Cell therapies for disrupting collagen and other ECM components

Abstract

Provided herein are methods of treating cancer comprising utilization of tumor infiltrating lymphocytes expressing tumor-targeted and membrane-anchored IL-12. Also provided herein are methods of treating cancer comprising utilization of T cells expressing tumor-targeted and membrane-anchored IL-12 with an additional therapeutic agent, particularly a therapeutic agent that inhibits CCKAR and / or CCKAR signaling pathways.

Description

CELL THERAPIES FOR DISRUPTING COLLAGEN AND OTHER ECM COMPONENTSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 623,620 filed January 22, 2024, the contents of which is hereby incorporated by reference in its entirety.GOVERNMENT SUPPORT

[0002] This invention was made with government support under CA120985 awarded by the National Institutes of Health. The government has certain rights in the invention.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted in ST26 format and is hereby incorporated by reference in its entirety. Said ST26 copy, created on January 19, 2024, is named MDAC_P1371USPl_Sequence_Listing.xml and is 12,499 bytes in size.BACKGROUND1. Field

[0004] The present invention relates at least generally to the fields of immunology and medicine. More particularly, it concerns engineered immune effector cell therapies for the treatment of diseases such as cancer.2. Description of Related Art

[0005] Autologous tumor-infiltrating lymphocyte (TIL) infusion has been a remarkable breakthrough in the treatment of patients with refractory melanoma and has resulted in higher response rates than has BRAF-targeted therapy or CTLA-4-blocking therapy. Most patients should experience a response to TIL transfer because TILs can be isolated from their tumors. However, in practice, the response rates are only about 50%, including a 10%- 15% complete response rate (Besser et al., 2010; Radvanyi et al., 2012; Dudley et al., 2005).

[0006] Major challenges in TIL therapy include the reduced tumor homing ability of TILs after reinfusion as well as the changes in the tumor microenvironment. In clinical trials, 1.5- 2xlOnTILs were infused to ensure enough tumor-targeting TILs and successful tumor remission (Radvanyi et al., 2012; Dudley et al., 2005). However, transferring such large numbers of TILs into cancer patients can cause off-target adverse effects. Approaches are needed that enable TILs to be delivered to tumor sites more efficiently and therefore requiremuch smaller numbers of infused TILs. Non-TIL T Cell therapies have also recently proven to be effective in many cancers, however, many cancers, particularly solid tumors, have proven resistant to T cell therapies.

[0007] In general, there are multiple challenges for using immune effector cell therapies (e.g., T cell CAR-T, TIL, and TCR-T, NK cell, etc.) for treatment of solid tumors, these challenges can contribute to or result in tumor cell resistance to immune effector cell therapies. Factors that contribute to tumor cell resistance include at least tumor heterogeneity to escape the antigen or target specific effector cell attack, failure of effector cell penetration into solid tumors, inactivation of the infiltrated effector cells by the immune suppressive environment, and / or the exhaustion of effector cells. Thus, there is an unmet need for improved immune cell therapies and associated treatment methodologies.SUMMARY

[0008] Provided herein are improved immunotherapy methods and compositions that can overcome tumor resistance. In particular, in certain embodiments provided herein are engineered Tumor Infiltrating Lymphocytes (TILs) and methods of using the engineered TILs to overcome cancers predicted to be resistant to immune effector cell therapies and / or other immunotherapies .

[0009] In some embodiments, provided herein are methods of treating immune effector cell resistant cancer in a subject with a tumor comprising, reducing and / or diminishing Cholecystokinin A receptor (CCKAR) protein and / or transcript expression in the tumor by contacting the tumor with tumor infiltrating lymphocytes (TILs) transgenically expressing a cell surface vimentin (CSV)-targeted and membrane-anchored cytokine.

[0010] In some embodiments, provided herein are methods of treating cancer in a subject with a tumor comprising, obtaining TILs from the subject, engineering the TILs to transgenically express a CSV-targeted and membrane- anchored cytokine, and administering the engineered TILs to the subject.

[0011] In some embodiments, provided herein are methods of treating cancer in a subject with a tumor comprising, i) determining levels of CCKAR, Sarcolipin (SLN), LARGE xylosyl- and glucuronyltransferase 2 (LARGE2), and / or Wnt family member 10A (WNT10A) expression in the tumor, and optionally determining the levels of izumo sperm-oocyte fusion 1 (IZUM01); ii) obtaining TILs from the subject; iii) engineering the TILs to transgenically express a CSV-targeted and membrane- anchored cytokine; and iv) if CCKAR, SLN, LARGE2, and / or WNT10A expression is greater than or equal to about 0.0001 Fragments Per Kilobaseof transcript per Million mapped reads (FPKM), and / or optionally if the expression of IZUMO 1 is less than or equal to about 0.0001 FPKM, administering to the subject the engineered TILs.

[0012] In some embodiments, provided herein are methods of treating cancer in a subject with a tumor comprising, i) determining levels of CCKAR, SLN, LARGE2, and / or WNT10A expression in the tumor, and optionally determining the levels of IZUMO 1 expression in the tumor; ii) obtaining TILs from the subject, or if no TILs are available, obtaining T cells from the subject; iii) engineering the TILs to transgenically express a CSV-targeted and membrane- anchored cytokine, or if no TILs are available, engineering the T cells to transgenically express a CSV-targeted and membrane- anchored cytokine; and iv) if the CCKAR, SLN, LARGE2, and / or WNT10A expression is greater than or equal to about 0.0001 FPKM, and / or optionally the expression of IZUMO1 is less than or equal to about 0.0001 FPKM, administering to the subject the engineered TILs, or if no TILs are available, administering to the subject the engineered T cells and one or more inhibitors of CCKAR and / or CCKAR regulated pathways.

[0013] In some embodiments, the expression of CCKAR in the tumor is greater than or equal to about 0.0001, 0.001, 0.01, 0.1, 1.0, or 10.0 FPKM. In some embodiments, the expression of at least two of CCKAR, SLN, LARGE2, and WNT10A in the tumor are greater than or equal to about 0.0001 FPKM. In some embodiments, the expression of at least two of CCKAR, SLN, LARGE2, and WNT10A in the tumor are greater than or equal to 0.0001, 0.001, 0.01, 0.1, 1.0, or 10.0 FPKM. In some embodiments, the expression of at least three of CCKAR, SLN, LARGE2, and WNT10A in the tumor are greater than or equal to about 0.0001 FPKM. In some embodiments, the expression of at least three of CCKAR, SLN, LARGE2, and WNT10A in the tumor are greater than or equal to about 0.0001, 0.001, 0.01, 0.1, 1.0, or 10.0 FPKM. In some embodiments, the expression of CCKAR, SLN, LARGE2, and WNT10A in the tumor are greater than or equal to about 0.0001 FPKM. In some embodiments, the expression of CCKAR, SLN, LARGE2, and WNT10A in the tumor are greater than or equal to about 0.0001, 0.001, 0.01, 0.1, 1.0, or 10.0 FPKM. In some embodiments, methods further comprise determining expression levels of IZUMO 1 in the tumor, and confirming expression of IZUMO1 is lower than 10.0, 1.0, 0.1, 0.001, or 0.0001 FPKM. In some embodiments, the methods further comprise determining expression levels of IZUMO 1 in the tumor, and confirming expression of IZUMO1 is lower than 0.0001 FPKM.

[0014] In some embodiments, methods provided herein include administration of one or more CCKAR and / or CCKAR regulated pathway inhibitors. In some embodiments, the CCKAR and / or CCKAR regulated pathway inhibitor comprises one or more of an inhibitor of CCKAR, an inhibitor of phosphorylated AKT serine / threonine kinase 1 (pAKT), an inhibitorof phosphorylated SMAD family member 3 (pSMAD3), and / or an inhibitor of spectrin beta non-erythrocytic 2 (SPTBN2). In some embodiments, the CCKAR and / or CCKAR regulated pathway inhibitor comprises an antibody directed to CCKAR, pAKT, pSMAD3, and / or SPTBN2. In some embodiments, the CCKAR and / or CCKAR regulated pathway inhibitor comprises an antibody directed to CCKAR. In some embodiments, the CCKAR and / or CCKAR regulated pathway inhibitor comprises a specific inhibitor of SMAD3 (SIS3).

[0015] In some embodiments, methods further comprise characterizing the proteome, transcriptome, genome, and / or histology of a tumor. In some embodiments, a tumor is characterized by abundant collagen, abundant extracellular matrix (ECM), and / or high levels of transforming growth factor beta (TGFP) (e.g., abundant levels relative to a comparable tumor, such as a comparable early stage tumor, and / or to corresponding healthy tissue). In some embodiments, abundant collagen, abundant extracellular matrix (ECM), and / or high levels of transforming growth factor beta (TGFP) are relative to a comparable tumor (e.g., a tumor from the same or a different individual that is of the same cancer type, e.g., pancreatic, ovarian, lung, etc., and / or that comprises similar tumor suppressor and / or oncogene genetic mutations). In some embodiments, abundant collagen, abundant extracellular matrix (ECM), and / or high levels of transforming growth factor beta (TGFP) are relative to corresponding healthy tissue.

[0016] In some embodiments, methods provided herein are directed against cancers wherein the cancer is resistant to one or more immune effector cell therapies. In some embodiments, a cancer is resistant to one or more tumor-targeted immune effector cell therapies. In some embodiments, the cancer is resistant to Chimeric Antigen Receptor (CAR) T cell therapy (CAR-T), T Cell Receptor (TCR) T cell therapy (TCR-T), TIL therapy, B cell therapy, and / or Natural Killer (NK) cell therapy. In some embodiments, methods provided herein increase the anti-tumor efficacy of endogenous immune cells and / or additional heterogeneous immune effector cell treatments. In some embodiments, methods provided herein increase the tumor penetrating capacity of endogenous immune cells and / or additional heterogeneous immune effector cell treatments. In some embodiments, methods provided herein comprise administering and / or contacting of the tumor with the TILs, wherein the administering and / or contacting facilitates entrance of endogenous immune cells and / or one or more additional exogenous agents into the tumor.

[0017] In some embodiments, methods provided herein result in a reduction of CCKAR in the tumor, and in turn result in a downregulation of collagen, ECM, and / or TGFP in the tumor. In some embodiments, a reduction of CCKAR in the tumor reduces one or more of pAKT and / or TGFP, and / or pAKT or TGFP signaling pathway transducers. In some embodiments, areduction of CCKAR inhibits pAKT and / or TGFP signal transducers rat sarcoma virus (RAS), pSMAD3, and / or SPTBN2.

[0018] In some embodiments, methods provided herein comprise TILs obtained from a tumor and / or blood circulating TILs. In some embodiments, methods provided herein comprise TILs obtained from a tumor. In some embodiments, methods provided herein comprise blood circulating TILs. In some embodiments, methods provided herein comprise ex-vivo expanding of the TILs before, after, and / or during the genetic engineering. In some embodiments, methods provided herein comprise ex-vivo expanding of the TILs before, after, and during the genetic engineering.

[0019] In some embodiments, methods provided herein comprise administering to the subject engineered TILs and engineered T cells. In some embodiments, methods provided herein comprise administering to the subject engineered TILs, engineered T cells, and one or more additional therapeutic agents, such as but not limited to an inhibitor of a CCKAR, pAKT, pSMAD3, and / or SPTBN2.

[0020] In some embodiments, TILs utilized in methods provided herein are engineered to express the CSV-targeted and membrane-anchored cytokine by lentiviral transduction. In some embodiments, the engineered membrane- anchored cytokine comprises interleukin 12 (IL- 12). In some embodiments, a tumor targeted in methods described herein comprises cells expressing CSV. In some embodiments, greater than about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of cells in the tumor express CSV. In some embodiments, greater than about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of tumor cells in the tumor express CSV. In some embodiments, the tumor targeting moiety specifically binds CSV and facilitates increased levels of interferon gamma (IFNy) and / or tumor necrosis factor alpha (TNFa) in the tumor. In some embodiments, the CSV-targeted moiety comprises a CSV binding peptide. In some embodiments, the CSV binding peptide comprises a sequence identical to SEQ ID NO: 1. In some embodiments, the CSV-targeted and membrane-anchored cytokine comprises attIL12. In some embodiments, the attIL12 comprises an amino acid sequence at least 90% identical to SEQ ID NOs: 4 and 5.

[0021] In some embodiments, following engineered TIL administration to a subject, relative to administration of non-engineered TILs, non-engineered T cells, and / or engineered T cells, levels of TNFa and / or IFNy in the tumor are enriched, and / or levels of TGFP in the tumor are reduced. In some embodiments, levels of TNFa and IFNy in the tumor are enriched, and levels of TGFP in the tumor are reduced. In some embodiments, following administration of engineered TILs to the subject, relative to administration of non-engineered TILs, non-engineered T cells, and / or engineered T cells, levels of CCKAR in the tumor are significantly reduced. In some embodiments, methods described herein comprise contacting tumor cells and / or cancer associated fibroblasts (CAFs) with the engineered TILs. In some embodiments, methods described herein comprise contacting tumor cells and CAFs with the engineered TILs.

[0022] In some embodiments, methods described herein comprise simultaneously reducing levels of collagen and ECM in the tumor. In some embodiments, methods described herein comprise simultaneously reducing levels of collagen, ECM, CCKAR, and TGEP in the tumor. In some embodiments, tumors targeted in methods described herein comprise tumor stroma, and the methods of treatment comprise breaking the tumor stroma. In some embodiments, methods of breaking the tumor stroma comprise reducing levels of collagen and / or ECM in the tumor stroma. In some embodiments, methods of breaking the tumor stroma comprise simultaneously reducing levels of collagen and ECM in the tumor stroma. In some embodiments, methods of breaking the tumor stroma comprise simultaneously reducing levels of collagen, ECM, CCKAR, and TGEP in the tumor stroma.

[0023] In some embodiments, methods of treatment described herein are directed to tumors characterized as stroma rich. In some embodiments, methods of treatment described herein are directed to cancers wherein the cancer is recurrent. In some embodiments, methods of treatment described herein are directed to cancers wherein a tumor comprises a solid tumor. In some embodiments, a tumor is characterized as collagen rich. In some embodiments, a tumor is characterized as heterogenous. In some embodiments, a tumor is characterized as fibronectin (FN) rich. In some embodiments, a tumor is characterized as fiber rich. In some embodiments, a tumor is characterized as collagen rich, heterogenous, fibronectin rich, and / or fiber rich. In some embodiments, a tumor classified as grade 2 or higher.

[0024] In some embodiments, methods described herein comprise TILs that are not derived from peripheral blood mononuclear cells (PBMCs). In some embodiments, methods described herein comprise TILs that are derived from a tumor. In some embodiments, methods described herein comprise TILs that are derived from a disassociated tumor biopsy. In some embodiments, methods described herein comprise TILs that are autologous for the subject to be treated.

[0025] In some embodiments, methods described herein comprise TILs that have functional expression of one or more endogenous T cell receptor (TCR) genes. In some embodiments, TILs have functional expression of TCR alpha chain and / or TCR beta chain encoding genes. In some embodiments, methods described herein comprise engineered TILs that have functional expression of TCR alpha chain and / or TCR beta chain encoding genes, butsaid TILs have reduced toxicity relative to non-engineered TILs through expression of an attIL12 construct. In some embodiments, methods described herein comprise TILs that are engineered to express one or more additional transgenes. In some embodiments, the one or more additional transgenes comprises CARs, heterologous TCRs, cytokines, cytokine receptors, and / or safety switches.

[0026] In some embodiments, methods described herein comprise targeting of a tumor wherein the tumor comprises cells expressing CCKAR, SLN, LARGE2, and / or WNT10A at levels greater than or equal to about 0.0001 to about 30 FPKM. In some embodiments, methods described herein comprise targeting of a tumor wherein the tumor comprises cells expressing CCKAR at greater than or equal to about 0.0001 to about 30 FPKM. In some embodiments, methods described herein comprise targeting of a tumor wherein the tumor comprises cells expressing at least two of CCKAR, SLN, LARGE2, and WNT10A at greater than or equal to about 0.0001 to about 30 FPKM. In some embodiments, methods described herein comprise targeting of a tumor wherein the tumor comprises cells expressing at least three of CCKAR, SLN, LARGE2, and WNT10A at greater than or equal to about 0.0001 to about 30 FPKM. In some embodiments, methods described herein comprise targeting of a tumor wherein the tumor comprises cells expressing CCKAR, SLN, LARGE2, and WNT10A at greater than or equal to about 0.0001 to about 30 FPKM. In some embodiments, methods described herein comprise targeting of a tumor wherein the tumor comprises cells expressing IZUMO1 at levels less than or equal to about 0.01, 0.001, or 0.0001 FPKM. In some embodiments, methods described herein comprise treatment of a cancer, wherein the cancer comprises glioblastoma, cervical cancer, pancreatic cancer, ovarian cancer, uterine cancer, esophageal cancer, melanoma cancer, head and neck cancer, colorectal cancer, bladder cancer, lung cancer, prostate cancer, sarcoma cancer, breast cancer, liver cancer, renal cancer, and / or acute myelogenous leukemia. In some embodiments, the cancer comprises sarcoma. In some embodiments, the sarcoma comprises liposarcoma. In some embodiments, methods described herein comprise treatment of a cancer, wherein the cancer is classified expressing high levels of fibronectin and / or high levels of collagen.

[0027] In some embodiments, methods described herein comprise administering one or more additional anticancer therapy to the subject. In some embodiments, one or more additional anticancer therapy comprises a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, immunotherapy, and / or cytokine therapy. In some embodiments, one or more additional anticancer therapy is a chemotherapy. In some embodiments, chemotherapy comprises cyclophosphamide, methotrexate, fluorouracil, doxorubicin, vincristine, ifosfamide,cisplatin, gemcitabine, busulfan, and / or ara-C. In some embodiments, chemotherapy comprises cyclophosphamide. In some embodiments, one or more additional anticancer therapy comprises immunotherapy or cytokine therapy. In some embodiments, one or more additional anticancer therapy comprises treatment with one or more TGFP inhibitors (e.g., one or more TGFP signaling pathway inhibitors). In some embodiments, one or more TGFP inhibitors comprises an antibody specific for TGFP and / or TGFP receptor(s). In some embodiments, one or more additional anticancer therapy comprises treatment with one or more antibody-drug- conjugates (ADCs). In some embodiments, one or more additional anticancer therapy comprises treatment with one or more immune effector cell therapy. In some embodiments, one or more immune effector cell therapy comprises attIL12 T cell therapy.

[0028] In some embodiments, methods described herein can comprise administration of TILs, T-cells, and / or at least one or more additional therapy, wherein the TILs, T-cells, and / or at least one or more additional therapy are administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, or by direct injection or perfusion. In some embodiments, TILs and / or at least one or more additional therapy are administered sequentially and are temporally disparate. In some embodiments, TILs and / or at least one or more additional therapy are administered concurrently. In some embodiments, TILs penetrate to or near the center of a tumor within the subject. In some embodiments, TILs are CD3+. In some embodiments, TILs are CD8+ and / or CD4+. In some embodiments, TILs are CD8+.

[0029] Also provided herein are compositions comprising any one or more component associated with methods described herein.

[0030] Also provided herein are kits comprising any one or more component associated with methods described herein.

[0031] Certain aspects of the present disclosure are characterized through the following enumerated aspects.

[0032] Aspect 1 is a method of treating immune effector cell resistant cancer in a subject with a tumor comprising, reducing and / or diminishing Cholecystokinin A receptor (CCKAR) protein and / or transcript expression in the tumor by contacting the tumor with tumor infiltrating lymphocytes (TILs) transgenically expressing a cell surface vimentin (CSV)-targeted and membrane- anchored cytokine.

[0033] Aspect 2 is a method of treating cancer in a subject with a tumor comprising, obtaining TILs from the subject, engineering the TILs to transgenically express a CSV-targeted and membrane- anchored cytokine, and administering the engineered TILs to the subject.

[0034] Aspect 3 is a method of treating cancer in a subject with a tumor comprising the steps of: i) determining levels of CCKAR, Sarcolipin (SLN), LARGE xylosyl- and glucuronyltransferase 2 (LARGE2), and / or Wnt family member 10A (WNT10A) expression in the tumor, and optionally determining the levels of izumo sperm-oocyte fusion 1 (IZUMO1); ii) obtaining TILs from the subject; iii) engineering the TILs to transgenically express a CSV- targeted and membrane-anchored cytokine; and iv) if CCKAR, SLN, LARGE2, and / or WNT10A expression is greater than or equal to about 0.0001 Fragments Per Kilobase of transcript per Million mapped reads (FPKM), and / or optionally if the expression of IZUMO 1 is less than or equal to about 0.0001 FPKM, administering to the subject the engineered TILs.

[0035] Aspect 4 is a method of treating cancer in a subject with a tumor comprising the steps of: i) determining levels of CCKAR, SLN, LARGE2, and / or WNT10A expression in the tumor, and optionally determining the levels of IZUMO 1 expression in the tumor; ii) obtaining TILs from the subject, or if no TILs are available, obtaining T cells from the subject; iii) engineering the TILs to transgenically express a CSV-targeted and membrane- anchored cytokine, or if no TILs are available, engineering the T cells to transgenically express a CSV- targeted and membrane- anchored cytokine; and iv) if the CCKAR, SLN, LARGE2, and / or WNT10A expression is greater than or equal to about 0.0001 FPKM, and / or optionally the expression of IZUMO1 is less than or equal to about 0.0001 FPKM, administering to the subject the engineered TILs, or if no TILs are available, administering to the subject the engineered T cells and one or more inhibitors of CCKAR and / or CCKAR regulated pathways.

[0036] Aspect 5 is a method of any one of aspects 1-4, wherein the expression of CCKAR in the tumor is greater than or equal to about 0.0001 FPKM.

[0037] Aspect 6 is a method of any one of aspects 1-5, wherein the expression of at least two of CCKAR, SLN, LARGE2, and WNT10A in the tumor are greater than or equal to about 0.0001 FPKM.

[0038] Aspect 7 is a method any one of aspects 1-6, wherein the expression of at least three of CCKAR, SLN, LARGE2, and WNT10A in the tumor are greater than or equal to about 0.0001 FPKM.

[0039] Aspect 8 is a method of any one of aspects 1-7, wherein the expression of CCKAR, SLN, LARGE2, and WNT10A in the tumor are greater than or equal to about 0.0001 FPKM.

[0040] Aspect 9 is a method of any one of aspects 1-8 further comprising determining expression levels of IZUMO 1 in the tumor, and confirming expression of IZUMO 1 is lower than 0.0001 FPKM.

[0041] Aspect 10 is a method of any one of aspects 3-9, wherein the CCKAR and / or CCKAR regulated pathway inhibitor comprises an inhibitor of CCKAR, phosphorylated AKT serine / threonine kinase 1 (pAKT), phosphorylated SMAD family member 3 (pSMAD3), and / or spectrin beta non-erythrocytic 2 (SPTBN2).

[0042] Aspect 11 is a method of any one of aspects 1-10, wherein the tumor is characterized by abundant collagen, abundant extracellular matrix (ECM), and / or high levels of transforming growth factor beta (TGFP) relative to a comparable tumor, optionally a comparable early stage tumor, and / or to corresponding healthy tissue.

[0043] Aspect 12 is a method of any one of aspects 1-11, wherein the cancer is resistant to tumor- targeted immune effector cell therapy.

[0044] Aspect 13 is a method of any one of aspects 1-12, wherein the cancer is resistant to Chimeric Antigen Receptor (CAR) T cell therapy (CAR-T), T Cell Receptor (TCR) T cell therapy (TCR-T), TIL therapy, B cell therapy, and / or NK cell therapy.

[0045] Aspect 14 is a method of any one of aspects 1-13, wherein the anti-tumor efficacy of endogenous immune cells and / or additional heterogeneous immune effector cell treatments are improved.

[0046] Aspect 15 is a method of any one of aspects 1-14, wherein the reduction of CCKAR results in downregulation of collagen, ECM, and / or TGFP in the tumor.

[0047] Aspect 16 is a method of any one of aspects 1-15, wherein the reduction of CCKAR inhibits one or more of pAKT, TGFP, and / or pAKT or TGFP signaling pathway transducers.

[0048] Aspect 17 is a method of any one of aspects 1-16, wherein the reduction of CCKAR inhibits pAKT and / or TGFP signal transducers rat sarcoma virus (RAS), pSMAD3, and / or SPTBN2.

[0049] Aspect 18 is a method of any one of aspects 1-15, wherein the TILs comprise TILs obtained from the tumor and / or blood circulating TILs.

[0050] Aspect 19 is a method of any one of aspects 1-18, wherein the method comprises ex-vivo expanding the TILs before, after, and / or during the genetic engineering.

[0051] Aspect 20 is a method of any one of aspects 1-19, wherein the method comprises administering to the subject the engineered TILs and engineered T cells.

[0052] Aspect 21 is a method of any one of aspects 1-20, wherein the TILs are engineered to express the CSV-targeted and membrane- anchored cytokine by lentiviral transduction.

[0053] Aspect 22 is a method of any one of aspects 1-21, wherein the membrane-anchored cytokine comprises interleukin 12 (IL- 12).

[0054] Aspect 23 is a method of any one of aspects 1-22, wherein the tumor comprises cells expressing cell- surface vimentin (CSV).

[0055] Aspect 24 is a method of any one of aspects 1-23, wherein greater than about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of cells in the tumor express CSV.

[0056] Aspect 25 is a method of aspect 24, wherein greater than about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of tumor cells in the tumor express CSV.

[0057] Aspect 26 is a method of any one of aspects 1-25, wherein the tumor targeting moiety specifically binds CSV and facilitates increased levels of interferon gamma (IFNy) and / or tumor necrosis factor alpha (TNFa) in the tumor.

[0058] Aspect 27 is a method of any one of aspects 1-26, wherein the CSV-targeted moiety comprises a CSV binding peptide.

[0059] Aspect 28 is a method of any one of aspects 1-27, wherein the CSV-targeted and membrane- anchored cytokine comprises attIL12.

[0060] Aspect 29 is a method of aspect 28, wherein the attIL12 comprises an amino acid sequence at least 90% identical to any one or more of SEQ ID NOs: 4-7.

[0061] Aspect 30 is a method of any one of aspects 1-29, wherein following engineered TILs administration to the subject, relative to administration of non-engineered TILs, nonengineered T cells, and / or engineered T cells, levels of TNFa and / or IFNy in the tumor are enriched, and / or levels of TGFP in the tumor are reduced.

[0062] Aspect 31 is a method of any one of aspects 1-30, wherein following engineered TILs administration to the subject, relative to administration of non-engineered TILs, nonengineered T cells, and / or engineered T cells, levels of CCKAR in the tumor are significantly reduced.

[0063] Aspect 32 is a method of any one of aspects 1-31, comprising contacting tumor cells and / or cancer associated fibroblasts (CAEs) with the engineered TILs.

[0064] Aspect 33 is a method of any one of aspects 1-32, comprising simultaneously reducing levels of collagen and ECM in the tumor.

[0065] Aspect 34 is a method of any one of aspects 1-33, comprising simultaneously reducing levels of collagen, ECM, CCKAR, and TGFP in the tumor.

[0066] Aspect 35 is a method of any one of aspects 1-33, wherein the tumor comprises tumor stroma and the method comprises breaking the tumor stroma.

[0067] Aspect 36 is a method of aspect 35, comprising reducing levels of collagen and / orECM in the tumor stroma.

[0068] Aspect 37 is a method of aspect 35 or 36, comprising simultaneously reducing levels of collagen and ECM in the tumor stroma.

[0069] Aspect 38 is a method of aspect 37, comprising simultaneously reducing levels of collagen, ECM, CCKAR, and TGFP in the tumor stroma.

[0070] Aspect 39 is a method of any one of aspects 35-38, wherein the tumor is characterized as stroma rich.

[0071] Aspect 40 is a method of any one of aspects 1-38, wherein the cancer is recurrent.

[0072] Aspect 41 is a method of any one of aspects 1-40, wherein the tumor comprises a solid tumor.

[0073] Aspect 42 is a method of any one of aspects 1-41, wherein the tumor is characterized as collagen rich relative to a comparable tumor, optionally a comparable early stage tumor, and / or to corresponding healthy tissue.

[0074] Aspect 43 is a method of any one of aspects 1-42, wherein the tumor is characterized as heterogenous.

[0075] Aspect 44 is a method of any one of aspects 1-43, wherein the tumor is characterized as fibronectin (FN) rich relative to a comparable tumor, optionally a comparable early stage tumor, and / or to corresponding healthy tissue.

[0076] Aspect 45 is a method of any one of aspects 1-44, wherein the tumor is characterized as fiber rich relative to a comparable tumor, optionally a comparable early stage tumor, and / or to corresponding healthy tissue.

[0077] Aspect 46 is a method of any one of aspects 1-45, wherein the tumor is a tumor classified as grade 2 or higher.

[0078] Aspect 47 is a method of any one of aspects 1-46, wherein the TILs are not derived from peripheral blood mononuclear cells (PBMCs).

[0079] Aspect 48 is a method of any one of aspects 1-47, wherein the TILs comprise alpha / beta T cells, Natural Killer T (NKT) cells, gamma / delta T cells, and / or NK cells.

[0080] Aspect 49 is a method of any one of aspects 1-48, wherein the TILs are CD3+.

[0081] Aspect 50 is a method of any one of aspects 1 -49, wherein the TILs are CD8+ and / or CD4+.

[0082] Aspect 51 is a method of any one of aspects 1-50, wherein the TILs are derived from a tumor.

[0083] Aspect 52 is a method of any one of aspects 1-51, wherein the TILs are autologous.

[0084] Aspect 53 is a method of any one of aspects 1-52, wherein the TILs have functional expression of one or more endogenous T cell receptor (TCR) genes.

[0085] Aspect 54 is a method of any one of aspects 1-53, wherein the TILs have functional expression of TCR alpha chain and / or TCR beta chain encoding genes.

[0086] Aspect 56 is a method of any one of aspects 1-54, wherein the TILs comprise greater than about 80% alpha / beta T cells.

[0087] Aspect 57 is a method of any one of aspects 1-55, wherein the TILs comprise greater than about 95% alpha / beta T cells.

[0088] Aspect 58 is a method of any one of aspects 1-56, wherein the TILs consist essentially of or consist of alpha / beta T cells.

[0089] Aspect 59 is a method of any one of aspects 1-57, wherein the TILs comprise TCR alpha chains and / or TCR beta chains targeting one or more tumor associated antigens.

[0090] Aspect 60 is a method of any one of aspects 1-58, where the TILs comprise tumor specific TCR-T cells.

[0091] Aspect 61 is a method of any one of aspects 1-59, wherein greater than 80% of the TILs comprise tumor specific TCR-T cells.

[0092] Aspect 62 is a method of any one of aspects 1-60, wherein greater than 95% of the TILs comprise tumor specific TCR-T cells.

[0093] Aspect 63 is a method of any one of aspects 1-61, wherein the TILs are engineered to express one or more additional transgenes.

[0094] Aspect 64 is a method of aspect 62, wherein the one or more additional transgenes comprises CARs, heterologous TCRs, cytokines, cytokine receptors, and / or safety switches.

[0095] Aspect 65 is a method of any one of aspects 1-63, wherein the tumor comprises cells expressing CCKAR, SLN, LARGE2, and / or WNT10A at levels greater than or equal to about 0.0001 to about 30 FPKM.

[0096] Aspect 66 is a method of any one of aspects 1-64, wherein the tumor comprises cells expressing CCKAR at greater than or equal to about 0.0001 to about 30 FPKM.

[0097] Aspect 67 is a method of any one of aspects 1-65, wherein the tumor comprises cells expressing at least two of CCKAR, SLN, LARGE2, and WNT10A at greater than or equal to about 0.0001 to about 30 FPKM.

[0098] Aspect 68 is a method of any one of aspects 1-66, wherein the tumor comprises cells expressing at least three of CCKAR, SLN, LARGE2, and WNT10A at greater than or equal to about 0.0001 to about 30 FPKM.

[0099] Aspect 69 is a method of any one of aspects 1-67, wherein the tumor comprises cells expressing CCKAR, SLN, LARGE2, and WNT10A at greater than or equal to about 0.0001 to about 30 FPKM.

[0100] Aspect 70 is a method of any one of aspects 1-68, wherein the tumor comprises cells expressing IZUMO1 at levels less than or equal to about 0.0001 FPKM.

[0101] Aspect 71 is a method of any one of aspects 1-69, wherein the cancer is glioblastoma, cervical cancer, pancreatic cancer, ovarian cancer, uterine cancer, esophageal cancer, melanoma cancer, head and neck cancer, colorectal cancer, bladder cancer, lung cancer, prostate cancer, sarcoma cancer, breast cancer, liver cancer, renal cancer, and / or acute myelogenous leukemia.

[0102] Aspect 72 is a method of any one of aspects 1-70, wherein the cancer comprises sarcoma.

[0103] Aspect 73 is a method of aspect 71, wherein the sarcoma comprises liposarcoma.

[0104] Aspect 74 is a method of any one of aspects 1-72, wherein the cancer is classified expressing high levels of fibronectin and / or high levels of collagen relative to a comparable tumor, optionally a comparable early stage tumor, and / or to corresponding healthy tissue.

[0105] Aspect 75 is a method of any one of aspects 1-73, wherein the administering and / or contacting of the tumor with the TILs facilitates entrance of endogenous immune cells and / or one or more additional exogenous agents into the tumor.

[0106] Aspect 76 is a method of any one of aspects 1-74, comprising administering one or more additional anticancer therapy to the subject.

[0107] Aspect 77 is a method of aspect 75, wherein the one or more additional anticancer therapy comprises a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, immunotherapy, and / or cytokine therapy.

[0108] Aspect 78 is a method of aspect 76, wherein the one or more additional anticancer therapy is a chemotherapy.

[0109] Aspect 79 is a method of aspect 77, wherein the chemotherapy comprises cyclophosphamide, methotrexate, fluorouracil, doxorubicin, vincristine, ifosfamide, cisplatin, gemcitabine, busulfan, and / or ara-C.

[0110] Aspect 80 is a method of aspect 78, wherein the chemotherapy comprises cyclopho sphamide .

[0111] Aspect 81 is a method of any one of aspects 75-79, wherein the one or more additional anticancer therapy comprises immunotherapy or cytokine therapy.

[0112] Aspect 82 is a method of aspect 80, wherein the one or more additional anticancer therapy comprises treatment with one or more TGFP inhibitors.

[0113] Aspect 83 is a method of aspect 81, wherein the one or more TGFP inhibitor comprises an antibody specific for TGFP and / or TGFP receptor(s).

[0114] Aspect 84 is a method of any one of aspects 75-82, wherein the one or more additional anticancer therapy comprises treatment with one or more antibody-drug-conjugates (ADCs).

[0115] Aspect 85 is a method of any one of aspects 75-83, wherein the one or more additional anticancer therapy comprises treatment with one or more immune effector cell therapy.

[0116] Aspect 86 is a method of aspect 84, wherein the one or more immune effector cell therapy comprises attIL12 T cell therapy.

[0117] Aspect 87 is a method of any one of aspects 75-85, wherein the TILs and / or at least one or more additional therapy is administered intravenously, intraperitoneally, intratracheally, intratumor ally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, or by direct injection or perfusion.

[0118] Aspect 88 is a method of any one of aspects 75-86, wherein the TILs and / or at least one or more additional therapy are administered sequentially and are temporally disparate.

[0119] Aspect 89 is a method of any one of aspects 75-86, wherein the TILs and / or at least one or more additional therapy are administered concurrently.

[0120] Aspect 90 is a method of any one of aspects 1-88, wherein the TILs penetrate to or near the center of a tumor within the subject.

[0121] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0122] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. Certain inventions described herein may be better understood by reference to one or more of these drawings in combination with the detailed description presented herein.

[0123] FIGs. 1A-1D, Engineered attIL12-tumor infiltrating lymphocytes (TILs) showed superior in vivo tumor infiltration when compared to control-TILs (“ctrl-TILs”) despite similar profiles of cell surface markers and effector molecules when measured in vitro. SA 127 TILs were left un-transduced (“SA127TIL”) or transduced with control lentivirus (“SA127TIL-ctrl)or attIL12 lentivirus (“SA127TIL-attIL12”) for 48 hours. FIG. 1A shows flow cytometry results for SA127TIL (left), SA127TIL-ctrl (middle), and SA127TIL-attIL12 (right) cells stained for IL12, CD3, CD4, CD8, CTLA4, PD1, CD28, CD69, Lag3, CD39 and NKG2D. FIG. IB shows flow cytometry results for isotype (FMO; left), SA127TIL-ctrl (middle), and SA127TIL-attIL12 (right) cells. Cells were treated with Brefeldin A (BFA) four hours before harvest. Cells were fixed and permeabilized, followed by staining for IFNy (top) and granzyme b (bottom) for flow cytometry analysis. FIG. 1C shows tumor staining from SA127 tumor bearing SCID mice. Mice were preconditioned with cyclophosphamide prior to two treatments of SA127TIL-ctrl or SA127TIL-attIL12. 4 days after the 2nd TIL transfer, tumors were collected for sectioning. Tumor sections were immunohistochemically stained for hCD3, which was detected with a Keyence microscope. FIG. ID shows flow cytometry results from the tumors presented in FIG. 1C, tumors were dissociated to single cell mixtures and stained with immune cell markers described in FIG. IB.

[0124] FIGs. 2A-2C, Engineered attIL12-TIL treatment boosted antitumor efficacy when treating autologous tumors. SA 127 tumor bearing SCID mice were preconditioned with cyclophosphamide (Dl l) prior to receiving two treatments with 2 x 10A6 ctrlTILs (per mouse per infusion) or 2 x 10A6 attIL12-TILs (per mouse per infusion), or no treatments, on indicated days D14 and D30 (black arrows). FIG. 2A shows tumor volume in the treated mice, monitored twice weekly. FIG. 2B shows a Kaplan-Meier survival curve for the animals. FIG. 2C shows the results of flow cytometry analysis of dissociated tumor cells obtained from the mice. Mice were treated with BFA 4 hours prior to tumor collection. Tumors were dissociated to single cell mixture and stained for effector molecular markers IFNy and GzmB. The percentage of total T cells positive for the effector molecules was determined. N = 4-6. Two way ANOVA was used for statistics analysis. attIL12-TIL vs. control TIL or no treatment: P < 0.0001, control TIL vs. no treatment: P = 0.0088.

[0125] FIGs. 3A-3E, High collagen deposition in human sarcoma tissues was associated with poor T cell scores and reduced survival. FIG. 3A provides imaging of an exemplary liposarcoma tissue microarray, where the samples were subjected to Sirius red staining to identify collagen density. FIG. 3B provides results of a probability of survival analysis for liposarcoma patients from the TCGA dataset when classified by ECM markers collagen (alpha-2 subunit of the fibril-forming type I collagen (CollA2), high or low) and Fibronectin (FN, high or low). High levels of Coll A2 and FN were associated with a significant reduction in probability of patient survival. FIG. 3C provides a Pearson correlation analysis of ECM markers and CD8b. FIG. 3D displays representative Sirius staining for collagen densityin SA127 tumors treated with sham, ctrl-TILs, or engineered attIL12-TILs. FIG. 3E displays representative immunofluorescence staining for collagen (pan-col) density in SA 127 tumors treated with ctrlTIL, attIL12TIL, or attIL12T respectively.

[0126] FIGs. 4A-4D, Cytokine balance shift regulated collagen production by sarcoma cells. FIG. 4A provides a Pearson correlation analysis of liposarcoma patient TCGA dataset, where transforming growth factor beta 1 (TGFpi) was correlated with the alpha- 1 subunit of the fibril-forming type I collagen (Coll Al) levels (left panel) and interferon gamma (IFNy) was correlated with CollAl and CollA2. FIG. 4B shows analysis of tumor lysates from FIG. 1C, where tumors were harvested by smashing frozen tumor samples in RIPA lysis and extraction buffer. 100 pg tumor lysates were used for ELISA to detect IFNy and TGFP levels in the tumor samples. The results showed significantly increased levels of IFNy and significantly decreased levels of TGFP in animals treated with attIL12-TILs. FIG. 4C, shows the levels of IFNy and TGFP in supernatant from cell culture of SA 127 cells with ctrl-TIL, attIL12-TIL, or sham. Cells were cocultured at an Effector to Target cell (E:T) ratio of 1:4 for 24 hours prior to harvesting and detection of IFNy and TGFP levels using ELISA. The results showed significantly increased levels of IFNy and significantly decreased levels of TGFP in supernatant from attIL12-TIL cocultures. FIG 4D, after coculture, T cells were removed (CD3), and tumor cells were stained with human anti-collagen antibody, the cells were then analyzed by flow cytometry. The results showed a significant decrease in the percentage of SA127 tumor cells staining positive for collagen when cocultured with attIL12-TILs relative to SA 127 tumor cells alone or cocultured with ctrl-TILs.

[0127] FIGs. 5A-5J, Dual signaling activation proved essential for the potency of attIL12-TILs. FIG. 5A, TILs were transduced with Ctrl (ctrlTIL), attIL12 (attIL12TIL), or attIL12 plus TCR knockout (e.g., SEQ ID NO: 8 sgRNA CRISPR / Cas system targeted to TCRa (“TCRaP- / -”)). TCRaP and IL12 were detected via flow cytometry, and TCRaP and / or IL12 expression profiles were confirmed. FIGs. 5B-5E, show results from SA127 tumor cells or SAI 17 tumor cells cocultured with ctrlTIL, attIL12TIL, or attIL12TCRaP- / -TILs without or with CSV blocking antibody (“+aCSV”). FIG. 5B shows the levels of IFNy and GzmB levels in TILs that were collected and analyzed via flow cytometry. The results showed decreased levels of IFNy and / or GzmB in +aCSV and TCRaP- / - test attIL12TIL populations. FIG. 5C shows the levels of collagen in tumor cells that were collected and analyzed via flow cytometry. The results showed decreased levels of collagen in tumor cells treated with attIL12TIL cells, but that this decrease was lost in +aCSV and / or TCRaP- / - test attIL12TIL populations. FIG. 5D shows the results of ELISA analysis TGFP and IFNy in supernatants from the cocultureexperiments. FIG. 5E shows the results of immunoblotting of tumor cells for detection of Collagen, SMAD family member 3 (SMAD3), phosphorylated SMAD3 (pSMAD3), or spectrin beta non-erythrocytic 2 (SPTBN2; “SPP2” or “SPb2”) with GAPDH as control. FIG. 5F shows the results of tumor volume analysis (measured twice weekly) in SA127 tumor bearing SCID mice that were pre-treated with cyclophosphamide on DI 1 and then treated twice (or not in no treatment control; D14 and D30) with ctrlTILs, attIL12-TILs, or attIL12-T cells. FIG. 5G shows the results of tumor volume analysis (measured twice weekly) in SA127 tumor bearing SCID mice that were pre-treated with cyclophosphamide on DI 1 and then treated twice (or not in no treatment control; D14 and D21) with ctrlTILs, attIL12-TILs, or attIL12TILTCRaP- / - cells. The results showed a significant increase in tumor volume in attIL12TCRaP- / -and ctrlTIL treatment groups relative to attIL12TIL treatments. FIGs. 5H-I, shows flow cytometry results from tumors and TILs described in FIG. 5G. Tumors were dissociated and cells were sorted via flow cytometry. IFNy and TGFP expression levels were detected in tumor infiltrating TILs (5H), and collagen was analyzed in tumor cells (51). FIG. 5J shows immunofluorescence staining of human collagen (green) and CD3 (red) from tumors excised from animals described in FIG. 5G, scale bar: 500 pm.

[0128] FIGs. 6A-6F, attIL12-TILs induced CCKAR downregulation to abolish its enhancer role in collagen regulation. FIG. 6A shows an RNAseq waterfall plot that indicated that CCKAR was the most significantly downregulated gene in attIL12-T cell treatment sensitive OS patient derived xenograft (PDX) models. FIGs. 6B-6F depict various immunoblots showing protein levels in SA127 or SAI 17 cells, or in transgenic SA127 cells or SAI 17 cells that were overexpressing CCKAR (SA127CCKARor SAI | 7CCKAR^when the tumor cells were cocultured with ctrlTIL or attIL12TILs at an E:T ratio of 1:4 in the presence or absence of indicated antibodies (e.g., anti-TGFp (aTGFP), anti-IFNy (alFNy), or anti-CCKAR (aCCKAR)) and / or cytokines / signaling molecules (e.g., IFNy, TGFp, or CCK) when treated for 24 hours. IFNy, TGFp, or CCK cultured at 100 ng / ml; anti-CCKAR, anti-IFNy, anti-TGFp antibodies at 10 pg / ml; SIS3 at 3 pM. Immune cells were removed, and tumor cells were lysed with RIPA buffer and denatured for immunoblot analysis. Collagen I, CCKAR, pSMAD3, Smad3, SPTBN2 (SPb2), CCK, pAkt, and Akt expression were detected in the tumor cell lysates. The results showed that attIL12TIL reduced CCKAR levels, collagen levels, pSMAD3 levels, and pAkt levels.

[0129] FIG. 7, shows how attIL12-TIL (brachyury T cells specific to UM-chor-1 cells) served as an inhibitor of pAKT and TGFP signaling pathway induction in UM-chor-1 cells when cultured at a 1:4 E:T ratio for 24 hours.

[0130] FIGs. 8A-8B, shows how attIL12-TILs expanded utilizing GMP protocols without anti-41BB antibody induced CCKAR downregulation and abolished its enhancer role in collagen regulation. FIG. 8A depict immunoblot results showing protein levels in SAI 17 cells when the tumor cells were cocultured with ctrlTIL, attIL12TIL, or attIL l 2I C R / “ TIL effector cells, at an E:T ratio of 1:4 in the presence or absence of indicated antibodies (e.g., anti-CSV (aCSV)), when treated for 24 hours. Immune cells were removed, and tumor cells were lysed with RIPA buffer and denatured for immunoblot analysis. Collagen I, CCKAR, pSMAD3, Smad3, SPTBN2 (SPb2), pAkt, and Akt expression (and GAPDH loading control) were detected in the tumor cell lysates. The results showed that attIL12TIL reduced CCKAR levels, collagen levels, SPTBN2, and pSMAD3 levels. FIG. 8B depict immunoblots showing protein levels in SAI 17 cells when the tumor cells were cocultured with ctrlTIL, or attIL12TIL, at an E:T ratio of 1:4 in the presence or absence of indicated antibodies (e.g., anti-TGFp (aTGFP), or anti-IFNy (alFNy)) and / or cytokines / signaling molecules (e.g., IFNy, or TGFP) when treated for 24 hours. Immune cells were removed, and tumor cells were lysed with RIPA buffer and denatured for immunoblot analysis. Collagen I, CCKAR, pSMAD3, Smad3, SPTBN2 (SPb2), pAkt, and Akt expression (and GAPDH loading control) were detected in the tumor cell lysates. The results showed that attIL12TIL reduced CCKAR levels, collagen levels, SPTBN2, and pSMAD3 levels.DETAILED DESCRIPTION

[0131] Rich extracellular matrix (ECM) in solid tumors, such as Osteosarcoma (OS) has been associated with invasiveness, metastases and / or reduced patient survival. This rich ECM may provide tumors with a protective shield that can impede immune cell infiltration into tumors. There is a pressing need for methods and / or compositions for improving immune cell infiltration into tumors and / or breaking the protective shield provided by ECM components and / or ECM related signaling pathways (for example but not limited to, collagen, fibronectin, TGFP, pSMAD3, pAKT, etc.).

[0132] In certain embodiments, the present disclosure provides methods of utilizing engineered effector cell immunotherapy comprising chimeric antigen-like (CAR-like) constructs, particularly CAR-like constructs comprising a membrane- anchored and tumor- targeted cytokine (e.g., IL-12) to treat diseases, such as cancers. The construct may comprise a tumor-targeting moiety, such as a peptide, antibody, or fragment thereof. An exemplary tumortargeting moiety is the cell surface vimentin (CSV) binding peptide (e.g., VNTANST (SEQ ID NO: 1) and / or an scFv. In some embodiments, the construct may be a retroviral vector or lentiviral vector. Further provided herein are cells engineered to express the construct, such asimmune cells, particularly Tumor Infiltrating Lymphocytes (TILs), which may be CD3+ T cells, and which may be CD8+ and / or CD4+ T cells. In some embodiments, engineered immune effector cells may be used to treat a disease or disorder, such as cancer, including solid tumors and / or blood cancers.

[0133] Specifically, in some embodiments, the CAR-like construct may comprise, consist, or consist essentially of a tumor-targeted membrane- anchored IL- 12 construct (“attIL12” or “attIL-12”). The attIL-12 can comprise an IL- 12 alpha subunit p35 (SEQ ID NO: 4) fused with a cell membrane anchoring domain (for example but not limited to, an EGFR transmembrane domain; e.g., SEQ ID NO: 2) directly or indirectly via a linker (for example but not limited to, e.g., SGGGGSGGGGSS; SEQ ID NO: 3), and an IL- 12 beta subunit p40 (SEQ ID NO: 5) fused with tumor-targeted peptide (e.g., a CSV binding peptide, e.g., VNTANST, SEQ ID NO: 1).

[0134] In certain embodiments, a tumor-targeted membrane- anchored construct may comprise heterologous intracellular signaling domains, such as those described in the inventors patent application publication U.S. 2022 / 0118015 Al published April 21, 2022, which is incorporated herein by reference in its entirety for the purposes described herein. In some embodiments, a tumor-targeted membrane- anchored construct may comprise a construct as described in the inventors patent publication U.S. 11,421,010 B2 granted August 23, 2022, which is incorporated herein by reference in its entirety for the purposes descried herein. In some embodiments, a tumor-targeted membrane- anchored construct may comprise a construct as described in the inventors publication Jiemiao Hu et al., “Cell Membrane anchored and tumor-targeted IL- 12 (attIL12)-T cell therapy for eliminating large and heterogeneous solid tumors”, Journal for ImmunoTherapy of Cancer, 2022, which is incorporated herein by reference in its entirety for the purposes described herein.

[0135] In certain embodiments, provided herein are naturally occurring tumor infiltrating lymphocytes (TILs) that have been engineered to express a tumor-targeted membrane- anchored construct (e.g., attIL12). In certain embodiments, the TILs are T cells that have infiltrated a subject’s tumor (e.g., a primary tumor, a secondary tumor, a metastasis, etc.). In some embodiments, the TILs are T cells that have infiltrated a subject’s tumor, left said tumor, and are collected from the blood of a subject. In some embodiments, TILs are ex vivo expanded. In some embodiments, TILs are activated ex vivo. In some embodiments, TILs have developed T cell Receptors (TCR) specific to a subject’s tumor neoantigens. In some embodiments, TILs have increased efficacy when the TCR alpha, beta, gamma, and / or delta genes are active and / or not ablated (e.g., not knocked out, knocked down, etc.). In some embodiments, TILs increasedefficacy requires activity of the TCR alpha, beta, gamma, and / or delta genes. In some embodiments, TILs increased efficacy requires activity of the TCR alpha and TCR beta genes. In some embodiments, engineered TILs provided herein can challenge tumors that are resistant to engineered cell therapies, such as but not limited to T cell therapies (e.g., CAR-T cells, attIL12-T cells, cytokine expressing T cells, etc.). In some embodiments, the TILs are not derived from peripheral blood monocytes (PBMC). In some embodiments, the TILs are cells that permeated a primary and / or secondary tumor, and are active against the tumor. In some embodiments, the TILs are obtained from a subject (e.g., from a biopsy of a tumor and / or from the blood of a patient), engineered, optionally expanded, optionally activated, and then reintroduced into the subject. In some embodiments, engineered TILs provided herein can reduce a tumors (inclusive of tumor cells, cancer associated fibroblasts, tumor associated cells, stroma, etc.) expression of TGFp, pSMAD3, pAkt, collagen, and / or CCKAR. In certain embodiments, engineered TIL treatment synergistically improves the impact of other types of endogenous or non-endogenous immune cells, such as endogenous T cell infiltration into the tumors and / or non-endogenous immune effector cell treatment infiltration into the tumors. In certain embodiments, engineered TIL treatment significantly reduces CCKAR expression in CCKAR expression high tumors, and breaks the resistance of said tumors to immunotherapies, such as but not limited to antibody therapies and / or immune effector cell therapies (e.g., CAR-T, TCR- T, NK cell, etc.).

[0136] As described herein, in certain embodiments that utilize attIL12, the construct comprises both the IL-12 p35 and p40 subunits. In some embodiments, the p40 subunit may be fused to the tumor-targeting moiety, such as a CSV binding peptide. In some embodiments, the p35 subunit may be fused to a transmembrane domain, such as EGFR transmembrane domain. In some embodiments, the p35 subunit may further be fused to a cell signaling domain, such as 4- IBB and / or CD3(^. In some embodiments, a construct can target tumors directly using the tumor-targeted peptide and / or scFv fused directly or indirectly with the p35 and / or p40 subunit; and can induce effector cell (e.g., TIL and / or T cell) proliferation through the membrane anchored p35 subunit. In certain embodiments, the membrane- anchored and tumor-targeted nature of the construct reduces toxicity concerns from effector cell therapy and / or IL- 12 therapy by rapidly targeting the effector cells to tumors. In certain embodiments, the membrane- anchored and tumor-targeted nature of the construct reduces toxicity concerns from TIL and / or T cell therapies that comprise active TCR alpha and / or beta genes by rapidly targeting the TILs and / or T cells to tumors. In some embodiments, engineered effector cell therapy described herein may be administered in tandem and / or in sequence with one or moretherapies, such as but not limited additional effector cell therapies, immunotherapies, and / or chemotherapies. In some embodiments, the engineered effector cell therapies act synergistically with the one or more additional therapies to provide improved therapeutic effects (e.g., increased tumor cell proliferation control, increased solid tumor penetration, decreased tumor and / or tumor supporting cell provided signaling molecules such as but not limited to TGFP).A. Definitions

[0137] As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and / or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01 %. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

[0138] As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.

[0139] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” As used herein “another” may mean at least a second or more. The terms “about”, “substantially” and “approximately” mean, in general, the stated value plus or minus 5%.

[0140] “Treating” or treatment of a disease or condition refers to executing a protocol, which may include administering one or more drugs to a patient, in an effort to alleviate signs or symptoms of the disease. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. Alleviation can occur prior to signs or symptoms of the disease or condition appearing, as well as after their appearance. Thus, “treating” or “treatment” may include “preventing” or “prevention” of disease or undesirable condition. In addition, “treating” or “treatment” does not require complete alleviation of signs or symptoms, does not require a cure, and specifically includes protocols that have only a marginal effect on the patient.

[0141] The term “therapeutic benefit” or “therapeutically effective” as used throughout this application refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of this condition. This includes, but is not limited to, areduction in the frequency or severity of the signs or symptoms of a disease. For example, treatment of cancer may involve, for example, a reduction in the size of a tumor, a reduction in the invasiveness of a tumor, reduction in the growth rate of the cancer, or prevention of metastasis. Treatment of cancer may also refer to prolonging survival of a subject with cancer.

[0142] “Subject” and “patient” refer to either a human or non-human, such as primates, mammals, and vertebrates. In particular embodiments, the subject is a human.

[0143] The phrases “pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, such as a human, as appropriate. The preparation of a pharmaceutical composition comprising an antibody or additional active ingredient will be known to those of skill in the art in light of the present disclosure. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biological Standards.

[0144] As used herein, “pharmaceutically acceptable carrier” includes any and all aqueous solvents (e.g., water, alcoholic / aqueous solutions, saline solutions, parenteral vehicles, such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters, such as ethyloleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, anti-oxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, fluid and nutrient replenishers, such like materials and combinations thereof, as would be known to one of ordinary skill in the art. The pH and exact concentration of the various components in a pharmaceutical composition are adjusted according to well-known parameters.

[0145] The term “membrane-anchored IL- 12” refers to an IL- 12 protein that comprises a transmembrane domain. The term “membrane- anchored tumor-targeted IL- 12 (attIL12)” refers to an IL- 12 protein that comprises both a transmembrane domain and a tumor-targeted domain.

[0146] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 80%, 85%, 90%, or 95%) of “percent similarity” or “sequence similarity” which refers to the degree by which one amino acid may substitute for another amino acid without loss of function. This percent similarity can be determined through the use of a matrix such as the PAM250 or BLOSUM62 matrix.

[0147] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 80%, 85%, 90%, or 95%) of "sequence identity" or “homology” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (E. M. Ausubel et al., eds., 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, using default parameters.

[0148] The term “corresponding healthy tissue” relative to a tumor tissue refers to the histologically normal tissue that is adjacent to a tumor but is not affected by cancer. This tissue is often used as a control in cancer research to compare the molecular and cellular characteristics of tumor tissues. In some aspects of the current disclosure, the tumor comprises at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or any range derivable therein or higher levels of one or more of collagen, fibronectin, fiber, extracellular matrix, and TGEP relative to corresponding healthy tissue. In some aspects of the current disclosure, the tumor comprises at least about 5-fold, or about 6- fold, or about 7-fold, or about 8-fold, or about 9-fold, or about 10-fold, or any range derivable therein or higher levels of one or more of collagen, fibronectin, fiber, extracellular matrix, and TGEP relative to corresponding healthy tissue. Methods of determining these levels are well known in the art.

[0149] The term “comparable tumor” or “reference tumor” refers to a tumor from the same or a different individual that is of the same cancer type, e.g., pancreatic, ovarian, lung, etc., and / or that comprises one or more similar tumor suppressor and / or oncogene genetic mutations (e.g., mutations in p53, pten, cdhl, nfl, nf2, egfr, vegfr, myc, ras, etc.). Such comparative tumor samples may be used as a control and / or reference in cancer research to compare the molecular and cellular characteristics of various tumor types / tissues. In some aspects, the comparable tumor is an early stage tumor of the same cancer type, for example an early stage sarcoma of the same tissue. In some aspects, an early-stage tumor refer to a malignant tumor that is localized and has not yet spread extensively to surrounding tissues or distant parts of the body. In some aspects, an early stage tumor is confined to the site of origin and small in size. In some aspects of the current disclosure, the tumor comprises at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or any range derivable therein or higher levels of one or more of collagen, fibronectin, fiber, extracellular matrix, and TGEP relative to a comparable early stage tumor. In some aspects of the currentdisclosure, the tumor comprises at least about 5-fold, or about 6-fold, or about 7-fold, or about 8-fold, or about 9-fold, or about 10-fold, or any range derivable therein, or higher levels of one or more of collagen, fibronectin, fiber, extracellular matrix, and TGFP relative to a comparable early stage tumor. Methods of determining these levels are well known in the art.

[0150] “Apheresis” is a medical procedure in which the blood of an individual is passed through an apparatus, a predominant constituent is collected (for example, mononuclear cells) and the other constituents are returned to the circulation. Apheresis is generally a three-step process comprising: (1) removing blood from the individual, (2) separating blood components (e.g., based on density) and (3) returning certain components of the blood to the individual. The blood is usually separated into three fractions: RBC (approximately 45% of the total blood), buffy coat (less than 1% of the total blood) and plasma (approximately 55% of the whole blood). Various types of apheresis procedures can be used depending on the blood component that is removed. For example, "plasmapheresis" refers, generally, to the separation and collection of blood plasma and "thrombocythepheresis" refers to the separation and collection of platelets, while "leukapheresis" refers, usually, to the separation and collection of leukocytes (WBC). In some embodiments, the leukocyte is a T-cell. In some embodiments, the leukocyte is a tumor infiltrating leukocyte (TIL). With the advancement of the medical sciences, apheresis can be carried out in a patient-connected, continuous-flow and closed- system manner. Devices used for this purpose include, for example, the following apheresis systems: COBE® Spectra, Trima, Spectra Optia systems (all marketed by Gambro BCT), and Amicus and CS-3000 + (marketed by Fenwal / Baxter).

[0151] “Leukapheresis” is used to collect a certain fraction of blood mononuclear cells (MNCs) for subsequent transplantation of a fraction thereof into a subject. In this instance, the buffy coat (which contains the majority of the WBCs (granulocytes, lymphocytes, monocytes), PBPC and some platelets) is first collected while the rest of the blood components (which include plasma, RBC, platelets and some WBC) is returned to the individual. Desired cells are then enriched and isolated, while the remaining fraction of the buffy coat (which makes up almost 99% of the buffy coat) is discarded. In some embodiments, the desired cells peripheral blood mononuclear cells (PBMCs), T-cells or TILs. In such embodiments, leukapheresis is conducted after mobilizing release of hematopoietic stem cells (HSCs) from the bone marrow by administering G-CSF to the subject (mobilized pheresis). In some embodiments, the cell is a peripheral blood mononuclear cell (PBMCs).

[0152] ‘Marker phenotyping” refers to identification of markers or antigens on cells for determining their phenotype (e.g., differentiation state and / or cell type). This may be done byimmunophenotyping, which uses antibodies that recognize antigens present on a cell. The antibodies may be monoclonal or polyclonal, but are generally chosen to have minimal cross reactivity with other cell markers. It is to be understood that certain cell differentiation or cell surface markers are unique to the animal species from which the cells are derived, while other cell markers will be common between species. These markers defining equivalent cell types between species are given the same marker identification even though there are species differences in structure (e.g., amino acid sequence). Cell markers include cell surfaces molecules, also referred to in certain situations as cell differentiation (CD) markers, and gene expression markers. The gene expression markers are those sets of expressed genes indicative of the cell type or differentiation state. In part, the gene expression profile will reflect the cell surface markers, although they may include non-cell surface molecules.

[0153] As used herein, “enriched” means that the percentage of marker phenotyped cells relative to other cells in a population is increased. In one embodiment, “purified” means that the percentage of marker phenotyped cells is substantially pure and excludes cells that are not marker phenotyped. A “substantially pure cell population” refers to a population of cells having a specified cell marker characteristic and differentiation potential that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more, or any value or range in between, of the cells making up the total cell population. Thus, a “substantially pure cell population” refers to a population of cells that contain fewer than about 50%, preferably fewer than about 20-25%, more preferably fewer than about 10-15%, and most preferably fewer than about 5% of cells that do not display a specified marker characteristic and differentiation potential under designated assay conditions.

[0154] In one embodiment, “isolated” refers to a product, compound, or composition which is separated from at least one other product, compound, or composition with which it is associated in its naturally occurring state, whether in nature or as made synthetically. In other embodiments, “isolated” means that desired marker phenotyped cells are physically separated from other cell populations. Methods for the enrichment, purification, and / or isolation of marker phenotyped cells are disclosed herein and are also well known in the art, such as by using fluorescence-activated cell scanning (FACS), magnetic cell sorting, and centrifugation (see, for example, U.S. Pat. Nos. 5,474,687, 5,677,136, and 6,004,743; and U.S. Pat. Publ. 2001 / 0039052).B. Engineered Immune Effector Cell Therapy

[0155] Certain embodiments of the present disclosure concern a CAR-like construct with a tumor-targeted and membrane- anchored IL- 12. In some aspects, the construct or expression vector is a retroviral expression vector, an adenoviral expression vector, a DNA plasmid expression vector, or an adeno associated virus (AAV) expression vector. In some embodiments, the construct is a viral vector, such as a retroviral vector or lentiviral vector. Specifically, in some embodiments, the IL- 12 comprises both the p35 and p40 subunits. In some embodiments, the p40 subunit is fused directly or indirectly to a tumor-targeting moiety. In some embodiments, the p35 subunit is fused directly or indirectly to a transmembrane domain. In some embodiments, the p35 subunit may be fused directly or indirectly to an intracellular cell signaling domain. In some embodiments, an intracellular cell signaling domain may comprise CD3(^, CD28, and / or 4-1BB signaling domains.

[0156] In some embodiments, the construct may comprise a tumor-targeting moiety, such as a peptide, antibody, or fragment thereof. In some embodiments, the tumor-targeting moiety may be the antigen-binding portion or portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb).

[0157] An exemplary tumor-targeting moiety is a cell surface vimentin (CSV) binding peptide or scFv targeting CSV. In some embodiments, the tumor-targeting moiety comprises, consists essentially of, or consists of a CSV targeting moiety. CSV is detected across many types of highly malignant tumors and is primarily found on highly malignant tumors such as metastatic and relapsed tumors. For example, studies have shown that CSV was present on 100% of metastatic tumor cell surfaces of sampled colon tumors, and 97-98% of drug or CAR-T cell resistant or relapsed acute lymphoblastic leukemia (ALL). In some embodiments, a CSV binding peptide comprises, consists essentially of, or consists of a CSV binding peptide sequence that is at least, exactly, or about 50%, 62.5%, 75%, 87.5%, or 100% identical to SEQ ID NO: 1.SEQ ID NO: 1 - CSV binding peptide amino acid sequenceVNTANST ( SEQ ID NO : 1 )

[0158] In some embodiments, a transmembrane domain for anchoring a tumor targeted cytokine to an effector cell may be any transmembrane domain known in the art. In some embodiments, a transmembrane domain comprises, consists essentially of, or consists of a EGFR transmembrane domain. In some embodiments, a transmembrane domain comprises, consists essentially of, or consists of a EGFR transmembrane domain that is at least, exactly,or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2.SEQ ID NO: 2 - EGER transmembrane domain amino acid sequenceIATGMVGALLLLLWALGIGLFMRRRHIVRKRTLRRLLQEREL ( SEQ ID NO : 2 )

[0159] In some embodiments, a transmembrane domain may comprise other transmembrane sequence known in the art such as disclosed in Kozma et al., Nucleic Acids Research 41 Database Issue, D524-D529, 2013. In some embodiments, the IL-12 p35 subunit is fused directly to a transmembrane domain. In some embodiments, a transmembrane domain may be derived from proteins having a transmembrane domain, such as but not limited to members of the integrin family, CD44, glycophorin, MHC Class I and II glycoproteins, EGF receptor, G protein coupled receptor (GPCR) family, receptor tyrosine kinases (such as insulinlike growth factor 1 receptor (IGFR) and platelet-derived growth factor receptor (PDGFR)), porin family and other transmembrane proteins. Certain embodiments of the present disclosure contemplate using a portion of a transmembrane polypeptide domain such as a truncated polypeptide having membrane-inserting characteristics as may be determined according to standard and well known methodologies.

[0160] In some embodiments, linker peptide can be utilized to indirectly fuse a transmembrane domain to a cytokine. In some embodiments, a linker sequence comprises, consists essentially of, or consists of an amino acid sequence that is at least, exactly, or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3.SEQ ID NO: 3 - Exemplified linker peptide amino acid sequenceSGGGGSGGGGSS ( SEQ ID NO : 3 )

[0161] The membrane- anchored IL- 12 protein sequences that can be used in various embodiments include the amino acid sequences of wild-type human IL- 12 (e.g., NCBI gene IDs 3592 and 3593), as well as analogues and / or derivatives thereof. In some embodiments, the analogues and / or derivatives can include, but are not limited to, additions or substitutions of amino acid residues within the amino acid sequences encoded by a nucleotide sequence, but that result in a silent change, thus producing a functionally equivalent gene product. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved. For example: nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic)amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0162] Amino acid substitutions may alternatively be made on the basis of the hydropathic index of amino acids. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The use of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art (Kyte and Doolittle, J. Mol. Biol. 157:105-132, 1982). It is known that in certain instances, certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, in certain embodiments the substitution of amino acids whose hydropathic indices are within + 2 is included, while in other embodiments amino acid substitutions that are within + 1 are included, and in yet other embodiments amino acid substitutions within + 0.5 are included.

[0163] Amino acid substitutions may alternatively be made on the basis of hydrophilicity, particularly where the biologically functional protein or peptide thereby created is intended for use in immunological embodiments. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, z.e., with a biological property of the protein. The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 + 1); glutamate (+3.0 + 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 + 1); alanine (- 0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4). In making changes based upon similar hydrophilicity values, in certain embodiments the substitution of amino acids whose hydrophilicity values are within + 2 is included, in certain embodiments those that are within + 1 are included, and in certain embodiments those within + 0.5 are included. One may also identify epitopes from primary amino acid sequences on the basis of hydrophilicity.

[0164] Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar shape and charge.Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Alternatively, substitutions may be non-conservative such that a function or activity of the polypeptide is affected. Nonconservative changes typically involve substituting a residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa.

[0165] In some embodiments, an IL- 12 p35 subunit comprises an amino acid sequence at least, exactly, or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4.

[0166] In some embodiments, an IL- 12 p40 subunit comprises an amino acid sequence at least, exactly, or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 5.

[0167] In some embodiments, an IL- 12 p35 subunit comprises an amino acid sequence at least, exactly, or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6. In some embodiments, an IL- 12 p35 subunit is encoded by a nucleic acid sequence at least, exactly, or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9.

[0168] In some embodiments, a tumor-targeted IL- 12 p40 subunit comprises an amino acid sequence at least, exactly, or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7. In some embodiments, a tumor-targeted IL- 12 p40 subunit is encoded by a nucleic acid sequence at least, exactly, or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10.SEQ ID NO: 4 - IL-12 p35 Amino Acid sequenceMWPPGSASQPPPSPAAATGLHPAARPVSLQCRLSMCPARSLLLVATLVLLHLSLARNLPVAT PDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELT KNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQI FLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSY LNA ( SEQ ID NO : 4 )SEQ ID NO: 5 - IL-12 p40 Amino Acid sequenceMCHQQLVISWFSLVFLASPLVAIWELKKDVYWELDWYPDAPGEMWLTCDTPEEDGITWTL DQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPK NKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKE YEYSVECQEDSACPAAEESLP IEVMVDAVHKLKYENYTSSFFIRDI IKPDPPKNLQLKPLKN SRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRA QDRYYSSSWSEWASVPCS ( SEQ ID NO : 5 )SEQ ID NO: 6 - Membrane-anchored IL-12 p35 Amino Acid sequenceMWPPGSASQPPPSPAAATGLHPAARPVSLQCRLSMCPARSLLLVATLVLLHLSLARNLPVAT PDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELT KNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQI FLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSY LNASGGGGSGGGGSSIATGMVGALLLLLWALGIGLFMRRRHIVRKRTLRRLLQEREL ( SEQ ID NO : 6 )SEQ ID NO: 9 - Membrane-anchored IL-12 p35 nucleic acid sequenceATGTGGCCCCCTGGGTCAGCCTCCCAGCCACCGCCCTCACCTGCCGCGGCCACAGGTCTGCA TCCAGCGGCTCGCCCTGTGTCCCTGCAGTGCCGGCTCAGCATGTGTCCAGCGCGCAGCCTCC TCCTTGTGGCTACCCTGGTCCTCCTGGACCACCTCAGTTTGGCCAGAAACCTCCCCGTGGCC ACTCCAGACCCAGGAATGTTCCCATGCCTTCACCACTCCCAAAACCTGCTGAGGGCCGTCAG CAACATGCTC C AG AAG G C C AG AC AAAC T C T AG AAT TTTACCCTTGCACTTCT G AAG AG AT T G ATCATGAAGATATCACAAAAGATAAAACCAGCACAGTGGAGGCCTGTTTACCATTGGAATTA ACCAAGAATGAGAGTTGCCTAAATTCCAGAGAGACCTCTTTCATAACTAATGGGAGTTGCCT GGCCTCCAGAAAGACCTCTTTTATGATGGCCCTGTGCCTTAGTAGTATTTATGAAGACTTGA AGATGTACCAGGTGGAGTTCAAGACCATGAATGCAAAGCTTCTGATGGATCCTAAGAGGCAG ATCTTTCTAGATCAAAACATGCTGGCAGTTATTGATGAGCTGATGCAGGCCCTGAATTTCAA C AG T G AG AC T G T G C C AC AAAAAT C C T C C C T T G AAG AAC C G G AT T T T T AT AAAAC T AAAAT C A AGCTCTGCATACTTCTTCATGCTTTCAGAATTCGGGCAGTGACTATTGATAGAGTGATGAGC TATCTGAATGCTTCCGGCGGCGGCGGCTCCGGAGGCGGAGGCTCTTCCATCGCCACTGGGAT GGTGGGGGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATGCGAAGGC GCCACATCGTTCGGAAGCGCACGCTGCGGAGGCTGCTGCAGGAGAGGGAGCTTTGA ( SEQ ID NO : 9 )SEQ ID NO: 7 - Tumor- targeted IL-12 p40 Amino Acid sequenceMCHQQLVISWFSLVFLASPLVAIWELKKDVYWELDWYPDAPGEMWLTCDTPEEDGITWTL DQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPK NKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKE YEYSVECQEDSACPAAEESLP IEVMVDAVHKLKYENYTSSFFIRDI IKPDPPKNLQLKPLKN SRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRA QDRYYSSSWSEWASVPCSVNTANST ( SEQ ID NO : 7 )SEQ ID NO: 10 - Tumor- targeted IL-12 p40 nucleic acid sequenceATGTGTCACCAGCAGTTGGTCATCTCTTGGTTTTCCCTGGTTTTTCTGGCATCTCCCCTCGT GGCCATATGGGAACTGAAGAAAGATGTTTATGTCGTAGAATTGGACTGGTATCCGGATGCCC CTGGAGAAATGGTGGTCCTCACCTGTGACACCCCTGAAGAAGATGGTATCACCTGGACCTTG GACCAGAGCAGTGAGGTCTTAGGCTCTGGCAAAACCCTGACCATCCAAGTCAAAGAGTTTGG AGATGCTGGCCAGTACACCTGTCACAAAGGAGGCGAGGTTCTAAGCCATTCGCTCCTGCTGC TTCACAAAAAGGAAGATGGAATTTGGTCCACTGATATTTTAAAGGACCAGAAAGAACCCAAAAATAAGACCTTTCTAAGATGCGAGGCCAAGAATTATTCTGGACGTTTCACCTGCTGGTGGCT GACGACAATCAGTACTGATTTGACATTCAGTGTCAAAAGCAGCAGAGGCTCTTCTGACCCCC AAGGGGTGACGTGCGGAGCTGCTACACTCTCTGCAGAGAGAGTCAGAGGGGACAACAAGGAG TATGAGTACTCAGTGGAGTGCCAGGAGGACAGTGCCTGCCCAGCTGCTGAGGAGAGTCTGCC CATTGAGGTCATGGTGGATGCCGTTCACAAGCTCAAGTATGAAAACTACACCAGCAGCTTCT TCATCAGGGACATCATCAAACCTGACCCACCCAAGAACTTGCAGCTGAAGCCATTAAAGAAT TCTCGGCAGGTGGAGGTCAGCTGGGAGTACCCTGACACCTGGAGTACTCCACATTCCTACTT CTCCCTGACATTCTGCGTTCAGGTCCAGGGCAAGAGCAAGAGAGAAAAGAAAGATAGAGTCT TCACGGACAAGACCTCAGCCACGGTCATCTGCCGCAAAAATGCCAGCATTAGCGTGCGGGCC CAGGACCGCTACTATAGCTCATCTTGGAGCGAATGGGCATCTGTGCCCTGCAGTGTCAACAC GGCTAACTCGACATAG ( SEQ ID NO : 10 )A. Tumor Infiltrating Lymphocyte (TIL) Cell Preparation

[0169] Provided herein are cells engineered to express a construct comprising a tumor- targeted and membrane- anchored cytokine (e.g., attIL12), such as immune effector cells. In some embodiments, the cell is a T-cell. In some particular embodiments, the cell is a Tumor Infiltrating Lymphocytes (TILs). In some embodiments, provided herein are methods and compositions comprising attIL12-TILs cells that may be used to treat a disease or disorder, such as a cancer, including solid tumor and / or blood cancers.

[0170] Certain embodiments of the present disclosure concern obtaining a starting population of T-cells, engineering the T-cells, optionally expanding the T-cells, and administering the engineered T cells to a subject as an immunotherapy to target cancer cells. Certain embodiments of the present disclosure concern obtaining a starting population of TILs, engineering the TILs, optionally expanding the TILs, and administering the engineered T cells to a subject as an immunotherapy to target cancer cells. In particular, in certain embodiments the TILs are engineered to express attIL12. Several basic approaches for the derivation, activation, and / or expansion of functional anti-tumor effector T cells have been described in the last two decades, and many of these approaches may be utilized for the derivation, activation, and / or expansion of functional anti-tumor effector TILs.

[0171] Cells used for the generation of transgenic cells of the current disclosure can be obtained from any suitable source. Cells can be primary cells. Cells can be recombinant cells. With reference to the subject Cells can be autologous / autogeneic ("self") or non-autologous ("non-self," e.g., allogeneic, syngeneic or xenogeneic). "Autologous," as used herein, refers to cells from the same subject. "Allogeneic," as used herein, refers to cells of the same species that differ genetically to the cell in comparison. "Syngeneic," as used herein, refers to cells of a different subject that are genetically identical to the cell in comparison. "Xenogeneic," as used herein, refers to cells of a different species to the cell in comparison. In some embodiments, the cells of the invention are allogeneic. In some embodiments, the cells of theinvention are autogenic. An "isolated cell" refers to a cell that has been obtained from an in vivo source and is substantially free of extracellular matrix. Cells can be derived from a healthy donor or from a patient diagnosed with cancer. In further embodiments, the subject may have been determined to have or be at risk for cancer. In some embodiments, with reference to the subject to be treated, the cells are autologous. In some embodiments, the methods include isolating cells from the subject, preparing, processing, culturing, and engineering them, and reintroducing them into the same patient, with or without an intermediate step of cryopreservation.

[0172] In some embodiments, the starting population of TILs and / or T cells are derived from a tumor, blood, bone marrow, lymph, and / or lymphoid organs. In some embodiments, the TILs and / or T cells are derived from a human and are utilized in autologous methods of treatment. The cells typically are primary cells, such as those isolated directly from a subject and / or isolated from a subject and directly utilized and / or frozen for future use.

[0173] With respect to T-cells, peripheral blood cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as centrifugation, filtration, sedimentation, apheresis, leukapheresis, Ficoll™ separation, cell sorting, marker phenotyping or any combination thereof. In some embodiments, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T-cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.

[0174] Similarly, TILs may be obtained by isolation methods known to the skilled artisan. In some embodiments, the method comprises, isolation of tumor tissues and further isolation and sorting of TILs. In some embodiments, tumor tissues can be mechanically separated to create a single-cell suspension for further processing, or cells can be separated using enzymatic digestion. One or more additional methods including centrifugation, filtration, sedimentation, Ficoll™ separation, cell sorting, marker phenotyping, or any combination thereof may be used to obtain suitable TILs as disclosed below.

[0175] In some embodiments, cell may be cryopreserved between one or more steps of the methods disclosed herein. In some embodiments, cell may be cryopreserved after isolation, after propagation, after transduction, or any combination thereof. In certain embodiments, cryopreserved cells may be thawed and washed as described herein and allowed to rest for one or more hours at room temperature prior to use in the methods of the present disclosure. Whilemany cryopreservation solutions and parameters are known in the art and may be useful in this context, some commonly used methods include using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, freezing the cells to -80° C and storing in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing can be used as well as uncontrolled freezing immediately at -20° C or in liquid nitrogen. Cells may also be stored at 4 °C for up to 48 hours, or at room temperature for up to 24 hours.

[0176] In some embodiments, the TILs include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+cells, CD8+cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and / or persistence capacities, antigen- specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. In some embodiments, T cells includes all types of immune cells expressing CD3 including T-helper cells, invariant natural killer T (iNKT) cells, cytotoxic T cells, T-regulatory cells (Treg) gamma-delta T cells, natural-killer (NK) cells, and neutrophils. The T cell may refer to a CD4+ or CD8+ T cell. Presence or absence of cell surface protein markers defined by monoclonal antibody recognition have been used to recognize and isolate T-cells and T-cell subtypes. Non-limiting examples of cell surface markers that may be used to identify T-cell include CD3, and CD45. In some embodiments, the T-cells for use in the current disclosure may comprise any T-cell subtype. In some embodiments, the T-cells for use in the current disclosure may comprise, consist essentially of, consist of CD4+CD8’ T cell, CD4’CD8+T cell, CD34+CD7+CDla+cell, CD3+ TCRab+, CD3+ TCRgd+, CD3+ TCRab+ CD4+ CD8-, CD3+ TCRab+ CD8+ CD4-, CD3+ TCRab+ CD4+ CD8- CD45RO- CD45RA+, CD3+ TCRab+ CD8+ CD4- CD45RO- CD45RA+, CD3+ TCRab+ CD4+ CD8- CD45RO- CD45RA+ CCR7+, CD3+ TCRab+ CD8+ CD4- CD45RO- CD45RA+ CCR7+, CD3+ TCRab+ CD4+ CD8- CD45RO- CD45RA+ CD27+, CD3+ TCRab+ CD8+ CD4- CD45RO- CD45RA+ CD27+, CD34+CD7+CDla+cell, CD34+CD5+CD7+, CD34+CD5+CD7-, natural killer T cell, regulatory T cell, antigenspecific T cell, intraepithelial lymphocyte T cell, or cell that are CD45+, CDl lb+, CD 11b-, CD15+, CD15-, CD24+, CD24-, CD114+, CD114-, CD182+, CD182-, CD4+, CD4-, CD14+, CD14-, CDl la+, CDl la-, CD91+, CD91-, CD16+, CD16-, CD3+, CD3-, CD25+, CD25-,Foxp3+, Fox3p-, CD8+, CD8-, CD19+, CD19-, CD20+, CD20-, CD24+, CD24, CD38+, CD38-, CD22+, CD22-, CD61+, CD61-, CD16+, CD16-, CD56+, CD56-, CD31+, CD31-, CD30+, CD30-, CD38+, and / or CD38- or combinations thereof. In some embodiments, the T- cell is a peripheral blood mononuclear T-cell identifiable by the expression of one or more of CD3, CD4, CD8, CD45, CD28, CD25, CD69, CD62L or any combination thereof. In some embodiments, the T-cell is not a peripheral blood mononuclear T-cell.

[0177] Among the sub-types and subpopulations of TILs that may be utilized include any lymphocyte that has infiltrated a tumor, such as but not limited to: T cells (e.g., CD4+and / or CD8+T cells), naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells. In certain embodiments, TILs that have infiltrated a tumor need not have remained in said tumor prior to isolation from a subject.

[0178] In some embodiments, one or more of the TIL and / or T cell populations are enriched for or depleted of cells that are positive for a specific marker, such as surface markers, or that are negative for a specific marker. In some cases, such markers are those that are absent or expressed at relatively low levels on certain populations of TILs and / or T cells (e.g., nonmemory cells) but are present or expressed at relatively higher levels on certain other populations of T cells (e.g., memory cells).

[0179] In some embodiments, TILs are not derived from a PBMC sample from a subject. In some embodiments, TILs are separated from a PBMC sample by negative selection of markers expressed on non-lymphocytes. In some embodiments, TILs are separated from a PBMC sample by negative selection of markers for B cells, monocytes, or other white blood cells, such as CD 14+ cells. In some aspects, a CD4+or CD8+selection step is used to separate CD4+helper and CD8+cytotoxic T cells and / or TILs. In some embodiments, CD4+and CD8+populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and / or effector T cell subpopulations.

[0180] In some embodiments, CD8+TILs cells are further enriched for or depleted of naive, central memory, effector memory, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. Insome embodiments, enrichment for TILs characterized as central memory T (TCM) cells is carried out, and may increase efficacy, such as to improve long-term survival, expansion, and / or engraftment following administration, which in some aspects is particularly robust in such sub-populations. See Terakura et al. (2012) Blood .1:72- 82; Wang et al. (2012) J Immunother. 35(9):689-701.

[0181] In some embodiments, the TILs are autologous T cells. In certain methods described herein, tumor samples may be obtained from patients, said tumors may be disassociated to single cells, and a single cell suspension may be obtained. In some embodiments, a single cell suspension can be obtained in any suitable manner, for example but not limited to mechanical and / or enzymatic disaggregation (e.g., disaggregating the tumor using a gentleMACS™ Dissociator, Miltenyi Biotec, Auburn, Calif., and / or collagenase or DNase). Single-cell suspensions of disaggregated tumor samples can be sorted and / or cultured in the presence of cytokines, such as interleukin-2 (IL-2). The cells can be cultured until confluence e.g., about 2xl06lymphocytes), e.g., from about 5 to about 21 days, preferably from about 10 to about 14 days. For example, the cells may be cultured from 5 days, 5.5 days, or 5.8 days to 21 days, 21.5 days, or 21.8 days, such as from 10 days, 10.5 days, or 10.8 days to 14 days, 14.5 days, or 14.8 days.

[0182] The cells may be cultured in the presence of a liquid culture medium. Typically, the medium may comprise a basal medium formulation as known in the art. Many basal media formulations can be used to culture cells herein, including but not limited to Eagle's Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), alpha modified Minimum Essential Medium (alpha-MEM), Basal Medium Essential (BME), Iscove's Modified Dulbecco's Medium (IMDM), BGJb medium, F-12 Nutrient Mixture (Ham), Liebovitz L-15, DMEM / F-12, Essential Modified Eagle's Medium (EMEM), RPMI-1640, AIM V™ and modifications and / or combinations thereof. Compositions of the above basal media are generally known in the art, and it is within the skill of one in the art to modify or modulate concentrations of media and / or media supplements as necessary for the cells cultured. In some embodiments, a culture medium formulation may be explants medium (CEM) which is composed of IMDM supplemented with 10% fetal bovine serum (FBS), 100 U / ml penicillin G, 100 pg / ml streptomycin and 2 mmol / L L-glutamine. Other embodiments may employ further basal media formulations, such as chosen from the ones above.

[0183] Any medium capable of supporting cells in vitro may be used to culture the cells.Media formulations that can support the growth of cells include, but are not limited to, Dulbecco's Modified Eagle's Medium (DMEM), alpha modified Minimal Essential Medium(aMEM), and Roswell Park Memorial Institute Media 1640 (RPMI Media 1640), AIM V™ and the like. Typically, up to 20% fetal bovine serum (FBS) or 1-20% horse serum, or human AB serum is added to the above medium in order to support the growth of cells. In some embodiments, the medium may be serum-free. A defined medium, however, also can be used if the growth factors, cytokines, and hormones necessary for culturing cells are provided at appropriate concentrations in the medium. Media useful in the methods of the disclosure may comprise one or more compounds of interest, including, but not limited to, antibiotics, mitogenic compounds, or differentiation compounds useful for the culturing of cells. The cells may be grown at temperatures between 27° C to 40° C, such as 31° C to 37° C, and may be in a humidified incubator. The carbon dioxide content may be maintained between 2% to 10% and the oxygen content may be maintained between 1% and 22%. The disclosure, however, should in no way be construed to be limited to any one method of isolating and culturing cells. Rather, any method of isolating and culturing cells should be construed to be included in the present disclosure.

[0184] For use in the cell culture, media can be supplied with one or more further components. For example, additional supplements can be used to supply the cells with the necessary trace elements and substances for optimal growth and expansion. Such supplements include insulin, transferrin, selenium salts, and combinations thereof. These components can be included in a salt solution such as, but not limited to, Hanks' Balanced Salt Solution (HBSS), Earle's Salt Solution. Further antioxidant supplements may be added, e.g., P-mercaptoethanol. While many media already contain amino acids, some amino acids may be supplemented later, e.g., E-glutamine, which is known to be less stable when in solution. A medium may be further supplied with antibiotic and / or antimycotic compounds, such as, typically, mixtures of penicillin and streptomycin, and / or other compounds, exemplified but not limited to, amphotericin, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tylosin, and zeocin. Also contemplated is supplementation of cell culture medium with cytokines. Non-limiting examples of suitable cytokines include stem cell factor (SCF), thrombopoietin (TPO), interleukin-3 (IE-3), interleukin-6 (IE-6), Fms-like tyrosine kinase 3 ligand (Flt3E), granulocyte colony- stimulating factor (G-CSF), and granulocyte-macrophage colony- stimulating factor (GM-CSF). Also contemplated is supplementation of cell culture medium with mammalian plasma or sera. Plasma or sera often contain cellular factors and components that are necessary for viability and expansion. The use of suitable serum replacements is also contemplated.

[0185] Reference to particular buffers, media, reagents, cells, culture conditions and the like, or to some subclass of same, is not intended to be limiting, but should be read to include all such related materials that one of ordinary skill in the art would recognize as being of interest or value in the particular context in which that discussion is presented. For example, it is often possible to substitute one buffer system or culture medium for another, such that a different but known way is used to achieve the same goals as those to which the use of a suggested method, material or composition is directed. In particular embodiments, cells are cultured in a cell culture system comprising a cell culture medium, preferably in a culture vessel, in particular a cell culture medium supplemented with a substance suitable and determined for protecting the cells from in vitro aging and / or inducing in an unspecific or specific reprogramming.

[0186] In certain embodiments, the cultured TILs can be pooled and rapidly expanded. Rapid expansion can provide an increase in the number of antigen- specific TILs of at least about 50-fold (e.g., 50-, 60-, 70-, 80-, 90-, or 100-fold, or greater) over a period of about 10 to about 14 days. More preferably, rapid expansion can provide an increase of at least about 200- fold (e.g., 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900-, or greater) over a period of about 10 to about 14 days.

[0187] Expansion can be accomplished by any of a number of methods as are known in the art. For example, TILs can be rapidly expanded using non-specific T-cell receptor stimulation in the presence of feeder lymphocytes and cytokines, such as interleukin-2 (IL-2) and / or interleukin- 15 (IL- 15). In some embodiments, non-specific T-cell receptor stimulus can include around 30 ng / ml of OKT3, a mouse monoclonal anti-CD3 antibody (available from Ortho-McNeil®, Raritan, N.J.). Alternatively, TILs cells can be rapidly expanded by stimulation in vitro with one or more antigens (including antigenic portions thereof, such as epitope(s), or a cell) of the cancer, which can be optionally expressed from a vector, such as a human leukocyte antigen A2 (HLA-A2) binding peptide, in the presence of a T-cell growth factor, such as IL-2 (e.g., 300 lU / ml IL-2). The TILs can be expanded by re-stimulation with the same antigen(s) of the cancer pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, the TILs can be re-stimulated with irradiated, autologous lymphocytes or with irradiated HLA-A2+allogeneic lymphocytes and IL-2, for example.

[0188] The autologous TILs can be engineered to express a T-cell growth factor that promotes the growth and activation of the autologous TILs. Suitable T-cell growth factors include, for example, interleukin (IL)-2, IL-7, IL- 15, and / or IL- 12. Suitable methods of modification are known in the art. See, for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rded., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; andAusubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. In particular aspects, engineered autologous TILs express the T-cell growth factor at high levels. In certain embodiments, engineering of TILs includes incorporation of a T-cell growth factor in a vector, such as a retroviral or lentiviral vector. The vector may be the same vector encoding attIL12, or a different vector. In some embodiments, T-cell growth factor coding sequences, such as that of IL- 12, are available in the art, as are promoters, the operable linkage of which to a T-cell growth factor coding sequence promote high-level expression.

[0189] In certain embodiments, TILs described herein retain functional expression of one or more endogenous T cell receptor (TCR) genes. In certain embodiments, the TILs have functional expression of TCR alpha chain and / or TCR beta chain encoding genes.B. TIL Cell Activation

[0190] In some embodiments, the present disclosure provides methods of activating TIL cells to increase expression of NKG2D receptor on the TILs, such as CD8+TILs. In some embodiments, a starting population of TILs may be pre-treated with an anti-CD3 antibody, such as anti-CD3 beads. Pre-treatment of TILs may be for about 12 hours to 3 days, such as about 24 hours. The expanded TILs may then be cultured with CD80 protein, such as CD80- Fc recombinant protein to induce CD28 activation and, thus, NKG2D expression. The culture with CD80 may be for about 1-6 days, such as about 1, 2, 3, 4, 5, or 6 days, particularly about 4 days. In some aspects, the TILs may be treated with anti-CD3 and CD80 simultaneously. In some embodiments, TILs may be activated and / or expanded with agonistic stimulation of CD137 / 4-1BB and exogenous IL-2. In some embodiments, TILs are not activated and / or expanded with agonistic stimulation of CD137 / 4-1BB. In some embodiments, TILs are activated and / or expanded as described in Fulbright, O .J. et al., 2022 (Fulbright, O .J., Forget, MA., Haymaker, C., Bernatchez, C. (2022). Isolation and Maintenance of Tumor- Infiltrating Lymphocytes for Translational and Clinical Applications: Established Methods and New Developments. In: McAllister, F. (eds) Cancer Immunoprevention. Methods in Molecular Biology, vol 2435. Humana, New York, NY. https: / / doi.org / 10.1007 / 978-l-0716-2014-4_5), which is incorporated herein by reference in its entirety for the purposes described herein. In some embodiments, TILs are activated and / or expanded as described in Fulbright O.J. et al., 2022, with the exclusion of the CD 137 antibody.C. Additional Genetic Engineering of TIL Cells

[0191] The TIL cells of the present disclosure can be genetically engineered to express the present attIL12 construct. In some embodiments, the TILs may be engineered with a construct comprising an extracellular antigen (or ligand) binding domain linked to one or more optional intracellular signaling components, in some aspects via linkers and / or transmembrane domain(s). Such molecules typically mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and / or a signal through a costimulatory receptor alone.

[0192] In some aspects, the antigen- specific binding, or recognition component is fused directly or indirectly to an IL- 12 p40 subunit, and the p40 subunit physiologically attaches to an IL- 12 p35 subunit extracellularly to form a heterodimer, the p35 subunit can be fused directly or indirectly to a transmembrane domain, and optionally fused directly or indirectly to an intracellular signaling domain.

[0193] In some embodiments, a transmembrane domain is derived from a natural and / or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions can include those derived from (z.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T- cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD 154. Alternatively or additionally, in some embodiments the transmembrane domain is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.

[0194] In some embodiments, an attIL12 construct can comprise one or more intracellular signaling components. In some embodiments, an attIL12 construct includes an intracellular component of the TCR complex, such as a TCR CD3+chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain. Thus, in some aspects, the antigen binding molecule is linked to one or more intracellular cell signaling modules. In some embodiments, cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and / or other CD transmembrane domains. In some embodiments, the attIL12 construct further includes a portion of one or more additional molecules such as Fc receptor y, CD8, CD4, CD25, or CD16. For example, in some aspects, the attIL-12 includes a chimeric molecule between CD3-zeta, CD3-Q or Fc receptor y, and CD8, CD4, CD25 or CD16.

[0195] In certain embodiments the TILs are engineered to express one or more additional transgenes. In some embodiments, the TILs are engineered to express a Chimeric Antigen Receptor (CAR). In some embodiments, a CAR comprises: a) one or more intracellular signaling domains, b) a transmembrane domain, and c) an extracellular domain comprising one or more antigen binding domains (e.g., one or more scFv). The extracellular antigen binding domain may be associated with a hinge of any kind, such as but not limited to IgGl, CD28, CD8alpha, and so forth. In some embodiments, an antigen- specific CAR polypeptide may comprise one or more epitope recognition domains that do not comprise an scFv. In some embodiments, an antigen-specific CAR polypeptide may comprise an antigen binding domain derived from one or more proteins selected from adnectins, affibodies, affillins, anticalins, atrimers, avimers, bicyclic peptides, centyrins, cys-knots, DARPins, FN3, Fynomers, Kunitz domains, Obodies, pronectins, and Tn3. In some embodiments, such antigen binding domains may be modified and / or optimized for human codon usage and / or expression in human cells.

[0196] In some embodiments, a CAR has specificity for a particular antigen (or marker or ligand), such as an antigen expressed in a particular cell type to be targeted, e.g., a cancer cell marker. In some embodiments, a CAR includes an antigen-binding portion or portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb).

[0197] In certain embodiments, a CAR may target (e.g., through its antigen-binding portion) to a tumor associated antigen or a pathogen- specific antigen binding domain. In some embodiments, antigens include carbohydrate antigens recognized by pattern-recognition receptors, such as Dectin- 1. In some embodiments, a tumor associated antigen targeted by a CAR may be of any kind, so long as it is expressed on the cell surface of tumor cells. In certain embodiments, an antigen may include CD19, CD70, HLA-G, CD38, CD123, CLL1, EBNA, CD 123, HER2, CA-125, TRAIL / DR4, CD20, carcinoembryonic antigen, alphafetoprotein, CD56, AKT, Her3, epithelial tumor antigen, CD319 (CS1), ROR1, folate binding protein, HIV- 1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, CD5, CD23, CD30, HERV-K, IL-l lRalpha, kappa chain, lambda chain, CSPG4, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, p53, mutated p53, Ras, mutated ras, c-Myc, cytoplasmic serine / threonine kinases (e.g., A-Raf, B-Raf, and C-Raf, cyclin-dependent kinases), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE- A12, MART-1, melanoma-associated antigen, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE- 3, -4, -5, -6, -7B, NA88-A, MC1R, mda-7, gp75, GplOO, PSA, PSM, Tyrosinase, tyrosinase- related protein, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1,MUC2, Phosphoinositide 3-kinases (PI3Ks), TRK receptors, PRAME, P15, RU1, RU2, SART- 1, SART-3, Wilms' tumor antigen (WT1), AFP, -catenin / m, Caspase-8 / m, CDK-4 / m, ELF2M, GnT-V, G250, HAGE, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, Myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP, Annexin II, CDC27 / m, TPVmbcr-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6 / AML, LDLR / FUT, Pml / RAR, Tumor- associated calcium signal transducer 1 (TACSTD1) TACSTD2, receptor tyrosine kinases (e.g., Epidermal Growth Factor receptor (EGFR) (in particular, EGFRvIII), platelet derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR)), VEGFR2, cytoplasmic tyrosine kinases (e.g., src-family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducers and activators of transcription STAT3, STATS, and STATE, hypoxia inducible factors (e.g., HIF-1 and HIF-2), Nuclear Factor- Kappa B (NF-B), Notch receptors (e.g., Notchl-4), NY ESO 1, c-Met, mammalian targets of rapamycin (mTOR), WNT, extracellular signal-regulated kinases (ERKs), and their regulatory subunits, PMSA, PR-3, MDM2, Mesothelin, renal cell carcinoma-5T4, SM22-alpha, carbonic anhydrases I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL / AML1, GD2, proteinase3, hTERT, sarcoma translocation breakpoints, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA 17, PAX3, ALK, androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC, GD3, fucosyl GM1, mesothelian, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SAGE, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP- 4, SSX2, XAGE 1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNCI, and LRRN1, or a combination thereof.

[0198] In certain embodiments, TILs and / or T cells are genetically engineered to express heterologous recombinant TCRs and / or heterologous TCRs cloned from naturally occurring T cells and / or TILs. A "T cell receptor" or "TCR" refers to a molecule that contains a variable a and P chains (also known as TCRa and TCRp, respectively) or a variable y and 5 chains (also known as TCRy and TCRS, respectively) and that is capable of specifically binding to an antigen peptide bound to a MHC receptor. In some embodiments, the TCR is in the aP form.

[0199] Typically, TCRs that exist in aP and y5 forms are generally structurally similar, but T cells expressing them may have distinct anatomical locations or functions. A TCR can be found on the surface of a cell or in soluble form. Generally, a TCR is found on the surface of T cells (or T lymphocytes) where it is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. In some embodiments, a TCR also can contain a constant domain, a transmembrane domain and / or a short cytoplasmic tail. Forexample, in some aspects, each chain of the TCR can possess one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminal end (see, e.g., Janeway et al., 1997). In some embodiments, a TCR is associated with invariant proteins of the CD3 complex involved in mediating signal transduction. Unless otherwise stated, the term "TCR" should be understood to encompass functional TCR fragments thereof. The term also encompasses intact or full-length TCRs, including TCRs in the aP form or y5 form.

[0200] Thus, for purposes herein, reference to a TCR includes any TCR or functional fragment, such as an antigen-binding portion of a TCR that binds to a specific antigenic peptide bound in an MHC molecule, i.e. MHC -peptide complex. An "antigen -binding portion" or antigen- binding fragment" of a TCR, which can be used interchangeably, refers to a molecule that contains a portion of the structural domains of a TCR, but that binds the antigen (e.g. MHC- peptide complex) to which the full TCR binds. In some cases, an antigen-binding portion contains the variable domains of a TCR, such as variable a chain and variable P chain of a TCR, sufficient to form a binding site for binding to a specific MHC-peptide complex, such as generally where each chain contains three complementarity determining regions.

[0201] In some embodiments, the variable domains of the TCR chains associate to form loops, or complementarity determining regions (CDRs) analogous to immunoglobulins, which confer antigen recognition and determine peptide specificity by forming the binding site of the TCR molecule and determine peptide specificity. Typically, like immunoglobulins, the CDRs are separated by framework regions (FRs) (see, e.g., Jores et al., 1990; Chothia et al., 1988; and Lefranc et al., 2003). In some embodiments, CDR3 is the main CDR responsible for recognizing processed antigen, although CDR1 of the alpha chain has also been shown to interact with the N-terminal part of the antigenic peptide, whereas CDR1 of the beta chain interacts with the C-terminal part of the peptide. CDR2 is thought to recognize the MHC molecule. In some embodiments, the variable region of the P-chain can contain a further hypervariability (HV4) region.

[0202] In some embodiments, the TCR chains contain a constant domain. For example, like immunoglobulins, the extracellular portion of TCR chains (e.g., a-chain, P-chain) can contain two immunoglobulin domains, a variable domain (e.g., Vaor Vp; typically amino acids 1 to 116 based on Kabat numbering Kabat et al., "Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5thed.) at the N-terminus, and one constant domain (e.g., a-chain constant domain or Ca, typically amino acids 117 to 259 based on Kabat, P-chain constant domain or Cp,typically amino acids 117 to 295 based on Kabat) adjacent to the cell membrane. For example, in some cases, the extracellular portion of the TCR formed by the two chains contains two membrane-proximal constant domains, and two membrane-distal variable domains containing CDRs. The constant domain of the TCR domain contains short connecting sequences in which a cysteine residue forms a disulfide bond, making a link between the two chains. In some embodiments, a TCR may have an additional cysteine residue in each of the a and P chains such that the TCR contains two disulfide bonds in the constant domains.

[0203] In some embodiments, the TCR chains can contain a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chains contains a cytoplasmic tail. In some cases, the structure allows the TCR to associate with other molecules like CD3. For example, a TCR containing constant domains with a transmembrane region can anchor the protein in the cell membrane and associate with invariant subunits of the CD3 signaling apparatus or complex.

[0204] Generally, CD3 is a multi-protein complex that can possess three distinct chains (y, 5, and a) in mammals and the ychain. For example, in mammals the complex can contain a CD3y chain, a CD38 chain, two CD3e chains, and a homodimer of CD3(^ chains. The CD3y, CD38, and CD3e chains are highly related cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain. The transmembrane regions of the CD3y, CD38, and CD3e chains are negatively charged, which is a characteristic that allows these chains to associate with the positively charged T cell receptor chains. The intracellular tails of the CD3y, CD38, and CD3e chains each contain a single conserved motif known as an immunoreceptor tyrosine -based activation motif or IT AM, whereas each CD3(^ chain has three. Generally, IT AMs are involved in the signaling capacity of the TCR complex. These accessory molecules have negatively charged transmembrane regions and play a role in propagating the signal from the TCR into the cell. The CD3- and (^-chains, together with the TCR, form what is known as the T cell receptor complex.

[0205] In some embodiments, the TCR may be a heterodimer of two chains a and P (or optionally y and 5) or it may be a single chain TCR construct. In some embodiments, the TCR is a heterodimer containing two separate chains (a and P chains or y and 5 chains) that are linked, such as by a disulfide bond or disulfide bonds. In some embodiments, a TCR for a target antigen (e.g., a cancer antigen) is identified and introduced into the cells. In some embodiments, nucleic acid encoding the TCR can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of publicly available TCR DNA sequences. Insome embodiments, the TCR is obtained from a biological source, such as from cells such as from a T cell (e.g. cytotoxic T cell), T cell hybridomas or other publicly available source. In some embodiments, the T cells can be obtained from in vivo isolated cells. In some embodiments, a high-affinity T cell clone can be isolated from a patient, and the TCR isolated. In some embodiments, the T cells can be a cultured T cell hybridoma or clone. In some embodiments, the TCR clone for a target antigen has been generated in transgenic mice engineered with human immune system genes (e.g., the human leukocyte antigen system, or HLA). See, e.g., tumor antigens (see, e.g., Parkhurst et al., 2009; and Cohen et al., 2005). In some embodiments, phage display is used to isolate TCRs against a target antigen (see, e.g., Varela-Rohena et al., 2008; and Li et al., 2005). In some embodiments, the TCR or antigenbinding portion thereof can be synthetically generated from knowledge of the sequence of the TCR.D. Methods of TIL Engineering

[0206] One of skill in the art would be well-equipped to construct a vector through standard recombinant techniques (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996, both incorporated herein by reference) for the expression of the antigen receptors of the present disclosure. Vectors include but are not limited to, plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs), such as retroviral vectors (e.g. derived from Moloney murine leukemia virus vectors (MoMLV), MSCV, SFFV, MPSV, SNV etc), lentiviral vectors (e.g. derived from HIV-1, HIV-2, SIV, BIV, FIV etc.), adenoviral (Ad) vectors including replication competent, replication deficient and gutless forms thereof, adeno-associated viral (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papilloma virus vectors, Epstein-Barr virus vectors, herpes virus vectors, vaccinia virus vectors, Harvey murine sarcoma virus vectors, murine mammary tumor virus vectors, Rous sarcoma virus vectors, parvovirus vectors, polio virus vectors, vesicular stomatitis virus vectors, maraba virus vectors and group B adenovirus enadenotucirev vectors. a. Viral Vectors

[0207] Viral vectors encoding a tumor-targeted membrane- anchored cytokine (e.g., attIL12) may be provided in certain aspects of the present disclosure. In generating recombinant viral vectors, non-essential genes are typically replaced with a gene or coding sequence for a heterologous (or non-native) protein. A viral vector is a kind of expression construct that utilizes viral sequences to introduce nucleic acid and possibly proteins into a cell. The ability of certain viruses to infect cells or enter cells via receptor mediated- endocytosis, and tointegrate into host cell genomes and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign nucleic acids into cells (e.g., mammalian cells). Non-limiting examples of virus vectors that may be used to deliver a nucleic acid of certain aspects of the present disclosure are described below.

[0208] Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. Lentiviral vectors are well known in the art (see, for example, U.S. Patents 6,013,516 and 5,994,136).

[0209] Recombinant lentiviral vectors are capable of infecting non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression of nucleic acid sequences. For example, recombinant lentivirus capable of infecting a non-dividing cell — wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat — is described in U.S. Patent 5,994,136, incorporated herein by reference. b. Regulatory Elements

[0210] Expression cassettes included in vectors useful in the present disclosure in particular contain (in a 5'-to-3' direction) a eukaryotic transcriptional promoter operably linked to a protein-coding sequence, splice signals including intervening sequences, and a transcriptional termination / polyadenylation sequence. The promoters and enhancers that control the transcription of protein encoding genes in eukaryotic cells are composed of multiple genetic elements. The cellular machinery is able to gather and integrate the regulatory information conveyed by each element, allowing different genes to evolve distinct, often complex patterns of transcriptional regulation. A promoter used in the context of the present disclosure includes constitutive, inducible, and tissue-specific promoters.(i) Promoter / Enhancers

[0211] The expression constructs provided herein comprise a promoter to drive expression of the antigen receptor. A promoter generally comprises a sequence that functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as, for example, the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation. Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30110 bp- upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. To bring a coding sequence“under the control of’ a promoter, one positions the 5' end of the transcription initiation site of the transcriptional reading frame “downstream” of (z.e., 3' of) the chosen promoter. The “upstream” promoter stimulates transcription of the DNA and promotes expression of the encoded RNA.

[0212] The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription. A promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

[0213] A promoter may be one naturally associated with a nucleic acid sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and / or exon. Such a promoter can be referred to as “endogenous.” Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other virus, or prokaryotic or eukaryotic cell, and promoters or enhancers not “naturally occurring,” i.e., containing different elements of different transcriptional regulatory regions, and / or mutations that alter expression. For example, promoters that are most commonly used in recombinant DNA construction include the |31actamase (penicillinase), lactose and tryptophan (trp-) promoter systems. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and / or nucleic acid amplification technology, including PCR, in connection with the compositions disclosed herein. Furthermore, it is contemplated that the control sequences that direct transcription and / or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

[0214] Naturally, it will be important to employ a promoter and / or enhancer that effectively directs the expression of the DNA segment in the organelle, cell type, tissue, organ, or organismchosen for expression. Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression, (see, for example Sambrook et al. 1989, incorporated herein by reference). The promoters employed may be constitutive, tissue-specific, inducible, and / or useful under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large- scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0215] Additionally, any promoter / enhancer combination (as per, for example, the Eukaryotic Promoter Data Base EPDB, through world wide web at epd.isb-sib.ch / ) could also be used to drive expression. Use of a T3, T7 or SP6 cytoplasmic expression system is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.

[0216] Non-limiting examples of promoters include early or late viral promoters, such as, SV40 early or late promoters, cytomegalovirus (CMV) immediate early promoters, Rous Sarcoma Virus (RSV) early promoters; eukaryotic cell promoters, such as, e. g., beta actin promoter, GAPDH promoter, metallothionein promoter; and concatenated response element promoters, such as cyclic AMP response element promoters (ere), serum response element promoter (sre), phorbol ester promoter (TPA) and response element promoters (tre) near a minimal TATA box. It is also possible to use human growth hormone promoter sequences (e.g. , the human growth hormone minimal promoter described at Genbank, accession no. X05244, nucleotide 283-341) or a mouse mammary tumor promoter (available from the ATCC, Cat. No. ATCC 45007). In certain embodiments, the promoter is CMV IE, dectin- 1, dectin-2, human CDl lc, F4 / 80, SM22, RSV, SV40, Ad MLP, beta-actin, MHC class I or MHC class II promoter, however any other promoter that is useful to drive expression of the therapeutic gene is applicable to the practice of the present disclosure.

[0217] In certain aspects, methods of the disclosure also concern enhancer sequences, i.e., nucleic acid sequences that increase a promoter’s activity and that have the potential to act in cis, and regardless of their orientation, even over relatively long distances (up to several kilobases away from the target promoter). However, enhancer function is not necessarily restricted to such long distances as they may also function in close proximity to a given promoter.(ii) Initiation Signals and Linked Expression

[0218] A specific initiation signal also may be used in the expression constructs provided in the present disclosure for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be “in-frame” with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.

[0219] Additionally, certain 2A sequence elements could be used to create linked- or coexpression of genes in the constructs provided in the present disclosure. For example, cleavage sequences could be used to co-express genes by linking open reading frames to form a single cistron. An exemplary cleavage sequence is the F2A (Foot-and-mouth disease virus 2A) or a “2A-like” sequence (e.g., Thosea asigna virus 2A; T2A).(iii) Origins of Replication

[0220] In order to propagate a vector in a host cell, it may contain one or more origins of replication sites (often termed “ori”), for example, a nucleic acid sequence corresponding to oriP of EBV as described above or a genetically engineered oriP with a similar or elevated function in programming, which is a specific nucleic acid sequence at which replication is initiated. Alternatively, a replication origin of other extra-chromosomally replicating virus as described above or an autonomously replicating sequence (ARS) can be employed. c. Selection and Screenable Markers

[0221] In some embodiments, cells containing a construct of the present disclosure may be identified in vitro or in vivo by including a marker in the expression vector. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector. Generally, a selection marker is one that confers a property that allows for selection. A positive selection marker is one in which the presence of the marker allows for its selection, while a negative selection marker is one in which its presence prevents its selection. An example of a positive selection marker is a drug resistance marker.

[0222] Usually the inclusion of a drug selection marker aids in the cloning and identification of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selection markers. In addition to markers conferring a phenotype that allows for the discrimination of transformantsbased on the implementation of conditions, other types of markers including screenable markers such as GFP, whose basis is colorimetric analysis, are also contemplated. Alternatively, screenable enzymes as negative selection markers such as herpes simplex virus thymidine kinase tk) or chloramphenicol acetyltransferase (CAT) may be utilized. One of skill in the art would also know how to employ immunologic markers, possibly in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selection and screenable markers are well known to one of skill in the art. d. Other Methods of Nucleic Acid Delivery

[0223] In addition to viral delivery of the nucleic acids encoding the tumor-targeted and membrane- anchored cytokine (e.g., attIL12), the following are additional methods of recombinant gene delivery to a given host cell and are thus considered in the present disclosure.

[0224] Introduction of a nucleic acid, such as DNA or RNA, into the immune cells of the current disclosure may use any suitable methods for nucleic acid delivery for transformation of a cell, as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by ex vivo transfection, by injection, including microinjection); by electroporation; by calcium phosphate precipitation; by using DEAE-dextran followed by polyethylene glycol; by direct sonic loading; by liposome mediated transfection and receptor-mediated transfection; by microprojectile bombardment; by agitation with silicon carbide fibers; by Agrobacterium-mediated transformation; by desiccation / inhibition-mediated DNA uptake, and any combination of such methods. Through the application of techniques such as these, organelle(s), cell(s), tissue(s) or organism(s) may be stably or transiently transformed.II. Methods of Treatment

[0225] Further provided herein are methods for treating or delaying progression of cancer in an individual comprising administering to the individual an effective amount of tumor- targeted and membrane- anchored cytokine engineered TIL and / or T cell therapy (e.g., attIL12- TIL and / or attIL12-T cell). Examples of cancers contemplated for treatment include but are not limited to lung cancer, head and neck cancer, breast cancer, pancreatic cancer, prostate cancer, renal cancer, bone cancer, testicular cancer, cervical cancer, gastrointestinal cancer, lymphomas, pre-neoplastic lesions in the lung, colon cancer, melanoma, and bladder cancer.

[0226] In some embodiments, the individual has cancer that is resistant (e.g., is recurrent and / or has been demonstrated to be resistant) to one or more anti-cancer therapies. In someembodiments, resistance to anti-cancer therapy includes recurrence of cancer or refractory cancer. Recurrence may refer to the reappearance of cancer, in the original site or a new site, after treatment. In some embodiments, resistance to anti-cancer therapy includes progression of the cancer during treatment with the anti-cancer therapy. In some embodiments, the cancer is at early stage or at late stage. In some embodiments, the cancer is at stage I, stage II, stage III, or stage IV.

[0227] In some embodiments, the subject is administered a chemotherapeutic in combination with the engineered TIL cell therapy. For example, the chemotherapeutic may be doxorubicin (Dox) and / or cyclophosphamide. In some embodiments, a subject may be pretreated with a chemotherapeutic agent such as doxorubicin and / or other immune cell recruiting inducers. In some embodiments, the pretreatment may be any suitable period of time prior to engineered TIL cell therapy, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, or greater than 48 hours to the TIL cell therapy.

[0228] In some embodiments, TILs are autologous. In some embodiments, the TILs retain functional copies of endogenous TCRs. In some embodiments, the TILs are isolated from the patient themself, so that the cells are autologous. In some embodiments, the cells are administered to the subject in need thereof in an amount sufficient to control, reduce, and / or eliminate symptoms and / or signs of the disease being treated (e.g., cancer).

[0229] The effectiveness of treatment can be measured by any suitable method known to those of skill in the art. In some embodiments, a white blood cell count (WBC) can be used to determine the responsiveness of a subject's immune system. A WBC measures the number of white blood cells in a subject. Using methods well known in the art, the white blood cells in a subject's blood sample are separated from other blood cells and counted. Normal values of white blood cells are about 4,500 to about 10,000 white blood cells / pl. Lower numbers of white blood cells can be indicative of a state of immunosuppression in the subject.

[0230] In another embodiment, immunosuppression in a subject may be determined using a T-lymphocyte count. Using methods well known in the art, the white blood cells in a subject's blood sample are separated from other blood cells. T-lymphocytes are differentiated from other white blood cells using standard methods in the art, such as, for example, immunofluorescence or FACS. Reduced numbers of T cells, or a specific population of T-cells can be used as a measurement of immunosuppression. A reduction in the number of T cells, or in a specific population of T cells, compared to the number of T cells (or the number of cells in the specificpopulation) prior to treatment can be used to indicate that immunosuppression has been induced.

[0231] In additional embodiments, tests to measure T cell activation, proliferation, or cytokine responses including those to specific antigens are performed. In some examples, the number of Treg or Breg cells can be measured in a sample from a subject. In additional examples, cytokines are measured in a sample, from a subject, such as IL- 10.

[0232] In other examples, to assess inflammation, neutrophil infiltration at the site of inflammation can be measured. In order to assess neutrophil infiltration myeloperoxidase activity can be measured. Myeloperoxidase is a hemoprotein present in azurophilic granules of polymorphonuclear leukocytes and monocytes. It catalyzes the oxidation of halide ions to their respective hypohalous acids, which are used for microbial killing by phagocytic cells. Thus, a decrease in myeloperoxidase activity in a tissue reflects decreased neutrophil infiltration, and can serve as a measure of inhibition of inflammation.

[0233] In another example, effective treatment of a subject can be assayed by measuring cytokine levels in the subject. Cytokine levels in body fluids or cell samples can be determined by conventional methods, for example, an immunospot assay, such as the enzyme-linked immunospot or “ELISPOT” assay, can be used. Immunospot assays can be highly sensitive and quantitative assays for detecting cytokine secretion at the single cell level. Immunospot methods and applications are well known in the art and are described, for example, in Czerkinsky et al., 1988; Olsson et al., 1990; and EP 957359. Variations of the standard immunospot assay are well known in the art and can be used to detect alterations in cytokine production in the methods of the disclosure (see, for example, U.S. Patent No. 5,939,281 and U.S. Patent No. 6,218,132).

[0234] In some embodiments, the subject can be administered nonmyeloablative lymphodepleting chemotherapy prior to the engineered TIL cell therapy. The nonmyeloablative lymphodepleting chemotherapy can be any suitable such therapy, which can be administered by any suitable route. The nonmyeloablative lymphodepleting chemotherapy can comprise, for example, the administration of cyclophosphamide and fludarabine, particularly if the cancer is melanoma, which can be metastatic. An exemplary route of administering cyclophosphamide and fludarabine is intravenously. Likewise, any suitable dose of cyclophosphamide and fludarabine can be administered. In particular aspects, around 60 mg / kg of cyclophosphamide is administered for two days after which around 25 mg / m2fludarabine is administered for five days.

[0235] In certain embodiments, cell growth factors that promotes the growth and activation of autologous TILs can be administered to the subject either concomitantly with the autologous TILs, prior to administration of the autologous TILs, and / or subsequently to the administration of the autologous TILs. The cell growth factor can be any suitable growth factor that promotes the growth and activation of the autologous TILs. Examples of suitable TIL cell growth factors include interleukin (IL)-2, IL-7, IL- 15, and IL- 12, which can be used alone or in various combinations, such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7 and IL-15, IL- 12 and IL-7, IL-12 and IL-15, or IL-12 and IL2. In certain embodiments, IL-12 is a preferred TIL cell growth factor. In certain embodiments, IL- 12 is provided through a transgenic construct, such as attIL12.

[0236] Local, regional or systemic administration may be appropriate. In some embodiments, for tumors of >4 cm, the volume to be administered can be about 4-10 ml (in particular 10 ml), while for tumors of <4 cm, a volume of about 1-3 ml can be used (in particular 3 ml). In some embodiments, multiple injections delivered as single dose can comprise about 0.1 to about 0.5 ml volumes.A. Overcoming Tumor Resistance

[0237] In some embodiments, provided herein are methods of treating a cancer in a subject, comprising overcoming tumor resistance using an engineered TIL. In some embodiments, provided herein are methods of treating a cancer in a subject comprising overcoming tumor resistance using an engineered T cell coupled with one or more therapeutic agents, such as one or more inhibitors of CCKAR and / or CCKAR regulated pathways.

[0238] In some embodiments, methods of overcoming tumor resistance comprise reducing, inhibiting, and / or targeting CCKAR and / or CCKAR regulated pathways. In some embodiments, methods of overcoming tumor resistance comprise administration of engineered autologous TILs. In some embodiments, the TILs are engineered to express attIL12. In some embodiments, the TILs comprise functional endogenous TCR alpha and / or beta genes. In some embodiments, the TILs are administered as part of a co-therapy with one or more agents.

[0239] In some embodiments, methods of treating immune effector cell resistant cancer in a subject with a tumor comprise, reducing and / or diminishing Cholecystokinin A receptor CCKAR protein and / or transcript expression in the tumor by contacting the tumor with TILs transgenically expressing a cell surface vimentin (CSV)-targeted and membrane-anchored cytokine.

[0240] In some embodiments, methods of treating cancer in a subject with a tumor comprise analyzing a tumor to determine the expression levels (transcript and / or protein) of one or more biomarkers. In some embodiments, methods of treatment are tailored dependent upon the expression levels of one or more tumor biomarkers. In some embodiments, engineered TIL therapy is utilized preferentially over other cell therapies (e.g., engineered T cell therapies) dependent upon the expression levels of one or more tumor biomarkers.1. Analyzing Tumor Samples

[0241] In some embodiments, a tumor sample is obtained from a patient, and the tumor sample is analyzed to determine the expression pattern of one or more biomarkers. In some embodiments, any suitable method known in the art for analyzing the expression pattern of one or more biomarkers may be utilized.

[0242] In some embodiments, the expression pattern of one or more biomarkers may be determined through nucleic acid sequencing methods. In some embodiments, nucleic acid sequencing may be directed to analysis of the genome of a bulk tumor mass, or the genomes of individual disassociated cells and / or populations of cells present in a tumor. In some embodiments, nucleic acid sequencing may be directed to analysis of the transcriptome of a bulk tumor mass, or the transcriptome of individual disassociated cells and / or populations of cells present in a tumor. In some embodiments, nucleic acid sequencing may be utilized to characterize the heterogeneity of a tumor.

[0243] In some embodiments, the expression pattern of one or more biomarkers may be determined through histological methods. In some embodiments, histological methods may be directed to analysis of a bulk tumor mass, or to individual disassociated cells and / or populations of cells present in a tumor. In some embodiments, histological analysis may be utilized to characterize the heterogeneity of a tumor. In some embodiments, histological analysis may comprise immunofluorescence staining. In some embodiments, histological analysis may comprise any staining suitable for the characterization of tumors, as known in the art.

[0244] In some embodiments, the expression pattern of one or more biomarkers may be determined through molecular level proteomics analysis, such as but not limited to mass spectrometry. In some embodiments, proteomic analysis may be conducted on a bulk tumor mass, or on individual disassociated cells and / or populations of cells present in a tumor.

[0245] In some embodiments, CCKAR, SLN, LARGE2, WNT10A, and / or IZUMO1 can act as markers (e.g., biomarkers), levels of which can be utilized for evaluating probability of immune effector cell therapy regimens. In some embodiments, CCKAR, SLN, LARGE2,WNT10A, and / or IZUMO1 can be used for evaluating likelihood of tumor resistance to immune effector cell therapy regimens.

[0246] In some embodiments, CCKAR, SLN, CDR1, LARGE2, WNT10A, BIRC7, IL1RL2, MYH14, FGFBP2 / PROM1, SCUBE1, GPR27, RAMP1, PIWIE2, TDRD9, ERRN2, RNF165, SYT13, AFAP1E2, GPR1, MATN4, PTPN20, CRTAC1, EAMA1, PRESS51, PENK, IHH, PLBD1, RAB9B, COL9A1, OSR1, PAX8, CD86, IL2RB, and / or IRAK3 can act as biomarkers, levels of which can be utilized for evaluating probability of immune effector cell therapy regimens.

[0247] In some embodiments, CCKAR, SLN, CDR1, LARGE2, WNT10A, BIRC7, IL1RL2, MYH14, FGFBP2 / PROM1, SCUBE1, GPR27, RAMP1, PIWIL2, TDRD9, LRRN2, RNF165, SYT13, AFAP1L2, GPR1, MATN4, PTPN20, CRTAC1, LAMA1, PRESS51, PENK, IHH, PLBD1, RAB9B, COL9A1, OSR1, PAX8, CD86, IL2RB, IRAK3, DDIT4L, TMEM178B, TBRESLC4A10, TMEM35A, CLIC5, CACNA1A, ST8SIA6, FGF12, BMP5, SFRP2, and / or IZUMO1 can act as markers, levels of which can be utilized for evaluating probability of immune effector cell therapy regimens.

[0248] In certain embodiments, high relative expression levels of CCKAR, SLN, CDR1, LARGE2, WNT10A, BIRC7, IL1RL2, MYH14, FGFBP2 / PROM1, SCUBE1, GPR27, RAMP1, PIWIL2, TDRD9, LRRN2, RNF165, SYT13, AFAP1L2, GPR1, MATN4, PTPN20, CRTAC1, LAMA1, PRESS51, PENK, IHH, PLBD1, RAB9B, COL9A1, OSR1, PAX8, CD86, IL2RB, and / or IRAK3 can be associated with tumor resistance to immune effector cell therapies, such as attIL12 T cell therapies.

[0249] In some embodiments, low relative expression of DDIT4L, TMEM178B, TBRESLC4A10, TMEM35A, CLIC5, CACNA1A, ST8SIA6, FGF12, BMP5, SFRP2, and / or IZUMO1 can be associated with sensitivity to immune effector cell therapies, such as attIL12 T cell therapies.

[0250] In some embodiments, engineered TIL therapies are preferentially utilized over alternative cell therapies when a tumor expresses CCKAR, SLN, LARGE2, and / or WNT10A at levels greater than or equal to about 0.0001 to about 30 FPKM. In some embodiments, engineered TIL therapies are preferentially utilized over alternative cell therapies when a tumor expresses IZUMO1 at levels less than or equal to about 0.1, 0.001, or 0.0001 FPKM.

[0251] In some embodiments, Cholecystokinin A receptor (CCKAR; NCBI Gene ID: 886) acts as a biomarker for determining what type of cell therapy and / or combination therapy would likely result in overcoming a therapy resistant tumor. The gene CCKAR (also known as CCK- A; CCK1R; CCKRA; CCK-1R; CCK1-R) encodes a G-protein coupled receptor that bindsnon-sulfated members of the cholecystokinin (CCK) family of peptide hormones. This receptor is a major physiologic mediator of pancreatic enzyme secretion and smooth muscle contraction of the gallbladder and stomach. In the central and peripheral nervous system this receptor regulates satiety and the release of beta-endorphin and dopamine. In some embodiments, when the expression of CCKAR in the tumor is greater than or equal to about 0.0001, 0.001, 0.01, 0.1, 1.0, or 10.0 FPKM, or greater than 10.0 FPKM, methods of treatment comprise preferential utilization of engineered TILs in place of alternative therapies, such as alternative monotherapies. In some embodiments, when the expression of CCKAR in the tumor is greater than or equal to about 0.0001, 0.001, 0.01, 0.1, 1.0, or 10.0 FPKM, or greater than 10.0 FPKM, and when subject derived TILs are not available, methods of treatment comprise preferential utilization of engineered T cells coupled with one or more inhibitors of CCKAR and / or CCKAR pathways in place of alternative therapies, such as monotherapies.

[0252] In some embodiments, Sarcolipin (SLN; NCBI Gene ID: 6588) acts as a biomarker for determining what type of cell therapy and / or combination therapy would likely result in overcoming a therapy resistant tumor. Sarcoplasmic reticulum Ca(2+)-ATPases are transmembrane proteins that catalyze the ATP-dependent transport of Ca(2+) from the cytosol into the lumen of the sarcoplasmic reticulum in muscle cells. The sin gene encodes a small proteolipid that regulates several sarcoplasmic reticulum Ca(2+)-ATPases. The transmembrane protein interacts with Ca(2+)-ATPases and reduces the accumulation of Ca(2+) in the sarcoplasmic reticulum without affecting the rate of ATP hydrolysis. In some embodiments, when the expression of SLN in the tumor is greater than or equal to about 0.0001, 0.001, 0.01, 0.1, 1.0, or 10.0 FPKM, or greater than 10.0 FPKM, methods of treatment comprise preferential utilization of engineered TILs in place of alternative therapies, such as alternative monotherapies. In some embodiments, when the expression of SLN in the tumor is greater than or equal to about 0.0001, 0.001, 0.01, 0.1, 1.0, or 10.0 FPKM, or greater than 10.0 FPKM, and when subject derived TILs are not available, methods of treatment comprise preferential utilization of engineered T cells coupled with one or more inhibitors of CCKAR and / or CCKAR pathways in place of alternative therapies, such as monotherapies.

[0253] In some embodiments, Large xylosyl- and glucuronyltransferase 2 (LARGE2; NCBI Gene ID: 120071) acts as a biomarker for determining what type of cell therapy and / or combination therapy would likely result in overcoming a therapy resistant tumor. LARGE2 is predicted to enable dystroglycan binding activity; glucuronosyltransferase activity; and xylosyltransferase activity. LARGE2 is involved in protein O-linked mannosylation, and is predicted to be located in intracellular membrane-bounded organelle, while being active in theGolgi apparatus. In some embodiments, when the expression of LARGE2 in the tumor is greater than or equal to about 0.0001, 0.001, 0.01, 0.1, 1.0, or 10.0 FPKM, or greater than 10.0 FPKM, methods of treatment comprise preferential utilization of engineered TIFs in place of alternative therapies, such as alternative mono-therapies. In some embodiments, when the expression of FARGE2 in the tumor is greater than or equal to about 0.0001, 0.001, 0.01, 0.1, 1.0, or 10.0 FPKM, or greater than 10.0 FPKM, and when subject derived TIFs are not available, methods of treatment comprise preferential utilization of engineered T cells coupled with one or more inhibitors of CCKAR and / or CCKAR pathways in place of alternative therapies, such as monotherapies.

[0254] In some embodiments, Wnt family member 10A (WNT10A; NCBI Gene ID: 80326) acts as a biomarker for determining what type of cell therapy and / or combination therapy would likely result in overcoming a therapy resistant tumor. The WNT gene family consists of structurally related genes which encode secreted signaling proteins. These proteins have been implicated in oncogenesis and in several developmental processes, including regulation of cell fate and patterning during embryogenesis. The gene WNT10A is a member of the WNT gene family. It is strongly expressed in the cell lines of promyelocytic leukemia and Burkitt's lymphoma. In addition, it and another family member, the WNT6 gene, are strongly co-expressed in colorectal cancer cell lines. In some cases, WNT10A gene overexpression may play key roles in carcinogenesis through activation of the WNT-beta- catenin-TCF signaling pathway. In some embodiments, when the expression of WNT10A in the tumor is greater than or equal to about 0.0001, 0.001, 0.01, 0.1, 1.0, or 10.0 FPKM, or greater than 10.0 FPKM, methods of treatment comprise preferential utilization of engineered TIFs in place of alternative therapies, such as alternative mono-therapies. In some embodiments, when the expression of WNT10A in the tumor is greater than or equal to about 0.0001, 0.001, 0.01, 0.1, 1.0, or 10.0 FPKM, or greater than 10.0 FPKM, and when subject derived TIFs are not available, methods of treatment comprise preferential utilization of engineered T cells coupled with one or more inhibitors of CCKAR and / or CCKAR pathways in place of alternative therapies, such as monotherapies.

[0255] In some embodiments, Izumo sperm-oocyte fusion 1 (IZUMO1; NCBI Gene ID 284359) acts as a biomarker for determining what type of cell therapy and / or combination therapy would likely result in overcoming a therapy resistant tumor. The sperm- specific protein Izumo, named for a Japanese shrine dedicated to marriage, is essential for sperm-egg plasma membrane binding and fusion. In some embodiments, when the expression of IZUMO 1 in the tumor is less than or equal to about 0.0001, 0.001, 0.01, 0.1, 1.0, or 10.0 FPKM, methods oftreatment comprise preferential utilization of engineered TILs in place of alternative therapies, such as alternative mono-therapies. In some embodiments, when the expression of IZUM01 in the tumor is less than or equal to about 0.0001, 0.001, 0.01, 0.1, 1.0, or 10.0 FPKM, and when subject derived TILs are not available, methods of treatment comprise preferential utilization of engineered T cells coupled with one or more inhibitors of CCKAR and / or CCKAR pathways in place of alternative therapies, such as monotherapies.2. Obtaining Immune Effector Cells

[0256] Tumor Infiltrating Lymphocytes

[0257] In some embodiments, TILs are obtained from tumor biopsies which have been disassociated and cell sorted. In some embodiments, TILs are obtained from blood and / or lymph from patients with tumors, where the TILs have previously infiltrated the tumor and subsequently left said tumor and entered the blood and / or lymph. In some embodiments, when TILs are not available in the tumor biopsies and / or subject blood or lymph, T Cells are obtained as an alternative.

[0258] In some embodiments, TILs are obtained using good manufacturing practice (GMP) protocols. In some embodiments, TILs are obtained without the use of 4-1BB and / or 4-1BBL antibodies. In some embodiments, with the exception of exclusion of the CD 137 antibody, TILs are isolated and maintained as described in Fulbright, O.J. et al., 2022 (Fulbright, O.J., Forget, MA., Haymaker, C., Bernatchez, C. (2022). Isolation and Maintenance of Tumor- Infiltrating Lymphocytes for Translational and Clinical Applications: Established Methods and New Developments. In: McAllister, F. (eds) Cancer Immunoprevention. Methods in Molecular Biology, vol 2435. Humana, New York, NY. https: / / doi.org / 10.1007 / 978-l-0716-2014-4_5), which is incorporated herein by reference in its entirety for the purposes described herein.3. Engineering and / or Expanding Immune Effector Cells

[0259] Immune effector cells can be engineered as described herein. In certain embodiments, immune effector cells comprise TILs and / or T cells that are engineered to express attIL12.

[0260] In some embodiments, artificial APC (AaPC) cell stimulated TILs are utilized. In some embodiments, an AaPC can comprise K562 AaPCs expressing CD64, CD86, CD137L modified to express IL15 / IL15Ra fusion protein (membrane-bound IL-15; mIL15). In some embodiments, AaPCs (e.g., K562 AaPCs) are cultured using any suitable means, such as but not limited to, in VueLife bags and / or a WAVE Bioreactor, and can be phenotyped to validateexpression of introduced transgenes / costimulatory molecules. In some embodiments, AaPCs (e.g., K562 AaPCs) can be loaded with anti-CD3 antibodies (0KT3, Orthoclone) via the CD64 / FcyR onto the cell surface (OKT3-K562 AaPC), irradiated (e.g., at 100 Gy), and frozen in aliquots for later use. In some embodiments, TILs are obtained from tumor tissue. In some embodiments, tumor tissues are enzymatically dissociated in media (such as but not limited to 10% RPMI) to create a single cell suspension. In some embodiments, TILS are then isolated using any suitable means (e.g., flow cytometry). In some embodiments, isolated cells can be co-cultured with OKT3-K562 AaPC along with addition of exogenous cytokines (e.g., IL-2, and IL-21) in a 7-day stimulation cycle for one, two, three, four, or more than four weeks.

[0261] In some embodiments, TILs are obtained and maintained largely as described in Fulbright, O.J., et al., 2022 (Fulbright, O .J., Forget, MA., Haymaker, C., Bernatchez, C. (2022). Isolation and Maintenance of Tumor-Infiltrating Lymphocytes for Translational and Clinical Applications: Established Methods and New Developments. In: McAllister, F. (eds) Cancer Immunoprevention. Methods in Molecular Biology, vol 2435. Humana, New York, NY. https: / / doi.org / 10.1007 / 978- 1-0716-2014-4_5), which is incorporated herein by reference in its entirety for the purposes described herein. In some embodiments, TILs are obtained an maintained largely as described in Fulbright, O .J., et al., 2022, except that TILs are not subject to CD 137 antibody exposure.4. Providing Immune Effector Cells

[0262] In certain embodiments, engineered TILs and / or T cells are provided to a subject in need thereof, such as an autologous subject with recurrent cancer.

[0263] In certain embodiments, providing subject with engineered TILs inhibits CCKAR, phosphorylated AKT serine / threonine kinase 1 (pAKT), phosphorylated SMAD family member 3 (pSMAD3), and / or spectrin beta non-erythrocytic 2 (SPTBN2) expression levels. In some embodiments, providing a subject with engineered TILs results in a reduction of CCKAR, and concatenate inhibition of one or more of pAKT and / or TGFp, and / or pAKT or TGFP signaling pathway transducers. In some embodiments, providing of engineered TILs results in levels of TNFa and / or IFNy being enriched in the tumor, and / or levels of TGFP in the tumor are reduced, relative to administration of non-engineered TILs, non-engineered T cells, and / or engineered T cells. In some embodiments, providing of engineered TILs to the subject results in significantly reduced levels of CCKAR in the tumor, relative to administration of nonengineered TILs, non-engineered T cells, and / or engineered T cells.

[0264] In some embodiments, providing engineered TILs comprises contacting tumor cells and / or cancer associated fibroblasts (CAFs) with the engineered TILs. In some embodiments, contacting of a tumor with engineered TILs results in simultaneous reduction of levels of collagen and ECM in the tumor. In some embodiments, contacting of a tumor with engineered TILs results in simultaneous reduction of levels of collagen, ECM, CCKAR, and TGFP in the tumor. In some embodiments, the providing of engineered TILs facilitates entrance of endogenous immune cells and / or one or more additional exogenous agents into the tumor.5. Additional Agents

[0265] In some embodiments, methods provided herein comprise administering one or more additional anticancer therapies to the subject. In some embodiments, methods provided herein include one or more additional anticancer therapy comprising a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, immunotherapy, and / or cytokine therapy.

[0266] In some embodiments, provided herein are methods of treating cancer comprising administration of engineered TILs and / or engineered T cells, and one or more inhibitors of CCKAR, phosphorylated AKT serine / threonine kinase 1 (pAKT), phosphorylated SMAD family member 3 (pSMAD3), and / or spectrin beta non-erythrocytic 2 (SPTBN2). In some exemplary aspects, an inhibitor of CCKAR is a CCKAR blocking antibody.

[0267] In some embodiments, provided herein are methods of treating cancer comprising administration of engineered TILs and / or engineered T cells and one or more additional anticancer therapies comprising one or more TGFP inhibitors. In some embodiments, one or more TGFP inhibitor can comprise an antibody specific for TGFP and / or TGFP receptor(s). In some embodiments, provided herein are methods of treating cancer comprising administration of engineered TILs and / or engineered T cells and one or more antibody-drug-conjugates (ADCs). In certain embodiments, provided herein are methods of treating cancer comprising administration of engineered TILs and one or more immune effector cell therapies. In some embodiments, provided herein are methods of treating cancer comprising administration of engineered TILs and engineered T cells.B. Pharmaceutical Compositions

[0268] Also provided herein are pharmaceutical compositions and formulations comprising an engineered TIL and / or engineered T cell therapy and a pharmaceutically acceptable carrier.

[0269] Pharmaceutical compositions and formulations as described herein can be prepared by mixing the active ingredients (such as an antibody or a polypeptide) having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 22nd edition, 2012), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn- protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include insterstitial drug dispersion agents such as soluble neutral- active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.C. Additional Therapy

[0270] In certain embodiments, the compositions and methods of the present embodiments involve an engineered TIL and / or T cell population in combination with at least one additional therapy. The additional therapy may be radiation therapy, surgery e.g., lumpectomy and a mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. The additional therapy may be in the form of adjuvant or neoadjuvant therapy.

[0271] An engineered TIL and / or engineered T cell therapy may be administered before, during, after, or in various combinations relative to an additional therapy, such as doxorubicin. The administrations may be in intervals ranging from concurrently to minutes to days to weeks. In embodiments where the TIL and / or T cell therapy is provided to a patient separately from an additional therapeutic agent, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the two compounds would still be able to exert an advantageously combined effect on the patient. In such instances, it is contemplated that one may provide a patient with the engineered TIL and / or engineered T cell therapy and the anti-cancer therapy within about 12 to 24 or 72 h of each other and, more particularly, within about 6-12 h of each other. In some situations it may be desirable to extend the time period for treatment significantly where several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) lapse between respective administrations.

[0272] The engineered TIL and / or T cell therapy and the additional therapeutic agent may be administered by the same route of administration or by different routes of administration. In some embodiments, the engineered TIL and / or engineered T cell therapy is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. An effective amount of the engineered TIL and / or T cell therapy and additional therapeutic agent may be administered for prevention or treatment of disease. The appropriate dosage of the engineered TIL and / or engineered T cell therapy and additional therapeutic agent be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.

[0273] In some embodiments, the additional therapy is the administration of small molecule enzymatic inhibitor or anti-metastatic agent. In some embodiments, the additional therapy is the administration of side-effect limiting agents (e.g., agents intended to lessen the occurrence and / or severity of side effects of treatment, such as anti-nausea agents, etc.). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is therapy targeting PBK / AKT / mTOR pathway, HSP90 inhibitor, tubulin inhibitor, apoptosis inhibitor, and / or chemopreventative agent. The additional therapy may be one or more of the chemotherapeutic agents known in the art.

[0274] Various combinations may be employed. For the example below a TIL cell therapy is “A” and an additional therapeutic agent is “B”:A / B / A B / A / B B / B / A A / A / B A / B / B B / A / A A / B / B / B B / A / B / BB / B / B / A B / B / A / B A / A / B / B A / B / A / B A / B / B / A B / B / A / AB / A / B / A B / A / A / B A / A / A / B B / A / A / A A / B / A / A A / A / B / A

[0275] Administration of any compound or therapy of the present embodiments to a patient will follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the agents. Therefore, in some embodiments there is a step of monitoring toxicity that is attributable to combination therapy.1. Chemotherapy

[0276] A wide variety of chemotherapeutic agents may be used in accordance with the present embodiments. The term “chemotherapy” refers to the use of drugs to treat cancer. A “chemotherapeutic agent” is used to connote a compound or composition that is administered in the treatment of cancer. These agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle. Alternatively, an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.

[0277] Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclo sphosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics, such as the enediyne antibiotics (e.g.. calicheamicin, especially calicheamicin gammall and calicheamicin omegall); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; anti-metabolites, such as methotrexate and 5- fluorouracil (5-FU); folic acid analogues, such as denopterin, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenals, such as mitotane and trilostane; folic acid replenisher, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PS Kpoly saccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.2. Radiotherapy

[0278] Other factors that cause DNA damage and have been used extensively include what are commonly known as y-rays, X-rays, and / or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated, such as microwaves, proton beam irradiation (U.S. Patents 5,760,395 and 4,870,287), and UV-irradiation. It is most likely that all of these factors affect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.3. Immunotherapy

[0279] The skilled artisan will understand that additional immunotherapies may be used in combination or in conjunction with methods of the embodiments. In the context of cancer treatment, immunotherapeutic s, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is such an example. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells can include cytotoxic T cells and NK cells.

[0280] Antibody-drug conjugates have emerged as a breakthrough approach to the development of cancer therapeutics. Cancer is one of the leading causes of deaths in the world. Antibody-drug conjugates (ADCs) comprise monoclonal antibodies (MAbs) that are covalently linked to cell-killing drugs. This approach combines the high specificity of MAbs against their antigen targets with highly potent cytotoxic drugs, resulting in “armed” MAbs that deliver the pay load (drug) to tumor cells with enriched levels of the antigen (Carter et al., 2008; Teicher et al., 2014; Leal et al., 2014). Targeted delivery of the drug also minimizes its exposure in normal tissues, resulting in decreased toxicity and improved therapeutic index. The approval of two ADC drugs, ADCETRIS® (brentuximab vedotin) in 2011 and KADCYLA® (trastuzumab emtansine or T-DM1) in 2013 by FDA validated the approach. There arecurrently more than 30 ADC drug candidates in various stages of clinical trials for cancer treatment (Leal et al., 2014). As antibody engineering and linker-payload optimization are becoming more and more mature, the discovery and development of new ADCs are increasingly dependent on the identification and validation of new targets that are suitable to this approach (Teicher et al., 2009) and the generation of targeting MAbs. Two criteria for ADC targets are upregulated / high levels of expression in tumor cells and robust internalization.

[0281] In some aspects of immunotherapy, the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present embodiments. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, laminin receptor, erb B, and pl 55. An alternative aspect of immunotherapy is to combine anticancer effects with immune stimulatory effects. Immune stimulating molecules also exist including: cytokines, such as IL- 2, IL-4, IL- 12, GM-CSF, gamma- IFN, chemokines, such as MIP-1, MCP-1, IL-8, and growth factors, such as FLT3 ligand.

[0282] Examples of immunotherapies currently under investigation or in use are immune adjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Patents 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapy, e.g., interferons ex, |3, and y, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapy, e.g., TNF, IL-1, IL-2, and p53 (Qin etal., 1998; Austin-Ward and Villaseca, 1998; U.S. Patents 5,830,880 and 5,846,945); and monoclonal antibodies, e.g., anti-CD20, anti-ganglioside GM2, and anti-pl85 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Patent 5,824,311). It is contemplated that one or more anti-cancer therapies may be employed with the antibody therapies described herein.

[0283] In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints are regulators in the immune system that either turn up a signal (e.g., costimulatory molecules) or turn down a signal. Inhibitory checkpoints that may be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD 152), indoleamine 2,3-dioxygenase (IDO), killer-cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T-cell immunoglobulin domain and mucin domain 3 (TIM-3) and V-domain Ig suppressor ofT cell activation (VISTA). In particular, the immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0284] The immune checkpoint inhibitors may be drugs such as small molecules, recombinant forms of ligand or receptors, or, in particular, are antibodies, such as human antibodies (e.g., International Patent Publication W02015016718; Pardoll, Nat Rev Cancer, 12(4): 252-64, 2012; both incorporated herein by reference). Known inhibitors of the immune checkpoint proteins or analogs thereof may be used, in particular chimerized, humanized or human forms of antibodies may be used. As the skilled person will know, alternative and / or equivalent names may be in use for certain antibodies mentioned in the present disclosure. Such alternative and / or equivalent names are interchangeable in the context of the present disclosure. For example it is known that lambrolizumab is also known under the alternative and equivalent names MK-3475 and pembrolizumab.

[0285] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a specific aspect, the PD-1 ligand binding partners are PDL1 and / or PDL2. In another embodiment, a PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partners. In a specific aspect, PDL1 binding partners are PD-1 and / or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partners. In a specific aspect, a PDL2 binding partner is PD- 1. The antagonist may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. US8735553, US8354509, and US8008449, all incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are known in the art such as described in U.S. Patent Application No. US20140294898, US2014022021, and US20110008369, all incorporated herein by reference.

[0286] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g. , an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP- 224. Nivolumab, also known as MDX- 1106-04, MDX- 1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in W02006 / 121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in W02009 / 114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in W02009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in W02010 / 027827 and WO2011 / 066342.

[0287] Another immune checkpoint that can be targeted in the methods provided herein is the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD 152. The complete cDNA sequence of human CTLA-4 has the Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an “off’ switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells. CTLA4 is similar to the T-cell co- stimulatory protein, CD28, and both molecules bind to CD80 and CD86, also called B7-1 and B7-2 respectively, on antigen-presenting cells. CTLA4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA- 4, an inhibitory receptor for B7 molecules.

[0288] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

[0289] Anti-human-CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-CTLA-4 antibodies can be used. For example, the anti- CTLA-4 antibodies disclosed in: US 8,119,129, WO 01 / 14424, WO 98 / 42752; WO 00 / 37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al. (1998) Proc Natl Acad Sci USA 95(17): 10067-10071; Camacho et al. (2004) J Clin Oncology 22(145): Abstract No. 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res 58:5301-5304 can be used in the methods disclosed herein. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 also can be used. For example, a humanized CTLA-4 antibody is described in International Patent Application No. W02001014424, W02000037504, and U.S. Patent No. US8017114; all incorporated herein by reference.

[0290] An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX- 010, MDX- 101, and Yervoy®) or antigen binding fragments and variants thereof (see, e.g., WOO 1 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRsor VRs of ipilimumab. Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, and the CDR1, CDR2 and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding with and / or binds to the same epitope on CTLA-4 as the above- mentioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above-mentioned antibodies (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).

[0291] Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors such as described in U.S. Patent Nos. US5844905, US5885796 and International Patent Application Nos. WO1995001994 and WO1998042752; all incorporated herein by reference, and immunoadhesins such as described in U.S. Patent No. US8329867, incorporated herein by reference.4. Surgery

[0292] Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and / or destroyed and may be used in conjunction with other therapies, such as the treatment of the present embodiments, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electro surgery, and microscopically-controlled surgery (Mohs’ surgery).

[0293] Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.5. Other Agents

[0294] It is contemplated that other agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase thesensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Increases in intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents can be used in combination with certain aspects of the present embodiments to improve the anti-hyperproliferative efficacy of the treatments. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiments. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with certain aspects of the present embodiments to improve the treatment efficacy.III. Articles of Manufacture or Kits

[0295] An article of manufacture or a kit is provided comprising engineered TILs and / or engineered T cells, such as engineered TILs and / or engineered T cells expressing attIL12. The article of manufacture or kit can further comprise a package insert comprising instructions for using the adoptive engineered TIL cell therapy optionally in conjunction with an additional therapeutic agent (e.g., doxorubicin) to treat or delay progression of cancer in an individual or to enhance immune function of an individual having cancer. Any of the adoptive engineered TIL and / or engineered T cells and / or additional therapeutic agents described herein may be included in the article of manufacture or kits. In some embodiments, the adoptive engineered TIL and / or engineered T cells and additional therapeutic agent are in the same container or separate containers. Suitable containers include, for example, bottles, vials, bags and syringes. The container may be formed from a variety of materials such as glass, plastic (such as polyvinyl chloride or polyolefin), or metal alloy (such as stainless steel or hastelloy). In some embodiments, the container holds the formulation and the label on, or associated with, the container may indicate directions for use. The article of manufacture or kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the article of manufacture further includes one or more of another agent (e.g., a chemotherapeutic agent, and anti-neoplastic agent). Suitable containers for the one or more agent include, for example, bottles, vials, bags and syringes.IV. Examples

[0296] The following examples are included to demonstrate exemplary embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of certain inventions, and thus in certain embodiments, can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the inventions described herein.Example 1 - Materials and MethodsTumor-targeted membrane-anchored cytokine construct

[0297] A tumor targeted anchored construct was developed with membrane-anchored IL- 12 being fused to a tumor-targeted peptide CSV. In brief, the two subunits of IL12 were cloned into a single vector. The p35 subunit (SEQ ID NO: 4) was fused with EGFR transmembrane domain (SEQ ID NO: 2) via a linker (SEQ ID NO: 3) providing the membrane- anchored p35 subunit (SEQ ID NO: 6), and the p40 subunit (SEQ ID NO: 5) was fused with a CSV binding peptide sequence (SEQ ID NO: 1) providing the tumor-targeted p40 subunit (SEQ ID NO: 7).

[0298] Human IL12 subunit P35 with and without a transmembrane domain and subunit P40 with and without a tumor-targeted peptide were synthesized by VectorBuilder Inc. and cloned into a third-generation self-inactivating lentiviral expression vector (VectorBuilder Inc.) under a murine stem cell virus and modified cytomegalovirus promoter.Animal Studies and Tumor Models

[0299] Six- to eight- week-old C.B-17SC scid- / - mice (“SCID” mice) of both sexes were purchased from The Jackson Laboratory. The mouse care and handling procedures were approved by the Institutional Animal Care and Use Committee of The University of Texas MD Anderson Cancer Center.

[0300] To generate PDX tumors in mice, tumor cells (e.g., soft tissue sarcoma cell lines SA127, or SAI 17) were implanted subcutaneously into C.B-17SC scid- / - mice. Mice were then preconditioned with cyclophosphamide (Baxter Healthcare) when tumors reached to 6~8 mm in diameter followed by 2 immune effector cell (e.g., TILs or T cells) infusions (2.5 x 106) 14 days apart. Tumors were measured with calipers twice weekly after implantation. Tumor volume was calculated by the formula V = (K / 8) X (a b2), where V = tumor volume in cubic centimeters, a = maximum tumor diameter, and b = diameter at 90° to a.

[0301] Mice were injected with Brefeldin A (Cell Signaling Technologies, 200 pg / mouse) via intraperitoneal injection 4 hours before euthanasia for IFNy and / or TGFP intracellular staining. Mice were sacrificed on notice and survival curves were generated by the Kaplan- Meier method.Cell Culture

[0302] Tumor cells were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% FBS and supplemented with antibiotics and nonessential amino acid solution, and maintained in an incubator at 5% CO2 and 37 °C. The tumor cell line was characterized by DNA fingerprinting at MD Anderson Cancer Center's Characterized Cell Line Core Facility within 6 months of initiating the experiments and treated with a mycoplasma removal agent from Bio-Rad.Human T cells

[0303] Buffy coats from deidentified healthy blood donors were purchased from the Gulf Coast Regional Blood Center; and their acquisition was approved by the MD Anderson Institutional Review Board. Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coat samples or bone marrow samples via centrifugation over Ficoll-Paque. Human T cells were enriched from PBMCs using an EasySep Human CD8C T-cell Isolation Kit (STEMCELL Technologies). Human T cells were cultured in 45% RPML1640 and 45% Click’ s medium containing 10% fetal bovine serum (FBS) and supplemented with recombinant human (rh) IL-2 (50 U / mL), rhIL-7 (10 ng / mL), and rhIL-15 (5 ng / mL).Tumor Infiltrating Lymphocytes

[0304] Artificial APC (aAPC) cell stimulated TILs: K562 aAPC expressing CD64, CD86, CD137L modified to express IL15 / IL15Ra fusion protein (membrane -bound IL- 15; mIL15) were used to expand TILs. K562 aAPC were cultured in VueLife bags and / or the WAVE Bioreactor and phenotyped to validate expression of introduced transgenes / costimulatory molecules. K562 aAPC were loaded with anti-CD3 antibodies (OKT3, Orthoclone) via the CD64 / FcyR onto the cell surface (OKT3-K562 AaPC), irradiated at 100 Gy, and frozen in aliquots for later use. For generation of TILs, tumor tissues were enzymatically dissociated in media (10% RPMI) to create a single cell suspension and isolated cells were co-cultured with OKT3-K562 aAPC along with addition of exogenous cytokines (IL-2, and IL-21) in a 7-day stimulation cycle for one or more weeks.

[0305] Except for exclusion of the CD 137 antibody, TILs were also isolated and maintained as described in Fulbright, O. J. et al., 2022 (Fulbright, O .J., Forget, MA., Haymaker, C., Bernatchez, C. (2022). Isolation and Maintenance of Tumor- Infiltrating Lymphocytes forTranslational and Clinical Applications: Established Methods and New Developments. In: McAllister, F. (eds) Cancer Immunoprevention. Methods in Molecular Biology, vol 2435. Humana, New York, NY. https: / / doi.org / 10.1007 / 978-l-0716-2014-4_5), which is incorporated herein by reference in its entirety for the purposes described herein.Generation of Lentivirus

[0306] High-titer replication-defective lentiviral vectors were produced and concentrated by the MD Anderson Functional Genomics Core Facility. Briefly, HEK293T human embryonic kidney cells were transfected with pVSV-G (a VSV glycoprotein expression plasmid), pCMV- Gag / Pol / Rev, and a transfer plasmid using Eipofectamine 2000 (Thermo Fisher Scientific). The viral supernatant was harvested 48 h after transfection. Viral particles were concentrated using a Eenti-X Concentrator (Takara Bio, Inc.).Lentiviral Transduction

[0307] Mouse T cells or TIEs were activated by adding CD3 / CD28 Dynabeads (Thermo Fisher Scientific) according to the manufacturer’s instructions and then transduced with lentiviral supernatants. Eentivirus was then added to RetroNectin (Takara)-coated non-tissue culture-treated plates to reach a total volume of 400 pF and mixed gently. Cells and virus were centrifuged at 1000 g for 2 hours and then incubated at 37 °C. After 3 days of culture, the medium was changed to complete RPMI-1640 medium (10% FBS, 2 mM GlutaMAX, 100 pM P-mercaptoethanol, 1% penicillin / streptomycin, 50 U / mE rmIE-2).

[0308] Human T cells or TIEs were Human T cells were stimulated by CD3 / CD28 Dynabeads (Thermo Fisher Scientific) according to the manufacturer’s instructions. On day 2, cells were plated in non-tissue culture-coated 24-well plates, and polybrene (8 pg / mL) was added to the medium. Lentiviral supernatant was first centrifuged at 1500 g for 2 hours on RetroNectin (Takara)-coated non-tissue culture-treated plates. Cells were then plated and centrifuged at 1000 g for 20 min and incubated at 37°C. After 3 days, the medium was changed to 45% RPMI-1640% and 45% Click’s medium containing 10% FBS and supplemented with rhIL-2 (50 U / mL), rhIL-7 (10 ng / mL), and rhIL-15 (5 ng / mL).Immunoblotting

[0309] Frozen tissue samples were smashed before being homogenized using a minibead beater with 5 to 8 silicone beads (BioSpec Products) in 0.4 mL of ice-cold radioimmunoprecipitation assay lysis buffer. The homogenized tumor cells were then subjected to lysis with this buffer. The protein extracts were separated from the tissue residues by centrifugation at the maximum speed for 20 minutes at 4 °C. Forty-microgram samples of total protein were fractionated by 10% sodium dodecyl sulfate-poly acrylamide gel electrophoresisand transferred to nitrocellulose membranes using a Trans-Blot Turbo transfer system (BioRad). The membranes were blotted with different primary and secondary antibodies to detect the proteins of interest.Immunohistochemistry and Immunofluorescence Staining

[0310] Frozen tumor sections were sequentially fixed with cold acetone, acetone plus chloroform (1:1), and acetone. Paraffin-embedded sections were deparaffinized and heated in antigen retrieval buffer. Tissue sections were blocked with 3% H2O2 in distilled water for 20 minutes and then in blocking buffer (5% normal horse serum and 1% normal goat serum in PBS). Slides were incubated with primary antibodies overnight at 4 °C and secondary antibodies for 1 h at room temperature. For immunohistochemistry staining, the secondary antibody was biotin conjugated, the sections were treated with ABC reagent (Vector Labs), and the nuclei were counterstained with hematoxylin (Sigma- Aldrich). Tumor sections were mounted with Cytoseal mounting medium (Life Technologies). Quantifications of immunohistochemistry images were assessed by examining 3 randomly selected low-power fields per slide. For immunofluorescence staining, tumor sections were mounted in an antifade fluorescence mounting medium with 4',6-diamidino-2-phenylindole. Slides were visualized under a Nikon Eclipse Ti fluorescence microscope.Enzyme-Linked Immunosorbent Assay (ELISA)

[0311] Culture medium / supematant was obtained (e.g., tumor lysates from tumor-bearing mice that had undergone the indicated treatments, or supernatant from the indicated coculture systems). The levels of IFNy, TGFp, Collagen, and / or fibronectin were measured by using ELISA Ready-SET-Go! kits (eBioscience) or ELISA Kit Picokine (bosterbio).Flow Cytometry

[0312] Cells were sequentially incubated with primary and secondary antibodies for 30 minutes each at 4 °C. Stained cells were analyzed using an Attune acoustic focusing cytometer (Applied Biosystems) or a BD LSR-Fortessa cell analyzer (BD Biosciences). Flow cytometry data were analyzed using the Flow Jo software program (Flow Jo, LLC).Tumor-cell Dissociation

[0313] Tumors were minced into 2-mm fragments, placed in 5 mL of dissociation buffer (RPML1640 medium with 100 U / mL collagenase type IV and 100 U / mL DNase I), and incubated at 37 °C while shaking at 120 rpm for 30 minutes to 1 h. The released cells were filtered with 70- pm strainers and centrifuged at 600 g for 5 minutes, followed by red blood cell lysis. Cells were then resuspended in fluorescence-activated cell sorting solution containing2% FBS. Single-tumor-cell suspensions were obtained after CD45 depletion using an Easy Sep Human CD45 Depletion Kit (Stem Cell Technologies).Statistical analysis

[0314] The directly measured outcomes were analyzed using a 2-sided Student t-test to compare 2 treatment groups or 1-way analysis of variance to compare more than 2 treatment groups. The statistical analyses were conducted using GraphPad Prism 8 software. All data values represent replicates and are shown as mean ± SEM. Significance was defined as *P<0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.Example 2 - attIL12TILs Displayed Superior Tumor Infiltration Relative to Control- TILs Despite Similar Profiles of Cell Surface Markers

[0315] The inventor’s attIL12 engineering of patient derived T cells has been shown to dramatically enhance T cell infiltration into and efficacy against many OS PDX tumor models. However, in certain of those models, particularly those with high levels of cancer associated fibroblasts (CAF) and / or high levels of extracellular matrix (ECM), the models failed to respond to attIL12-T cell treatment. As disclosed herein, the inventors have discovered that in these resistant models, the TGFP dominant tumor microenvironment promoted ECM development and inhibited IFNy function. Therefore, the IFNy production as the result of attIL12-T cell contact with CSV+tumor cells and / or CAFs was insufficient to clear the ECM barrier in the tumor stroma and facilitate T cell infiltration.

[0316] To overcome the ECM rich solid tumors and overcome the TGFP dominant immune suppressive tumor microenvironment, the inventors sought to discover an approach which, besides the attIL12-T and CSV+ pathway, could stimulate an additional pathway to trigger IFNy elevation, and biomarkers that could help differentiate treatment options to overcome these resistances. Sarcoma tumors lack universal cell surface markers, as such, the inventors utilized the CSV+PDX tumor SA127 and autologous TILs to answer this question and potentially identify biomarkers that could influence treatment regimens (e.g., would the TILs endogenous TCRs / anti-cancer receptors prove sufficient to overcome the TGFP dominant immune suppressive tumor microenvironment).

[0317] The inventors found that TILs obtained from SA127 PDX tumors (“SA127TILs”) were mostly CD8+T cells. These TILs could be transformed with attIL12 (referenced as “attIL12TIL”, “attIL12-TIL”, or “SA127attIL12TILs”), and these engineered attIL12TILs expressed high levels of IL12 on the cell surface (FIG. 1A). When comparing the costimulatory and exhaustion markers on control-TILs (SA127TIL, or lentiviral transducedcontrol SA127TIL-ctrl) and attIL12-TILs, the inventors found that attIL12 transduction did not remarkably change the expression profiles of TILs when analyzed in vitro (FIG. 1A), for example with CD3+ cell populations expressing similar levels of Lag3+, CD69+, NKG2D+, CTLA4+, PD1+, and CD39+. To assess the effector function of the TILs, the inventors also measured levels of cytokine production (e.g., IFNy and Granzyme B) from the TILs and found that both SA127TIL-ctrl and SA127attIL12-TILs expressed these cytokine molecules at similar levels (FIG. IB). These results showed that engineering TILs with attIL12 did not significantly alter the TIL characteristics in vitro.

[0318] The inventors then sought to determine TIL effector function in vivo, for example, could SA127TIL-ctrl and / or attIL12-TILs successfully re-infiltrate into the ECM rich autologous sarcoma tumor models? As noted above, penetration of immunological agents, such as effector cells, into solid tumors, particularly ECM rich tumors, has proven to be a significant challenge for the field. SA127 tumors were transplanted subcutaneously to SCID mice and control- or attIL12- autologous TILs were then infused into the mice intravenously when the PDX SA127 tumors reach 6 mm. A second TIL infusion was performed 14 days after the first infusion. Four days after the second TIL cell treatment, the tumors were harvested to detect human CD3+ cells in the tumor sections. When examining SA 127 PDX tumor models that received ctrl-TIL cell treatment, very few CD3+ cells were found to have infiltrated into the tumors. However, engineering of TILs with attIL12 resulted in significant accumulation of CD3+ T cells in the tumors (FIG. 1C). These results demonstrated that control TILs partially lost their tumor penetration ability due to rich ECM in the stroma, whereas attIL12-TILs maintained the ability to infiltrate the tumor in the autologous tumor model. The cell surface marker profile for these tumor infiltrated TILs was analyzed, interestingly, ctrl-TIL treated tumors were found to contain more CD4+T cells than CD8+T cells, whereas attIL12-TIL treated tumors were found to contain majority CD8+T cells (FIG. ID). The ctrl-TIL and attIL12-TILs showed very similar T cell marker profiles, except for a slight decrease of CTLA4 on the attIL12TILs (FIG. ID). These results showed that engineering of TILs with attIL12 resulted in increased levels of successful TIL entrance into autologous tumors.Example 3 - Engineered attIL12TIL Boosted Antitumor Efficacy When Treating Autologous Tumors

[0319] In solid tumor treatment models, high levels of immune effector cell (e.g., T cell) infiltration into a tumor serves as a predictive indicator for successful responses, a fact-pattern that was recapitulated in the immediate experiments comprising engineered attIL12TIL therapyfor autologous SA127 tumor clearance. After two infusions of attIL12-TILs, SA127 tumors regressed, and surprisingly, 2 out of 9 treated mice were found to have complete tumor clearance (FIG. 2A). Significantly, no long-term adverse effects were observed in the tumor free survivors (FIG. 2B). However, the control-TILs only temporarily inhibited tumor progression (Fig. 2A-2B). Despite strong effector molecule production before the infusions (FIG. IB), the control-TILs showed low efficacy against tumor cells in vivo in contrast to the high IFNy and / or Granzyme B producing attIL12-TILs in the tumors in vivo (FIG. 2C). Together, these results suggested that the tumor infiltrated attIL12-TILs exhibited high levels of antitumor efficacy against the autologous tumor cells, and could eradicate the autologous tumors.Example 4 - High Collagen Deposition in Human Sarcoma Tissues was Associated With Poor T Cell Scores and Reduced Patient Survival

[0320] The inventors found that in ECM rich OS PDX models, the ability of attIL12-T cells to successfully destroy CAFs / ECM structures led to enhanced T cell infiltration. This discovery prompted the inventors to determine whether ECM would also be heavily deposited in soft tissue sarcoma tumors, and whether the destruction of CAFs / ECM structures would lead to enhanced T cell and / or TIL infiltration into said tumors. To analyze the levels of ECM in soft tissue sarcomas, a liposarcoma tissue microarray containing 5 normal adipose tissues, 2 stage I tissues (mixed type liposarcoma IB), 4 stage II tissues (dedifferentiated liposarcoma II), and 7 stage III malignant liposarcoma clinical samples (5 dedifferentiated liposarcoma IIIA and 2 dedifferentiated liposarcoma IIIB) were examined. Sirius staining showed very low levels of collagen in the normal and stage I tumor tissues, and much higher densities of collagen deposition in stage III liposarcoma tissues (FIG. 3A). Such high collagen density in advanced stage liposarcoma was significantly associated with poor survival, as determined by a survival analysis from a soft tissue sarcoma dataset from The Cancer Genome Atlas (TCGA) (FIG. 3B). The top 50% of patients expressing the highest Colla2 and Fnl levels were compared with the 50% of patients expressing the lowest levels of Colla2 and Fnl. Log-rank (Mantel- Cox) test showed that patients with high levels of these ECM factors had worse probabilities of survival over time when compared to patients with low ECM expression profiles (P = 0.03) (FIG. 3B). The median survival time of the ECM high patients was 54 months whereas the ECM low patients was 89 months. The inventors further analyzed the mRNA (z-scores) of Cd8b and ECM factors Col2al and Fnl) from this dataset, and demonstrated that these markers negatively correlated (P < 0.0001, Pearson r: -0.2701), suggesting that effector T cellswere excluded from the tumors with rich ECM (FIG. 3C). To determine if the ECM density also played as apparently critical of a role in SA 127 PDX tumors as it did in the clinical samples, the inventors assessed the collagen density of SA127 PDX tumors after control-TIL or attIL12-TIL treatment. Sirius red staining was utilized to detect collagen deposition in the SA127 PDX tumor models, and the attIL12-TILs, but not the control-TILs, were found to remarkably reduce collagen expression in the autologous tumors (FIG. 3D). These results showed that attIL12-TILs were able to penetrate into autologous tumors and reduce collagen deposition levels.Example 5 - Cytokine Balance Shift Regulated Collagen Production by Sarcoma Cells

[0321] The inventors determined that failure of attIL12-T cell therapy for some ECM rich OS PDX tumors could be due to high basal levels of TGFP in the tumor micro environment (TME). These high levels of TGFP in the TME could promote dense ECM deposition that may impede attIL12-T cell infiltration into the tumor, and the resulting IFNy elevation observed following attIL12 activation upon CSV stimulation could prove insufficient to overcome the effects of TGFp. If attIL12-TILs inhibited collagen produced by sarcoma cells, the inventors determined that it would likely be regulated by the cytokine network around the tumor cells. TCGA analysis of the sarcoma dataset showed that the Tgffll mRNA (z-scores) expression was positively correlated to Collal gene expression (P <0.0001, Pearson r: 0.3991), whereas the Ifiiy gene expression was negatively correlated to Collal and Col2al gene expression (P = 0.0388, Pearson r: -0.2091) (FIG. 4A). These results from clinical samples were consistent with the inventors discoveries in OS PDX models. To test the above hypothesis, the inventors processed SA 127 PDX tumors which were untreated, or treated with control-TIL or attIL12- TILs. The data showed that TGFP levels were high in untreated SA127 tumors, slighted decreased in control-TIL treated tumors, and significantly reduced by attIL12-TIL treatment. In striking contrast, levels of IFNy in these tumors were completely the opposite of TGFP levels, with a dramatic elevation in IFNy levels observed in the attIL12-TIL treated tumors (FIG. 4B). These data clearly showed that unlike the attIL12-T cells, attIL12-TILs successfully reshaped the cytokine environment in ECM rich and T-cell resistant SA127 tumors. To validate the role of the TGFP to IFNy balance shift in collagen expression, the inventors set up an in vitro model by coculturing the SA 127 tumor cells with the autologous control- or attIL12-TILs at an Effector : Target (E:T) ratio of 1:4 (1 TIL : 4 tumor cells) for 24 hours. The coculture supernatant was collected for ELISA analysis, results of which showed the TGFP dominant toIFNy dominant cytokine balance shift after attIL12-TIL coculture (FIG. 4C), results which aligned well with the observed in vivo results. The tumor cells from these coculture experiments were harvested to determine collagen production via flow cytometry. The results confirmed that control-TILs could partially reduce collagen levels, and that attIL12-TILs were sufficient to significantly reduce collagen production, and in these conditions, completely inhibit collagen positive tumor cells (FIG. 4D). Collectively, the results showed that attIL12-TILs stimulated significant increases in IFNy levels and significantly restrained TGFP levels, leading to significantly decreased collagen expression by tumor cells.Example 6 - Dual Signaling Activation was Necessary for the Enhanced Potency of attIL12-TILs

[0322] AttIL12-TILs can be distinguished from attIL12-T cells or TILs, as attIL12-TILs comprise dual signaling activity including both the engineered attIL12 binding via the CSV targeting moiety and the endogenous TIL recognizing autologous tumor cell neoantigens (e.g., via endogenous TCRs), both of which can be activated simultaneously. However, whether the dual signaling can contribute to attIL12-TIL induced cytokine balance changes and / or collagen reduction had not yet been clarified.

[0323] To investigate engineered TIL signaling, the inventors generated the appropriate control conditions. Lentivirus containing a TCRafi KO gene construct using the Crispr / Cas9 technology were generated, with a gRNA targeting TCRa (TCTCTCAGCTGGTACACGGC; SEQ ID NO: 8). AttILI 2TCRa^ / _TILs were generated and exhibited IL- 12 expression on the cell surface, and displayed little-to-no TCRaP expression (FIG. 5A). To control for attIL12 CSV mediated signaling, the inventors utilized the 84-1 antibody, which can block CSV signaling (see e.g., Jiemiao Hu et al., “attIL12-T cell therapy destructs cancer-associated fibroblasts and extracellular matrix in heterogenous osteosarcoma xenograft models”, 2023, inpress). In both SA127 (sarcoma) and SAI 17 (osteosarcoma) models, autologous attIL12-TIL coculture dramatically stimulated IFNy and granzyme B production by the TIL cells (FIG. 5B). However, by blocking CSV binding (e.g., with 10 pg / mL antibody 84-1) or knocking out TCRafi (e.g., with SEQ ID NO: 8), the expression of IFNy and granzyme B production by the TIL cells was greatly diminished (FIG. 5B), when both CSV binding and TCRafi signaling mediated pathways were inhibited, the expression of effector cytokines was even further diminished (FIG. 5B). These results suggested that both CSV-attIL12 and tumor antigen-TIL activation were required to trigger the observed high levels of inflammatory cytokine secretion. As a result, of the conditions tested (e.g., ctrlTIL; attIL12-TIL; attIL12-TIL +anti-CSV;attILI 2-TILI CR_ / _; and attILl 2-TILl ( R_ / _+anti-CSV), only coculture of attIL12-TIL with the tumor cells (e.g., SA127 or SAI 17) resulted in high levels of inhibition of tumor cell collagen production. If either or both of these signaling pathways were removed, the immune effector cells failed to reduce tumor cell collagen levels (FIG. 5C).

[0324] As TGFP was highly expressed by these tumor cells (FIG. 4C), and TGFP is a potent inducer of collagen, the inventors sought to determine whether the collagen level changes were associated with TGFP release. The coculture supernatant was collected from tumor cells cocultured with ctrl-TILs, attIL12-TILs, or attIL12-TILs with one or both of the signaling pathways blocked (e.g., attIL12-TIL +anti-CSV; attIL l 2-TIL, C R / “; and attIL12- TILTCR_ / ' +anti-CSV). The results showed that TGFP was mostly suppressed after attIL12-TIL coculture, but that these levels recovered after blocking CSV and / or impairing TCR activation (FIG. 5D). In contrast, IFNy was markedly elevated in the attIL12-TIL coculture conditions, but was significantly suppressed when either or both of the CSV and TCR mediated signaling pathways were inhibited (FIG. 5D). Levels of the TGFP downstream signaling pathway proteins beta non-erythrocytic 2 (SPTBN2; “SPP2” or “SPb2”) and phosphorylated mothers against decapentaplegic homolog 3 (pSMAD3) were measured with immunoblotting. Levels of TGFP and SPb2 were found to be in line with collagen expression levels (reduced) in the tumor cells following coculture with attIL12-TILs, but that this reduction was suppressed when CSV and / or TCR mediated signaling pathways were inhibited (FIG. 5E). These data further demonstrated that the dual signaling activation of attIL12-TIL and tumor cell interaction reshaped the TGFP to IFNy cytokine balance shift, which in turn inhibited collagen expression from the tumor cells.

[0325] In some ECM rich PDX tumor cell models, attIL12-T cell therapy failed to penetrate into the tumor (see e.g., Jiemiao Hu et al., “attIL12-T cell therapy destructs cancer- associated fibroblasts and extracellular matrix in heterogenous osteosarcoma xenograft models”, 2023, in-press), a finding that suggested treating some ECM rich PDX tumors with effector cells comprising attIL12-CSV activation alone was insufficient to overcome TGFP high immunosuppressive tumor environments. When attIL12-TILs encounter an autologous tumor cell, this triggered additional signaling pathway s / activation (e.g., through endogenous TCRs against neoantigens), a trait which lead to dramatic TGFP reduction and collagen downregulation. These results suggested that in certain conditions, when utilized alone, attIL12-TIL transfer could achieve better antitumor efficacy against ECM rich models when compared to other immune effector cell therapies, such as attIL12-T cell therapies utilized alone. To test this, the inventors treated SA127 tumor bearing mice with control TILs(“ctrlTIL”), attIL12-T cells, or attIL12-TILs. Ctrl-TILs and attIL12-T cells which were stimulated by single signaling pathway (e.g., tumor antigens or CSV signaling, respectively), resulted in delayed tumor development when compared to no treatment controls (FIG. 5F). Significantly, attIL12-TILs that were activated by both CSV and tumor antigens showed increased inhibition of the autologous tumor growth when compared to controls (FIG. 5F). This potent tumor inhibition was impaired by TCRaP knockout, suggesting that the tumor antigen induced activation and the second signaling stimulation (e.g., CSV-attIL12) was necessary to treat these ECM rich solid tumors (FIG. 5G). These tumors were dissociated following sacrifice of the mice, and changes of both the tumor infiltrated TILs and tumor cells were assessed. The results showed that control TILs failed to inhibit TGFP production from the tumor, significantly increased TGFP levels in T cells, and significantly lost effector function (e.g., low levels of IFNy), in sharp contrast to the significantly higher levels of IFNy produced by almost all the attIL12-TILs and reduced TGFP levels in both T cells and non-T cells (FIG. 5H). Ablation of TCRaP in attIL12-TILs impaired the cytokine release from these T cells. The tumor cells also released high TGFP after control-TIL treatment (FIG. 5H), leading to high expression of collagen (FIG. 51), but attIL12-TILs were able to completely suppress TGFP production and collagen expression, which was abrogated with ablation of TCRaP (FIGs. 5H and 51). Intriguingly, as observed in FIG. 5J, T cells (shown in red) displayed high levels of infiltration into tumor core regions after attIL12-TIL treatment, a finding in contrast to the observed T cell exclusion in from the tumor micro environment (TME) filled with high density of collagen (shown in green) after control TIL treatment (FIG. 5J). Consistent with the previously observed results, TCRaP ablation impaired attIL12-TIL mediated collagen reduction and T cell infiltration, which further compromised the antitumor efficacy (FIG. 5J). These in vivo data were in line with the in vitro discoveries discussed above. Together, these data showed that the dual signaling activation of attIL12-TILs promoted a IFNy dominant TME, resulting in a suppression of collagen expression in the tumor, and effector cells being rendered capable of overcoming ECM rich solid tumors.Example 7 - AttIL12-TILs Induced CCKAR Downregulation and Abolished its Enhancer Role in Collagen Regulation

[0326] In the inventors studies directed to attIL12-T cell therapy for treating OS PDX tumors, the transcriptome profiles of models were analyzed, three OS PDX tumors that were sensitive / responsive to the treatment where compared to the transcriptomes of another three OS PDX tumors that were resistant to attIL12-T cell therapy. Using the bulk RNA sequencingresults, the inventors identified the ECM-receptor interaction pathway to be a significantly differentiated pathway when compared between these two groups (sensitive vs resistant). The data showed that enriched gene sets were mainly associated with ECM composition and functions, and were downregulated in responsive tumors, and the most significantly downregulated gene was identified to be Cckar (FIG. 6A). Cckar encodes a G-protein coupled receptor binding to CCK (cholecystokinin) family protein (CCKAR). The role of CCKAR in collagen expression, such as its function in TGFP mediated collagen expression, had not previously been identified / determined.

[0327] Given that attIL12-TIL induced collagen reduction was found to be driven by changes in the TME cytokine balance (e.g., IFNy vs TGFP), the inventors cocultured tumor cells (e.g., SA127 or SAI 17) with autologous control TIL or attIL12-TILs in the presence or absence of exogenously added IFNy, TGFP, or blocking antibodies directed to IFNy or TGFP signaling pathways (FIG. 6B). These studies were designed to elucidate the role of CCKAR in collagen regulation. Consistent with the results reported above, attIL12-TIL coculture with SA127 or SAI 17 dramatically decreased collagen expression in these tumor cells when compared to coculturing with the control-TIL cells. Additionally, exogenously added IFNy or the blocking of TGFP promoted collagen reduction (FIG. 6B), whereas additional TGFP or blocking of IFNy promoted collagen expression (FIG. 6B). The CCKAR levels showed a similar trend to that of the collagen levels, suggesting that CCKAR may be a crucial regulator of collagen creation expression. The results also showed that pSMAD3 and SPP2 levels were in line with and confirmatory of TGFP downstream signaling (FIG. 6B). It has been reported that TGFP stimulates pSMAD3-pAKT signaling to induce collagen. To further decipher how CCKAR regulated collagen expression, the inventors added a CCKAR blocking antibody and / or additional CCK ligand to the SA 127 or SAI 17 coculture with control-TIL or attIL12- TILs (FIGs. 6C and 6D). CCKAR blockade in the control-TIL coculture (middle rows) was sufficient to cause collagen reduction and pAKT downregulation (FIG. 6C). Recombinant CCK in attIL12-TIL coculture stimulated CCKAR mediated pAKT and collagen expression (FIG. 6D). These results suggested that CCKAR activation was triggered by TGFP to enhance collagen production through pAKT activation. This conclusion was further supported by the results observed when CCKAR was overexpressed in transgenic tumor cells (SA127CCKARor SA117CCKAR) cocultured with control-TIL or attIL12-TILs (FIG. 6E). CCKAR high expression in the tumor cells amplified pAKT induced collagen expression even with attIL12- TIL coculture, suggesting that under low levels of TGFP and pSMAD3 activation, redundant CCKAR functioned as a robust inducer of pAKT signaling and collagen expression in sarcomatumor cells. pSMAD3 serves as an intracellular mediator of TGFp. The inventors noticed that CCKAR appeared to have minimal effects on pSMAD3 levels, but whether pSMAD3 regulated CCKAR was unclear. To decipher this relationship, the inventors utilized a pSMAD3 inhibitor, specific inhibitor of Smad3 (SIS3), during coculture of SA127 or SAI 17 cells with control- TIL or attIL12-TILs (FIG. 6F). Inhibition of pSMAD3 activation dramatically decreased CCKAR and downstream pAKT-collagen expression when compared to trials utilizing vehicle control, showing that CCKAR was regulated by pSMAD3 signaling (FIG. 6F). As shown in FIG. 7, when control T cells or attIL12-T cells were cocultured with UM-chor-l:Brachyury cells, attIL12-T cells induced a reduction in collagen, integrin bl & b5, CCKAR, SPb2, pSMAD3, and pAKT levels relative to controls. Shown in FIGs. 8A-8B are results from experiments similar to those provided in FIG. 6B, but wherein the TILs were derived from SAI 17 tumors, and obtained and expanded for two weeks in the absence of 41BB targeted antibody. The effector cells were then co-cocultured with SAI 17 cells at an E:T ratio of 1:4 for 24 hours, followed by protein extraction. The results show that attIL12-TILs obtained / expanded with multiple different protocols can reduce CCKAR, TGFp, Collagen, and / or pSMAD3 levels relative to controls.

[0328] Collectively, these results demonstrated that tumor microenvironments were TGFP dominant after tumor cell coculture with control-TILs, that pSMAD3 was activated to induce CCKAR, and that CCKAR in turn enhanced the pAKT signaling pathway promoting expression of collagen. In contrast, when coculturing tumor cells with attIL12-TILs, the tumor cell-effector cell interaction reshaped the tumor microenvironment to become IFNy dominant, which in turn resulted in deactivation of pSMAD3 and CCKAR, and reduced expression of collagen in the tumors.* * *

[0329] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.REFERENCESThe following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.Austin-Ward and Villaseca, Revista Medica de Chile, 126(7):838-845, 1998.Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994.Besser et al. Clin Cancer Res. 16(9):2646-55, 2010.Bukowski et al., Clinical Cancer Res., 4(10):2337-2347, 1998.Camacho et al. J Clin Oncology 22(145): Abstract No. 2505, 2004.Carter et al. Cancer J. 14(3): 154-69, 2008.Christodoulides et al., Microbiology, 144(Pt l l):3027-3037, 1998.Czerkinsky et al., J. Immunol. Methods 1988;110:29-36.Davidson et al., J. Immunother., 21(5):389-398, 1998.Dudley et al. Clinical Oncology. 23(10):2346-57, 2005.European Patent Publication No. EP957359Hanibuchi et al., Int. J. Cancer, 78(4):480-485, 1998.Hellstrand et al., Acta Oncologica, 37(4):347-353, 1998.Hollander, Front. Immun., 3:3, 2012.Hui and Hashimoto, Infection Immun., 66(l l):5329-5336, 1998.Hurwitz et al. Proc Natl Acad Sci USA 95(17): 10067-10071, 1998.International Patent Publication No. WO2014005566International Patent Publication No. WO1995001994International Patent Publication No. WO1998042752International Patent Publication No. W02000037504International Patent Publication No. WO2001014424International Patent Publication No. WO2014055668International Patent Publication No. WO2014031687International Patent Publication No. W02015016718Kozma et al., Nucleic Acids Research 41 Database Issue, D524-D529, 2013.Kyte and Doolittle, J. Mol. Biol. 157:105-132, 1982.Leal et al., Ann N Y Acad Sci. 2014;1321:41-54, 2014.Li Nat Biotechnol. 23:349-354, 2005.Mokyr et al. Cancer Res 58:5301-5304, 1998.Olsson et al. J. Clin. Invest. 1990;86:981-985.Pardoll Nat Rev Cancer, 12(4): 252-64, 2012.Qin et al., Proc. Natl. Acad. Sci. USA, 95(24): 14411-14416, 1998.Radvanyi et al. Clin Cancer Res. 18(24):6758-70, 2012.Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rded., Cold Spring HarborPress, Cold Spring Harbor, N.Y. 2001.Teicher BA. Current cancer drug targets. 9(8):982-1004, 2009.Teicher BA. Current opinion in oncology. 26(5):476-83, 2014.Terakura et al. BloodAYl - 82, 2012.U.S. Patent No. 4,870,287U.S. Patent No. 4,897,355U.S. Patent No. 4,946,787U.S. Patent No. 5,049,386U.S. Patent No. 5,739,169U.S. Patent No. 5,760,395U.S. Patent No. 5,801,005U.S. Patent No. 5,824,311U.S. Patent No. 5,830,880U.S. Patent No. 5,844,905U.S. Patent No. 5,846,945U.S. Patent No. 5,885,796U.S. Patent No. 5,939,281U.S. Patent No. 6,207,156U.S. Patent No. 6,218,132U.S. Patent No. 6,225,042U.S. Patent No. 6,355,479U.S. Patent No. 6,362,001U.S. Patent No. 6,410,319U.S. Patent No. 6,451,995U.S. Patent No. 6,790,662U.S. Patent No. 7,070,995U.S. Patent No. 7,265,209U.S. Patent No. 7,354,762U.S. Patent No. 7,446,179U.S. Patent No. 7,446,190U.S. Patent No. 7,446,191U.S. Patent No. 8,008,449U.S. Patent No. 8,017,114U.S. Patent No. 8,119,129U.S. Patent No. 8,252,592U.S. Patent No. 8,324,353U.S. Patent No. 8,329,867U.S. Patent No. 8,339,645U.S. Patent No. 8,354,509U.S. Patent No. 8,398,282U.S. Patent No. 8,479,118U.S. Patent No. 8,735,553U.S. Patent Publication No. 20050260186U.S. Patent Publication No. 20060104968U.S. Patent Publication No. 20110008369U.S. Patent Publication No. 20140022021U.S. Patent Publication No. 20140294898Wang et al. J Immunother. 35(9):689-701, 2012.

Claims

WHAT IS CLAIMED IS:

1. A method of treating immune effector cell resistant cancer in a subject with a tumor comprising, reducing and / or diminishing Cholecystokinin A receptor (CCKAR) protein and / or transcript expression in the tumor by contacting the tumor with tumor infiltrating lymphocytes (TILs) transgenically expressing a cell surface vimentin (CSV)-targeted and membrane- anchored cytokine.

2. The method of claim 1, wherein the TILs are autologous, allogenic or syngeneic.

3. A method of treating cancer in a subject with a tumor comprising, obtaining TILs from the subject, engineering the TILs to transgenically express a CSV-targeted and membrane- anchored cytokine, and administering the engineered TILs to the subject.

4. A method of treating cancer in a subject with a tumor comprising, i) determining levels of CCKAR, Sarcolipin (SLN), LARGE xylosyl- and glucuronyltransferase 2 (LARGE2), and / or Wnt family member 10A (WNT10A) expression in the tumor, and optionally determining the levels of izumo sperm-oocyte fusion 1 (IZUM01); ii) obtaining TILs from the subject; iii) engineering the TILs to transgenically express a CSV-targeted and membrane- anchored cytokine; and iv) if CCKAR, SLN, LARGE2, and / or WNT10A expression is greater than or equal to about 0.0001 Fragments Per Kilobase of transcript per Million mapped reads (FPKM), and / or optionally if the expression of IZUMO 1 is less than or equal to about 0.0001 FPKM, administering to the subject the engineered TIEs.

5. A method of treating cancer in a subject with a tumor comprising, i) determining levels of CCKAR, SEN, LARGE2, and / or WNT10A expression in the tumor, and optionally determining the levels of IZUMO 1 expression in the tumor; ii) obtaining T cells and / or TILs from the subject;iii) engineering the T cells and / or TILs to transgenically express a CSV-targeted and membrane- anchored cytokine; and iv) if the CCKAR, SLN, LARGE2, and / or WNT10A expression is greater than or equal to about 0.0001 FPKM, and / or optionally the expression of IZUM01 is less than or equal to about 0.0001 FPKM, administering to the subject the engineered T cells and / or TIFs and one or more inhibitors of CCKAR and / or CCKAR regulated pathways.

6. The method of claim 5, wherein step (ii) comprises obtaining TIFs from the subject.

7. The method of claim 4 or 5, wherein the expression of CCKAR in the tumor is greater than or equal to about 0.0001 FPKM.

8. The method of claim 4 or 5, wherein the expression of at least two of CCKAR, SFN, FARGE2, and WNT10A in the tumor are greater than or equal to about 0.0001 FPKM.

9. The method of claim 8, wherein the expression of at least three of CCKAR, SEN, EARGE2, and WNT10A in the tumor are greater than or equal to about 0.0001 FPKM.

10. The method of claim 9, wherein the expression of CCKAR, SEN, EARGE2, and WNT10A in the tumor are greater than or equal to about 0.0001 FPKM.

11. The method of claim 4 or 5 further comprising determining expression levels of IZUM01 in the tumor, and confirming expression of IZUM01 is lower than 0.0001 FPKM.

12. The method of claim 5, wherein the CCKAR and / or CCKAR regulated pathway inhibitor comprises an inhibitor of CCKAR, phosphorylated AKT serine / threonine kinase 1 (pAKT), phosphorylated SMAD family member 3 (pSMAD3), and / or spectrin beta non- erythrocytic 2 (SPTBN2).

13. The method of claim 12, wherein the inhibitor of CCKAR is an anti-CCKAR antibody.

14. The method of any one of claims 1-5, wherein the tumor is characterized by higher collagen, higher extracellular matrix (ECM), and / or higher levels of transforming growth factor beta (TGFP) relative to a comparable early stage tumor and / or to corresponding healthy tissue.

15. The method of claim 14, wherein the cancer is resistant to tumor- targeted immune effector cell therapy.

16. The method of claim 15, wherein the cancer is resistant to Chimeric Antigen Receptor (CAR) T cell therapy (CAR-T), T Cell Receptor (TCR) T cell therapy (TCR-T), TIL therapy, B cell therapy, and / or NK cell therapy.

17. The method of any one of claims 1-5, wherein the anti-tumor efficacy of endogenous immune cells and / or additional heterogeneous immune effector cell treatments are improved.

18. The method of any one of claims 1-5, wherein reduction of CCKAR results in downregulation of collagen, ECM, and / or TGFP in the tumor.

19. The method of any one of claims 1-5, wherein reduction of CCKAR inhibits one or more of pAKT, TGFP, and / or pAKT or TGFP signaling pathway transducers.

20. The method of any one of claims 1-5, wherein the reduction of CCKAR inhibits pAKT and / or TGFP signal transducers rat sarcoma virus (RAS), pSMAD3, and / or SPTBN2.

21. The method of any one of claims 1-5, wherein the TILs comprise TILs obtained from the tumor and / or blood circulating TILs.

22. The method of claim 21, wherein the blood circulating TILs are obtained by a method comprising leukapheresis.

23. The method of claim 21, wherein the TILs are obtained from the tumor by a method comprising mechanical or enzymatic disruption of the tumor.

24. The method of any one of claims 1-5, wherein the method comprises ex-vivo expanding the TILs before, after, and / or during the genetic engineering.

25. The method of any one of claims 1-5, wherein the method comprises administering to the subject the engineered TILs and / or engineered T cells.

26. The method of any one of claims 1-5, wherein the TILs and / or T-cells are engineered to express the CSV-targeted and membrane- anchored cytokine by viral transduction.

27. The method of any one of claims 1-5, wherein the membrane- anchored cytokine comprises interleukin 12 (IL- 12).

28. The method of any one of claims 1-5, wherein the tumor comprises cells expressing cell-surface vimentin (CSV).

29. The method of any one of claims 1-5, wherein greater than about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of cells in the tumor express CSV.

30. The method of claim 29, wherein greater than about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of tumor cells in the tumor express CSV.

31. The method of any one of claims 1-5, wherein the CSV-targeted moiety of the CSV targeted and membrane-anchored cytokine specifically binds CSV and facilitates increased levels of interferon gamma (IFNy) and / or tumor necrosis factor alpha (TNFa) in the tumor.

32. The method of claim 31, wherein the CSV-targeted moiety of the CSV targeted and membrane- anchored cytokine comprises a CSV binding peptide.

33. The method of claims 31, wherein the CSV-targeted and membrane- anchored cytokine comprises attIL12.

34. The method of claim 33, wherein the attIL12 comprises an amino acid sequence at least 90% identical to any one or more of SEQ ID NOs: 4-7.

35. The method of claim 25, wherein following engineered TILs administration to the subject, relative to administration of non-engineered TILs, non-engineered T cells, and / or engineered T cells, levels of TNFa and / or IFNy in the tumor are enriched, and / or levels of TGFP in the tumor are reduced.

36. The method of claim 25, wherein following engineered TILs administration to the subject, relative to administration of non-engineered TILs, non-engineered T cells, and / or engineered T cells, levels of CCKAR in the tumor are significantly reduced.

37. The method of any one of claims 1-5, comprising contacting tumor cells and / or cancer associated fibroblasts (CAFs) with the engineered TILs.

38. The method of any one of claims 1-5, comprising simultaneously reducing levels of collagen and ECM in the tumor.

39. The method of any one of claims 1-5, comprising simultaneously reducing levels of collagen, ECM, CCKAR, and TGFP in the tumor.

40. The method of any one of claims 1-5, wherein the tumor comprises tumor stroma and the method comprises breaking the tumor stroma.

41. The method of claim 40, comprising reducing levels of collagen and / or ECM in the tumor stroma.

42. The method of claim 41, comprising simultaneously reducing levels of collagen and ECM in the tumor stroma.

43. The method of claim 42, comprising simultaneously reducing levels of collagen, ECM, CCKAR, and TGFP in the tumor stroma.

44. The method of any one of claims 1-5, wherein the tumor is characterized as stroma rich relative to a comparable early stage tumor and / or to corresponding healthy tissue.

45. The method of any one of claims 1-5, wherein the cancer is recurrent.

46. The method of any one of claims 1-5, wherein the tumor comprises a solid tumor.

47. The method of any one of claims 1-5, wherein the tumor is characterized as collagen rich relative to a comparable early stage tumor and / or to corresponding healthy tissue.

48. The method of any one of claims 1-5, wherein the tumor is characterized as heterogenous.

49. The method of any one of claims 1-5, wherein the tumor is characterized as fibronectin (FN) rich relative to a comparable early stage tumor and / or to corresponding healthy tissue.

50. The method of any one of claims 1-5, wherein the tumor is characterized as fiber rich relative to a comparable early stage tumor and / or to corresponding healthy tissue.

51. The method of any one of claims 1-5, wherein the tumor is a tumor classified as grade 2 or higher.

52. The method of any one of claims 1-5, wherein the TILs are not derived from peripheral blood mononuclear cells (PBMCs).

53. The method of any one of claims 1-5, wherein the TILs comprise alpha / beta T cells, Natural Killer T (NKT) cells, gamma / delta T cells, and / or NK cells.

54. The method of any one of claims 1-5, wherein the TILs are CD3+.

55. The method of any one of claims 1-5, wherein the TILs are CD 8+ and / or CD4+.

56. The method of any one of claims 1-5, wherein the TILs are derived from a tumor.

57. The method of any one of claims 1-5, wherein the TILs have functional expression of one or more endogenous T cell receptor (TCR) genes.

58. The method of any one of claims 1-5, wherein the TILs have functional expression of TCR alpha chain and / or TCR beta chain encoding genes.

59. The method of any one of claims 1-5, wherein the TILs comprise greater than about 80% alpha / beta T cells.

60. The method of claim 59, wherein the TILs comprise greater than about 95% alpha / beta T cells.

61. The method of claim 60, wherein the TILs consist essentially of or consist of alpha / beta T cells.

62. The method of claim 58, wherein the TILs comprise TCR alpha chains and / or TCR beta chains targeting one or more tumor associated antigens.

63. The method of any one of claims 1-5, where the TILs comprise tumor specific TCR-T cells.

64. The method of claim 63, wherein greater than 80% of the TILs comprise tumor specific TCR-T cells.

65. The method of claim 64, wherein greater than 95% of the TILs comprise tumor specific TCR-T cells.

66. The method of claim 58, wherein the TILs are engineered to express one or more additional transgenes.

67. The method of claim 66, wherein the one or more additional transgenes comprises CARs, heterologous TCRs, cytokines, cytokine receptors, and / or safety switches.

68. The method of any one of claims 1-5, wherein the tumor comprises cells expressing CCKAR, SLN, LARGE2, and / or WNT10A at levels greater than or equal to about 0.0001 to about 30 FPKM.

69. The method of claim 68, wherein the tumor comprises cells expressing CCKAR at greater than or equal to about 0.0001 to about 30 FPKM.

70. The method of claim 68, wherein the tumor comprises cells expressing at least two of CCKAR, SLN, LARGE2, and WNT10A at greater than or equal to about 0.0001 to about 30 FPKM.

71. The method of claim 70, wherein the tumor comprises cells expressing at least three of CCKAR, SLN, LARGE2, and WNT10A at greater than or equal to about 0.0001 to about 30 FPKM.

72. The method of claim 71, wherein the tumor comprises cells expressing CCKAR, SLN, LARGE2, and WNT10A at greater than or equal to about 0.0001 to about 30 FPKM.

73. The method of any one of claims 1-5, wherein the tumor comprises cells expressing IZUM01 at levels less than or equal to about 0.0001 FPKM.

74. The method of any one of claims 1-5, wherein the cancer is glioblastoma, cervical cancer, pancreatic cancer, ovarian cancer, uterine cancer, esophageal cancer, melanoma cancer, head and neck cancer, colorectal cancer, bladder cancer, lung cancer, prostate cancer, sarcoma cancer, breast cancer, liver cancer, renal cancer, and / or acute myelogenous leukemia.

75. The method of any one of claims 1-5, wherein the cancer comprises sarcoma.

76. The method of claim 75, wherein the sarcoma comprises liposarcoma.

77. The method of any one of claims 1-5, wherein the cancer is classified expressing high levels of fibronectin and / or high levels of collagen relative to a comparable early stage tumor and / or to corresponding healthy tissue.

78. The method of claim 37, wherein contacting of the tumor with the TILs facilitates entrance of endogenous immune cells and / or one or more additional exogenous agents into the tumor.

79. The method of any one of claims 1-5, comprising administering one or more additional anticancer therapy to the subject.

80. The method of claim 79, wherein the one or more additional anticancer therapy comprises a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, immunotherapy, and / or cytokine therapy.

81. The method of claim 80, wherein the one or more additional anticancer therapy is a chemotherapy.

82. The method of claim 81, wherein the chemotherapy comprises cyclophosphamide, methotrexate, fluorouracil, doxorubicin, vincristine, ifosfamide, cisplatin, gemcitabine, busulfan, and / or ara-C.

83. The method of claim 82, wherein the chemotherapy comprises cyclophosphamide.

84. The method of claim 79, wherein the one or more additional anticancer therapy comprises immunotherapy or cytokine therapy.

85. The method of claim 84, wherein the one or more additional anticancer therapy comprises treatment with one or more TGFP inhibitors.

86. The method of claim 85, wherein the one or more TGFP inhibitor comprises an antibody specific for TGFP and / or TGFP receptor(s).

87. The method of claim 79, wherein the one or more additional anticancer therapy comprises treatment with one or more antibody-drug-conjugates (ADCs).

88. The method of claim 79, wherein the one or more additional anticancer therapy comprises treatment with one or more immune effector cell therapy.

89. The method of claim 88, wherein the one or more immune effector cell therapy comprises attIL12 T cell therapy.

90. The method of claim 79, wherein the TILs and / or at least one or more additional therapy is administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, or by direct injection or perfusion.

91. The method of claim 79, wherein the TILs and / or at least one or more additional therapy are administered sequentially and are temporally disparate.

92. The method of claim 79, wherein the TILs and / or at least one or more additional therapy are administered concurrently.

93. The method of any one of claims 1-5, wherein the TILs penetrate to or near the center of a tumor within the subject.

94. A kit comprising means for performing the method of any one of claims 1-93.

95. A composition comprising means for performing the method of any one of claims 1- 93.

96. A method of manufacturing a medicament for the treatment of a tumor, the method comprising, i) determining levels of CCKAR, Sarcolipin (SLN), LARGE xylosyl- and glucuronyltransferase 2 (LARGE2), and / or Wnt family member 10A (WNT10A) expression in the tumor, and optionally determining the levels of izumo sperm-oocyte fusion 1 (IZUMO1); ii) obtaining TILs from a subject; iii) engineering the TILs to transgenically express a CSV-targeted and membrane- anchored cytokine; and iv) if CCKAR, SLN, LARGE2, and / or WNT10A expression is greater than or equal to about 0.0001 Fragments Per Kilobase of transcript per Million mapped reads (FPKM), and / or optionally if the expression of IZUMO 1 is less than or equal to about 0.0001 FPKM, preparing the engineered TIEs for administration to the subject.

97. A method of manufacturing a medicament for the treatment of a tumor, the method comprising, i) determining levels of CCKAR, SLN, LARGE2, and / or WNT10A expression in the tumor, and optionally determining the levels of IZUM01 expression in the tumor; ii) obtaining T cells and / or TILs from a subject; iii) engineering the T cells and / or TILs to transgenically express a CSV-targeted and membrane- anchored cytokine; and iv) if the CCKAR, SLN, LARGE2, and / or WNT10A expression is greater than or equal to about 0.0001 EPKM, and / or optionally the expression of IZUMO1 is less than or equal to about 0.0001 EPKM, preparing the engineered T cells and / or TILs for administration to the subject in combination with one or more inhibitors of CCKAR and / or CCKAR regulated pathways.

Images