomatrix.

[0190] In some embodiments, the CD3 agonist and the CD28 agonist are conjugated to the same polymer chain. In some embodiments, the CD3 agonist and the CD28 agonist are conjugated to different polymer chains. In some embodiments, the CD3 agonist, or fragment thereof, is conjugated to the nanomatrix at 25 μg per mg of nanomatrix. In some embodiments, the CD3 agonist, or fragment thereof, is conjugated to the nanomatrix at about 5 μg to about 10 μg per mg of nanomatrix. In some embodiments, the CD3 agonist, or fragment thereof, is conjugated to the nanomatrix at about 10 μg to about 15 μg per mg of nanomatrix. In some embodiments, the CD3 agonist, or fragment thereof, is conjugated to the nanomatrix at about 15 μg to about 20 μg per mg of nanomatrix. In some embodiments, the CD3 agonist, or fragment thereof, is conjugated to the nanomatrix at about 20 μg to about 25 μg per mg of nanomatrix. In some embodiments, the CD3 agonist or fragment thereof is bound to the nanomatrix at about 25 μg to about 30 μg per mg of nanomatrix. In some embodiments, the CD3 agonist or fragment thereof is bound to the nanomatrix at about 30 μg to about 35 μg per mg of nanomatrix. In some embodiments, the CD3 agonist or fragment thereof is bound to the nanomatrix at about 35 μg to about 40 μg per mg of nanomatrix. In some embodiments, the CD3 agonist or fragment thereof is bound to the nanomatrix at about 40 μg to about 45 μg per mg of nanomatrix. In some embodiments, the CD3 agonist or fragment thereof is bound to the nanomatrix at about 45 μg to about 50 μg per mg of nanomatrix.

[0191] In some embodiments, the CD28 agonist or fragment thereof is bound to the nanomatrix at 25 μg per mg of nanomatrix. In some embodiments, the CD28 agonist or fragment thereof is bound to the nanomatrix at about 5 μg to about 10 μg per mg of nanomatrix. In some embodiments, the CD28 agonist or fragment thereof is bound to the nanomatrix at about 10 μg to about 15 μg per mg of nanomatrix. In some embodiments, the CD28 agonist or fragment thereof is bound to the nanomatrix at about 15 μg to about 20 μg per mg of nanomatrix. In some embodiments, the CD28 agonist or fragment thereof is bound to the nanomatrix at about 20 μg to about 25 μg per mg of nanomatrix. In some embodiments, the CD28 agonist or fragment thereof is bound to the nanomatrix at about 25 μg to about 30 μg per mg of nanomatrix. In some embodiments, the CD28 agonist or fragment thereof is bound to the nanomatrix at about 30 μg to about 35 μg per mg of nanomatrix. In some embodiments, the CD28 agonist or fragment thereof is bound to the nanomatrix at about 35 μg to about 40 μg per mg of nanomatrix. In some embodiments, the CD28 agonist or fragment thereof is bound to the nanomatrix at about 40 μg to about 45 μg per mg of nanomatrix. In some embodiments, the CD28 agonist or fragment thereof is bound to the nanomatrix at about 45 μg to about 50 μg per mg of nanomatrix.

[0192] In some embodiments, the nanomatrix further comprises magnetic, paramagnetic, or superparamagnetic nanocrystals embedded between or within the matrix of polymer chains. In some embodiments, the matrix of polymer chains comprises a dextran polymer. In some embodiments, the polymer chains are colloidal polymer chains.

[0193] In some embodiments, the ratio of the volume of the nanomatrix to the volume of the TIL is 1:5 or greater. In some embodiments, the ratio of the volume of the nanomatrix to the volume of the TIL is 1:10 or greater. In some embodiments, the ratio of the volume of the nanomatrix to the volume of the TIL is 1:25 or greater. In some embodiments, the ratio of the volume of the nanomatrix to the volume of the TIL is 1:50 or greater. In some embodiments, the ratio of the volume of the nanomatrix to the volume of the TIL is 1:100 or greater. In some embodiments, the ratio of the volume of the nanomatrix to the volume of the TIL is 1:200 or greater. In some embodiments, the ratio of the volume of the nanomatrix to the volume of the TIL is 1:300 or greater. In some embodiments, the ratio of the volume of the nanomatrix to the volume of the TIL is 1:400 or greater. In some embodiments, the ratio of the volume of the nanomatrix to the volume of the TIL is 1:500 or greater. In some embodiments, the ratio of the volume of the nanomatrix to the volume of the TIL is 1:600 ​​or greater. In some embodiments, the ratio of the volume of the nanomatrix to the volume of the TIL is 1:700 or greater. In some embodiments, the ratio of the volume of the nanomatrix to the volume of the TIL is 1:800 or greater. In some embodiments, the ratio of the volume of the nanomatrix to the volume of the TIL is 1:900 or greater. In some embodiments, the ratio of the volume of the nanomatrix to the volume of the TIL is 1:1,000 or greater.

[0194] In some embodiments, the ratio of the number of matrix to TILs is 1:500 or greater. In some embodiments, the ratio of the number of matrix to TILs is 1:500 to 1:750. In some embodiments, the ratio of the number of matrix to TILs is 1:750 to 1:1,000. In some embodiments, the ratio of the number of matrix to TILs is 1:1,000 to 1:1,250. In some embodiments, the ratio of the number of matrix to TILs is 1:1,250 to 1:1,500. In some embodiments, the ratio of the number of matrix to TILs is 1:1,500 to 1:1,750. In some embodiments, the ratio of the number of matrix to TILs is 1:1,750 to 1:2,000. In some embodiments, the ratio of the number of matrix to TILs is 1:2,000 to 1:2,250. In some embodiments, the ratio of the number of matrix to TILs is 1:2,250 to 1:2,500. In some embodiments, the ratio of the number of matrix to TILs is 1:2,500 to 1:2,750. In some embodiments, the ratio of the number of matrix to TILs is 1:2,750 to 1:3,000. In some embodiments, the ratio of the number of matrix to TILs is 1:3,000 to 1:3,500. In some embodiments, the ratio of the number of matrix to TILs is 1:3,500 to 1:4,000. In some embodiments, the ratio of the number of matrix to TILs is 1:4,000 to 1:5,000.

[0195] In some embodiments, the agonist is a recombinant agonist. In some embodiments, the agonist is an antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the CD3 agonist is an OKT3 antibody or a UCHT1 antibody.

[0196] C. Soluble monospecific complexes In another embodiment of the methods disclosed herein, a method of expanding a population of TILs comprises contacting the population of TILs with a composition comprising first, second, and third soluble monospecific complexes, each soluble monospecific complex comprising two antibodies or fragments thereof linked together, each antibody or fragment thereof of each soluble monospecific complex specifically binding to the same antigen on the population of TILs, the first soluble monospecific complex comprising an anti-CD3 antibody, the second soluble monospecific complex comprising an anti-CD28 antibody, and the third soluble monospecific complex comprising an anti-CD2 antibody, and the method does not comprise the use of feeder cells during the expansion of the population of TILs.

[0197] In some embodiments, the TCR agonist comprises a soluble monospecific complex comprising two linked anti-CD3 antibodies, hi some embodiments, the CD28 agonist comprises a soluble monospecific complex comprising two linked anti-CD28 antibodies.

[0198] In some embodiments, the medium comprises a CD2 agonist, hi some embodiments, the CD2 agonist comprises a soluble monospecific complex comprising two linked anti-CD2 antibodies.

[0199] In some embodiments, the soluble monospecific complex has a concentration of 0.2 to 25 μl / ml. In some embodiments, the soluble monospecific complex has a concentration of 0.2 to 1 μl / ml. In some embodiments, the soluble monospecific complex has a concentration of 1 to 2 μl / ml. In some embodiments, the soluble monospecific complex has a concentration of 2 to 5 μl / ml. In some embodiments, the soluble monospecific complex has a concentration of 5 to 10 μl / ml. In some embodiments, the soluble monospecific complex has a concentration of 10 to 15 μl / ml. In some embodiments, the soluble monospecific complex has a concentration of 15 to 20 μl / ml. In some embodiments, the soluble monospecific complex has a concentration of 20 to 25 μl / ml. In some embodiments, the soluble monospecific complex is a tetrameric antibody complex (TAC). In some embodiments, each TAC comprises two antibodies from a first animal species joined by two antibody molecules from a second species that specifically bind to the Fc portion of the antibodies from the first animal species. In some embodiments, the anti-CD3 antibodies are OKT3 or UCHT1 antibodies. In some embodiments, the soluble monospecific complexes are particularly effective in expanding central memory T cell phenotypes.

[0200] d. TIL proliferation In some embodiments, the TILs are expanded for a total of up to 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days from the time of initial tumor fragmentation or dissociation. In some embodiments, the TILs are expanded for a total of 9-25 days, 9-21 days, or 9-14 days. In some embodiments, the TILs are expanded for a total of up to 9 days. In some embodiments, the TILs are expanded for a total of up to 10 days. In some embodiments, the TILs are expanded for a total of up to 11 days. In some embodiments, the TILs are expanded for a total of up to 12 days. In some embodiments, the TILs are expanded for a total of up to 13 days. In some embodiments, the TILs are expanded for a total of up to 14 days. In some embodiments, the TILs are expanded for a total of up to 15 days. In some embodiments, the TILs are expanded for a total of up to 16 days. In some embodiments, the TILs are expanded for up to a total of 17 days. In some embodiments, the TILs are expanded for up to a total of 18 days. In some embodiments, the TILs are expanded for up to a total of 19 days. In some embodiments, the TILs are expanded for up to a total of 20 days. In some embodiments, the TILs are expanded for up to a total of 21 days. In some embodiments, the TILs are expanded for up to a total of 22 days. In some embodiments, the TILs are expanded for up to a total of 23 days. In some embodiments, the TILs are expanded for up to a total of 24 days. In some embodiments, the TILs are expanded for up to a total of 25 days.

[0201] In some embodiments, the population of TILs is expanded 500 to 500,000 fold. In some embodiments, the population of TILs is expanded 500 to 1,000 fold. In some embodiments, the population of TILs is expanded 1,000 to 2,500 fold. In some embodiments, the population of TILs is expanded 2,500 to 5,000 fold. In some embodiments, the population of TILs is expanded 5,000 to 10,000 fold. In some embodiments, the population of TILs is expanded 10,000 to 20,000 fold. In some embodiments, the population of TILs is expanded 20,000 to 30,000 fold. In some embodiments, the population of TILs is expanded 30,000 to 40,000 fold. In some embodiments, the population of TILs is expanded 40,000 to 50,000 fold. In some embodiments, the population of TILs is expanded 50,000-100,000 fold. In some embodiments, the population of TILs is expanded 100,000-150,000 fold. In some embodiments, the population of TILs is expanded 150,000-200,000 fold. In some embodiments, the population of TILs is expanded 200,000-250,000 fold. In some embodiments, the population of TILs is expanded 250,000-300,000 fold. In some embodiments, the population of TILs is expanded 300,000-350,000 fold. In some embodiments, the population of TILs is expanded 350,000-400,000 fold. In some embodiments, the population of TILs is expanded 400,000-450,000 fold. In some embodiments, the population of TILs is expanded 450,000-500,000 fold.

[0202] In some embodiments, the population of TILs is expanded from an initial population of 100 to 100,000 TILs. In some embodiments, the population of TILs is expanded from an initial population of 100 to 1,000 TILs. In some embodiments, the population of TILs is expanded from an initial population of 1,000 to 2,500 TILs. In some embodiments, the population of TILs is expanded from an initial population of 2,500 to 5,000 TILs. In some embodiments, the population of TILs is expanded from an initial population of 5,000 to 7,500 TILs. In some embodiments, the population of TILs is expanded from an initial population of 7,500 to 10,000 TILs. In some embodiments, the population of TILs is expanded from an initial population of 10,000 to 20,000 TILs. In some embodiments, the population of TILs is expanded from an initial population of 20,000 to 30,000 TILs. In some embodiments, the population of TILs is expanded from an initial population of 30,000-40,000 TILs. In some embodiments, the population of TILs is expanded from an initial population of 40,000-50,000 TILs. In some embodiments, the population of TILs is expanded from an initial population of 50,000-60,000 TILs. In some embodiments, the population of TILs is expanded from an initial population of 60,000-70,000 TILs. In some embodiments, the population of TILs is expanded from an initial population of 70,000-80,000 TILs. In some embodiments, the population of TILs is expanded from an initial population of 80,000-90,000 TILs. In some embodiments, the population of TILs is expanded from an initial population of 90,000-100,000 TILs.

[0203] In some embodiments, the population of TILs is expanded at least 150-fold at 10 days of expansion. In some embodiments, the population of TILs is expanded at least 500-fold at 10 days of expansion. In some embodiments, the population of TILs is expanded at least 750-fold at 10 days of expansion. In some embodiments, the population of TILs is expanded at least 1000-fold at 10 days of expansion. In some embodiments, the population of TILs is expanded at least 1500-fold at 10 days of expansion. In some embodiments, the population of TILs is expanded at least 2000-fold at 10 days of expansion. In some embodiments, the population of TILs is expanded at least 2500-fold at 10 days of expansion. In some embodiments, the population of TILs is expanded at least 3000-fold at 10 days of expansion. In some embodiments, the population of TILs is expanded at least 4000-fold at 10 days of expansion. In some embodiments, the population of TILs is expanded at least 5000-fold at day 10 of expansion. In some embodiments, the population of TILs is expanded at least 6000-fold at day 10 of expansion. In some embodiments, the population of TILs is expanded at least 7000-fold at day 10 of expansion. In some embodiments, the population of TILs is expanded at least 8000-fold at day 10 of expansion. In some embodiments, the population of TILs is expanded at least 9000-fold at day 10 of expansion. In some embodiments, the population of TILs is expanded at least 10,000-fold at day 10 of expansion. In some embodiments, these fold expansions at day 10 occur in TILs derived from failure of pre-REP.

[0204] In some embodiments, the population of TILs is expanded at least 1,500-fold at 14 days of expansion. In some embodiments, the population of TILs is expanded at least 5,000-fold at 14 days of expansion. In some embodiments, the population of TILs is expanded at least 7,500-fold at 14 days of expansion. In some embodiments, the population of TILs is expanded at least 10,000-fold at 14 days of expansion. In some embodiments, the population of TILs is expanded at least 15,000-fold at 14 days of expansion. In some embodiments, the population of TILs is expanded at least 20,000-fold at 14 days of expansion. In some embodiments, the population of TILs is expanded at least 25,000-fold at 14 days of expansion. In some embodiments, the population of TILs is expanded at least 30,000-fold at 14 days of expansion. In some embodiments, the population of TILs is expanded at least 40,000-fold at day 14 of expansion. In some embodiments, the population of TILs is expanded at least 50,000-fold at day 14 of expansion. In some embodiments, the population of TILs is expanded at least 60,000-fold at day 14 of expansion. In some embodiments, the population of TILs is expanded at least 70,000-fold at day 14 of expansion. In some embodiments, the population of TILs is expanded at least 80,000-fold at day 14 of expansion. In some embodiments, the population of TILs is expanded at least 90,000-fold at day 14 of expansion. In some embodiments, the population of TILs is expanded at least 100,000-fold at day 14 of expansion. In some embodiments, the population of TILs is expanded at least 110,000-fold at day 14 of expansion. In some embodiments, the population of TILs is expanded by at least 120,000-fold at day 14 of expansion. In some embodiments, the population of TILs is expanded by at least 130,000-fold at day 14 of expansion. In some embodiments, the population of TILs is expanded by at least 140,000-fold at day 14 of expansion.In some embodiments, these fold expansions at day 14 occur in TILs derived from failure of pre-REP.

[0205] In some embodiments, the population of TILs is expanded up to 150,000-fold at day 14 of expansion. In some embodiments, the population of TILs is expanded up to 5,000-fold at day 14 of expansion. In some embodiments, the population of TILs is expanded up to 7,500-fold at day 14 of expansion. In some embodiments, the population of TILs is expanded up to 10,000-fold at day 14 of expansion. In some embodiments, the population of TILs is expanded up to 15,000-fold at day 14 of expansion. In some embodiments, the population of TILs is expanded up to 20,000-fold at day 14 of expansion. In some embodiments, the population of TILs is expanded up to 25,000-fold at day 14 of expansion. In some embodiments, the population of TILs is expanded up to 30,000-fold at day 14 of expansion. In some embodiments, the population of TILs is expanded up to 40,000-fold at 14 days of expansion. In some embodiments, the population of TILs is expanded up to 50,000-fold at 14 days of expansion. In some embodiments, the population of TILs is expanded up to 60,000-fold at 14 days of expansion. In some embodiments, the population of TILs is expanded up to 70,000-fold at 14 days of expansion. In some embodiments, the population of TILs is expanded up to 80,000-fold at 14 days of expansion. In some embodiments, the population of TILs is expanded up to 90,000-fold at 14 days of expansion. In some embodiments, the population of TILs is expanded up to 100,000-fold at 14 days of expansion. In some embodiments, the population of TILs is expanded up to 110,000-fold at 14 days of expansion. In some embodiments, the population of TILs is expanded by up to 120,000-fold at day 14 of expansion. In some embodiments, the population of TILs is expanded by up to 130,000-fold at day 14 of expansion. In some embodiments, the population of TILs is expanded by up to 140,000-fold at day 14 of expansion. In some embodiments, these fold expansions at day 14 occur in TILs derived from failure of pre-REP.

[0206] In some embodiments, the population of TILs is expanded at least 15,000-fold at day 21 of expansion. In some embodiments, the population of TILs is expanded at least 20,000-fold at day 21 of expansion. In some embodiments, the population of TILs is expanded at least 25,000-fold at day 21 of expansion. In some embodiments, the population of TILs is expanded at least 30,000-fold at day 21 of expansion. In some embodiments, the population of TILs is expanded at least 40,000-fold at day 21 of expansion. In some embodiments, the population of TILs is expanded at least 50,000-fold at day 21 of expansion. In some embodiments, the population of TILs is expanded at least 60,000-fold at day 21 of expansion. In some embodiments, the population of TILs is expanded at least 70,000-fold at day 21 of expansion. In some embodiments, the population of TILs is expanded at least 80,000-fold at day 21 of expansion. In some embodiments, the population of TILs is expanded at least 90,000-fold at day 21 of expansion. In some embodiments, the population of TILs is expanded at least 100,000-fold at day 21 of expansion. In some embodiments, the population of TILs is expanded at least 110,000-fold at day 21 of expansion. In some embodiments, the population of TILs is expanded at least 120,000-fold at day 21 of expansion. In some embodiments, the population of TILs is expanded at least 130,000-fold at day 21 of expansion. In some embodiments, the population of TILs is expanded at least 140,000-fold at day 21 of expansion. In some embodiments, the population of TILs is expanded at least 150,000-fold at day 21 of expansion. In some embodiments, the population of TILs is expanded by at least 200,000-fold at day 21 of expansion. In some embodiments, the population of TILs is expanded by at least 300,000-fold at day 21 of expansion. In some embodiments, the population of TILs is expanded by at least 400,000-fold at day 21 of expansion.In some embodiments, these fold expansions at day 21 occur in TILs derived from failure of pre-REP.

[0207] In some embodiments, the population of TILs is expanded up to 500,000-fold at day 21 of expansion. In some embodiments, the population of TILs is expanded up to 20,000-fold at day 21 of expansion. In some embodiments, the population of TILs is expanded up to 25,000-fold at day 21 of expansion. In some embodiments, the population of TILs is expanded up to 30,000-fold at day 21 of expansion. In some embodiments, the population of TILs is expanded up to 40,000-fold at day 21 of expansion. In some embodiments, the population of TILs is expanded up to 50,000-fold at day 21 of expansion. In some embodiments, the population of TILs is expanded up to 60,000-fold at day 21 of expansion. In some embodiments, the population of TILs is expanded up to 70,000-fold at day 21 of expansion. In some embodiments, the population of TILs is expanded up to 80,000-fold at 21 days of expansion. In some embodiments, the population of TILs is expanded up to 90,000-fold at 21 days of expansion. In some embodiments, the population of TILs is expanded up to 100,000-fold at 21 days of expansion. In some embodiments, the population of TILs is expanded up to 110,000-fold at 21 days of expansion. In some embodiments, the population of TILs is expanded up to 120,000-fold at 21 days of expansion. In some embodiments, the population of TILs is expanded up to 130,000-fold at 21 days of expansion. In some embodiments, the population of TILs is expanded up to 140,000-fold at 21 days of expansion. In some embodiments, the population of TILs is expanded up to 150,000-fold at 21 days of expansion. In some embodiments, the population of TILs is expanded by up to 200,000-fold at day 21 of expansion. In some embodiments, the population of TILs is expanded by up to 300,000-fold at day 21 of expansion. In some embodiments, the population of TILs is expanded by up to 400,000-fold at day 21 of expansion. In some embodiments, these fold expansions at day 21 occur in TILs derived from failure of pre-REP.

[0208] In some embodiments, members of the population of TILs are genetically modified. In some embodiments, the population of TILs is genetically modified using an RNA-guided nuclease. In some embodiments, the population of TILs is genetically modified using Cas9 and at least one guide RNA. In some embodiments, members of the population of TILs are epigenetically modified.

[0209] In some embodiments, the population of TILs is expanded to produce a population of expanded TILs in which at least 2% of the expanded population have a central memory T cell phenotype. In some embodiments, the population of TILs is expanded to produce a population of expanded TILs in which at least 3% of the expanded population have a central memory T cell phenotype. In some embodiments, the population of TILs is expanded to produce a population of expanded TILs in which at least 4% of the expanded population have a central memory T cell phenotype. In some embodiments, the population of TILs is expanded to produce a population of expanded TILs in which at least 5% of the expanded population have a central memory T cell phenotype. In some embodiments, the population of TILs is expanded to produce a population of expanded TILs in which at least 6% of the expanded population have a central memory T cell phenotype. In some embodiments, the population of TILs is expanded to produce a population of expanded TILs in which at least 7% of the expanded population have a central memory T cell phenotype. In some embodiments, the population of TILs is expanded to produce a population of expanded TILs in which at least 8% of the expanded population have a central memory T cell phenotype. In some embodiments, the population of TILs is expanded to produce a population of expanded TILs in which at least 9% of the expanded population have a central memory T cell phenotype. In some embodiments, the population of TILs is expanded to produce a population of expanded TILs in which at least 10% of the expanded population have a central memory T cell phenotype. In some embodiments, the population of TILs is expanded to produce a population of expanded TILs in which at least 11% of the expanded population have a central memory T cell phenotype. In some embodiments, the population of TILs is expanded to produce a population of expanded TILs in which at least 12% of the expanded population have a central memory T cell phenotype. In some embodiments, the population of TILs is expanded to produce a population of expanded TILs in which at least 13% of the expanded population have a central memory T cell phenotype. In some embodiments, the population of TILs is expanded to produce a population of expanded TILs, wherein at least 14% of the expanded population has a central memory T cell phenotype.In some embodiments, the population of TILs is expanded to produce a population of expanded TILs, wherein at least 15% of the expanded population has a central memory T cell phenotype.

[0210] In some embodiments, the population of TILs is expanded to produce a population of expanded TILs in which, at 14 days after expansion, 5-50% of the expanded population have a central memory T cell phenotype. In some embodiments, the population of TILs is expanded to produce a population of expanded TILs in which, at 14 days after expansion, 10-25% of the expanded population have a central memory T cell phenotype. In some embodiments, the population of TILs is expanded to produce a population of expanded TILs in which, at 14 days after expansion, 5-10% of the expanded population have a central memory T cell phenotype. In some embodiments, the population of TILs is expanded to produce a population of expanded TILs in which, at 14 days after expansion, 10-15% of the expanded population have a central memory T cell phenotype. In some embodiments, the population of TILs is expanded to produce a population of expanded TILs in which, at 14 days after expansion, 15-20% of the expanded population have a central memory T cell phenotype. In some embodiments, the population of TILs is expanded to produce a population of expanded TILs in which, at 14 days of expansion, 20-25% of the expanded population have a central memory T cell phenotype. In some embodiments, the population of TILs is expanded to produce a population of expanded TILs in which, at 14 days of expansion, 25-30% of the expanded population have a central memory T cell phenotype. In some embodiments, the population of TILs is expanded to produce a population of expanded TILs in which, at 14 days of expansion, 30-35% of the expanded population have a central memory T cell phenotype. In some embodiments, the population of TILs is expanded to produce a population of expanded TILs in which, at 14 days of expansion, 35-40% of the expanded population have a central memory T cell phenotype. In some embodiments, the population of TILs is expanded to produce a population of expanded TILs in which, at 14 days of expansion, 40-45% of the expanded population have a central memory T cell phenotype. In some embodiments, the population of TILs is expanded to produce a population of expanded TILs in which 45-50% of the expanded population has a central memory T cell phenotype at 14 days of expansion.

[0211] In some embodiments, the population of TILs is expanded to produce an expanded population of TILs with increased abundance of CD8+ cells. In some embodiments, the population of TILs is enriched by 10% after expansion compared to the starting population of TILs. In some embodiments, the population of TILs is enriched by 20% after expansion compared to the starting population of TILs. In some embodiments, the population of TILs is enriched by 30% after expansion compared to the starting population of TILs. In some embodiments, the population of TILs is enriched by 40% after expansion compared to the starting population of TILs. In some embodiments, the population of TILs is enriched by 50% after expansion compared to the starting population of TILs. In some embodiments, the population of TILs is enriched by 60% after expansion compared to the starting population of TILs. In some embodiments, the population of TILs is enriched by 70% after expansion compared to the starting population of TILs. In some embodiments, the population of TILs is enriched by 80% after expansion compared to the starting population of TILs. In some embodiments, the population of TILs is enriched by 90% after expansion compared to the starting population of TILs. In some embodiments, the population of TILs is 100% enriched after expansion compared to the starting population of TILs.

[0212] In another aspect, the invention disclosed herein relates to a composition comprising a population of expanded TILs produced by any of the methods disclosed herein.

[0213] B. Phenotypic characteristics of expanded TILs Optionally, the expanded TILs are analyzed for expression of a number of phenotypic markers, including those described herein. Optionally, the markers are selected from TCRα / β, CD57, CD28, CD4, CD27, CD56, CD8a, CD45RA, CD45RO, CD8a, CCR7, CD4, CD3, CD38, and HLA-DR. Optionally, expression of one or more from the group of regulatory markers, i.e., CD137, CD8a, Lag3, CD4, CD3, PD-1, TIM-3, CD69, CD8a, TIGIT, CD4, CD3, KLRG1, and CD154, is measured.

[0214] In some cases, the memory marker is CCR7 or CD62L. Optionally, restimulated TILs can also be evaluated for cytokine release using a cytokine release assay. Optionally, TILs can be evaluated for interferon-gamma (IFN-gamma) secretion in response to stimulation with OKT3 or co-culture with autologous tumor digest.

[0215] In some cases, the TILs are assessed for various regulatory markers, such as TCRα / β, CD56, CD27, CD28, CD57, CD45RA, CD45RO, CD25, CD127, CD95, IL-2R, CCR7, CD62L, KLRG1, and CD122.

[0216] C. Genetic modification of TILs In some cases, the TILs are genetically engineered to include additional functionality, including, but not limited to, a high-affinity T cell receptor (TCR), e.g., a TCR that targets a tumor-associated antigen such as MAGE-1, HER2, or NY-ESO-1, or a chimeric antigen receptor (CAR) that binds to a tumor-associated cell surface molecule (e.g., mesothelin) or a lineage-restricted cell surface molecule (e.g., EGFR, CD19, or HER2).

[0217] In some embodiments, the present disclosure provides engineered TILs, including TILs comprising one or more genomic modifications that result in decreased expression and / or function of one or more endogenous target genes, and immune effector cells comprising a gene regulatory system capable of decreasing the expression and / or function of one or more endogenous target genes. In some embodiments, these endogenous genes include ANKRD11, BCL2L11, BCL3, BCOR, CALM2, CBLB, CHIC2, CTLA4, DHODH, E2F8, EGR2, FLI1, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, LAG3, MAP4K, NFKBIA, NR4A3, NRP1, PBRM1, PCBP1, PDCD1, PELI1, PIK3CD, PPP2R2D, PTPN1, PTPN 2, PTPN22, PTPN6, RBM39, RC3H1, SEMA7A, SERPINA3, SETD5, SH2B3, SH2D1A, SMAD2, SOCS1, TANK, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TNIP1, TRAF6, UMPS, WDR6, and ZC3H12A (see International Publication Nos. WO2019 / 178422, WO2019 / 178420, and WO2019 / 178421, which are incorporated by reference in their entireties). In some embodiments, these genes include SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, NFKBIA. In some embodiments, these genes include SOCS1 and at least one, two, or more genes selected from PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA.

[0218] As used herein, the term "modified TIL" encompasses TILs that contain one or more genomic modifications that arise by non-natural means that result in reduced expression and / or function of one or more endogenous target genes, as well as TILs that contain non-naturally occurring gene regulatory systems that are capable of reducing the expression and / or function of one or more endogenous target genes. The term "modified TIL" is used interchangeably with the term "engineered TIL" or "eTIL™."

[0219] As used herein, "unmodified TILs" or "control TILs" refers to a cell or population of cells whose genome has not been modified using exogenous means and which does not contain an exogenous gene regulatory system or which contains a control gene regulatory system (e.g., an empty vector control, a non-targeting gRNA, a scrambled siRNA, etc.). Exemplary modifications that can be made to TILs are set forth in International Publication Nos. WO 2019 / 178422, WO 2019 / 178420, and WO 2019 / 178421, which are incorporated by reference in their entireties. Naturally occurring TILs with reduced expression and / or function of one or more endogenous genes are included in the term unmodified TILs or control TILs.

[0220] Without wishing to be bound by theory, TILs are believed to have increased specificity for tumor antigens (Radvanyi et al., 2012 Clin Canc Res 18:6758-6770, incorporated herein by reference in its entirety) and therefore are believed to be able to mediate tumor antigen-specific immune responses (e.g., activation, proliferation, and cytotoxic activity against cancer cells), resulting in the destruction of cancer cells without the introduction of exogenous engineered receptors (Brudno et al., 2018 Nat Rev Clin Onc 15:31-46, incorporated herein by reference in its entirety). Thus, in some embodiments, TILs are isolated from a subject's tumor, expanded ex vivo, and reinfused into the subject. In some embodiments, TILs are modified to express one or more exogenous receptors specific for self-tumor antigens, expanded ex vivo, and reinfused into the subject. Such an embodiment can be embodied using an in vivo mouse model in which mice are implanted with a cancer cell line expressing a cancer antigen (e.g., CD19) and treated with engineered T cells expressing an exogenous receptor specific for the cancer antigen.

[0221] In some embodiments, the modified TIL comprises one or more modifications (e.g., one or more nucleic acid insertions, deletions, or mutations) in the genomic DNA sequence of an endogenous target gene that reduce expression and / or function of the endogenous gene. Such modifications are referred to herein as "inactivating mutations," and an endogenous gene comprising an inactivating mutation is referred to as a "modified endogenous target gene." In some embodiments, the inactivating mutation reduces or inhibits transcription of mRNA, thereby reducing expression levels of the encoded mRNA transcript and protein. In some embodiments, the inactivating mutation reduces or inhibits translation of mRNA, thereby reducing expression levels of the encoded protein. In some embodiments, the inactivating mutation encodes a modified endogenous protein (e.g., a dominant-negative mutant, as described below) that has reduced or altered function compared to the unmodified (i.e., wild-type) form of the endogenous protein. Exemplary modifications that can be made to TILs are set forth in International Publication Nos. WO2019 / 178422, WO2019 / 178420, and WO2019 / 178421, which are incorporated by reference in their entireties. In some embodiments, the modified TILs comprise at least one, two, or more modified endogenous target genes selected from SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA. In some embodiments, the modified TILs comprise modified endogenous target genes SOCS1 and at least one, two, or more modified endogenous target genes selected from PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA.

[0222] In some embodiments, the modified TILs comprise one or more genomic modifications at a genomic location other than the endogenous target gene that reduce the expression and / or function of the endogenous target gene or that result in the expression of a modified endogenous protein. For example, in some embodiments, a polynucleotide sequence encoding a gene regulatory system is inserted at one or more locations within the genome, such that upon expression of the gene regulatory system, the expression and / or function of the endogenous target gene is reduced. In some embodiments, a polynucleotide sequence encoding a modified endogenous protein is inserted at one or more locations within the genome, such that the function of the modified protein is reduced compared to the unmodified or wild-type protein (e.g., a dominant-negative mutant, as described below).

[0223] In some embodiments, the modified TILs described herein comprise one or more modified endogenous target genes, and the one or more modifications result in reduced expression and / or function of the gene product (i.e., mRNA transcript or protein) encoded by the endogenous target gene(s) compared to unmodified TILs. For example, in some embodiments, the modified TILs exhibit reduced expression of mRNA transcripts and / or reduced protein expression. In some embodiments, the expression of the gene product in the modified TILs is reduced by at least 5% compared to the expression of the gene product in unmodified TILs. In some embodiments, the expression of the gene product in the modified TILs is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to the expression of the gene product in unmodified TILs. In some embodiments, the modified TILs described herein exhibit reduced expression and / or function of gene products encoded by multiple (e.g., two or more) endogenous target genes compared to the expression of the gene product in unmodified TILs. For example, in some embodiments, the modified TILs exhibit reduced expression and / or function of gene products from two, three, four, five, six, seven, eight, nine, ten, or more endogenous target genes compared to the expression of those gene products in unmodified TILs.

[0224] In some embodiments, the present disclosure provides modified TILs in which one or more endogenous target genes, or portions thereof, have been deleted (i.e., "knocked out") so that the modified TILs do not express mRNA transcripts or proteins. In some embodiments, the modified TILs comprise deletions of multiple endogenous target genes, or portions thereof. In some embodiments, the modified TILs comprise deletions of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more endogenous target genes.

[0225] In some embodiments, the modified TILs described herein comprise one or more modified endogenous target genes, wherein one or more modifications to the target DNA sequences result in the expression of proteins (e.g., "modified endogenous proteins") that have reduced or altered function compared to the function of the corresponding protein (e.g., "unmodified endogenous protein") expressed in an unmodified TIL. In some embodiments, the modified TILs described herein comprise two, three, four, five, six, seven, eight, nine, ten, or more modified endogenous target genes that encode two, three, four, five, six, seven, eight, nine, ten, or more modified endogenous proteins. In some embodiments, the modified endogenous proteins exhibit reduced or altered binding affinity for another protein expressed by the modified TIL or by another cell, reduced or altered signaling ability, reduced or altered enzymatic activity, reduced or altered DNA-binding activity, or reduced or altered ability to function as a scaffolding protein.

[0226] In some embodiments, the modified endogenous target gene comprises one or more dominant-negative mutations. As used herein, a "dominant-negative mutation" refers to a substitution, deletion, or insertion of one or more nucleotides in a target gene such that the encoded protein acts antagonistically to the protein encoded by the unmodified target gene. The mutation is dominant-negative because this negative phenotype is genetically dominant over the positive phenotype of the corresponding unmodified gene. Genes containing one or more dominant-negative mutations and the proteins encoded by them are referred to as "dominant-negative mutants," e.g., dominant-negative genes and dominant-negative proteins. In some embodiments, the dominant-negative mutant protein is encoded by an exogenous transgene inserted at one or more locations in the genome of the TIL.

[0227] Various dominant-negative mechanisms are known. Typically, the gene product of a dominant-negative mutant retains some of the functions of the unmodified gene product but lacks one or more other important functions of the unmodified gene product. This allows the dominant-negative mutant to competitively inhibit the unmodified gene product. For example, in an exemplary embodiment, a dominant-negative mutant of a transcription factor may lack a functional activation domain but retain a functional DNA-binding domain. In this example, the dominant-negative transcription factor cannot activate DNA transcription like the unmodified transcription factor, but it can indirectly inhibit gene expression by preventing the unmodified transcription factor from binding to the transcription factor binding site. As another exemplary embodiment, a dominant-negative mutant of a protein that functions as a dimer is known. A dominant-negative mutant of such a dimeric protein may retain the ability to form a dimer with the unmodified protein but may be unable to function otherwise. The dominant-negative monomer forms a heterodimer by dimerizing with the unmodified monomer, preventing the unmodified monomer from forming a functional homodimer. Dominant-negative mutations of the SOCS1 gene are known in the art and include the mouse F59D mutant (see, for example, Hanada et al., J Biol Chem, 276:44:2 (2001), 40746-40754 and Suzuki et al., J Exp Med, 193:4 (2001), 471-482) and the human F58D mutant, which were identified by sequence alignment of the amino acid sequences of human and mouse SOCS1.

[0228] In some embodiments, the modified TIL comprises a gene regulation system that can reduce the expression or function of one or more endogenous target genes. In some embodiments, the one or more target genes include ANKRD11, BCL2L11, BCL3, BCOR, CALM2, CBLB, CHIC2, CTLA4, DHODH, E2F8, EGR2, FLI1, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, LAG3, MAP4K, NFKBIA, NR4A3, NRP1, PBRM1, PCBP1, PDCD1, PELI1, PIK3CD, PPP2R2D, PTPN1, PTPN2, (See International Publication Nos. WO2019 / 178422, WO2019 / 178420, and WO2019 / 178421, which are incorporated by reference in their entireties.) In some embodiments, the modified TILs described herein comprise a gene regulatory system capable of reducing the expression and / or function of one or more endogenous target genes selected from SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA. In some embodiments, the modified TILs described herein comprise a gene regulatory system capable of reducing the expression and / or function of one or more endogenous target genes selected from SOCS1 and at least one, two or more modified endogenous target genes selected from PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA.

[0229] In some embodiments, the modified TILs described herein comprise a gene regulatory system capable of reducing the expression and / or function of two or more endogenous target genes, in some embodiments, the two or more target genes are ANKRD11, BCL2L11, BCL3, BCOR, CALM2, CBLB, CHIC2, CTLA4, DHODH, E2F8, EGR2, FLI1, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, LAG3, MAP4K, NFKBIA, NR4A3, NRP1, PBRM1, PCBP1, PD In some embodiments, the modified TILs described herein comprise a gene regulatory system capable of reducing the expression and / or function of two or more endogenous target genes selected from: CD1, PELI1, PIK3CD, PPP2R2D, PTPN1, PTPN2, PTPN22, PTPN6, RBM39, RC3H1, SEMA7A, SERPINA3, SETD5, SH2B3, SH2D1A, SMAD2, SOCS1, TANK, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TNIP1, TRAF6, UMPS, WDR6, and ZC3H12A. In some embodiments, the modified TILs described herein comprise a gene regulatory system capable of reducing the expression and / or function of two or more endogenous target genes selected from: SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA. In some embodiments, the modified TILs described herein comprise a gene regulatory system capable of reducing the expression and / or function of at least one, two, or more modified endogenous target genes selected from SOCS1 and PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA. The gene regulatory system can reduce the expression and / or function of the endogenous target genes by various mechanisms, such as by modifying the genomic DNA sequence of the endogenous target genes (e.g., by inserting, deleting, or mutating one or more nucleic acids in the genomic DNA sequence), by modulating the transcription of the endogenous target genes (e.g., by inhibiting or suppressing mRNA transcription), and / or by modulating the translation of the endogenous target genes (e.g., by mRNA degradation).

[0230] In some embodiments, the modified TILs described herein comprise a gene regulatory system (e.g., a nucleic acid-based gene regulatory system, a protein-based gene regulatory system, or a combination protein / nucleic acid-based gene regulatory system). In such embodiments, the gene regulatory system comprised in the modified TILs can modify one or more endogenous target genes. In some embodiments, the modified TILs described herein comprise a gene regulatory system comprising:

[0231] a. one or more nucleic acid molecules capable of reducing the expression of or altering the function of a gene product encoded by one or more endogenous target genes; b. one or more polynucleotides encoding nucleic acid molecules capable of reducing the expression of or altering the function of a gene product encoded by one or more endogenous target genes; c. one or more proteins capable of reducing the expression of or altering the function of a gene product encoded by one or more endogenous target genes; d. one or more polynucleotides encoding proteins capable of reducing the expression of or altering the function of a gene product encoded by one or more endogenous target genes; e. one or more guide RNAs (gRNAs) capable of binding to a target DNA sequence in an endogenous gene; f. one or more polynucleotides encoding one or more gRNAs capable of binding to a target DNA sequence in an endogenous gene; g. one or more site-specific modifying polypeptides capable of interacting with the gRNA and modifying a target DNA sequence in the endogenous gene; h. one or more polynucleotides encoding a site-specific modifying polypeptide capable of interacting with the gRNA and modifying a target DNA sequence in the endogenous gene; i. one or more guide DNAs (gDNAs) capable of binding to a target DNA sequence in an endogenous gene; j. one or more polynucleotides encoding one or more gDNAs capable of binding to a target DNA sequence in an endogenous gene; k. one or more site-specific modifying polypeptides capable of interacting with gDNA and modifying a target DNA sequence in an endogenous gene; l. one or more polynucleotides encoding site-specific modifying polypeptides capable of interacting with gDNA and modifying a target DNA sequence in an endogenous gene; m. one or more gRNAs capable of binding to a target mRNA sequence encoded by an endogenous gene; n. one or more polynucleotides encoding one or more gRNAs capable of binding to a target mRNA sequence encoded by an endogenous gene; o. one or more site-specific modifying polypeptides capable of interacting with the gRNA and modifying the target mRNA sequence encoded by the endogenous gene; p. one or more polynucleotides encoding a site-specific modifying polypeptide capable of interacting with the gRNA and modifying a target mRNA sequence encoded by an endogenous gene; or q. Any combination of the above.

[0232] In some embodiments, the modified TILs described herein comprise a gene regulatory system comprising: a. one or more nucleic acid molecules capable of reducing the expression and / or altering the function of a gene product encoded by one or more endogenous target genes selected from SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA; b. one or more polynucleotides encoding one or more nucleic acid molecules capable of reducing the expression and / or altering the function of a gene product encoded by one or more endogenous target genes selected from SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA; c. one or more proteins capable of reducing the expression and / or altering the function of a gene product encoded by one or more endogenous target genes selected from SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA; d. one or more polynucleotides encoding one or more proteins capable of reducing the expression and / or altering the function of a gene product encoded by one or more endogenous target genes selected from SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA; e. one or more guide RNAs (gRNAs) capable of binding to a target DNA sequence in one or more endogenous genes selected from SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA; f. one or more polynucleotides encoding one or more gRNAs capable of binding to a target DNA sequence in one or more endogenous genes selected from SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA; g. one or more site-directed modifying polypeptides capable of interacting with a gRNA and modifying a target DNA sequence in an endogenous gene selected from SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA; h. one or more polynucleotides encoding site-specific modifying polypeptides capable of interacting with a gRNA and modifying a target DNA sequence in an endogenous gene selected from SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA; i. one or more guide DNAs (gDNAs) capable of binding to target DNA sequences in two or more endogenous genes selected from SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA; j. one or more polynucleotides encoding one or more gDNAs capable of binding to target DNA sequences in two or more endogenous genes selected from SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA; k. one or more site-directed modifying polypeptides capable of interacting with gDNA and modifying a target DNA sequence in an endogenous gene selected from SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA; l. one or more polynucleotides encoding site-directed modifying polypeptides capable of interacting with gDNA and modifying a target DNA sequence in an endogenous gene selected from SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA; m. one or more gRNAs capable of binding to a target mRNA sequence encoded by one or more endogenous genes selected from SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA; n. one or more polynucleotides encoding one or more gRNAs capable of binding to target mRNA sequences encoded by two or more endogenous genes selected from SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA; o. one or more site-directed modifying polypeptides capable of interacting with the gRNA and modifying a target mRNA sequence encoded by an endogenous gene selected from SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA; p. one or more polynucleotides encoding site-directed modifying polypeptides capable of interacting with the gRNA and modifying a target mRNA sequence encoded by an endogenous gene selected from SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA; or q. Any combination of the above.

[0233] In some embodiments, the modified TILs described herein comprise a gene regulatory system comprising: a. two or more nucleic acid molecules capable of reducing the expression and / or altering the function of gene products encoded by two or more endogenous target genes selected from SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA; b. one or more polynucleotides encoding two or more nucleic acid molecules capable of reducing the expression and / or altering the function of gene products encoded by two or more endogenous target genes selected from SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA; c. two or more proteins capable of reducing the expression and / or altering the function of gene products encoded by two or more endogenous target genes selected from SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA; d. one or more polynucleotides encoding two or more proteins capable of reducing the expression and / or altering the function of gene products encoded by two or more endogenous target genes selected from SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA; e. two or more guide RNAs (gRNAs) capable of binding to target DNA sequences in two or more endogenous genes selected from SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA; f. one or more polynucleotides encoding two or more gRNAs capable of binding to target DNA sequences in two or more endogenous genes selected from SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA; g. Two or more guide DNAs (gDNAs) capable of binding to target DNA sequences in two or more endogenous genes selected from SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA; h. one or more polynucleotides encoding two or more gDNAs capable of binding to target DNA sequences in two or more endogenous genes selected from SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA; i. two or more gRNAs capable of binding to target mRNA sequences encoded by two or more endogenous genes selected from SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA; j. One or more polynucleotides encoding two or more gRNAs capable of binding to target mRNA sequences encoded by two or more endogenous genes selected from SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA; k. Any combination of the above.

[0234] In some embodiments, one, two, or more polynucleotides encoding the gene regulatory system are inserted into the genome of the TIL. In some embodiments, one or more polynucleotides encoding the gene regulatory system are expressed episomally and are not inserted into the genome of the TIL.

[0235] In some embodiments, the modified TILs described herein comprise reduced expression and / or function of one or more endogenous target genes and further comprise one or more exogenous transgenes inserted into one or more genomic loci (e.g., gene "knock-ins"). In some embodiments, the one or more exogenous transgenes encode a detectable tag, a safety switch system, a chimeric switch receptor, and / or an engineered antigen-specific receptor.

[0236] In some embodiments, the modified TILs described herein further comprise an exogenous transgene encoding a detectable tag, including, but not limited to, a FLAG tag, a polyhistidine tag (e.g., 6xHis), a SNAP tag, a Halo tag, a cMyc tag, a glutathione-S-transferase tag, avidin, an enzyme, a fluorescent protein, a luminescent protein, a chemiluminescent protein, a bioluminescent protein, and a phosphorescent protein.In some embodiments, the fluorescent protein is a blue / UV protein (such as BFP, TagBFP, mTagBFP2, Azurite, EBFP2, mKalama1, Sirius, Sapphire, and T-Sapphire), a cyan protein (such as CFP, eCFP, Cerulean, SCFP3A, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP, and mTFP1), a green protein (such as GFP, eGFP, meGFP (A208K mutation), Emerald, Superfolder GFP, monomeric Azami), or the like. Green, TagGFP2, mUKG, mWasabi, Clover, and mNeonGreen, etc.), yellow proteins (YFP, eYFP, Citrine, Venus, SYFP2, and TagYFP, etc.), orange proteins (monomeric Kusabira-Orange, mKOκ, mKO2, mOrange, and mOrange2, etc.), red proteins (RFP, mRaspberry, mCherry, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, and mRuby2, etc.), far-red proteins (mPlum, HcRed-Tandem, mKate2, mNeptune, and NirFP, etc.), near-infrared proteins (TagRFP657, IFP1.4, and iRFP, etc.), long Stokes shift proteins (mKeima Red, LSS-mKate1, LSS-mKate2, and mBeRFP, etc.), photoactivatable proteins (PA-GFP, PAmCherry1, and PATagRFP, etc.), photoconvertible proteins (Kaede(green), Kaede(red), KikGR1(green), KikGR1(red), PS-CFP2, PS-CFP2, mEos2(green), mEos2(red), mEos3.2(green), mEos3.2(red), PSmOrange, and PSmOrange, etc.), and photoswitchable proteins (Dronpa, etc.).In some embodiments, the detectable tag may be selected from AmCyan, AsRed, DsRed2, DsRed Express, E2-Crimson, HcRed, ZsGreen, ZsYellow, mCherry, mStrawberry, mOrange, mBanana, mPlum, mRasberry, tdTomato, DsRed Monomer, and / or AcGFP, all of which are available from Clontech.

[0237] In some embodiments, the modified TILs described herein further comprise an exogenous transgene encoding a safety switch system. A safety switch system (also referred to in the art as a suicide gene system) comprises an exogenous transgene encoding one or more proteins that enable the elimination of modified TILs after administration to a subject. Examples of safety switch systems are known in the art. For example, a safety switch system comprises a gene encoding a protein that converts a non-toxic prodrug into a toxic compound, such as the herpes simplex thymidine kinase (Hsv-tk) and ganciclovir (GCV) system (Hsv-tk / GCV). Hsv-tk converts non-toxic GCV into a cytotoxic compound that leads to cell apoptosis. Thus, administering GCV to a subject treated with modified TILs containing a transgene encoding the Hsv-tk protein can selectively eliminate the modified TILs while sparing endogenous TILs. See, for example, Bonini, J. Immunol. 1999, 103:111-114, which is incorporated herein by reference in its entirety. See, e.g., et al., Science, 1997, 276(5319):1719-1724; Ciceri et al., Blood, 2007, 109(11):1828-1836; Bondanza et al., Blood 2006, 107(5):1828-1836.

[0238] Further safety switch systems include genes encoding cell surface markers, allowing for ADCC-mediated removal of modified TILs by administration of a monoclonal antibody specific for the cell surface marker. In some embodiments, the cell surface marker is CD20, and the modified TILs can be removed by administration of an anti-CD20 monoclonal antibody, such as rituximab (see, e.g., Introna et al., Hum Gene Ther, 2000, 11(4):611-620; Serafini et al., Hum Gene Ther, 2004, 14, 63-76; van Meerten et al., Gene Ther, 2006, 13, 789-797, incorporated herein by reference in its entirety. A similar system using EGF-R and cetuximab or panitumumab is described in International PCT Publication No. WO2018006880, incorporated herein by reference in its entirety. An additional safety switch system includes a transgene encoding a pro-apoptotic molecule containing one or more binding sites for a chemical inducer of dimerization (CID), allowing for the removal of modified TILs by administration of a CID that induces oligomerization of the pro-apoptotic molecule and activation of the apoptotic pathway. In some embodiments, the pro-apoptotic molecule is Fas (also known as CD95) (Thomis et al., Blood, 2001, 97(5), 1249-1257, incorporated herein by reference in its entirety). In some embodiments, the pro-apoptotic molecule is caspase-9 (Straathof et al., Blood, 2005, 105(11), 4247-4254, incorporated herein by reference in its entirety).

[0239] In some embodiments, the modified TILs described herein further comprise an exogenous transgene encoding a chimeric switch receptor. A chimeric switch receptor is an engineered cell surface receptor comprising an extracellular domain derived from an endogenous cell surface receptor and a heterologous intracellular signaling domain, such that upon ligand recognition by the extracellular domain, a signaling cascade is activated that differs from that activated by the wild-type form of the cell surface receptor. In some embodiments, the chimeric switch receptor comprises the extracellular domain of an inhibitory cell surface receptor fused to an intracellular domain, which transduces an activating signal rather than the inhibitory signal normally transduced by the inhibitory cell surface receptor. In certain embodiments, an extracellular domain derived from a cell surface receptor known to inhibit TIL activation can be fused to an intracellular activation domain. Binding of the corresponding ligand activates a signaling cascade that enhances, rather than inhibits, TIL activation. For example, in some embodiments, the modified TILs described herein comprise a transgene encoding a PD1-CD28 switch receptor, in which the extracellular domain of PD1 is fused to the intracellular signaling domain of CD28 (see, e.g., Liu et al., Cancer Res 76:6 (2016), 1578-1590 and Moon et al., Molecular Therapy 22 (2014), S201, incorporated herein by reference in their entireties). In some embodiments, the modified TILs described herein comprise a transgene encoding the extracellular domain of CD200R and the intracellular signaling domain of CD28 (see, Oda et al., Blood 130:22 (2017), 2410-2419, incorporated herein by reference in their entireties). In some embodiments, the modified TILs described herein further comprise an engineered antigen-specific receptor that recognizes a protein target expressed by a target cell, such as a tumor cell or an antigen-presenting cell (APC), referred to herein as an "modified receptor-transduced cell" or "modified RE cell."The term "engineered antigen receptor" refers to a non-naturally occurring antigen-specific receptor, such as a chimeric antigen receptor (CAR) or a recombinant T cell receptor (TCR). In some embodiments, the engineered antigen receptor is a CAR comprising an extracellular antigen-binding domain fused to a cytoplasmic domain comprising a signaling domain via a hinge domain and a transmembrane domain. In some embodiments, the extracellular domain of the CAR binds to an antigen expressed by a target cell in an MHC-independent manner, leading to RE cell activation and proliferation. In some embodiments, the extracellular domain of the CAR recognizes a tag fused to an antibody or antigen-binding fragment thereof. In such embodiments, the antigen specificity of the CAR depends on the antigen specificity of the labeled antibody, such that a single CAR construct can be used to target multiple different antigens by replacing one antibody with another (see, e.g., U.S. Pat. Nos. 9,233,125 and 9,624,279; U.S. Patent Application Publication Nos. 20150238631 and 20180104354). In some embodiments, the extracellular domain of the CAR may comprise an antigen-binding fragment derived from an antibody. Antigen-binding domains useful in the present disclosure include, for example, scFvs, antibodies, antigen-binding regions of antibodies, heavy / light chain variable regions, and single-chain antibodies.

[0240] In some embodiments, the intracellular signaling domain of the CAR can be derived from a TCR complex zeta chain domain (such as a CD3ξ signaling domain), an FcγRIII domain, an FcεRI domain, or a T lymphocyte activation domain. In some embodiments, the intracellular signaling domain of the CAR further comprises a costimulatory domain, such as a 4-1BB domain, a CD28 domain, a CD40 domain, a MyD88 domain, or a CD70 domain. In some embodiments, the intracellular signaling domain of the CAR comprises two costimulatory domains, such as any two of a 4-1BB domain, a CD28 domain, a CD40 domain, a MyD88 domain, or a CD70 domain. Exemplary CAR structures and intracellular signaling domains are known in the art (see, e.g., WO2009 / 091826, US20130287748, WO2015 / 142675, WO2014 / 055657, and WO2015 / 090229, which are incorporated herein by reference).

[0241] As for CARs specific for tumor antigens, as is known in the art, examples include CD171-specific CAR (Park et al., Mol Ther (2007) 15(4):825-833), EGFRvIII-specific CAR (Morgan et al., Hum Gene Ther (2012) 23(10):1043-1053), EGF-R-specific CAR (Kobold et al., J Natl Cancer Inst (2014) 107(1):364), carbonyl dehydratase K-specific CAR (Lamers et al., Biochem Soc Trans (2016) 44(3):951-959), FR-α-specific CAR (Kershaw et al., Clin Cancer Res(2006)12(20):6106-6015), HER2-specific CAR (Ahmed et al., J Clin Oncol(2015)33(15):1688-1696, Nakazawa et al., Mol Ther(2011)19(12):2133-2143, Ahmed et al., Mol Ther(2009)17(10):1779-1787, Luo et al., Cell Res(2016)26(7):850-853, Morgan et al., Mol Ther(2010)18(4):843-851, Grada et al., Mol Ther Nucleic Acids(2013)9(2):32), CEA-specific CAR (Katz et al., Clin Cancer Res (2015) 21(14): 3149-3159), IL13Rα2-specific CAR (Brown et al., Clin Cacner Res (2015) 21(18): 4062-4072), GD2-specific CAR (Louis et al., Blood (2011) 118(23): 6050-6056, Caruana et al., Nat Med (2015) 21(5): 524-529), ErbB2-specific CAR (Wilkie et al., J Clin Immunol (2012) 32(5):1059-1070), VEGF-R-specific CARs (Chinnasamy et al., Cancer Res (2016) 22(2):436-447), FAP-specific CARs (Wang et al., Cancer Immunol Res (2014) 2(2):154-166), MSLN-specific CARs (Moon et al., Clin Cancer Res (2011) 17(14):4719-30), NKG2D-specific CARs (VanSeggelen et al., Mol Ther (2015) 23(10):1600-1610), and CD19-specific CARs (axicabtadin ciloreucel (Yescarta®) and tisagenlecleucel (Kymriah®). Clinical trials of tumor-specific CARs are reviewed in Li et al., J Hematol and See also Oncol (2018) 11(22). Exemplary CARs suitable for use in accordance with the present disclosure are listed in Table 2 below. [Table 2]

[0242] In some embodiments, the engineered antigen receptor is a recombinant TCR. The recombinant TCR comprises a TCR alpha and / or TCR beta chain isolated and cloned from a T cell population that recognizes a specific target antigen. For example, the TCR alpha and / or TCR beta genes (i.e., TRAC and TRBC) can be cloned from a T cell population isolated from an individual with a specific malignancy or from a T cell population isolated from a humanized mouse immunized with a specific tumor antigen or tumor cells. The recombinant TCR recognizes antigen through the same mechanism as its endogenous counterpart (e.g., by recognizing its cognate antigen presented in the context of major histocompatibility complex (MHC) proteins expressed on the surface of target cells). This antigen binding stimulates endogenous signaling pathways, leading to activation and proliferation of the TCR-engineered cells.

[0243] Recombinant TCRs specific for tumor antigens are known in the art, and include, for example, WT1-specific TCR (JTCR016, Juno Therapeutics, WT1-TCRc4 described in U.S. Patent Application Publication No. 20160083449), MART-1-specific TCR (DMF4T clone described in Morgan et al., Science 314 (2006) 126-129, DMF5T clone described in Johnson et al., Blood 114 (2009) 535-546, and van den Berg et al., J. Immunol. 1999, 114 (2009) 545-556). al., Mol. Ther. 23 (2015) 1541-1550), gp100-specific TCR (Johnson et al., Blood 114 (2009) 535-546), CEA-specific TCR (Parkhurst et al., Mol Ther. 19 (2011) 620-626), NY-ESO and LAGE-1-specific TCR (Robbins et al., J Clin Oncol 26 (2011) 917-924, Robbins et al., Clin Cancer Res 21 (2015) 1019-1027 and Rapoport et al., Nature Medicine 21 (2015) 914-921), and MAGE-A3-specific TCR (Morgan et al., J Immunother 36 (2013) 133-151 and Linette et al. al., Blood 122 (2013) 227-242) (see also Debets et al., Seminars in Immunology 23 (2016) 10-21).

[0244] To generate a recombinant TCR, the native TRAC (SEQ ID NO: 882) and TRBC (SEQ ID NO: 883) protein sequences are fused to the C-terminus of the variable regions of the TCR-α and TCR-β chains specific for a protein or peptide of interest. For example, the engineered TCR may recognize the NY-ESO peptide (SLLMWITQC, SEQ ID NO: 884), e.g., the 1G4 TCR or 95:LY TCR (Robbins et al., Journal of Immunology 2008 180:6116-6131). In such an exemplary embodiment, the α / β chain pair of the 1G4-TCR comprises SEQ ID NOs: 885 and 886, respectively, and the α / β chain pair of the 95:LY-TCR comprises SEQ ID NOs: 887 and 888, respectively. The recombinant TCR may recognize the MART-1 peptide (AAGIGILTV, SEQ ID NO: 889), e.g., DMF4 and DMF5 TCRs (Robbins et al., Journal of Immunology 2008 180:6116-6131). In such exemplary embodiments, the α / β chain pair of the DMF4-TCR comprises SEQ ID NOs: 890 and 891, respectively, and the α / β chain pair of the DMF5-TCR comprises SEQ ID NOs: 892 and 893, respectively. The recombinant TCR may recognize the WT-1 peptide (RMFPNAPYL, SEQ ID NO: 894), e.g., DLT TCR (Robbins et al., Journal of Immunology 2008 180:6116-6131). In such exemplary embodiments, the α / β chain pair of the high-affinity DLT-TCR comprises SEQ ID NOs: 895 and 896, respectively.

[0245] Codon-optimized DNA sequences encoding the recombinant TCR α and TCR β chain proteins can be generated to drive expression of both TCR chains stoichiometrically from a single promoter. In such embodiments, a P2A sequence (SEQ ID NO: 897) can be inserted between the DNA sequences encoding the TCR β and TCR α chains, such that the expression cassette encoding the recombinant TCR chains comprises the configuration TCR β-P2A-TCR α. In exemplary embodiments, the protein sequence of the IG4 NY-ESO-specific TCR expressed from such a cassette comprises SEQ ID NO: 898, the protein sequence of the 95:LY NY-ESO-specific TCR expressed from such a cassette comprises SEQ ID NO: 899, the protein sequence of the DMF4 MART1-specific TCR expressed from such a cassette comprises SEQ ID NO: 900, the protein sequence of the DMF5 MART1-specific TCR expressed from such a cassette comprises SEQ ID NO: 901, and the protein sequence of the DLT WT1-specific TCR expressed from such a cassette comprises SEQ ID NO: 902.

[0246] In some embodiments, the engineered antigen receptor is a cluster of differentiation molecule, e.g., CD3, CD4, CD8, CD16, CD24, CD25, CD33, CD34, CD45, CD64, CD71, CD78, CD80 (also known as B7-1), CD86 (also known as B7-2), CD96, CD116, CD117, CD123, CD133, and CD138, CD371 (also known as CLL1), tumor-associated surface antigens, e.g., 5T4, BCMA (also known as CD269 and TNFRSF17), Unknown. iProt number Q02223), carcinoembryonic antigen (CEA), carbonic anhydrase 9 (CAIX or MN / CAIX), CD19, CD20, CD22, CD30, CD40, disialogangliosides, e.g., GD2, ELF2M, ductal epithelial mucin, ephrin B2, epithelial cell adhesion molecule (EpCAM), ErbB2 (HER2 / neu), FCRL5 (UniProt number Q68SN8), FKBP11 (UniProt number Q9NYL4), glioma-associated antigen, glycosphingolipids, gp36, GPRC5D (UniProt number Q9NZD1), mut hsp70-2, intestinal carboxylesterase, IGF-I receptor, ITGA8 (UniProt number P53708), KAMP3, LAGE-1a, MAGE, mesothelin, neutrophil elastase, NKG2D, Nkp30, NY-ESO-1, PAP, prostase, prostate cancer tumor antigen-1 (PCTA-1), prostate-specific antigen (PSA), PSMA, prostein, RAGE-1, ROR1, RU1 (SFMBT1), RU2 (DCDC2), SLAMF7 (UniProt number Q9NQ25), survivin, Tag-72, and telomerase, major histocompatibility complex (MHC) molecules presenting tumor-specific peptide epitopes, tumor stromal antigens such as extra domain A (EDA) and extra domain B (EDB) of fibronectin, and the A1 domain of tenascin-C (TnCA1) and a target antigen selected from fibroblast-associated proteins (FAPs), cytokine receptors such as epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), TFGβ-R or components thereof such as endoglin, major histocompatibility complex (MHC) molecules, virus-specific surface antigens such as HIV-specific antigens (such as HIV gp120), EBV-specific antigens, CMV-specific antigens, HPV-specific antigens, Lassa virus-specific antigens, influenza virus-specific antigens, and derivatives or variants of any of these surface antigens.

[0247] In some embodiments, the present disclosure provides modified TILs comprising reduced expression and / or function of one, two, or more endogenous target genes, including, but not limited to, ANKRD11, BCL2L11, BCL3, BCOR, CALM2, CBLB, CHIC2, CTLA4, DHODH, E2F8, EGR2, FLI1, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, LAG3, MAP4K, NFKBIA, NR4A3, NRP1, PBRM1, PCBP1, PDCD1, PELI1, PIK3CD, PPP2R2D, PTPN1, and PTPN 2, PTPN22, PTPN6, RBM39, RC3H1, SEMA7A, SERPINA3, SETD5, SH2B3, SH2D1A, SMAD2, SOCS1, TANK, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TNIP1, TRAF6, UMPS, WDR6, and ZC3H12A (see International Publication Nos. WO2019 / 178422, WO2019 / 178420, and WO2019 / 178421, which are incorporated by reference in their entireties).

[0248] In some embodiments, the present disclosure provides modified TILs that comprise a gene regulatory system that comprises reduced expression and / or function of SOCS1 and PTPN2 or that is capable of reducing the expression and / or function of SOCS1 and PTPN2, and further comprise a CAR or recombinant TCR expressed on the cell surface. In some embodiments, the modified TILs comprise a gene regulatory system that comprises reduced expression and / or function of SOCS1 and PTPN2 or that is capable of reducing the expression and / or function of SOCS1 and PTPN2, and further comprise a recombinant expression vector encoding a CAR or recombinant TCR.

[0249] In some embodiments, the present disclosure provides modified TILs that comprise reduced expression and / or function of SOCS1 and ZC3H12A or a gene regulatory system capable of reducing the expression and / or function of SOCS1 and ZC3H12A, and further comprise a CAR or recombinant TCR expressed on the cell surface. In some embodiments, the modified TILs comprise reduced expression and / or function of SOCS1 and ZC3H12A or a gene regulatory system capable of reducing the expression and / or function of SOCS1 and ZC3H12A, and further comprise a recombinant expression vector encoding a CAR or recombinant TCR.

[0250] In some embodiments, the present disclosure provides modified TILs that comprise reduced expression and / or function of PTPN2 and ZC3H12A or a gene regulatory system capable of reducing the expression and / or function of PTPN2 and ZC3H12A, and further comprise a CAR or recombinant TCR expressed on the cell surface. In some embodiments, the modified TILs comprise reduced expression and / or function of PTPN2 and ZC3H12A or a gene regulatory system capable of reducing the expression and / or function of PTPN2 and ZC3H12A, and further comprise a recombinant expression vector encoding a CAR or recombinant TCR.

[0251] In some embodiments, the present disclosure provides modified TILs that comprise a gene regulatory system that comprises reduced expression and / or function of PTPN2 and CBLB or that can reduce the expression and / or function of PTPN2 and CBLB, and further comprise a CAR or recombinant TCR expressed on the cell surface. In some embodiments, the modified TILs comprise a gene regulatory system that comprises reduced expression and / or function of PTPN2 and CBLB or that can reduce the expression and / or function of PTPN2 and CBLB, and further comprise a recombinant expression vector encoding a CAR or recombinant TCR.

[0252] In some embodiments, the present disclosure provides modified TILs that comprise reduced expression and / or function of ZC3H12A and CBLB or that comprise a gene regulatory system capable of reducing the expression and / or function of ZC3H12A and CBLB, and further comprise a CAR or recombinant TCR expressed on the cell surface. In some embodiments, the modified TILs comprise reduced expression and / or function of ZC3H12A and CBLB or that comprise a gene regulatory system capable of reducing the expression and / or function of ZC3H12A and CBLB, and further comprise a recombinant expression vector encoding a CAR or recombinant TCR.

[0253] In some embodiments, the present disclosure provides modified TILs that comprise a gene regulatory system that comprises reduced expression and / or function of SOCS1 and CBLB or that can reduce the expression and / or function of SOCS1 and CBLB, and further comprise a CAR or recombinant TCR expressed on the cell surface. In some embodiments, the modified TILs comprise a gene regulatory system that comprises reduced expression and / or function of SOCS1 and CBLB or that can reduce the expression and / or function of SOCS1 and CBLB, and further comprise a recombinant expression vector encoding a CAR or recombinant TCR.

[0254] In some embodiments, the present disclosure provides modified TILs that comprise a gene regulatory system that comprises reduced expression and / or function of PTPN2 and RC3H1 or that is capable of reducing the expression and / or function of PTPN2 and RC3H1, and further comprise a CAR or recombinant TCR expressed on the cell surface. In some embodiments, the modified TILs comprise a gene regulatory system that comprises reduced expression and / or function of PTPN2 and RC3H1 or that is capable of reducing the expression and / or function of PTPN2 and RC3H1, and further comprise a recombinant expression vector encoding a CAR or recombinant TCR.

[0255] In some embodiments, the present disclosure provides modified TILs that comprise reduced expression and / or function of ZC3H12A and RC3H1 or that comprise a gene regulatory system capable of reducing the expression and / or function of ZC3H12A and RC3H1, and further comprise a CAR or recombinant TCR expressed on the cell surface. In some embodiments, the modified TILs comprise reduced expression and / or function of ZC3H12A and RC3H1 or that comprise a gene regulatory system capable of reducing the expression and / or function of ZC3H12A and RC3H1, and further comprise a recombinant expression vector encoding a CAR or recombinant TCR.

[0256] In some embodiments, the present disclosure provides modified TILs that comprise a gene regulatory system that comprises reduced expression and / or function of SOCS1 and RC3H1 or that is capable of reducing the expression and / or function of SOCS1 and RC3H1, and further comprise a CAR or recombinant TCR expressed on the cell surface. In some embodiments, the modified TILs comprise a gene regulatory system that comprises reduced expression and / or function of SOCS1 and RC3H1 or that is capable of reducing the expression and / or function of SOCS1 and RC3H1, and further comprise a recombinant expression vector encoding a CAR or recombinant TCR.

[0257] In some embodiments, the present disclosure provides modified TILs that comprise a gene regulatory system that comprises reduced expression and / or function of CBLB and RC3H1 or that is capable of reducing the expression and / or function of CBLB and RC3H1, and further comprise a CAR or recombinant TCR expressed on the cell surface. In some embodiments, the modified TILs comprise a gene regulatory system that comprises reduced expression and / or function of CBLB and RC3H1 or that is capable of reducing the expression and / or function of CBLB and RC3H1, and further comprise a recombinant expression vector encoding a CAR or recombinant TCR.

[0258] In some embodiments, the present disclosure provides modified TILs that comprise a gene regulatory system that comprises reduced expression and / or function of PTPN2 and NFKBIA or that is capable of reducing the expression and / or function of PTPN2 and NFKBIA, and further comprise a CAR or recombinant TCR expressed on the cell surface. In some embodiments, the modified TILs comprise a gene regulatory system that comprises reduced expression and / or function of PTPN2 and NFKBIA or that is capable of reducing the expression and / or function of PTPN2 and NFKBIA, and further comprise a recombinant expression vector encoding a CAR or recombinant TCR.

[0259] In some embodiments, the present disclosure provides modified TILs that comprise reduced expression and / or function of ZC3H12A and NFKBIA or that comprise a gene regulatory system capable of reducing the expression and / or function of ZC3H12A and NFKBIA, and further comprise a CAR or recombinant TCR expressed on the cell surface. In some embodiments, the modified TILs comprise reduced expression and / or function of ZC3H12A and NFKBIA or that comprise a gene regulatory system capable of reducing the expression and / or function of ZC3H12A and NFKBIA, and further comprise a recombinant expression vector encoding a CAR or recombinant TCR.

[0260] In some embodiments, the present disclosure provides modified TILs that comprise a gene regulatory system that comprises reduced expression and / or function of SOCS1 and NFKBIA or that is capable of reducing the expression and / or function of SOCS1 and NFKBIA, and further comprise a CAR or recombinant TCR expressed on the cell surface. In some embodiments, the modified TILs comprise a gene regulatory system that comprises reduced expression and / or function of SOCS1 and NFKBIA or that is capable of reducing the expression and / or function of SOCS1 and NFKBIA, and further comprise a recombinant expression vector encoding a CAR or recombinant TCR.

[0261] In some embodiments, the present disclosure provides modified TILs that comprise a gene regulatory system that comprises reduced expression and / or function of CBLB and NFKBIA or that is capable of reducing the expression and / or function of CBLB and NFKBIA, and further comprise a CAR or recombinant TCR expressed on the cell surface. In some embodiments, the modified TILs comprise a gene regulatory system that comprises reduced expression and / or function of CBLB and NFKBIA or that is capable of reducing the expression and / or function of CBLB and NFKBIA, and further comprise a recombinant expression vector encoding a CAR or recombinant TCR.

[0262] In some embodiments, the present disclosure provides modified TILs that include a gene regulatory system that includes reduced expression and / or function of RC3H1 and NFKBIA or that is capable of reducing the expression and / or function of RC3H1 and NFKBIA, and further include a CAR or recombinant TCR expressed on the cell surface. In some embodiments, the modified TILs include a gene regulatory system that includes reduced expression and / or function of RC3H1 and NFKBIA or that is capable of reducing the expression and / or function of RC3H1 and NFKBIA, and further include a recombinant expression vector encoding a CAR or recombinant TCR.

[0263] In some embodiments, the present disclosure provides modified TILs comprising a gene regulatory system capable of reducing the expression and / or function of one or more endogenous target genes, including ANKRD11, BCL2L11, BCL3, BCOR, CALM2, CBLB, CHIC2, CTLA4, DHODH, E2F8, EGR2, FLI1, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, LAG3, MAP4K, NFKBIA, NR4A3, NRP1, PBRM1, PCBP1, PDCD1, PELI1, PIK3CD, PPP2R2D, PTPN1, and PTPN 2, PTPN22, PTPN6, RBM39, RC3H1, SEMA7A, SERPINA3, SETD5, SH2B3, SH2D1A, SMAD2, SOCS1, TANK, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TNIP1, TRAF6, UMPS, WDR6, and ZC3H12A (see International Publication Nos. WO2019 / 178422, WO2019 / 178420, and WO2019 / 178421, which are incorporated by reference in their entireties).

[0264] In some embodiments, the present disclosure provides modified TILs comprising a gene regulatory system that comprises reduced expression and / or function of SOCS1 and PTPN2 or that is capable of reducing the expression and / or function of SOCS1 and PTPN2, wherein the immune effector cells are TILs.

[0265] In some embodiments, the present disclosure provides modified TILs comprising reduced expression and / or function of SOCS1 and ZC3H12A or comprising a gene regulatory system capable of reducing the expression and / or function of SOCS1 and ZC3H12A, wherein the immune effector cells are TILs.

[0266] In some embodiments, the present disclosure provides modified TILs comprising reduced expression and / or function of PTPN2 and ZC3H12A or comprising a gene regulatory system capable of reducing the expression and / or function of PTPN2 and ZC3H12A, wherein the immune effector cells are TILs.

[0267] In some embodiments, the present disclosure provides modified TILs comprising a gene regulatory system that includes reduced expression and / or function of PTPN2 and CBLB or that can reduce the expression and / or function of PTPN2 and CBLB, wherein the immune effector cells are TILs.

[0268] In some embodiments, the present disclosure provides modified TILs comprising reduced expression and / or function of ZC3H12A and CBLB or comprising a gene regulatory system capable of reducing the expression and / or function of ZC3H12A and CBLB, wherein the immune effector cells are TILs.

[0269] In some embodiments, the present disclosure provides modified TILs comprising a gene regulatory system that comprises reduced expression and / or function of SOCS1 and CBLB or that can reduce the expression and / or function of SOCS1 and CBLB, wherein the immune effector cells are TILs.

[0270] In some embodiments, the present disclosure provides modified TILs comprising reduced expression and / or function of PTPN2 and RC3H1 or comprising a gene regulatory system capable of reducing the expression and / or function of PTPN2 and RC3H1, wherein the immune effector cells are TILs.

[0271] In some embodiments, the present disclosure provides modified TILs comprising reduced expression and / or function of ZC3H12A and RC3H1 or comprising a gene regulatory system capable of reducing the expression and / or function of ZC3H12A and RC3H1, wherein the immune effector cells are TILs.

[0272] In some embodiments, the present disclosure provides modified TILs comprising a gene regulatory system that comprises reduced expression and / or function of SOCS1 and RC3H1 or that is capable of reducing the expression and / or function of SOCS1 and RC3H1, wherein the immune effector cells are TILs.

[0273] In some embodiments, the present disclosure provides modified TILs comprising reduced expression and / or function of CBLB and RC3H1 or comprising a gene regulatory system capable of reducing the expression and / or function of CBLB and RC3H1, wherein the immune effector cells are TILs.

[0274] In some embodiments, the present disclosure provides modified TILs comprising a gene regulatory system that includes reduced expression and / or function of PTPN2 and NFKBIA or that can reduce the expression and / or function of PTPN2 and NFKBIA, wherein the immune effector cells are TILs.

[0275] In some embodiments, the present disclosure provides modified TILs comprising reduced expression and / or function of ZC3H12A and NFKBIA or comprising a gene regulatory system capable of reducing the expression and / or function of ZC3H12A and NFKBIA, wherein the immune effector cells are TILs.

[0276] In some embodiments, the present disclosure provides modified TILs comprising a gene regulatory system that comprises reduced expression and / or function of SOCS1 and NFKBIA or that is capable of reducing the expression and / or function of SOCS1 and NFKBIA, wherein the immune effector cells are TILs.

[0277] In some embodiments, the present disclosure provides modified TILs comprising a gene regulatory system that includes reduced expression and / or function of CBLB and NFKBIA or that can reduce the expression and / or function of CBLB and NFKBIA, wherein the immune effector cells are TILs.

[0278] In some embodiments, the present disclosure provides modified TILs comprising a gene regulatory system that includes reduced expression and / or function of RC3H1 and NFKBIA or that can reduce the expression and / or function of RC3H1 and NFKBIA, wherein the immune effector cells are TILs.

[0279] D. Effector Function In some embodiments, the modified TILs described herein are selected from the group consisting of ANKRD11, BCL2L11, BCL3, BCOR, CALM2, CBLB, CHIC2, CTLA4, DHODH, E2F8, EGR2, FLI1, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, LAG3, MAP4K, NFKBIA, NR4A3, NRP1, PBRM1, PCBP1, PDCD1, PELI1, PIK3CD, PPP2R2D, PTPN1, PTPN2, PTPN22, PTPN6, RBM39, RC3H1, SEMA7A, SERPINA3, SET D5, SH2B3, SH2D1A, SMAD2, SOCS1, TANK, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TNIP1, TRAF6, UMPS, WDR6, and ZC3H12A (see International Publication Nos. WO2019 / 178422, WO2019 / 178420, and WO2019 / 178421, each of which is incorporated by reference in its entirety), and exhibits increased effector function of one or more immune cells.

[0280] In some embodiments, the modified TILs described herein comprise reduced expression and / or function (or a gene regulatory system capable of reducing the expression and / or function) of one or more endogenous target genes selected from SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA, and exhibit one or more increased immune cell effector functions. As used herein, the term "effector function" refers to an immune cell function associated with generating, maintaining, and / or enhancing an immune response to a target cell or target antigen. In some embodiments, the modified TILs described herein exhibit, compared to unmodified TILs, increased tumor infiltration or migration, increased proliferation, increased or prolonged cell survival, improved resistance to inhibitory factors in the surrounding microenvironment resulting in a prolonged or increased activation state of the cells, increased production of proinflammatory immune factors (e.g., inflammatory cytokines, chemokines, and / or enzymes), increased cytotoxicity, improved exhaustion resistance, and / or increased T cell proliferation. cm indicates one or more of the characteristics for which the percentage increases.

[0281] In some embodiments, the modified TILs described herein exhibit increased tumor infiltration compared to unmodified TILs. In some embodiments, increased tumor infiltration by modified TILs refers to an increase in the number of modified TILs infiltrating a tumor over a given period of time compared to the number of unmodified TILs infiltrating a tumor over the same period of time. In some embodiments, the modified TILs exhibit a 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or higher increase in tumor infiltration compared to unmodified immune cells. Tumor infiltration can be measured by isolating one or more tumors from a subject and assessing the number of modified immune cells in the sample by flow cytometry, immunohistochemistry and / or immunofluorescence.

[0282] In some embodiments, the modified TILs described herein exhibit increased cell proliferation compared to unmodified TILs. In these embodiments, the result is that after a predetermined period of time, the number of modified TILs present is increased compared to unmodified TILs. For example, in some embodiments, the modified TILs exhibit an increased proliferation rate compared to unmodified TILs, in which the modified TILs divide at a faster rate than unmodified TILs. In some embodiments, the modified TILs exhibit a 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or higher increase in proliferation rate compared to unmodified immune cells. In some embodiments, the modified TILs exhibit an extended period of proliferation compared to unmodified TILs, where the modified and unmodified TILs divide at the same rate but the modified TILs remain in a proliferative state for a longer period of time. In some embodiments, the modified TILs remain in a proliferative state for 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold or more times longer than unmodified immune cells.

[0283] In some embodiments, the modified TILs described herein exhibit increased or prolonged cell survival compared to unmodified TILs. In such embodiments, the result is that after a predetermined period of time, an increased number of modified TILs are present compared to unmodified TILs. For example, in some embodiments, the modified TILs described herein remain viable and persist for 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more times longer than unmodified immune cells.

[0284] In some embodiments, the modified TILs described herein exhibit improved resistance to suppressive factors compared to unmodified TILs. Exemplary suppressive factors include signaling by immune checkpoint molecules (e.g., PD1, PDL1, CTLA4, LAG3, IDO) and / or suppressive cytokines (e.g., IL-10, TGFβ).

[0285] In some embodiments, the modified T cells described herein exhibit increased resistance to T cell exhaustion compared to unmodified T cells. T cell exhaustion is a state of antigen-specific T cell dysfunction characterized by reduced effector function and subsequent exhaustion of antigen-specific T cells. In some embodiments, exhausted T cells lack the ability to proliferate in response to antigen, exhibit reduced cytokine production, and / or exhibit reduced cytotoxicity against target cells, such as tumor cells. In some embodiments, exhausted T cells are identified by altered expression of cell surface markers and transcription factors, e.g., decreased cell surface expression of CD122 and CD127, increased expression of inhibitory cell surface markers, e.g., PD1, LAG3, CD244, CD160, TIM3, and / or CTLA4, and / or increased expression of transcription factors, e.g., Blimp1, NFAT, and / or BATF. In some embodiments, exhausted T cells exhibit altered sensitivity to cytokine signaling, e.g., increased sensitivity to TGFβ signaling and / or decreased sensitivity to IL-7 signaling. T cell exhaustion can be determined, for example, by co-culturing the T cells with a target cell population and measuring T cell proliferation, cytokine production, and / or target cell lysis. In some embodiments, modified TILs described herein are co-cultured with a population of target cells (e.g., autologous tumor cells or an autologous cell line engineered to express a target tumor antigen), and effector cell proliferation, cytokine production, and / or target cell lysis are measured. These results are then compared to results obtained by co-culturing the target cells with a population of control immune cells (e.g., unmodified TILs, or immune effector cells with a control modification).

[0286] In some embodiments, resistance to T cell exhaustion is indicated by increased production of one or more cytokines (e.g., IFNγ, TNFα, or IL-2) from the modified TILs compared to cytokine production observed in a control immune cell population. In some embodiments, a 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or higher increase in cytokine production from the modified TILs compared to cytokine production from a control immune cell population indicates increased resistance to T cell exhaustion. In some embodiments, resistance to T cell exhaustion is indicated by increased proliferation of the modified TILs compared to proliferation observed in a control immune cell population. In some embodiments, a 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or higher increase in proliferation of the modified TILs compared to proliferation in a control immune cell population indicates increased resistance to T cell exhaustion. In some embodiments, resistance to T cell exhaustion is indicated by increased lysis of target cells by the engineered TILs compared to the lysis of target cells observed in a control immune cell population. In some embodiments, a 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or higher increase in lysis of target cells by the engineered TILs compared to the lysis of target cells by a control immune cell population indicates increased resistance to T cell exhaustion.

[0287] In some embodiments, the exhaustion of the modified TILs compared to a control immune cell population is measured during the in vitro or ex vivo manufacturing process. For example, in some embodiments, TILs isolated from tumor fragments are modified according to the methods described herein and then expanded in one or more rounds of expansion to generate a modified TIL population. In such embodiments, the exhaustion of the modified TILs can be determined immediately after collection and before the first round of expansion, after the first round of expansion and before the second round of expansion, and / or after the first and second rounds of expansion. In some embodiments, the exhaustion of the modified TILs compared to a control immune cell population is measured at one or more time points after the modified TILs are transferred into a subject. For example, in some embodiments, the modified cells are produced according to the methods described herein and administered to a subject. After transfer, samples can then be taken from the subject at various time points to determine the exhaustion of the modified TILs over time in vivo.

[0288] In some embodiments, the modified TILs described herein exhibit increased expression or production of proinflammatory immune factors compared to unmodified TILs, including cytolytic factors such as granzyme B, perforin, and granulysin, and inflammatory cytokines such as interferons (IFNα, IFNβ, IFNγ), TNFα, IL-1β, IL-12, IL-2, IL-17, CXCL8, and / or IL-6.

[0289] In some embodiments, the modified TILs described herein exhibit increased cytotoxicity against target cells compared to unmodified TILs. In some embodiments, the modified TILs exhibit a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or higher increase in cytotoxicity against target cells compared to unmodified immune cells. Assays for measuring immune effector function are known in the art. For example, tumor infiltration can be measured by isolating a tumor from a subject and determining the total number and / or phenotype of lymphocytes present in the tumor by flow cytometry, immunohistochemistry, and / or immunofluorescence. Cell surface receptor expression can be determined by flow cytometry, immunohistochemistry, immunofluorescence, Western blot, and / or qPCR. Cytokine and chemokine expression and production can be measured by flow cytometry, immunohistochemistry, immunofluorescence, Western blot, ELISA, and / or qPCR. Responsiveness or sensitivity to extracellular stimuli (e.g., cytokines, inhibitory ligands, or antigens) can be measured by assaying cell proliferation and / or activation of downstream signaling pathways (e.g., phosphorylation of downstream signaling intermediates) in response to the stimuli. Cytotoxicity can be measured by target cell lysis assays known in the art, including in vitro or ex vivo co-culture of modified TILs with target cells and in vivo mouse tumor models as described throughout the Examples.

[0290] E. Regulation of Endogenous Pathways and Genes In some embodiments, the modified TILs described herein exhibit reduced expression and / or function of one, two, or more endogenous target genes selected from SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA. Further details of the endogenous target genes are provided below in Table 3. In such embodiments, the reduced expression or function of the one, two, or more endogenous target genes enhances one or more effector functions of the immune cells.

[0291] In some embodiments, the modified TILs described herein comprise reduced expression and / or function of the cytokine signaling suppressor SOCS 1 (SOCS1) gene. The SOCS1 protein contains a C-terminal SOCS box motif, an SH2 domain, an ESS domain, and an N-terminal KIR domain. A 12-amino acid residue region, termed the kinase inhibitory region (KIR), has been shown to be essential for SOCS1's ability to negatively regulate the tyrosine kinase function of JAK1, TYK2, and JAK2.

[0292] In some embodiments, the modified TILs described herein comprise reduced expression and / or function of the PTPN2 gene. Protein tyrosine phosphatase family (PTPs) dephosphorylate phosphotyrosine residues through their phosphatase catalytic domains. PTPN2 functions as a brake on both TCR and cytokine signaling through the JAK / STAT signaling complex, thereby serving as a checkpoint for both signal 1 and signal 3. After T cells bind antigen and activate the TCR, phosphorylation of tyrosine residues results in downstream amplification of positive signals by the kinases Lck and Fyn. PTPN2 acts to dephosphorylate both Lck and Fyn, thereby attenuating TCR signaling. In addition, after T cells encounter cytokines, signal through the common gamma chain receptor complex, and transmit positive signals through JAK / STAT signaling, PTPN2 also attenuates STAT1 and STAT3 by dephosphorylating them. The overall functional impact of PTPN2 deletion on T cell function is a lowering of the activation threshold required to acutely activate T cells through the TCR and hypersensitivity to growth- and differentiation-promoting cytokines.

[0293] Furthermore, systemic deletion of PTPN2 in mice results in increased cytokine levels, lymphocytic infiltration in non-lymphoid tissues, and early signs of rheumatoid arthritis-like symptoms, and these mice only survive to 5 weeks of age. Thus, PTPN2 has been identified as essential for postnatal development in mice. Consistent with this autoimmune phenotype, deletion of Ptpn2 in the T cell lineage from birth also increases lymphocytic infiltration in non-lymphoid tissues. Importantly, inducible knockout of Ptpn2 in adult mouse T cells did not result in any autoimmune manifestations. In addition to its role in autoimmunity, Ptpn2 deletion has been identified in humans to be associated with a small proportion of T-cell acute lymphoblastic leukemia (ALL) and to enhance skin tumor growth in two-stage chemically induced carcinogenesis.

[0294] In some embodiments, the modified TILs described herein comprise reduced expression and / or function of the ZC3H12A gene. Zc3h12, also known as MCPIP1 and Regnase-1, is an RNase with an RNase domain immediately upstream of a CCCH-type zinc finger motif. Through its nuclease activity, Zc3h12a targets mRNAs of transcripts such as IL-6 and destabilizes them by binding to a conserved stem-loop structure in the 3'UTR of these genes. In T cells, Zc3h12a controls the transcription levels of several pro-inflammatory genes, including c-Rel, Ox40, and IL-2. Regnase-1 activation is transient and is subject to negative feedback mechanisms, including proteasome-mediated degradation or mucosa-associated lymphoid tissue 1 (MALT1)-mediated cleavage. The primary function of Regnase-1 is to promote mRNA degradation through its ribonuclease activity by specifically targeting a subset of genes in different cell types. In monocytes, Regnase-1 downregulates IL-6 and IL-12B mRNA, thereby attenuating inflammation. In T cells, Regnase-1 limits T cell activation by targeting c-Rel, Ox40, and IL-2 transcripts. In cancer cells, Regnase-1 promotes apoptosis by inhibiting anti-apoptotic genes, including Bcl2L1, Bcl2A1, RelB, and Bcl3.

[0295] In some embodiments, the modified TILs described herein comprise reduced expression and / or function of the CBLB gene. This gene encodes CBL-B, also known as RNF56, Nbla00127, and Cbl proto-oncogene B. CBL-B is an E3 ubiquitin-protein ligase and a member of the CBL gene family. CBL-B functions as a negative regulator of T cell activation. Expression of CBL-B in T cells results in ligand-induced downregulation of the T cell receptor, controlling the degree of T cell activation during antigen presentation. Mutations in the CBLB gene are associated with autoimmune conditions, such as type 1 diabetes.

[0296] In some embodiments, the modified TILs described herein comprise reduced expression and / or function of the RC3H1 gene. This gene encodes the RING finger and CCCH-type domain 1, also known as Roquin-1. Roquin-1 recognizes and binds to constitutive decay elements (CDEs) present in the 3'UTR of mRNAs, resulting in deadenylation and degradation of the mRNA. Alternative splicing generates multiple transcript variants.

[0297] In some embodiments, the modified TILs described herein comprise reduced expression and / or function of the NFKBIA gene. This gene encodes IκBα, also known as NFKB inhibitor alpha, MAD-3, NFKBI, and EDAID2. IκBα is a member of a family of cellular proteins that function to inhibit NF-κB transcription factors. IκBα inhibits NF-κB by masking the nuclear localization signal (NLS) of NF-κB proteins, sequestering them in the cytoplasm in an inactive state. Furthermore, IκBα blocks the ability of NF-κB transcription factors to bind to DNA, which is necessary for proper NF-κB function. The NFKBIA gene is mutated in some Hodgkin's lymphoma cells. Such mutations inactivate the IκBα protein, thereby rendering NF-κB chronically active in these tumor cells, and this activity contributes to the malignant state of these tumor cells. [Table 3]

[0298] In some embodiments, the modified TILs comprise reduced expression and / or function of any one or more of SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, or NFKBIA. In some embodiments, the modified TILs comprise reduced expression and / or function of at least one endogenous target gene selected from SOCS1, PTPN2, ZC3H12A, RC3H1, and NFKBIA, and further comprise reduced expression and / or function of CBLB. In some embodiments, the modified TILs comprise reduced expression and / or function of at least two endogenous target genes selected from SOCS1, PTPN2, ZC3H12A, RC3H1, and NFKBIA, and further comprise reduced expression and / or function of CBLB.

[0299] In some embodiments, the modified TILs comprise reduced expression and / or function of at least one endogenous target gene selected from CBLB, PTPN2, ZC3H12A, RC3H1, and NFKBIA, and further comprise reduced expression and / or function of SOCS1. In some embodiments, the modified TILs comprise reduced expression and / or function of at least two endogenous target genes selected from CBLB, PTPN2, ZC3H12A, RC3H1, and NFKBIA, and further comprise reduced expression and / or function of SOCS1.

[0300] In some embodiments, the modified TILs comprise reduced expression and / or function of at least one endogenous target gene selected from CBLB, SOCS1, ZC3H12A, RC3H1, and NFKBIA, and further comprise reduced expression and / or function of PTPN2. In some embodiments, the modified TILs comprise reduced expression and / or function of at least two endogenous target genes selected from CBLB, SOCS1, ZC3H12A, RC3H1, and NFKBIA, and further comprise reduced expression and / or function of PTPN2.

[0301] In some embodiments, the modified TILs comprise reduced expression and / or function of at least one endogenous target gene selected from CBLB, SOCS1, PTPN2, RC3H1, and NFKBIA, and further comprise reduced expression and / or function of ZC3H12A. In some embodiments, the modified TILs comprise reduced expression and / or function of at least two endogenous target genes selected from CBLB, SOCS1, PTPN2, RC3H1, and NFKBIA, and further comprise reduced expression and / or function of ZC3H12A.

[0302] In some embodiments, the modified TILs comprise reduced expression and / or function of at least one endogenous target gene selected from CBLB, SOCS1, PTPN2, ZC3H12A, and NFKBIA, and further comprise reduced expression and / or function of RC3H1. In some embodiments, the modified TILs comprise reduced expression and / or function of at least two endogenous target genes selected from CBLB, SOCS1, PTPN2, ZC3H12A, and NFKBIA, and further comprise reduced expression and / or function of RC3H1.

[0303] In some embodiments, the modified TILs comprise reduced expression and / or function of at least one endogenous target gene selected from CBLB, SOCS1, PTPN2, ZC3H12A, and RC3H1, and further comprise reduced expression and / or function of NFKBIA. In some embodiments, the modified TILs comprise reduced expression and / or function of at least two endogenous target genes selected from CBLB, SOCS1, PTPN2, ZC3H12A, and RC3H1, and further comprise reduced expression and / or function of NFKBIA.

[0304] II. Gene Regulatory Systems As used herein, the term "gene regulation system" refers to a protein, nucleic acid, or combination thereof that, when introduced into a cell, can regulate the expression or function of the encoded gene product by modifying the endogenous target DNA sequence. Many gene regulation systems suitable for use in the methods of the present disclosure are known in the art, including, but not limited to, shRNA, siRNA, zinc finger nuclease systems, TALEN systems, and CRISPR / Cas systems. In some embodiments, the gene regulation system is a gene editing system. Gene editing systems suitable for use in the methods of the present disclosure are known in the art, including, but not limited to, zinc finger nuclease systems, TALEN systems, and CRISPR / Cas systems.

[0305] As used herein, "modulate" when used in reference to the effect of a gene regulatory system on an endogenous target gene includes any change in the sequence of the endogenous target gene, any change in the epigenetic state of the endogenous target gene, and / or any change in the expression or function of the protein encoded by the endogenous target gene.

[0306] In some embodiments, the gene regulation system can mediate the change of the sequence of the endogenous target gene by introducing one or more mutations into the endogenous target sequence, for example, by inserting or deleting one or more nucleic acids in the endogenous target sequence.The exemplary mechanism that can mediate the change of the endogenous target sequence includes but is not limited to non-homologous end joining (NHEJ) (for example, classical or alternative), microhomology-mediated end joining (MMEJ), homology-directed repair (for example, endogenous donor template-mediated), SDSA (synthesis-dependent single-strand annealing), single-strand annealing or single-strand invasion.

[0307] In some embodiments, the gene regulation system can mediate changes in the epigenetic state of the endogenous target sequence. For example, in some embodiments, the gene regulation system can mediate covalent modifications of the DNA of the endogenous target gene (e.g., cytosine methylation and hydroxymethylation), or covalent modifications of associated histone proteins (e.g., lysine acetylation, lysine and arginine methylation, serine and threonine phosphorylation, and lysine ubiquitination and sumoylation).

[0308] In some embodiments, the gene regulatory system can mediate the change in expression of the protein encoded by the endogenous target gene. In such embodiments, the gene regulatory system can regulate the expression of the encoded protein by modifying the endogenous target DNA sequence or by acting on the mRNA product encoded by the DNA sequence. In some embodiments, the gene regulatory system can express a modified endogenous protein. In such embodiments, the modification of the endogenous DNA sequence mediated by the gene regulatory system results in the expression of an endogenous protein that exhibits reduced function compared to the corresponding endogenous protein in unmodified TILs. In such embodiments, the expression level of the modified endogenous protein can be increased or decreased compared to the expression level of the corresponding endogenous protein in unmodified immune cells, or can be the same as or substantially similar to the expression level of the corresponding endogenous protein.

[0309] A. Nucleic Acid-Based Gene Regulation Systems In some embodiments, the present disclosure provides a method for the detection of inflammatory cytokines including, but not limited to, ANKRD11, BCL2L11, BCL3, BCOR, CALM2, CBLB, CHIC2, CTLA4, DHODH, E2F8, EGR2, FLI1, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, LAG3, MAP4K, NFKBIA, NR4A3, NRP1, PBRM1, PCBP1, PDCD1, PELI1, PIK3CD, PPP2R2D, PTPN1, PTPN2, PTPN22, PTPN6, RBM39, RC3H1, SEMA7A, SERPINA3, SETD

[0010] In some embodiments, the present disclosure provides nucleic acid gene regulatory systems comprising one, two, or more nucleic acids capable of reducing the expression and / or function of at least one endogenous gene selected from SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA. In some embodiments, the present disclosure provides a nucleic acid gene regulatory system comprising a nucleic acid capable of reducing the expression and / or function of at least one, two, or more endogenous target genes selected from SOCS1 and PTPN2, ZC3H12A, CBLB, RC3H1, and NFKBIA. In some embodiments, the present disclosure provides modified TILs produced by the methods described herein comprising such a gene regulatory system. As used herein, a nucleic acid-based gene regulatory system is a system comprising one or more nucleic acid molecules capable of regulating the expression of endogenous target genes without the need for exogenous proteins. In some embodiments, the gene regulatory system comprises an RNA interference molecule or an antisense RNA molecule complementary to a target nucleic acid sequence.

[0310] "Antisense RNA molecule" refers to an RNA molecule, regardless of length, that is complementary to an mRNA transcript.Antisense RNA molecule refers to a single-stranded RNA molecule that can be introduced into a cell, tissue, or subject and reduces the expression of an endogenous target gene product through a mechanism that relies on RNase H-mediated degradation of the target mRNA transcript, rather than relying on the endogenous gene silencing pathway.In some embodiments, the antisense nucleic acid may contain a modified backbone, such as phosphorothioate, phosphorodithioate, or other backbone known in the art, or may contain non-naturally occurring internucleoside linkages.In some embodiments, the antisense nucleic acid may comprise a locked nucleic acid (LNA).

[0311] As used herein, "RNA interference molecule" refers to an RNA polynucleotide that mediates the reduction of expression of an endogenous target gene product by degrading the target mRNA through the endogenous gene silencing pathway (e.g., Dicer and RNA-induced silencing complex (RISC)). Exemplary RNA interference agents include microRNAs (also referred to herein as "miRNAs"), short hairpin RNAs (shRNAs), small interfering RNAs (siRNAs), RNA aptamers, and morpholinos.

[0312] In some embodiments, the gene regulation system comprises one or more miRNAs. miRNAs are naturally occurring, small, non-coding RNA molecules approximately 21-25 nucleotides in length. miRNAs are at least partially complementary to one or more target mRNA molecules. miRNAs can down-regulate (e.g., decrease) expression of endogenous target gene products through translational repression, mRNA cleavage, and / or deadenylation.

[0313] In some embodiments, the gene regulation system comprises one or more shRNAs. shRNAs are single-stranded RNA molecules approximately 50-70 nucleotides long that form a stem-loop structure and result in degradation of complementary mRNA sequences. shRNAs can be cloned into plasmids or non-replicating recombinant viral vectors that are introduced into cells, allowing the shRNA coding sequence to be integrated into the genome. Thus, shRNAs can result in stable and robust inhibition of translation and expression of endogenous target genes.

[0314] In some embodiments, the nucleic acid-based gene regulation system comprises one or more siRNAs. siRNA refers to a double-stranded RNA molecule, typically about 21-23 nucleotides in length. The siRNA associates with a multiprotein complex called the RNA-induced silencing complex (RISC), during which the "passenger" sense strand is enzymatically cleaved. The antisense "guide" strand contained in the activated RISC then guides the RISC to the corresponding mRNA by sequence homology, where the same nuclease cleaves the target mRNA, resulting in specific gene silencing. Optimally, the siRNA is 18, 19, 20, 21, 22, 23, or 24 nucleotides in length and has a two-base overhang at its 3' end. The siRNA can be introduced into individual cells and / or culture systems to cause degradation of the target mRNA sequence. siRNAs and shRNAs are further described in Fire et al., Nature, 391:19, 1998, and U.S. Patent Nos. 7,732,417, 8,202,846, and 8,383,599.

[0315] In some embodiments, the gene regulation system comprises one or more morpholinos.As used herein, " morpholinos " refers to modified nucleic acid oligomers in which standard nucleic acid bases are linked to morpholine rings and are linked through phosphorodiamidate bonds.Similar to siRNA and shRNA, morpholinos bind to complementary mRNA sequences.However, morpholinos do not target complementary mRNA sequences for degradation, but function by sterically inhibiting the translation of mRNA and changing mRNA splicing.

[0316] In some embodiments, the gene regulatory system comprises a nucleic acid molecule that binds to a target RNA sequence that is at least 90% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates set forth in Tables 4, 5, 9-12, and 17-22. Throughout this application, reference genomic coordinates are based on the genome annotation of the GRCh38 (also referred to as hg38) assembly of the human genome obtained from the Genome Reference Consortium, which is incorporated by reference in its entirety by the National Center for Molecular Biology. Tools and methods for converting genomic coordinates between one assembly and another are known in the art and can be used to convert the genomic coordinates provided herein to corresponding coordinates in another assembly of the human genome (including converting to a previous assembly produced by the same institution or using the same algorithm (e.g., GRCh38 to GRCh37), and converting assemblies produced by a different institution or algorithm (e.g., GRCh38 to NCBI33, produced by the International Human Genome Sequencing Consortium)). Available methods and tools known in the art include the NCBI Genome Sequencing Kit, available at the website of the National Center for Biotechnology Information, and the National Center for Biotechnology Information. These include, but are not limited to, the Remapping Service, UCSC LiftOver available on the UCSC Genome Brower website, and the Assembly Converter available on the Ensembl.org website.

[0317] In some embodiments, the nucleic acid-based gene regulation system comprises at least one nucleic acid molecule (e.g., siRNA, shRNA, RNA aptamer, or morpholino), and the at least one nucleic acid molecule is a nucleic acid molecule that targets SOCS1. In some embodiments, the at least one nucleic acid molecule that targets SOCS1 binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA sequence encoded by the SOCS1 gene (SEQ ID NO: 1) or Socs1 gene (SEQ ID NO: 2). In some embodiments, the at least one nucleic acid molecule that targets SOCS1 binds to a target RNA sequence that is 100% identical to the RNA sequence encoded by the SOCS1 gene (SEQ ID NO: 1) or Socs1 gene (SEQ ID NO: 2). In some embodiments, the at least one nucleic acid molecule that targets SOCS1 is an siRNA molecule or an shRNA molecule. In some embodiments, the at least one siRNA or shRNA molecule targeting SOCS1 binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA sequence encoded by the SOCS1 gene (SEQ ID NO: 1) or Socs1 gene (SEQ ID NO: 2). In some embodiments, the at least one siRNA or shRNA molecule targeting SOCS1 binds to a target RNA sequence that is 100% identical to the RNA sequence encoded by the SOCS1 gene (SEQ ID NO: 1) or Socs1 gene (SEQ ID NO: 2).

[0318] In some embodiments, the at least one SOCS1-targeting nucleic acid molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by a DNA sequence defined by the set of genomic coordinates set forth in Table 4 (human genome) or Table 5 (mouse genome). In some embodiments, the at least one SOCS1-targeting nucleic acid molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by a DNA sequence defined by the set of genomic coordinates set forth in Table 4 or Table 5. In some embodiments, the at least one SOCS1-targeting nucleic acid molecule binds to a human target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by one of SEQ ID NOs: 23-200. In some embodiments, the at least one SOCS1-targeting nucleic acid molecule binds to a human target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a human RNA sequence encoded by one of SEQ ID NOs: 23-35 and 56-187. In some embodiments, the at least one SOCS1-targeting nucleic acid molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by one of SEQ ID NOs: 23-200. In some embodiments, the at least one SOCS1-targeting nucleic acid molecule binds to a target human RNA sequence that is 100% identical to a human RNA sequence encoded by one of SEQ ID NOs: 23-35 and 56-187.

[0319] In some embodiments, the at least one SOCS1-targeting nucleic acid molecule is an shRNA or siRNA molecule that targets SOCS1. In some embodiments, the at least one SOCS1-targeting shRNA or siRNA molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by a DNA sequence defined by the set of genomic coordinates set forth in Table 4 or Table 5. In some embodiments, the at least one SOCS1-targeting shRNA or siRNA molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by a DNA sequence defined by the set of genomic coordinates set forth in Table 4 or Table 5. In some embodiments, the at least one SOCS1-targeting shRNA or siRNA molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by one of SEQ ID NOs: 23-55 or 23-200. In some embodiments, the at least one SOCS1-targeting shRNA or siRNA molecule binds to a target human RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a human RNA sequence encoded by one of SEQ ID NOs: 23-35 and 56-187. In some embodiments, the at least one SOCS1-targeting shRNA or siRNA molecule binds to a target human RNA sequence that is 100% identical to a human RNA sequence encoded by one of SEQ ID NOs: 23-55 or 23-200. In some embodiments, the at least one SOCS1-targeting shRNA or siRNA molecule binds to a target human RNA sequence that is 100% identical to a human RNA sequence encoded by one of SEQ ID NOs: 23-35 and 56-187.

[0320] In some embodiments, the nucleic acid-based gene regulation system comprises at least one siRNA or shRNA molecule targeting SOCS1 selected from those known to those of skill in the art. For example, in some embodiments, the nucleic acid molecule targeting SOCS1 is an siRNA targeting SOCS1 comprising a nucleic acid sequence selected from SEQ ID NOs: 13-22 (see International PCT Publication Nos. WO2017120996, WO2018137295, WO2017120998, and WO2018137293, which are incorporated herein by reference in their entireties) (Table 6). In some embodiments, the siRNA or shRNA molecule targeting SOCS1 is encoded by a nucleic acid sequence selected from SEQ ID NOs: 13-200. In some embodiments, the siRNA or shRNA molecule targeting SOCS1 is encoded by a human nucleic acid sequence selected from SEQ ID NOs: 23-35 and 56-187. In some embodiments, the nucleic acid molecule targeting SOCS1 is an shRNA or siRNA molecule targeting SOCS1 that binds to a human target sequence selected from SEQ ID NOs: 23-35 (see U.S. Patent No. 8,324,369, incorporated herein by reference in its entirety) (Table 7). In some embodiments, the nucleic acid molecule targeting SOCS1 is an shRNA or siRNA molecule targeting SOCS1 that binds to a mouse target sequence selected from SEQ ID NOs: 36-55 (see U.S. Patent No. 9,944,931, incorporated herein by reference in its entirety) (Table 8). [Table 4-1] [Table 4-2] [Table 4-3] [Table 5] [Table 6] [Table 7] [Table 8]

[0321] In some embodiments, the nucleic acid-based gene regulation system comprises at least one nucleic acid molecule (e.g., siRNA, shRNA, RNA aptamer, or morpholino), and the at least one nucleic acid molecule is a nucleic acid molecule targeting PTPN2. In some embodiments, the at least one PTPN2-targeting nucleic acid molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA sequence encoded by the PTPN2 gene (SEQ ID NO: 3) or Ptpn2 gene (SEQ ID NO: 4). In some embodiments, the at least one PTPN2-targeting nucleic acid molecule binds to a target RNA sequence that is 100% identical to the RNA sequence encoded by the PTPN2 gene (SEQ ID NO: 3) or Ptpn2 gene (SEQ ID NO: 4). In some embodiments, the at least one PTPN2-targeting nucleic acid molecule is an siRNA molecule or an shRNA molecule. In some embodiments, the at least one PTPN2-targeting siRNA or shRNA molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA sequence encoded by the PTPN2 gene (SEQ ID NO: 3) or Ptpn2 gene (SEQ ID NO: 4). In some embodiments, the at least one PTPN2-targeting siRNA or shRNA molecule binds to a target RNA sequence that is 100% identical to the RNA sequence encoded by the PTPN2 gene (SEQ ID NO: 3) or Ptpn2 gene (SEQ ID NO: 4).

[0322] In some embodiments, the at least one PTPN2-targeting nucleic acid molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by a DNA sequence defined by the set of genomic coordinates set forth in Table 9 (human genome) or Table 10 (mouse genome). In some embodiments, the at least one PTPN2-targeting nucleic acid molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by a DNA sequence defined by the set of genomic coordinates set forth in Table 9 or Table 10. In some embodiments, the at least one PTPN2-targeting nucleic acid molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by one of SEQ ID NOs: 201-327. In some embodiments, the at least one PTPN2-targeting nucleic acid molecule binds to a human target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a human RNA sequence encoded by one of SEQ ID NOs: 201-314. In some embodiments, the at least one PTPN2-targeting nucleic acid molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by one of SEQ ID NOs: 201-327. In some embodiments, the at least one PTPN2-targeting nucleic acid molecule binds to a human target RNA sequence that is 100% identical to a human RNA sequence encoded by one of SEQ ID NOs: 201-314.

[0323] In some embodiments, the at least one PTPN2-targeting nucleic acid molecule is an shRNA or siRNA molecule that targets SOCS1. In some embodiments, the at least one PTPN2-targeting shRNA or siRNA molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by a DNA sequence defined by the set of genomic coordinates set forth in Table 9 or Table 10. In some embodiments, the at least one PTPN2-targeting shRNA or siRNA molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by a DNA sequence defined by the set of genomic coordinates set forth in Table 9 or Table 10. In some embodiments, the at least one PTPN2-targeting shRNA or siRNA molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by one of SEQ ID NOs: 201-327. In some embodiments, the at least one shRNA or siRNA molecule targeting PTPN2 binds to a human target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a human RNA sequence encoded by one of SEQ ID NOs: 201-314. In some embodiments, the at least one shRNA or siRNA molecule targeting PTPN2 binds to a human target RNA sequence that is 100% identical to a human RNA sequence encoded by one of SEQ ID NOs: 201-327. In some embodiments, the at least one shRNA or siRNA molecule targeting PTPN2 binds to a target RNA sequence that is 100% identical to a RNA sequence encoded by one of SEQ ID NOs: 201-314. [Table 9-1] [Table 9-2] [Table 9-3] [Table 10]

[0324] In some embodiments, the nucleic acid-based gene regulation system comprises at least one nucleic acid molecule (e.g., siRNA, shRNA, RNA aptamer, or morpholino), and the at least one nucleic acid molecule is a nucleic acid molecule that targets ZC3H12A. In some embodiments, the at least one ZC3H12A-targeting nucleic acid molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA sequence encoded by the ZC3H12A gene (SEQ ID NO: 5) or Zc3h12A gene (SEQ ID NO: 6). In some embodiments, the at least one ZC3H12A-targeting nucleic acid molecule binds to a target RNA sequence that is 100% identical to the RNA sequence encoded by the ZC3H12A gene (SEQ ID NO: 5) or Zc3h12a gene (SEQ ID NO: 6). In some embodiments, the at least one ZC3H12A-targeting nucleic acid molecule is an siRNA molecule or an shRNA molecule. In some embodiments, the at least one ZC3H12A-targeting siRNA or shRNA molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA sequence encoded by the ZC3H12A gene (SEQ ID NO: 5) or Zc3h12a gene (SEQ ID NO: 6). In some embodiments, the at least one ZC3H12A-targeting siRNA or shRNA molecule binds to a target RNA sequence that is 100% identical to the RNA sequence encoded by the ZC3H12A gene (SEQ ID NO: 5) or Zc3h12a gene (SEQ ID NO: 6).

[0325] In some embodiments, the at least one ZC3H12A-targeting nucleic acid molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by a DNA sequence defined by the set of genomic coordinates set forth in Table 11 (human genome) or Table 12 (mouse genome). In some embodiments, the at least one ZC3H12A-targeting nucleic acid molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by a DNA sequence defined by the set of genomic coordinates set forth in Table 11 or Table 12. In some embodiments, the at least one ZC3H12A-targeting nucleic acid molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by one of SEQ ID NOs: 331-337 or 331-797. In some embodiments, the at least one ZC3H12A-targeting nucleic acid molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by one of SEQ ID NOs: 331-337 or 331-797.

[0326] In some embodiments, the at least one ZC3H12A-targeting nucleic acid molecule is a ZC3H12A-targeting shRNA or siRNA molecule. In some embodiments, the at least one ZC3H12A-targeting shRNA or siRNA molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by a DNA sequence defined by the set of genomic coordinates shown in Table 11 or Table 12. In some embodiments, the at least one ZC3H12A-targeting shRNA or siRNA molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by a DNA sequence defined by the set of genomic coordinates shown in Table 11 or Table 12. In some embodiments, the ZC3H12A-targeting nucleic acid molecule is a ZC3H12A-targeting siRNA comprising a nucleic acid sequence selected from SEQ ID NOs: 328-330 or 329 and 330 (human) (see Liu et al., Scientific Reports (2016), 6, Article #24073 and Mino et al., Cell (2015) 161(5), 1058-1073, incorporated herein by reference in their entireties). In some embodiments, the at least one ZC3H12A-targeting shRNA or siRNA molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by one of SEQ ID NOs: 331-797. In some embodiments, the at least one ZC3H12A-targeting shRNA or siRNA molecule binds to a human target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a human RNA sequence encoded by one of SEQ ID NOs: 336-789. In some embodiments, the at least one ZC3H12A-targeting shRNA or siRNA molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by one of SEQ ID NOs: 331-797.In some embodiments, the at least one ZC3H12A-targeting shRNA or siRNA molecule binds to a human target RNA sequence that is 100% identical to a human RNA sequence encoded by one of SEQ ID NOs: 336-789. In some embodiments, the ZC3H12A-targeting nucleic acid molecule is a ZC3H12A-targeting shRNA molecule encoded by a nucleic acid sequence selected from SEQ ID NOs: 331-337 (see Huang et al., J Biol Chem (2015) 290(34), 20782-20792, incorporated herein by reference in its entirety). [Table 11-1] [Table 11-2] [Table 11-3] [Table 11-4] [Table 11-5] [Table 11-6] [Table 11-7] [Table 11-8] [Table 11-9] [Table 11-10] [Table 11-11] [Table 12] [Table 13] [Table 14] [Table 15] [Table 16]

[0327] In some embodiments, the nucleic acid-based gene regulation system comprises at least one nucleic acid molecule (e.g., siRNA, shRNA, RNA aptamer, or morpholino), and the at least one nucleic acid molecule is a nucleic acid molecule that targets CBLB. In some embodiments, the at least one nucleic acid molecule that targets CBLB binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA sequence encoded by the CBLB gene (SEQ ID NO: 7) or the Cblb gene (SEQ ID NO: 8). In some embodiments, the at least one nucleic acid molecule that targets CBLB binds to a target RNA sequence that is 100% identical to the RNA sequence encoded by the CBLB gene (SEQ ID NO: 7) or the Cblb gene (SEQ ID NO: 8). In some embodiments, the at least one nucleic acid molecule that targets CBLB is an siRNA molecule or an shRNA molecule. In some embodiments, the at least one CBLB-targeting siRNA or shRNA molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA sequence encoded by the CBLB gene (SEQ ID NO: 7) or Cblb gene (SEQ ID NO: 8). In some embodiments, the at least one CBLB-targeting siRNA or shRNA molecule binds to a target RNA sequence that is 100% identical to the RNA sequence encoded by the CBLB gene (SEQ ID NO: 7) or Cblb gene (SEQ ID NO: 8).

[0328] In some embodiments, the nucleic acid molecule targeting at least one CBLB binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by a DNA sequence defined by the set of genomic coordinates shown in Table 17 (human genome) or Table 18 (mouse genome). In some embodiments, the nucleic acid molecule targeting at least one CBLB binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by a DNA sequence defined by the set of genomic coordinates shown in Table 17 or Table 18. In some embodiments, the nucleic acid molecule targeting at least one CBLB binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by one of SEQ ID NOs: 798-823. In some embodiments, the at least one CBLB-targeting nucleic acid molecule binds to a human target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a human RNA sequence encoded by one of SEQ ID NOs: 798-808. In some embodiments, the at least one CBLB-targeting nucleic acid molecule binds to a target RNA sequence that is 100% identical to a human RNA sequence encoded by one of SEQ ID NOs: 798-823. In some embodiments, the at least one CBLB-targeting nucleic acid molecule binds to a human target RNA sequence that is 100% identical to a human RNA sequence encoded by one of SEQ ID NOs: 798-808.

[0329] In some embodiments, the nucleic acid molecule targeting at least one CBLB is an shRNA molecule or siRNA molecule targeting a CBLB. In some embodiments, the shRNA molecule or siRNA molecule targeting at least one CBLB binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA sequence encoded by the DNA sequence defined by the set of genomic coordinates shown in Table 17 or Table 18. In some embodiments, the shRNA molecule or siRNA molecule targeting at least one CBLB binds to a target RNA sequence that is 100% identical to the RNA sequence encoded by the DNA sequence defined by the set of genomic coordinates shown in Table 17 or Table 18. In some embodiments, the shRNA molecule or siRNA molecule targeting at least one CBLB binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA sequence encoded by one of SEQ ID NOs: 798-823. In some embodiments, the at least one shRNA or siRNA molecule targeting a CBLB binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA sequence encoded by one of SEQ ID NOs: 798-823. In some embodiments, the at least one shRNA or siRNA molecule targeting a CBLB binds to a human target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the human RNA sequence encoded by one of SEQ ID NOs: 798-808. In some embodiments, the at least one shRNA or siRNA molecule targeting a CBLB binds to a target RNA sequence that is 100% identical to the RNA sequence encoded by one of SEQ ID NOs: 798-823. In some embodiments, the at least one shRNA or siRNA molecule targeting a CBLB binds to a human target RNA sequence that is 100% identical to the human RNA sequence encoded by one of SEQ ID NOs: 798-808. [Table 17] [Table 18]

[0330] In some embodiments, the nucleic acid-based gene regulation system comprises at least one nucleic acid molecule (e.g., siRNA, shRNA, RNA aptamer, or morpholino), and the at least one nucleic acid molecule is a nucleic acid molecule that targets RC3H1. In some embodiments, the at least one nucleic acid molecule that targets RC3H1 binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA sequence encoded by the RC3H1 gene (SEQ ID NO: 9) or the Rc3h1 gene (SEQ ID NO: 10). In some embodiments, the at least one nucleic acid molecule that targets RC3H1 binds to a target RNA sequence that is 100% identical to the RNA sequence encoded by the RC3H1 gene (SEQ ID NO: 9) or the Rc3h1 gene (SEQ ID NO: 10). In some embodiments, the at least one nucleic acid molecule that targets RC3H1 is an siRNA molecule or an shRNA molecule. In some embodiments, the at least one RC3H1-targeting siRNA or shRNA molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA sequence encoded by the RC3H1 gene (SEQ ID NO: 9) or the Rc3h1 gene (SEQ ID NO: 10). In some embodiments, the at least one RC3H1-targeting siRNA or shRNA molecule binds to a target RNA sequence that is 100% identical to the RNA sequence encoded by the RC3H1 gene (SEQ ID NO: 9) or the Rc3h1 gene (SEQ ID NO: 10).

[0331] In some embodiments, the at least one RC3H1-targeting nucleic acid molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by a DNA sequence defined by the set of genomic coordinates set forth in Table 19 (human genome) or Table 20 (mouse genome). In some embodiments, the at least one RC3H1-targeting nucleic acid molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by a DNA sequence defined by the set of genomic coordinates set forth in Table 19 or Table 20. In some embodiments, the at least one RC3H1-targeting nucleic acid molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by one of SEQ ID NOs: 824-844. In some embodiments, the at least one RC3H1-targeting nucleic acid molecule binds to a human target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a human RNA sequence encoded by one of SEQ ID NOs: 824-836. In some embodiments, the at least one RC3H1-targeting nucleic acid molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by one of SEQ ID NOs: 824-844. In some embodiments, the at least one RC3H1-targeting nucleic acid molecule binds to a human target RNA sequence that is 100% identical to a human RNA sequence encoded by one of SEQ ID NOs: 824-836.

[0332] In some embodiments, the at least one RC3H1-targeting nucleic acid molecule is an shRNA molecule or siRNA molecule that targets RC3H1. In some embodiments, the at least one RC3H1-targeting shRNA molecule or siRNA molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Table 19 or Table 20. In some embodiments, the at least one RC3H1-targeting shRNA molecule or siRNA molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Table 19 or Table 20. In some embodiments, the at least one RC3H1-targeting shRNA molecule or siRNA molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by one of SEQ ID NOs: 824-844. In some embodiments, the at least one shRNA or siRNA molecule targeting RC3H1 binds to a human target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a human RNA sequence encoded by one of SEQ ID NOs: 824-836. In some embodiments, the at least one shRNA or siRNA molecule targeting RC3H1 binds to a target RNA sequence that is 100% identical to a human RNA sequence encoded by one of SEQ ID NOs: 824-844. In some embodiments, the at least one shRNA or siRNA molecule targeting RC3H1 binds to a human target RNA sequence that is 100% identical to a human RNA sequence encoded by one of SEQ ID NOs: 824-836. [Table 19] [Table 20]

[0333] In some embodiments, the nucleic acid-based gene regulation system comprises at least one nucleic acid molecule (e.g., siRNA, shRNA, RNA aptamer, or morpholino), wherein the at least one nucleic acid molecule is a nucleic acid molecule targeting NFKBIA. In some embodiments, the at least one nucleic acid molecule targeting NFKBIA binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA sequence encoded by the NFKBIA gene (SEQ ID NO: 11) or the Nfkbia gene (SEQ ID NO: 12). In some embodiments, the at least one nucleic acid molecule targeting NFKBIA binds to a target RNA sequence that is 100% identical to the RNA sequence encoded by the NFKBIA gene (SEQ ID NO: 11) or the Nfkbia gene (SEQ ID NO: 12). In some embodiments, the at least one nucleic acid molecule targeting NFKBIA is an siRNA molecule or an shRNA molecule. In some embodiments, the at least one NFKBIA-targeting siRNA or shRNA molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA sequence encoded by the NFKBIA gene (SEQ ID NO: 11) or the Nfkbia gene (SEQ ID NO: 12). In some embodiments, the at least one NFKBIA-targeting siRNA or shRNA molecule binds to a target RNA sequence that is 100% identical to the RNA sequence encoded by the NFKBIA gene (SEQ ID NO: 11) or the Nfkbia gene (SEQ ID NO: 12).

[0334] In some embodiments, the at least one NFKBIA-targeting nucleic acid molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by a DNA sequence defined by the set of genomic coordinates set forth in Table 21 (human genome) or Table 22 (mouse genome). In some embodiments, the at least one NFKBIA-targeting nucleic acid molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by a DNA sequence defined by the set of genomic coordinates set forth in Table 21 or Table 22. In some embodiments, the at least one NFKBIA-targeting nucleic acid molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by one of SEQ ID NOs: 845-875. In some embodiments, the at least one NFKBIA-targeting nucleic acid molecule binds to a human target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a human RNA sequence encoded by one of SEQ ID NOs: 845-856. In some embodiments, the at least one NFKBIA-targeting nucleic acid molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by one of SEQ ID NOs: 845-875. In some embodiments, the at least one NFKBIA-targeting nucleic acid molecule binds to a human target RNA sequence that is 100% identical to a human RNA sequence encoded by one of SEQ ID NOs: 845-856.

[0335] In some embodiments, the at least one NFKBIA-targeting nucleic acid molecule is an shRNA or siRNA molecule that targets NFKBIA. In some embodiments, the at least one NFKBIA-targeting shRNA or siRNA molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by a DNA sequence defined by the set of genomic coordinates shown in Table 21 or Table 22. In some embodiments, the at least one NFKBIA-targeting shRNA or siRNA molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by a DNA sequence defined by the set of genomic coordinates shown in Table 21 or Table 22. In some embodiments, the at least one NFKBIA-targeting shRNA or siRNA molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by one of SEQ ID NOs: 845-875. In some embodiments, the at least one shRNA or siRNA molecule targeting NFKBIA binds to a human target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a human RNA sequence encoded by one of SEQ ID NOs: 845-856. In some embodiments, the at least one shRNA or siRNA molecule targeting NFKBIA binds to a target RNA sequence that is 100% identical to a human RNA sequence encoded by one of SEQ ID NOs: 845-875. In some embodiments, the at least one shRNA or siRNA molecule targeting NFKBIA binds to a human target RNA sequence that is 100% identical to a human RNA sequence encoded by one of SEQ ID NOs: 845-856. [Table 21] [Table 22]

[0336] In some embodiments, the siRNA or shRNA molecule targeting at least one of SOCS1, PTPN2, ZC3H12A, CBLB, RC3H1, or NFKBIA is obtained from a commercial supplier, e.g., Sigma Aldrich®, Dharmacon®, ThermoFisher®, etc. In some embodiments, the siRNA molecule targeting at least one of SOCS1, PTPN2, or ZC3H12A is one shown in Table 23. In some embodiments, the shRNA molecule targeting at least one of SOCS1, PTPN2, or ZC3H12A is one shown in Table 24. [Table 23] [Table 24]

[0337] In some embodiments, the nucleic acid-based gene regulation system comprises at least two nucleic acid molecules (e.g., siRNA, shRNA, RNA aptamer, or morpholino), wherein at least one nucleic acid molecule is a nucleic acid molecule targeting SOCS1 and at least one nucleic acid molecule is a nucleic acid molecule targeting PTPN2. In some embodiments, the at least one nucleic acid molecule targeting SOCS1 binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA sequence encoded by the SOCS1 gene (SEQ ID NO: 1) or Socs1 gene (SEQ ID NO: 2), and the at least one nucleic acid molecule targeting PTPN2 binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA sequence encoded by the PTPN2 gene (SEQ ID NO: 3) or Ptpn2 gene (SEQ ID NO: 4). In some embodiments, the at least one nucleic acid molecule targeting SOCS1 binds to a target RNA sequence that is 100% identical to the RNA sequence encoded by the SOCS1 gene (SEQ ID NO: 1) or the Socs1 gene (SEQ ID NO: 2), and the at least one nucleic acid molecule targeting PTPN2 binds to a target RNA sequence that is 100% identical to the RNA sequence encoded by the PTPN2 gene (SEQ ID NO: 3) or the Ptpn2 gene (SEQ ID NO: 4).

[0338] In some embodiments, the at least one SOCS1-targeting nucleic acid molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates set forth in Table 4 or Table 5, and the at least one PTPN2-targeting nucleic acid molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates set forth in Table 9 or Table 10. In some embodiments, the at least one SOCS1-targeting nucleic acid molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates set forth in Table 4 or Table 5, and the at least one PTPN2-targeting nucleic acid molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates set forth in Table 9 or Table 10.

[0339] In some embodiments, the at least one SOCS1-targeting nucleic acid molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by one of SEQ ID NOs: 23-200 or 23-55, and the at least one PTPN2-targeting nucleic acid molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by one of SEQ ID NOs: 201-327. In some embodiments, the at least one SOCS1-targeting nucleic acid molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by one of SEQ ID NOs: 23-200 or 23-55, and the at least one PTPN2-targeting nucleic acid molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by one of SEQ ID NOs: 201-327.

[0340] In some embodiments, the nucleic acid-based gene regulation system comprises at least two siRNA molecules or shRNA molecules, wherein at least one siRNA molecule or shRNA molecule is a siRNA molecule or shRNA molecule that targets SOCS1, and at least one siRNA molecule or shRNA molecule is a siRNA molecule or shRNA molecule that targets PTPN2. In some embodiments, the nucleic acid molecule that targets at least one SOCS1 is a siRNA molecule or shRNA molecule, and the nucleic acid molecule that targets at least one PTPN2 is a siRNA molecule or shRNA molecule. In some embodiments, the at least one siRNA or shRNA molecule targeting SOCS1 binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA sequence encoded by the SOCS1 gene (SEQ ID NO: 1) or Socs1 gene (SEQ ID NO: 2), and the at least one siRNA or shRNA molecule targeting PTPN2 binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA sequence encoded by the PTPN2 gene (SEQ ID NO: 3) or Ptpn2 gene (SEQ ID NO: 4). In some embodiments, the at least one siRNA or shRNA molecule targeting SOCS1 binds to a target RNA sequence that is 100% identical to the RNA sequence encoded by the SOCS1 gene (SEQ ID NO: 1) or the Socs1 gene (SEQ ID NO: 2), and the at least one siRNA or shRNA molecule targeting PTPN2 binds to a target RNA sequence that is 100% identical to the RNA sequence encoded by the PTPN2 gene (SEQ ID NO: 3) or the Ptpn2 gene (SEQ ID NO: 4).

[0341] In some embodiments, the at least one siRNA or shRNA molecule targeting SOCS1 binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates set forth in Table 4 or Table 5, and the at least one siRNA or shRNA molecule targeting PTPN2 binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates set forth in Table 9 or Table 10. In some embodiments, the at least one siRNA or shRNA molecule targeting SOCS1 binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates set forth in Table 4 or Table 5, and the at least one siRNA or shRNA molecule targeting PTPN2 binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates set forth in Table 9 or Table 10.

[0342] In some embodiments, the at least one siRNA or shRNA molecule targeting SOCS1 binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by one of SEQ ID NOs: 23-200 or 23-55, and the at least one siRNA or shRNA molecule targeting PTPN2 binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by one of SEQ ID NOs: 201-327. In some embodiments, the at least one siRNA or shRNA molecule targeting SOCS1 binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by one of SEQ ID NOs: 23-200 or 23-55, and the at least one siRNA or shRNA molecule targeting PTPN2 binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by one of SEQ ID NOs: 201-327.

[0343] In some embodiments, the nucleic acid-based gene regulation system comprises at least two nucleic acid molecules (e.g., siRNA, shRNA, RNA aptamer, or morpholino), wherein at least one nucleic acid molecule is a nucleic acid molecule targeting SOCS1, and at least one nucleic acid molecule is a nucleic acid molecule targeting ZC3H12A. In some embodiments, the at least one nucleic acid molecule targeting SOCS1 binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA sequence encoded by the SOCS1 gene (SEQ ID NO: 1) or Socs1 gene (SEQ ID NO: 2), and the at least one nucleic acid molecule targeting ZC3H12A binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA sequence encoded by the ZC3H12A gene (SEQ ID NO: 5) or Zc3h12a gene (SEQ ID NO: 6). In some embodiments, the at least one nucleic acid molecule targeting SOCS1 binds to a target RNA sequence that is 100% identical to the RNA sequence encoded by the SOCS1 gene (SEQ ID NO: 1) or the Socs1 gene (SEQ ID NO: 2), and the at least one nucleic acid molecule targeting ZC3H12A binds to a target RNA sequence that is 100% identical to the RNA sequence encoded by the ZC3H12A gene (SEQ ID NO: 5) or the Zc3h12a gene (SEQ ID NO: 6).

[0344] In some embodiments, the at least one SOCS1-targeting nucleic acid molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates set forth in Table 4 or Table 5, and the at least one ZC3H12A-targeting nucleic acid molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates set forth in Table 11 or Table 12. In some embodiments, the at least one SOCS1-targeting nucleic acid molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates set forth in Table 4 or Table 5, and the at least one ZC3H12A-targeting nucleic acid molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates set forth in Table 11 or Table 12.

[0345] In some embodiments, the at least one SOCS1-targeting nucleic acid molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by one of SEQ ID NOs: 23-200 or 23-55, and the at least one ZC3H12A-targeting nucleic acid molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by one of SEQ ID NOs: 331-797 or 331-337. In some embodiments, the at least one SOCS1-targeting nucleic acid molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by one of SEQ ID NOs: 23-200 or 23-55, and the at least one ZC3H12A-targeting nucleic acid molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by one of SEQ ID NOs: 331-797 or 331-337.

[0346] In some embodiments, the nucleic acid-based gene regulation system comprises at least two siRNA molecules or shRNA molecules, wherein at least one siRNA molecule or shRNA molecule is the siRNA molecule or shRNA molecule that targets SOCS1, and at least one siRNA molecule or shRNA molecule is the siRNA molecule or shRNA molecule that targets ZC3H12A.In some embodiments, the nucleic acid molecule that targets at least one SOCS1 is a siRNA molecule or shRNA molecule, and the nucleic acid molecule that targets at least one ZC3H12A is a siRNA molecule or shRNA molecule. In some embodiments, the at least one siRNA or shRNA molecule targeting SOCS1 binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA sequence encoded by the SOCS1 gene (SEQ ID NO: 1) or Socs1 gene (SEQ ID NO: 2), and the at least one siRNA or shRNA molecule targeting ZC3H12A binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA sequence encoded by the ZC3H12A gene (SEQ ID NO: 5) or Zc3h12a gene (SEQ ID NO: 6). In some embodiments, the at least one siRNA or shRNA molecule targeting SOCS1 binds to a target RNA sequence that is 100% identical to the RNA sequence encoded by the SOCS1 gene (SEQ ID NO: 1) or the Socs1 gene (SEQ ID NO: 2), and the at least one siRNA or shRNA molecule targeting ZC3H12A binds to a target RNA sequence that is 100% identical to the RNA sequence encoded by the ZC3H12A gene (SEQ ID NO: 5) or the Zc3h12a gene (SEQ ID NO: 6).

[0347] In some embodiments, the at least one siRNA or shRNA molecule targeting SOCS1 binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Table 4 or Table 5, and the at least one siRNA or shRNA molecule targeting ZC3H12A binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Table 11 or Table 12. In some embodiments, the at least one siRNA or shRNA molecule targeting SOCS1 binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Table 4 or Table 5, and the at least one siRNA or shRNA molecule targeting ZC3H12A binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Table 11 or Table 12.

[0348] In some embodiments, the at least one siRNA or shRNA molecule targeting SOCS1 binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by one of SEQ ID NOs: 23-200 or 23-55, and the at least one siRNA or shRNA molecule targeting ZC3H12A binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by one of SEQ ID NOs: 331-797 or 331-337. In some embodiments, the at least one siRNA or shRNA molecule targeting SOCS1 binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by one of SEQ ID NOs: 23-200 or 23-55, and the at least one siRNA or shRNA molecule targeting ZC3H12A binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by one of SEQ ID NOs: 331-797 or 331-337.

[0349] In some embodiments, the nucleic acid-based gene regulation system comprises at least two nucleic acid molecules (e.g., siRNA, shRNA, RNA aptamer, or morpholino), wherein at least one nucleic acid molecule is a nucleic acid molecule targeting PTPN2 and at least one nucleic acid molecule is a nucleic acid molecule targeting ZC3H12A. In some embodiments, the at least one PTPN2-targeting nucleic acid molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA sequence encoded by the PTPN2 gene (SEQ ID NO: 3) or the Ptpn2 gene (SEQ ID NO: 4), and the at least one ZC3H12A-targeting nucleic acid molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA sequence encoded by the ZC3H12A gene (SEQ ID NO: 5) or the Zc3h12a gene (SEQ ID NO: 6). In some embodiments, the at least one nucleic acid molecule targeting PTPN2 binds to a target RNA sequence that is 100% identical to the RNA sequence encoded by the PTPN2 gene (SEQ ID NO: 3) or the Ptpn2 gene (SEQ ID NO: 4), and the at least one nucleic acid molecule targeting ZC3H12A binds to a target RNA sequence that is 100% identical to the RNA sequence encoded by the ZC3H12A gene (SEQ ID NO: 5) or the Zc3h12a gene (SEQ ID NO: 6).

[0350] In some embodiments, the at least one PTPN2-targeting nucleic acid molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates set forth in Table 9 or Table 10, and the at least one ZC3H12A-targeting nucleic acid molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates set forth in Table 11 or Table 12. In some embodiments, the at least one PTPN2-targeting nucleic acid molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates set forth in Table 9 or Table 10, and the at least one ZC3H12A-targeting nucleic acid molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates set forth in Table 11 or Table 12.

[0351] In some embodiments, the at least one PTPN2-targeting nucleic acid molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by one of SEQ ID NOs: 201-327, and the at least one ZC3H12A-targeting nucleic acid molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by one of SEQ ID NOs: 331-797 or 331-337. In some embodiments, the at least one PTPN2-targeting nucleic acid molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by one of SEQ ID NOs: 201-327, and the at least one ZC3H12A-targeting nucleic acid molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by one of SEQ ID NOs: 331-797 or 331-337.

[0352] In some embodiments, the nucleic acid-based gene regulation system comprises at least two siRNA or shRNA molecules, wherein at least one siRNA or shRNA molecule is a PTPN2-targeting siRNA or shRNA molecule, and at least one siRNA or shRNA molecule is a ZC3H12A-targeting siRNA or shRNA molecule. In some embodiments, the at least one PTPN2-targeting siRNA or shRNA molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA sequence encoded by the PTPN2 gene (SEQ ID NO: 3) or Ptpn2 gene (SEQ ID NO: 4), and the at least one ZC3H12A-targeting siRNA or shRNA molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA sequence encoded by the ZC3H12A gene (SEQ ID NO: 5) or Zc3h12a gene (SEQ ID NO: 6). In some embodiments, the at least one siRNA or shRNA molecule targeting PTPN2 binds to a target RNA sequence that is 100% identical to the RNA sequence encoded by the PTPN2 gene (SEQ ID NO: 3) or the Ptpn2 gene (SEQ ID NO: 4), and the at least one siRNA or shRNA molecule targeting ZC3H12A binds to a target RNA sequence that is 100% identical to the RNA sequence encoded by the ZC3H12A gene (SEQ ID NO: 5) or the Zc3h12a gene (SEQ ID NO: 6).

[0353] In some embodiments, the at least one PTPN2-targeting siRNA or shRNA molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Table 9 or Table 10, and the at least one ZC3H12A-targeting siRNA or shRNA molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Table 11 or Table 12. In some embodiments, the at least one PTPN2-targeting siRNA or shRNA molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Table 9 or Table 10, and the at least one ZC3H12A-targeting siRNA or shRNA molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Table 11 or Table 12.

[0354] In some embodiments, the at least one PTPN2-targeting siRNA or shRNA molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by one of SEQ ID NOs: 201-327, and the at least one ZC3H12A-targeting siRNA or shRNA molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by one of SEQ ID NOs: 331-797 or 331-337. In some embodiments, the at least one PTPN2-targeting siRNA or shRNA molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by one of SEQ ID NOs: 201-327, and the at least one ZC3H12A-targeting siRNA or shRNA molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by one of SEQ ID NOs: 331-797 or 331-337.

[0355] In some embodiments, the nucleic acid-based gene regulation system comprises at least two nucleic acid molecules (e.g., siRNA, shRNA, RNA aptamer, or morpholino), wherein at least one nucleic acid molecule is a CBLB-targeting nucleic acid molecule and at least one nucleic acid molecule is a PTPN2-targeting nucleic acid molecule. In some embodiments, the at least one CBLB-targeting nucleic acid molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA sequence encoded by the CBLB gene (SEQ ID NO:7) or Cblb gene (SEQ ID NO:8), and the at least one PTPN2-targeting nucleic acid molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA sequence encoded by the PTPN2 gene (SEQ ID NO:3) or Ptpn2 gene (SEQ ID NO:4). In some embodiments, the at least one CBLB-targeting nucleic acid molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by the CBLB gene (SEQ ID NO: 7) or the Cblb gene (SEQ ID NO: 8), and the at least one PTPN2-targeting nucleic acid molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by the PTPN2 gene (SEQ ID NO: 3) or the Ptpn2 gene (SEQ ID NO: 4).

[0356] In some embodiments, the at least one CBLB-targeting nucleic acid molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Table 17 or Table 18, and the at least one PTPN2-targeting nucleic acid molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Table 9 or Table 10. In some embodiments, the at least one CBLB-targeting nucleic acid molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Table 17 or Table 18, and the at least one PTPN2-targeting nucleic acid molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Table 9 or Table 10.

[0357] In some embodiments, the at least one CBLB-targeting nucleic acid molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by one of SEQ ID NOs: 798-823, and the at least one PTPN2-targeting nucleic acid molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by one of SEQ ID NOs: 201-327. In some embodiments, the at least one CBLB-targeting nucleic acid molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by one of SEQ ID NOs: 798-823, and the at least one PTPN2-targeting nucleic acid molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by one of SEQ ID NOs: 201-327.

[0358] In some embodiments, the nucleic acid-based gene regulation system comprises at least two siRNA molecules or shRNA molecules, wherein at least one siRNA molecule or shRNA molecule is a siRNA molecule or shRNA molecule that targets CBLB, and at least one siRNA molecule or shRNA molecule is a siRNA molecule or shRNA molecule that targets PTPN2. In some embodiments, the nucleic acid molecule that targets at least one CBLB is a siRNA molecule or shRNA molecule, and the nucleic acid molecule that targets at least one PTPN2 is a siRNA molecule or shRNA molecule. In some embodiments, the at least one CBLB-targeting siRNA or shRNA molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by the CBLB gene (SEQ ID NO: 7) or the Cblb gene (SEQ ID NO: 8), and the at least one PTPN2-targeting siRNA or shRNA molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by the PTPN2 gene (SEQ ID NO: 3) or the Ptpn2 gene (SEQ ID NO: 4). In some embodiments, the at least one CBLB-targeting siRNA or shRNA molecule binds to a target RNA sequence that is 100% identical to the RNA sequence encoded by the CBLB gene (SEQ ID NO: 7) or the Cblb gene (SEQ ID NO: 8), and the at least one PTPN2-targeting siRNA or shRNA molecule binds to a target RNA sequence that is 100% identical to the RNA sequence encoded by the PTPN2 gene (SEQ ID NO: 3) or the Ptpn2 gene (SEQ ID NO: 4).

[0359] In some embodiments, the at least one CBLB-targeting siRNA or shRNA molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Table 17 or Table 18, and the at least one PTPN2-targeting siRNA or shRNA molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Table 9 or Table 10. In some embodiments, the at least one CBLB-targeting siRNA or shRNA molecule binds to a target RNA sequence that is 100% identical to the RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Table 17 or Table 18, and the at least one PTPN2-targeting siRNA or shRNA molecule binds to a target RNA sequence that is 100% identical to the RNA sequence encoded by a DNA sequence defined by a set of genomic coordinates shown in Table 9 or Table 10.

[0360] In some embodiments, the at least one CBLB-targeting siRNA or shRNA molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by one of SEQ ID NOs: 798-823, and the at least one PTPN2-targeting siRNA or shRNA molecule binds to a target RNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an RNA sequence encoded by one of SEQ ID NOs: 201-327. In some embodiments, the at least one CBLB-targeting siRNA or shRNA molecule binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by one of SEQ ID NOs: 798-823, and the at least one PTPN2-targ...

Claims

[Claim 1] The invention described in the specification.

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