Compositions and methods for engineering t cells for improved tumor infiltration and uses thereof

Abstract

Embodiments of the instant disclosure relate to compositions and methods for making and using engineered T cells or other immune-related cells. In certain embodiments, engineered T cells disclosed herein include up-regulated or increased expression of one or more Formin family proteins in the engineered T cells. In some embodiments, the engineered T cells include at least one of CD8+ cells, CD4+ T cells, chimeric antigen receptor (CAR)-T cell or other T cell of use to treat a health condition. In certain embodiments, engineered T cells disclosed herein can be used to treat cancer, autoimmunity, or other health condition in a subject.

Description

COMPOSITIONS AND METHODS FOR ENGINEERING T CELLS FOR IMPROVED TUMOR INFILTRATION AND USES THEREOF PRIORITY

[0001] This International Application claims the benefit of U.S. Provisional Application No. 63 / 465,164 filed May 09, 2023. This provisional application is incorporated herein in its entirety for all purposes. FIELD

[0002] Embodiments of the instant disclosure relate to compositions and methods for making and using engineered T cells or other immune cells. In certain embodiments, engineered T cells disclosed herein include up-regulated or increased expression of one or more Formin family proteins in the engineered T cells. In certain embodiments, engineered T cells disclosed herein can be used to treat cancer, autoimmune, or other health condition in a subject. BACKGROUND

[0003] Adoptive T cell transfer immunotherapies, such as chimeric antigen receptor (CAR)- T cell therapy and tumor-infiltrating lymphocyte (TIL) therapy have illustrated significant promise in the treatment of cancers. However, these therapies have been unsuccessful in a majority of subjects having solid tumors. These failures are due in part to the fact that these solid tumors are devoid of T cells and / or that T cells are only present in the surrounding stroma of the tumor but typically excluded from the tumor mass. Furthermore, lack of T cells or their exclusion from the tumor mass correlate with poor prognosis for a subject having solid tumors. Therefore, a need exists to identify ways to increase T cell presence in solid tumors to enhance tumor cell killing and improve patient outcome.

[0004] Further, in reference to autoimmune disorders, lack of, or insufficient number of regulatory T cells can create an environment inadequate to control and / or stop progression or onset of an autoimmune condition. Therefore, a need exists to increase the number of regulatory T cells at autoimmune condition sites for treating the autoimmune condition. SUMMARY

[0005] Embodiments of the instant disclosure relate to compositions and methods for making and using engineered T cells or other immune-related cells. In certain embodiments, engineered T cells or other immune-related cells of use in inventions disclosed herein can include up-regulating or increasing expression of one or more Formin family proteins in the 1 94952371.1engineered T cells or other immune-related T-cells. In some embodiments, engineered T cells or other immune-related cells of use in inventions disclosed herein can include up-regulating or increasing expression of one or more Formin family proteins in combination with upregulating at least one other protein in the engineered T cells or other immune cell. In certain embodiments, upregulation, or overexpression of the at least one Formin family protein can for example, enhance cell migration and / or enhance cell function. In certain embodiments, upregulation, or overexpression of the at least one Formin family protein can for example, enhance anti-tumor activities or anti-autoimmune activities of the engineered T cells or other immune cell.

[0006] In some embodiments and further to paragraph

[0005] above, the engineered T cells or other immune-related cells can include, but it are not limited to, CD8+ T cells, CD4+ T cells, CD8+ / CD4+ T cells, chimeric antigen receptor (CAR)-T cell, tumor-infiltrating lymphocyte (TIL) transfer therapy, regulatory T cells (Tregs), natural killer (NK) cells, other immunologic cell, or other T cell. In other embodiments, the engineered T cells or other immune-related cells can include, but it is not limited to, CD8+ T cells, CD4+ T cells, CD8+ / CD4+ T cells, chimeric antigen receptor (CAR)-T cell, tumor-infiltrating lymphocyte (TIL) transfer therapy, regulatory T cells (Tregs), natural killer (NK) cells, other immunologic cell, or other T cell having increased expression of one or more Formin family protein for use to treat a health condition in a subject. In certain embodiments, engineered T cells or other immune-related cells disclosed herein can be used to treat cancer, an immune-related condition, or other health condition in a subject.

[0007] In certain embodiments and further to paragraphs

[0005] -

[0006] above, engineered T cells disclosed herein can include upregulated or increased expression or overexpression of at least one Formin-like-1 (e.g., FMNL1, or FRL1) or Diaphanous-related Formin 1 (e.g., mDia1, or Drf1, or Diaph1) or mutants thereof having biological activity thereof or mutants thereof capable of increasing migration and function of the engineered T cells. In accordance with these embodiments, these proteins can be from human or other mammals. In some embodiments, these engineered T cells having upregulated or increased expression or overexpression of at least FMNL1, mDia1, or mutants thereof having biological activity thereof or mutants thereof capable of increasing migration of the engineered T cells have enhanced anti-tumor activity and / or enhanced tumor infiltration, and / or increased accumulation in tumor capabilities to 2 94952371.1induce enhanced tumor cell killing or supplement other anti-tumor therapies in treating a subject. In some embodiments, engineered T cells having upregulated or increased expression or overexpression of at least FMNL1, or mDia1, or mutants thereof having biological activity thereof, or mutants thereof capable of increasing migration of the engineered regulatory T cells have enhanced migration to sites of autoimmune disease and immune suppression capabilities. In some embodiments, a mutant FMNL1 or mDia1 includes, but is not limited to, DID and DAD domain mutants or other mutants capable of inducing constitutively active Formin proteins in T cell populations contemplated herein.

[0008] In some embodiments and further to paragraphs

[0005] -

[0007] above, overexpression of Formin proteins in engineered T cells disclosed herein can be used in therapies or combination therapies. In certain embodiments, engineered T cells disclosed herein can be used in combination with radiation, chemotherapy and / or surgery such as excision surgery or other anti-cancer therapy to treat cancer in a subject. In certain embodiments, the engineered T cells are Chimeric Antigen Receptor (CAR) T cells directed to treat cancer such as a solid tumor. In other embodiments, engineered T cells include T cells expressing one or more tumor-specific antigen receptors, such as CAR or T cell receptors (TCRs). In other embodiments, engineered T cells include T cells expressing one or more tumor-specific antigen receptors, such as CAR or T cell receptors (TCRs) where these engineered T cells disclosed herein can be independent of CAR or TCR receptor composition or antigen specificity. In other embodiments, the engineered T cells disclosed herein can be a tumor-infiltrating lymphocyte (TIL) in which the TILs are engineered to overexpress at least one Formin protein. In yet other embodiments, engineered T cells overexpressing at least one Formin protein disclosed herein can be used in combination with checkpoint inhibitor therapies to enhance anti-cancer effects and promote tumor cell killing (e.g., anti-PD-L1, anti-PD-1, and / or anti-CTLA-4 immunotherapy). In some embodiments, a subject can be treated with one or more immune checkpoint inhibitor therapy (e.g., anti-PD-L1 and / or anti-CTLA4 antibodies), a chemotherapeutic agent, radiation, or surgery before, simultaneously, or after being treated with engineered T cells disclosed herein. One advantage of using engineered T cells overexpressing at least one Formin protein disclosed herein for T cell infiltration enhancement provided by Formin family protein overexpression is that these therapies are not limited to a particular tumor antigen expressed on a targeted tumor and are not MHC restricted. Therefore, these T cell 3 94952371.1engineering strategies can be applied to any T cell-based tumor immunotherapy, or CAR-T cell therapy or other immune-related cell therapy regardless of the tumor antigen specificity and MHC restriction, or the specific CAR construct used.

[0009] In some embodiments and further to paragraphs

[0005] -

[0008] above, engineered T cells overexpressing at least one Formin family protein disclosed herein can be tumor- infiltrating lymphocytes (TILs) derived from a subject or CAR-T cells selected to bind tumor cells of a subject such as solid tumor cells. In accordance with these embodiments, engineered CAR-T or TIL cells disclosed herein can overexpress one or more Formin family protein alone or in combination with overexpression of at least one other protein. In certain embodiments, the at least one overexpressed Formin family protein includes at least one of FMNL1 and Diaphanous-related Formin 1 (e.g., mDia1 or Diaph1 or Drf1).

[0010] In some embodiments and further to paragraphs

[0005] -

[0009] above, engineered T cells overexpressing at least one Formin protein disclosed herein can be generated using, for example, transductions with polynucleotides or plasmids harboring polynucleotides contemplated herein to express one or more Formin family protein in the T cells or other immune-related cells. In certain embodiments, polynucleotides disclosed herein can be transduced in a target cell using viral, lentiviral, retroviral, or mammalian transduction systems known in the art harboring polynucleotides for encoding one or more Formin proteins or Formin-like proteins or mutants of these proteins having biological activity or enhanced biological activity thereof. In certain embodiments, polynucleotides disclosed herein can be electroporated as plasmids expressing one or more Formin or Formin-like protein or mutants of these proteins having biological activity or enhanced biological activity thereof.

[0011] In certain embodiments and further to paragraphs

[0005] -

[0010] above, a subject contemplated herein is a subject capable of developing cancer, having cancer, an autoimmune condition, or other condition related to uncontrolled cell growth. In some embodiments, the subject is a human subject or other mammalian subject. In accordance with these embodiments, a human subject can include a fetus, an infant, a toddler, a child, an adolescent, a young adult, an adult, or an elderly adult.

[0012] In some embodiments and further to paragraphs

[0005] -

[0011] above, cancers to be treated by compositions and methods disclosed herein can include, but are not limited to, blood cancer, solid tumors, and non-solid tumors. In certain embodiments, solid tumors can include, 4 94952371.1but are not limited to brain, lung, heart, stomach, liver, kidney, pancreatic, skin (e.g., melanoma), intestinal, lymph, esophageal, ovarian, prostate, spleen, breast, bone, or other type of solid tumor. In some embodiments, autoimmune conditions to be treated with engineered regulatory T cells disclosed herein can include, but are not limited to, multiple sclerosis, type 1 diabetes, lupus, rheumatoid arthritis, scleroderma, Crohn's disease, psoriasis, inflammatory bowel disease (IBD, e.g., ulcerative colitis or other), Hashimoto’s thyroiditis, and alopecia or other autoimmune condition.

[0013] In some embodiments and further to paragraphs

[0005] -

[0012] above, methods for administering compositions disclosed herein to a subject can include any suitable mode of administration. In some embodiments, methods of administering compositions can include intravenous, subcutaneous, intraperitoneal, by infusion, by bolus introduction, intratumoral introduction, intrathecal, or other suitable modes of administration.

[0014] In certain embodiments and further to paragraphs

[0005] -

[0013] above, the present disclosure provides kits for storing or transporting compositions disclosed herein and for use in practicing any of the methods disclosed herein. In some embodiments, kits disclosed herein can include combination compositions for administration to a subject in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The following drawings form part of the present specification and are included to further demonstrate certain embodiments of the present disclosure. Certain embodiments can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0016] FIGS.1A-1B illustrate in 1A exemplary bar graphs and a linear graph representing an in vitro 3D migration in collagen gels of wild type (WT) and experimental T cells lacking a Formin protein; and 1B illustrates in an exemplary graph, in vivo interstitial migration of WT and experimental T cells lacking a Formin protein according to some embodiments disclosed herein.

[0017] FIG.2 illustrates a timeline of exposure of an acceptable mouse model of autoimmunity receiving wild type and Formin family protein knock out T cells where the bar graphs represent the ratio of wild type and Formin protein knock out T cells in the blood and pancreatic islets (left) and average number of wild type and Formin protein knock out T cells per islet (right) as an indication of migration of the cells under the various conditions according to some embodiments disclosed herein. 5 94952371.1

[0018] FIG.3 illustrates exemplary immunofluorescence gel electrophoresis protein separation of a control cell population and a Formin family protein overexpressed in engineered T cells according to some embodiments disclosed herein.

[0019] FIG.4 represents an exemplary bar graph of percent migration of control T cells versus Formin family protein overexpressing T cells in vitro according to some embodiments disclosed herein.

[0020] FIG.5 illustrates a timeline of exposure using an acceptable mouse tumor model receiving T cell co-transfer of wild type and Formin family overexpressing T cells according to some embodiments disclosed herein.

[0021] FIGS.6A-6C represent exemplary photographic images of T cell tracking using fluorescence techniques (6A and 6B) under various conditions; and in 6C, displacement or migration of control tumor-specific T cells at a solid tumor site according to some embodiments disclosed herein.

[0022] FIG.7 represents a schematic diagram model projection of Formin overexpression in a population of engineered T cells leading to an increase in engineered T cell numbers at a tumor site and induced function of the engineered T cells according to some embodiments disclosed herein.

[0023] FIG.8A represents an exemplary flow cytometry output and gating scheme for sorting (using Fluorescence-activated cell sorting (FACS)) of high and low Formin protein overexpressing cells according to some embodiments disclosed herein.

[0024] FIG.8B represents an exemplary bar graph of ratios of accumulation at the solid tumor site of low Formin protein overexpressing T cells compared to control T cells, and high Formin protein overexpressing T cells compared to control T cells, according to some embodiments disclosed herein.

[0025] FIGS.9A-9B represent exemplary bar graphs illustrating accumulation of control and Formin protein overexpressing tumor infiltrating lymphocytes (TILs) at the solid tumor site (FIG.9A) and overall accumulation in different tissues in a test animal bearing a solid tumor after a predetermined period (FIG.9B) according to some embodiments disclosed herein.

[0026] FIGS.10A-10B represent exemplary bar graphs illustrating accumulation of control and Formin protein overexpressing Chimeric Antigen Receptor (CAR) T-cells at the solid tumor 6 94952371.1site (FIG.10A) and overall accumulation in different tissues in a test animal bearing a solid tumor after a predetermined period (FIG.10B) according to some embodiments disclosed herein.

[0027] FIGS.11A-11B represent an exemplary plot of tumor size over time (FIG.11A) and an exemplary survival curve (FIG.11B) of test animals after injection with control or Formin overexpressing CAR-T cells according to some embodiments disclosed herein.

[0028] FIGS.12A-12B represent exemplary bar graphs illustrating accumulation of control CAR-T cells and CAR-T cells overexpressing a constitutively active mutant of a Formin protein at a solid tumor site (FIG.12A) and overall accumulation in different tissues in a test animal bearing a solid tumor after a predetermined period (FIG.12B) according to some embodiments disclosed herein.

[0029] FIGS.13A-13B represent exemplary bar graphs illustrating control CAR-T cells and CAR-T cells overexpressing a different form of Formin protein accumulating at a tumor site (FIG.13A) and overall accumulation in different tissues in a test animal bearing a solid tumor after a predetermined period (FIG.13B) according to some embodiments disclosed herein.

[0030] FIGS.14A-14B represent exemplary bar graphs illustrating control CAR-T cells and CAR-T cells overexpressing a Formin protein at a predetermined expression level accumulating in a lung carcinoma tumor (FIG.14A) and overall accumulation in different tissues in a test animal after a predetermined period (FIG.14B) according to some embodiments disclosed herein.

[0031] FIGS.15A-15B represent exemplary bar graphs illustrating control regulatory T cells and regulatory T cells overexpressing a Formin protein accumulating in targeted tissues (FIG.15A) and overall accumulation in different tissues (FIG.15B) after a predetermined period in a test animal that functions as an autoimmune model of a disease condition, according to some embodiments disclosed herein. DEFINITIONS

[0032] Terms, unless defined herein, have meanings as commonly understood by a person of ordinary skill in the art relevant to certain embodiments disclosed herein or as applicable.

[0033] Unless otherwise indicated, all numbers expressing quantities of agents and / or compounds, properties such as molecular weights, reaction conditions, and as disclosed herein are contemplated as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters in the specification and claims are 7 94952371.1approximations that can vary from about 10% to about 15% plus and / or minus depending upon the desired properties sought as disclosed herein. Numerical values as represented herein inherently contain standard error of the mean that necessarily result from errors found in the numerical value's testing measurements.

[0034] As used herein, “individual”, “subject”, “host”, and “patient” can be used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, prophylaxis, or therapy is desired, for example, humans, or other mammals. DETAILED DESCRIPTION OF THE INVENTION

[0035] In the following sections, certain exemplary compositions and methods are described to detail certain embodiments of the invention. It will be obvious to one skilled in the art that practicing the certain embodiments does not require the employment of all or even some of the specific details outlined herein, but rather that concentrations, times, and other specific details can be modified through routine experimentation. In some cases, well known methods, or components have not been included in the description.

[0036] One problem for treating solid tumors in a subject having one or more solid tumors is that solid tumors often create a microenvironment that is hostile to the positive effects of anti- tumor T cells and other immune cells activities. Additionally, tumors are often devoid of T cells and tissue barriers surrounding the tumor can prevent the entry of T cells into the tumor mass to permit killing of the tumor cells. It has further been observed that the vasculature at the tumor site is frequently abnormal and limits T cell extravasation and trafficking to the tumor. Furthermore, T cell migration can be blocked or obstructed by restrictive tumor-derived extra cellular matrices (such as fibrosis), resulting in T cell retention in dense stroma that surrounds the tumor. As one consequence, T cells often fail to infiltrate the tumor mass and are thus ineffective in reducing or killing tumor cells in these environments. This can be a cause of failure of T cell-based therapies and of immune checkpoint inhibitor therapies when T cells do not enter the tumor site. Therefore, novel, and improved treatments for solid tumors are urgently needed. As disclosed herein one approach to alleviate this problem is to increase the migratory capacity and accumulation at the tumor site of anti-tumor T cells in the context of adoptive T cell transfer therapy, such as CAR-T cell and TIL-based therapy.

[0037] In certain embodiments, in autoimmune related conditions, regulatory T cells have a key role in maintaining immune homeostasis and preventing autoimmunity. In some 8 94952371.1embodiments, in subjects having an autoimmune condition, it has been demonstrated that, compared to healthy subjects, these subject can have a reduced number of regulatory T cells. As suggested by previous studies in animal models of autoimmunity, increasing the activity and / or number of active regulatory T cells can be beneficial to a subject having an autoimmune or other similar condition. Some embodiments disclosed herein relate to increasing the number of regulatory T cells at an autoimmune condition site to prevent, delay onset or progression, or treat the autoimmune condition.

[0038] To date, strategies to increase T cell trafficking to the tumor have been mainly limited to exploiting chemokine signaling. However, while this approach can be effective, due to the heterogenicity of chemokine expression in different tumor microenvironments and the shared expression of chemokines in tumors and other anatomical sites, targeting specific chemokine signaling pathways can have limited broad applicability. Further, while the chemokine signaling can affect T cell homing to the tumor, it does not necessarily enhance the ability of T cells to migrate through physical barriers at the tumor site to penetrate deeper into the tumor mass. In certain embodiments, one mechanism of action of Formin family proteins (e.g., FMNL1) overexpression in T cell migration is to enable squeezing of T cells through small pores and confined tissue environments at the tumor site to increase the number of T cells within a tumor.

[0039] Embodiments disclosed herein address an unmet need to improve migration and accumulation of T cells into tumors and regulatory T cells into the autoimmune tissue site by creating T cells with increased capacity to migrate through restrictive environments. In certain embodiments, at least one Formin family protein (e.g., Formin-like-1 (FMNL1)) as a cytoskeletal effector protein enhances extravasation and / or trafficking of T cells when modified in T cell populations contemplated herein. In some embodiments, an implanted mouse melanoma tumor model can be used to enable verification of T cell migration into a tumor for proof of concept. These models are comparable, or an acceptable model compared to human tumors and tumors in other subjects such as other mammals. Studies disclosed herein permitted in situ live imaging of the localization and migration of engineered T cells to a tumor site in addition to analysis by flow cytometry. In certain embodiments disclosed herein, it was demonstrated that verification that overexpression of at least one Formin family protein (e.g., FMNL1) enhances T cell migration and significantly increases the number of tumor-specific T cells present within a tumor site. In some embodiments, autoimmune conditions to be treated with engineered gene 9 94952371.1overexpressing regulatory T cells disclosed herein can include, but are not limited to, multiple sclerosis (MS), type 1 diabetes, lupus, rheumatoid arthritis, scleroderma, Crohn's disease, psoriasis, inflammatory bowel disease (IBD, e.g., ulcerative colitis or other), Hashimoto’s thyroiditis, and alopecia or other autoimmune condition.

[0040] Embodiments of the instant application are directed to engineered T cells overexpressing at least one target protein imparting T cells with improved ability to infiltrate and / or accumulate in tumors or autoimmune tissue sites. In certain embodiments, engineered T cells overexpressing at least one target protein of the instant application overexpress cytoskeletal proteins. In some embodiments, engineered T cells of the instant application overexpress one or more Formin family proteins or Formin-like family proteins. In accordance with these embodiments, cytoskeletal protein Formin-like-1 (FMNL1) or its mutants can be overexpressed in engineered T cells disclosed herein to enhance their migration and / or accumulation into tumors or autoimmune tissue sites. In other embodiments, Diaphanous-related Formin 1 (mDia1, Dia1, or Diaph1 or Drf1) or its mutants can be overexpressed in engineered T cells disclosed herein to enhance their migration into tumors or autoimmune tissue sites. In other embodiments, cytoskeletal protein Formin-like-1 (FMNL1) and Diaphanous-related Formin 1 (mDia1, Dia1, or Diaph1 or Drf1), FMNL2, FMNL3, mDia2, mDia3, Daam1, Daam2, FHOD1, FHOD3, FMN1, FMN2, INF1, INF2, or Delphilin can be overexpressed in engineered T cells disclosed herein to enhance their migration and / or into tumors or autoimmune tissue sites. In other embodiments, it is understood that recitation of FMNL1 and mDia1 disclosed herein can include any variant, such as a splice variant or any isoform thereof, such as the alpha, beta or gamma FMNL1 or mDia1, 2 or 3 or other isoform. In certain embodiments, enhanced migration of the engineered T cells increases their anti-tumor activity in tumors; for example, facilitating increased penetration into a tumor mass to enhance anti-tumor activities of the engineered T cells (or other immune- related cells) to treat a subject having a tumor (e.g., solid tumor). In some embodiments, further to above, enhanced migration of the engineered T cells increases their anti-tumor activity in tumors where T cells are normally excluded from the tumor mass. In other embodiments, enhanced migration of the engineered regulatory T cells can increase their ability to act on autoimmunity and to suppress autoimmunity or autoimmune incidences in a subject.

[0041] Certain embodiments and further to paragraphs

[0035] to

[0040] above, concern compositions including, but not limited to, engineered isolated T cells over-expressing at least 10 94952371.1one Formin family member protein or mutant thereof (e.g., DID or DAD mutants or other biologically active mutant) where over-expression of the at least one Formin family member protein includes at least a 10% increase, at least a 20% increase, at least a 30% increase, at least a 40% increase, at least a 50% increase, at least a 60% increase, at least a 70% increase or more in the level of expression of the at least one Formin family member protein in the engineered isolated T cell population compared to a control not over-expressing the at least one Formin family member protein; and a medium. In some embodiments, compositions disclosed herein of use to reduce onset of, reduce progression of, or treat a condition can include, but is not limited to, engineered isolated T cells over-expressing at least one Formin family member protein or mutant thereof (e.g., DID or DAD mutants or other biologically active mutant) where over- expression of the at least one Formin family member protein includes about a 30% increase, about a 35% increase, about a 40% increase, about a 45% increase, about a 50% increase, about a 60% increase, about a 70% increase or more in the level of expression of the at least one Formin family member protein in the engineered isolated T cell (or other immune-related cell) population compared to a control T cell not over-expressing the at least one Formin family member protein; and a medium. In some embodiments, compositions disclosed herein of use to reduce onset of, reduce progression of, or treat a condition can include, but is not limited to, engineered isolated T cells or other immune-related cell over-expressing at least one Formin family member protein or mutant thereof (e.g., DID or DAD mutants or other biologically active mutant) where over-expression of the at least one Formin family member protein includes at least about a 30% increase in the level of expression of the at least one Formin family member protein in an engineered isolated T cell (or other immune-related cell) population compared to a control T cell not over-expressing the at least one Formin family member protein; and a medium. In accordance with these embodiments, the at least one Formin family member protein includes, but is not limited to, at least one of Formin-like-1 (FMNL1), Diaphanous homolog-1 (mDia1 or Dia1 or or Diaph1 or Drf1), Formin-like-2 (FMNL2), Formin-like-3 (FMNL3), Diaphanous homolog-2 (mDia2), or Diaphanous homolog-3 (mDia3) or variant (e.g., splice variant thereof) or other Formin family proteins such as Daam1, Daam2, FHOD1, FHOD3, FMN1, FMN2, INF1, INF2, or Delphilin, or mutants thereof. In certain embodiments, mutants of Formin proteins of use in overexpressing in immune-related cells disclosed herein can include, but are not limited to, mutants that prevent the interaction of the DID and DAD domains where these mutants force 11 94952371.1the Formin protein to remain in an open more active conformation or in other words, reduce or prevent the Formin protein from closing by reducing DID / DAD interactions. Examples of these mutations include, but are not limited to, FMNL1 amino acid Valine 281(human Valine 282) to Glutamic acid, or amino acid Leucine 1062 (human Isoleucine 1072) to Aspartic Acid, Isoleucine 1058 (human Isoleucine 1068) to Glutamic acid, Isoleucine 1059 (human Isoleucine 1068) to Glutamic acid, or mDia1 Leucine 260 (human Leucine 269) to Glutamic acid, or mDia1 Leucine 1189 (human Leucine 1206) to Aspartic Acid or Alanine (amino acid identification numbers are based on a mouse sequence but the equivalent amino acid positions would be mutated in other mammalian equivalent protein positions including, but not limited to, humans). In some embodiments, these mutations can be used as single mutations or combinations of mutations to enhance their effect to reduce interaction and for the Formin protein to remain in an open conformation. In other embodiments, Formin mutants that have truncations of the DID and / or DAD domain and remain in a more open conformation are contemplated to be overexpressed in engineered T cells disclosed herein. In some embodiments, a truncated FMNL1 missing the DAD C-terminal domain is contemplated of use herein. In other embodiments, FMNL1 truncations lacking the DAD, or lacking the DID domain are contemplated of use herein that can confer active conformation to intact, whole length Formin. In yet other embodiments, it is contemplated that NK cells like T cells as provided here can be engineered to overexpress at least one Formin protein by compositions and methods disclosed herein to induce NK cell migration into tumor for improved anti-tumor activity and improved NK cell function. In yet other embodiments, immunosuppressive cells can be inhibited at the time of, before, during or after engineered T cells or NK cells are used to treat a health condition disclosed herein.

[0042] In some embodiments and further to paragraphs

[0035] to

[0041] above, T cells contemplated herein can include isolated CD3+ T cells expressing at least one of CD8+, CD4+, CD8+ CD4+ markers, a combination thereof, or other marker combinations thereof. In some embodiments, the T cells contemplated herein can include, but are not limited to, regulatory T cells expressing at least CD4+ CD25+ markers and in certain embodiments these T cells can also be CD127 dim, CD49d, CD45RA+, or a combination thereof. In some embodiments, the T cells include, but are not limited to, chimeric antigen receptor (CAR)-T cells or tumor infiltrating lymphocytes (TIL), or regulatory T cells or combination thereof. In some embodiments, T cells are harvested from a subject to be treated or other subject and engineered to overexpress at least 12 94952371.1one Formin family protein disclosed herein. In some embodiments, engineered T cells or other immune cells overexpressing at least one Formin protein disclosed herein can include at least one marker or agent for tracking the isolated T cells over-expressing at least one Formin family member protein. In certain embodiments, the at least one marker can include at least one fluorescent marker or a maker capable of fluorescing under certain conditions, a surface protein, or other selection marker known in the art. In other embodiments, engineered T or NK cells or other immune cells disclosed herein can be selected or enriched using selection of membrane proteins co-expressed in a transduction vector marking the cell surface of a desired engineered cell or by antibiotic selection marker systems in a vector to identify transduced or engineered T or NK cells disclosed herein.

[0043] In some embodiments and further to paragraphs

[0035] to

[0042] above, migration of the engineered isolated T cell population can be characterized in that the engineered isolated T cell population or other immune cells include at least one of enhanced migration capabilities, increased accumulation at a tumor site, and enhanced anti-tumor activity compared to a control T cell population not overexpressing the at least one Formin family member protein. In some embodiments, migration of the engineered isolated T cell population can be characterized in that the engineered isolated T cell population include at least one of enhanced migration capabilities and enhanced immune suppression capabilities compared to a control T cell population not overexpressing the at least one Formin family member protein. In some embodiments, these engineered immune cell population composition can be a pharmaceutical composition and further include at least one pharmaceutically acceptable excipient. It is contemplated herein that any formulation or media for expanding, preserving, storing, or delivering an engineered T cell population disclosed herein to a subject is included in this disclosure.

[0044] In other embodiments and further to paragraphs

[0035] to

[0043] above, methods for reducing onset of, preventing, or treating a health condition in a subject are disclosed. In accordance with these embodiments, upregulating, or inducing expression or overexpression of at least one Formin family member protein in a population of T cells to create engineered population T cells; and administering the engineered T cells to the subject are disclosed to reduce onset of, prevent, or treat the health condition in the subject. In certain embodiments, the health condition includes cancer. In some embodiments, the cancer includes a solid tumor. 13 94952371.1

[0045] In other embodiments and further to paragraphs

[0035] to

[0044] above, combination therapies are contemplated of use to treat a health condition. In some embodiments, a health condition including cancer can be treated with a standard anti-cancer treatment to reduce or eliminate the cancer in the subject. In some embodiments, other standard anti-cancer treatments can include, but are not limited to, at least one of radiation, chemotherapy, immune checkpoint inhibitor therapy, surgical excision, or other surgical procedure or the like. In certain embodiments, these procedures can be performed at least one of before, during or after administering engineered T cells to a subject in need of such a treatment.

[0046] In some embodiments and further to paragraphs

[0035] to

[0045] above, a single bolus of engineered T cells or other immune-related cell population disclosed herein can be administered to a subject having a tumor or autoimmune disorder. In other embodiments, more than one engineered T cell population or other immune cell population application can be administered to a subject depending on need, size of tumors, number of tumors, condition of the subject, T cell survival or other factors. In some embodiments, a treatment of engineered T cells or other immune cell population contemplated herein can include from about 1.0 X 106to about 2.0 x 109T cells or other immune-related cells per treatment or up to about 1.0 x 1010T cells or other immune-related cells per day of infused cells to the subject.

[0047] In some embodiments and further to paragraphs

[0035] to

[0046] above, methods disclosed herein include in vitro methods for generating a T cell population or other immune cell population with enhanced migration capabilities by upregulating or inducing expression or overexpression of at least one Formin family member protein in a plurality of T cells or other immune cells to create an engineered population of T cells. Then, selecting the T cells with upregulated, induced expression or overexpression of the at least one Formin family member protein for further expansion, for immediate implantation in a subject, or to store for later use. In certain embodiments, the at least one Formin family member protein includes, but is not limited to, at least one of Formin-like-1 (FMNL1) and Diaphanous homolog-1 (mDia1 or Diaph1 or Drf1), Formin-like-2 (FMNL2), Formin-like-3 (FMNL3), Diaphanous homolog2 (mDia2), or Diaphanous homolog-3 (mDia3) or similar protein thereof or mutant thereof having biological activity. In some embodiments, T cells are isolated or harvested from a subject in need of a treatment disclosed herein or are harvested from a different source such as a relative or donor or unrelated species. In accordance with these embodiments, these collected cell populations can 14 94952371.1then be expanded to a desirable density and later transfected (by a viral or mammalian system or by a plasmid), induced, or other method for upregulating or overexpressing at least one Formin family member protein to create an engineered T cell or other immune cell population. In other embodiments, collected cell populations can be activated and then be expanded (e.g., for example to a desirable density) and then transfected (by a viral or mammalian system or by a plasmid), induced, or other method for upregulating or overexpressing at least one Formin family member protein to create an engineered T cell or other immune cell population. In certain embodiments, the engineered T cells or other immune cell population are identified and harvested to generate an enriched population of engineered T cells. In some embodiments, the engineered T cell population or enriched engineered population of T cells can be plated into a medium, suspended in a medium or buffering solution and the engineered T cell population is expanded or cell growth is induced or accelerated before, at the time of transduction, and / or after transduction. In certain embodiments, a Chimeric Antigen Receptor or T cell receptors can be transduced into T cells in at least one of before, during or after the transfection or induction of the at least one Formin protein. In some embodiments, regarding retroviral transduction, one thing for successful outcome is to having proliferating cells; therefore, T cells or other immune- related cells are activated by methods known in the art and then transduced days later (e.g., 1 to about a week later) at a time of rapid division of the cells to maximize transduction efficiency. In certain embodiments, the engineered T cell population can be used in a pharmaceutical composition to treat a subject immediately or soon after generation or stored for later use under conditions appropriate for maintaining the cells. In some embodiments, the T cell populations of use herein include at least one of CD8+ T cells, CD4+ T cells, CD8+ CD4+ T cells, CAR-T cells, regulatory CD4+, CD25+ T cells, or a combination thereof.

[0048] In other embodiments and further to paragraphs

[0035] to

[0047] above, kits are contemplated. In accordance with these embodiments, kits can include components for generating an engineered T cell population contemplated herein. In other embodiments, kits can include engineered T cells for use to treat, ameliorate or prevent a health condition in a subject. In some embodiments, the kits can include at least one container appropriate for storing components of a kit disclosed herein or for storing and or dispersing cells produced by methods disclosed herein. 15 94952371.1

[0049] In certain embodiments and further to paragraphs

[0035] -

[0048] above, a subject contemplated herein is a human subject or other mammalian subject. In accordance with these embodiments, a human subject can include a fetus, an infant, a toddler, a child, an adolescent, a young adult, an adult, or an elderly adult.

[0050] In some embodiments and further to paragraphs

[0035] -

[0049] above, methods for administering compositions disclosed herein to a subject can include any suitable mode of administration. In some embodiments, methods of administering compositions can include intravenous, oral, subcutaneous. intraperitoneal, intrathecal, intratumoral, or other suitable mode of administration.

[0051] In some embodiments and further to paragraphs

[0035] -

[0050] above, pharmaceutical compositions herein can include engineered T cells disclosed herein and at least one pharmaceutically acceptable excipient or carrier. As used herein, the term “pharmaceutically acceptable carrier” can refer to solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic and absorption delaying agents, or the like that are physiologically compatible. Pharmaceutically acceptable carriers suitable for use herein, include, but are not limited to, buffers that are well known in the art, and can be phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; amino acids; hydrophobic polymers; monosaccharides; disaccharides; and other carbohydrates; metal complexes; and / or non-ionic surfactants.

[0052] In some embodiments and further to paragraphs

[0035] -

[0051] above, engineered T cells disclosed herein can be generated using transductions with polynucleotides or plasmids harboring polynucleotides contemplated herein to express one or more Formin family protein in T cells. In certain embodiments, polynucleotides disclosed herein can be transduced using a viral, lentiviral, retroviral, or mammalian transduction systems known in the art harboring or able to produce polynucleotides encoding one or more Formin proteins or Formin-like proteins or mutants of these proteins having biological activity or enhanced biological activity thereof (e.g., DID and / or DAD mutants). In certain embodiments, polynucleotides disclosed herein can be electroporated as plasmids expressing one or more Formin or Formin-like protein or mutants of these proteins having biological activity or enhanced biological activity thereof. 16 94952371.1

[0053] In some embodiments and further to paragraphs

[0035] -

[0052] above, T cells include, but are not limited to, TILs, CAR T cells, CD8+ and / or CD4+ T cells. In certain embodiments, these cells can be harvested from a subject to be treated or from a related subject and purified using for example, a negative selection kits that yield pure T cell populations (e.g., CD8+ only, CD4+ only or a mix of CD8+ and CD4+ or combination cells). In some embodiments, isolated T cells can be combined in vitro with anti-CD3 and anti-CD28 antibodies for activation and expansion (e.g., on beads, on a plate, or soluble matrices or other known surface, or in solution or medium, etc.). In some embodiments, the activation and expansion of T cells can be enhanced by the presence of growth factors, including but not limited to, interleukins such as IL-2, IL-7, or IL-15 or other cytokines or combinations thereof. In some embodiments, isolated T cells from a subject to be engineered or manipulated by compositions and methods disclosed herein, can include, but is not limited to, T cells derived from or isolated from blood, tumor biopsy, or other tissue (including but not limited to, lymph node or spleen), surgically removed tumor, or from bone marrow or the like.

[0054] In some embodiments and further to paragraphs

[0035] -

[0053] above, pharmaceutical compositions for use herein can be formulated for parenteral administration, such as intravenous or intravascular, bolus infusion, intrarenal introduction, intracerebroventricular injection, intra- cisterna magna injection, intra-parenchymal injection, or a combination thereof. In some embodiments, pharmaceutical compositions for use herein can be formulated for infusion of engineered T cells disclosed herein. In some embodiments, pharmaceutical compositions for use herein be formulated for parenteral / infusion administration can include pharmaceutically acceptable carriers including sterile liquids, such as water and oil, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and the like. Saline solutions and aqueous dextrose, polyethylene glycol (PEG) and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. In some embodiments, pharmaceutical compositions for use herein can further include additional ingredients, for example preservatives, buffers, tonicity agents, antioxidants and stabilizers, nonionic wetting or clarifying agents, viscosity-increasing agents, and the like. In some embodiments, pharmaceutical compositions described herein can be packaged in single unit dosages or in multidosage forms. 17 94952371.1

[0055] In some embodiments and further to paragraphs

[0035] -

[0054] above, formulations suitable for parenteral / infusion administration can include aqueous and non-aqueous sterile injection solutions which can contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non- aqueous sterile suspensions which can include suspending agents and thickening agents. In accordance with some embodiments herein, aqueous solutions can be suitably buffered (e.g., a pH of from 3 to 9). The preparation of suitable parenteral / infusion formulations for use herein under sterile conditions can be readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

[0056] In some embodiments and further to paragraphs

[0035] -

[0055] above, pharmaceutical compositions herein can further include one or more pharmaceutically acceptable salts. Non- limiting examples of pharmaceutically acceptable salts include acid addition salts (formed from a free amino group of a polypeptide with an inorganic acid, or an organic acid. In some embodiments, the salt formed with the free carboxyl groups is derived from an inorganic base, or an organic base. In some embodiments, any of the pharmaceutical compositions herein can be used in therapeutic applications, for example, treating a disease and / or a disorder in human patients, which are disclosed herein. In some embodiments, any of the pharmaceutical compositions disclosed herein can be used in therapeutic applications, for example, treating a brain disorder.

[0057] In certain embodiments and further to paragraphs

[0035] -

[0056] above, methods of treating or ameliorating a health condition and / or a disorder in a subject are disclosed. In some embodiments, methods of treating or ameliorating a health condition and / or a disorder in a subject include, but are not limited to, administration of an effective amount of engineered T cells as described herein. “An effective amount” as used herein refers to a bolus or dose of engineered T cells that is sufficient to confer a therapeutic effect on a subject having or suspected of having a condition, injury, disease and / or a disorder herein. In certain embodiments, a therapeutic effect for a subject having or suspected of having a disease and / or a disorder herein can include reducing the symptoms or consequences of the health condition (e.g., cancer, autoimmune conditions, and the like).

[0058] In some embodiments and further to paragraphs

[0035] -

[0057] above, a subject can be treated with engineered T cells or combination therapies including engineered T cells 18 94952371.1disclosed herein over the course of a day, for a few hours, daily, every other day, 2 times per week, weekly, every other week, monthly, or other appropriate treatment regimen. In some embodiments, a subject to any of the methods disclosed herein can be any subject for whom treatment or therapy is desired. In some embodiments, a subject to any of the methods herein can be any subject having or is suspected of having a health condition in need of treatment with engineered T cells or combination therapies. In other embodiments, a subject to any of the methods disclosed herein can be any subject having or suspected of having cancer.

[0059] In some embodiments and further to paragraphs

[0035] -

[0059] above, cancers to be treated by compositions and methods disclosed herein can include, but are not limited to, blood cancer, solid tumors, and non-solid tumors. In certain embodiments, solid tumors can include, but are not limited to brain, lung, heart, stomach, liver, kidney, pancreatic, skin (e.g., melanoma), intestinal, lymph, esophageal, ovarian, prostate, spleen, breast, bone, or other type of solid tumor. In some embodiments, autoimmune conditions to be treated with engineered regulatory T cells or other engineered immune-related cells disclosed herein can include, but are not limited to, multiple sclerosis, type 1 diabetes, lupus, rheumatoid arthritis, scleroderma, Crohn's disease, psoriasis, inflammatory bowel disease (IBD, e.g., ulcerative colitis or other), Hashimoto’s thyroiditis, and alopecia or other autoimmune condition.

[0060] In some embodiments and further to paragraphs

[0035] -

[0059] above, kits are provided for use in treating or alleviating a targeted disease or condition treatable by use of engineered T cells or combination therapies disclosed herein. In some embodiments, kits can include instructions for use in accordance with any of the methods described herein. The included instructions can include a description of administration of engineered T cells or combination therapies and / or pharmaceutical compositions described herein and optionally one or more additional therapies to treat, delay the onset, or alleviate a target disease (e.g., cancer or autoimmune condition) as those described herein. In some embodiments, kits disclosed herein can further include a description of selecting an individual suitable for treatment based on identifying whether that individual has the target disease, e.g., applying one or more diagnostic methods. In some embodiments, the instructions can include a description of administering engineered T cells or combination therapies to a subject having a targeted health condition.

[0061] In certain embodiments and further to paragraphs

[0035] -

[0060] above, kits disclosed herein include suitable packaging. Suitable packaging includes, but is not limited to, syringes, 19 94952371.1vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated herein are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump. A kit can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container can also have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). EXAMPLES

[0062] The following examples are included to illustrate certain embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered to function in the practice of the claimed methods, compositions, and apparatus. However, those of skill in the art should, in light of the present disclosure, appreciate that changes can be made to certain examples or some embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Example 1

[0063] In one exemplary method, it was determined that overexpression of FMNL1 enhanced tumor-specific T cell migration and accumulation within tumors (See for example, FIGS.9 and 10). In other examples, it was determined (FIG.12), and it is predicted that biologically active mutants of Formins can further enhance tumor-specific T cell migration and accumulation at tumor sites. Mutants of Formin proteins can include mutants that prevent the interaction of the DID and DAD domains and thus forcing the Formin protein to remain in an open more active conformation. Examples of these mutations are: FMNL1 amino acid Valine 281 (human Valine 282) to Glutamic acid, or amino acid Leucine 1062 (human Isoleucine 1072) to Aspartic Acid, Isoleucine 1058 (human Isoleucine 1068) to Glutamic acid, Isoleucine 1059 (human Isoleucine 1068) to Glutamic acid, or mDia1 Leucine 260 (human Leucine 269) to Glutamic acid, or mDia1 Leucine 1189 (human Leucine 1206) to Aspartic Acid or Alanine (amino acid numbers are based on mouse sequence and the equivalent amino acid positions would be mutated in the human proteins or other mammalian equivalent sites in these proteins). In some examples, these mutations can be used as single mutations or combination of mutations to enhance their effect. Formin mutants that have truncations of the DID and / or DAD domain 20 94952371.1and in an open conformation are contemplated of use in methods disclosed herein. Flow cytometry analysis of FMNL1-overexpressing T cell trafficking was used to demonstrate increased numbers of T cells at the tumor site, example, melanoma tumor site (FIGS.9 and 10). Time-course experiments of T cell and CAR-T cell trafficking and accumulation in the tumor site were also performed (FIG.9A and 10A) It is noted that flow cytometry data do not provide feedback on the location of the T cells in relation to a targeted tumor mass and the surrounding stroma and ECM. Therefore, using 2-photon microscopy, localization of FMNL1-overexpressing T cells at the tumor site can be analyzed. It is proposed that increasing the number of engineered T cells at the tumor site, by Formin family protein, such as FMNL1, overexpression can improve T cell migration through the tumor collagen capsule and infiltration into the tumor mass. In another exemplary method, engineered T cell migration into tumors will be tested for the ability to be further enhanced by overexpressing biologically active mutants of proteins disclosed herein, for example, a Leucine 1062 to Aspartic Acid or equivalent amino acid thereof, of FMNL1 compared to overexpression of wild-type (WT) FMNL1. It is contemplated that this change can enhance its anti-tumor and / or anti-autoimmune activities.

[0064] FIG.2 illustrates a timeline of exposure of an acceptable mouse model of autoimmunity receiving wild type and Formin family protein knock out T cells where the bar graphs represent the ratio of wild type and FMNL1 knock out T cells in the blood and pancreatic islets (left) and average number of wild type and FMNL1 knock out T cells per islet (right) under the various conditions according to some embodiments disclosed herein. This analysis was used to assess potential use of overexpression of these proteins in T cells to be used to treat autoimmune disease, such as, Type 1 diabetes. Example 2

[0065] In another example, it was demonstrated that engineered T cells overexpressing FMNL1 can have increased anti-tumor activity (FIG.11). This data suggests that overexpression of FMNL1 (WT or active mutants as described herein) in adoptively transferred tumor-specific T cells can provide better tumor infiltration and anti-tumor responses compared to control tumor- specific T cells.

[0066] In another example, transfer of engineered T cells overexpressing FMNL1 constructs will be analyzed for enhancing the efficacy of anti-PD-L1, anti-PD-1, and / or anti-CTLA-4 immunotherapy in a mouse model of melanoma or other solid tumor model. Control engineered 21 94952371.1T cells or engineered T cells overexpressing FMNL1 constructs will be transferred into a mouse tumor model and these mice will concomitantly receive antibodies against PD-L1 and / or CTLA- 4 as combination therapy examples. These immune checkpoint inhibitor antibodies will be administered at the time of engineered T cells transfer or starting 1-2 days later and repeated every 3 days, but other regimens are contemplated to increase anti-tumoral activity of these combination therapies.

[0067] In another example, the data presented herein will support the concept that FMNL1 promotes efficient T cell migration through collagen matrices in vitro as well as interstitial migration in vivo (Figs.1A-1B). Increasing the expression of FMNL1 in T cells can enable them to have enhanced migratory capabilities, particularly through restrictive environments such as those at the tumor site. In support of this, overexpression of FMNL1 in effector T cells was demonstrated to enhance their migration through small transwell pores (Fig.4). FMNL1 overexpression greatly increases the number of T cells at the tumor site as discovered herein (See for example, Figs.9 and 10). Overall, these observations support the rationale for using engineered T cells disclosed herein for improving T cell-based cancer therapies.

[0068] FIGS.1A-1B illustrate that FMNL1 regulates 3D migration in collagen matrices in vitro and interstitial motility in lymph nodes in vivo.1A) WT control and FMNL1 KO (FKO) activated T cells embedded in 3D collagen matrices were imaged by confocal time-lapse microscopy. The average speed, Arrest Coefficient (% of time spent moving at less than 2 ^m / min) and mean square displacement over time of WT and FMNL1 KO T cells are illustrated. 1B) Ex vivo activated WT control and FMNL1 KO T cells were differentially fluorescently- labeled and co-transferred IV into WT recipient mice.18-24 hours post-transfer lymph nodes were explanted and imaged by 2-photon microscopy. Quantification of T cell mean square displacement over time is illustrated. Data are the average (+SEM) from 3 independent experiments.

[0069] As demonstrated in one example, using a mouse model for melanoma, based on the B16 melanoma FMNL1 overexpression in T cells a significant increase in the number of tumor- specific T cells at the tumor site by approximately 3-fold compared to control tumor-specific T cells was observed (Fig.9). For these experiments, ovalbumin-specific OT-I T cells were transduced with a control empty vector or a FMNL1 WT expressing vector. The transduced T cells were mixed 1:1 and co-transferred into ovalbumin-expressing B78ChOva tumor bearing 22 94952371.1mice and 3-6 days later the tumors were harvested to quantify the number of transferred T cells. This fluorescent melanoma model is amenable to in situ live 2-photon imaging, allowing visualization of the tumor site and determination of the localization and migration behavior of T cells relative to the tumor mass and the collagen capsule surrounding the tumor (Figs.6A-6C). This experiment will permit quantification of effects of overexpressing WT FMNL1 or active FMNL1 or active FMNL1 in enhancing effector T cell migration, infiltration, and localization into the tumor mass. With this system, the ability of FMNL1-overexpressing tumor-specific T cells to control tumor growth can and will be assessed.

[0070] FIGS.6A-6C illustrates in situ imaging of WT T cell infiltration and migration at melanoma tumor sites.6A) Representative 3D side view reconstruction (left) and top-down maximum Z-projection view (right) of a melanoma (red) tumor site with collagen fibers (blue) and WT OT-I T cells (green).6B) Representative zoomed-in imaging of WT T cell motility at the tumor site. White tracks demonstrate the movement of T cells over 10 minutes.6C) Example of quantification of mean square displacement of WT OT-I T cells at the periphery of the tumor. Materials and Methods

[0071] To mimic T cell-based immunotherapy settings, such as TIL or CAR-T cell therapy, for some of these studies anti-CD3 / anti-CD28 antibody activated effector T cells were used. Naïve CD8 T cells from Ovalbumin (OVA)-specific OT-I transgenic mice are purified using magnetic bead negative selection kits (Stem Cell Technologies) and then activated ex vivo using plate-bound CD3 and CD28 antibodies, or alternatively activated using their cognate ovalbumin- derived peptide. On day 2 and 3 post-activation, the T cells were transduced with retroviral vectors to express FMNL1 WT or FMNL1 active mutants and fluorescent markers (e.g., GFP or CFP). As a control ‘empty’ vectors are used that only contain the GFP or CFP fluorescent marker. The control and FMNL1 transduced T cells were then sorted based on the fluorescent markers and co-transferred into tumor bearing mice 5-6 days after their initial activation. In each experiment, to correlate the level of T cell migration and tumor infiltration with the amount of FMNL1 overexpression, an aliquot of the sorted control and FMNL1-overexpressing T cells were lysed and FMNL1 protein levels measured by western blotting as previously used.

[0072] In these experiments, B78ChOva mouse melanoma model derived from B16 melanoma was used. This melanoma model is poorly immunogenic and relatively low in T cell infiltration. B78ChOva melanoma cells have been transduced to stably express the model antigen 23 94952371.1Ovalbumin (OVA) and the red fluorescence protein mCherry. For this transplantable melanoma model, 1x105B78ChOva cells are injected subcutaneously into the flank of recipient mice and 6 days later 1x105tumor-specific OT-I effector CD8 T cells are adoptively transferred into the recipient mice. CD45.1.1 congenically marked recipient mice were used to distinguish endogenous T cells from transferred T cells, which are marked by the CD45.2 isoform. At various timepoints after T cell transfer, intact tumors are harvested and imaged by live 2-photon microscopy to determine the localization and migration of T cells in situ in explanted tumors (Figs.6A-6C). Alternatively, harvested tumors were digested and dissociated to single cell suspensions for flow cytometry analysis to quantify the number (Fig.9) and phenotype of the transferred T cells. Example 3

[0073] In another example, compositions and methods disclosed herein were used to determine if overexpression of FMNL1 or its active mutants enhances tumor-specific engineered T cell migration and accumulation within tumors. Different tumors have varying levels of T cell presence at the tumor site (often referred to as ‘hot’ or ‘cold’ tumors). Increased T cell presence at the tumor site correlates with improved treatment outcomes. Therefore, using a time-course analysis, overexpressing FMNL1 in tumor-specific T cells was identified to increase recruitment and numbers at the tumor site. In these experiments, OT-I T cells were activated, transduced with a control CFP vector or a FMNL1 GFP vector, and then sorted by methods known in the art. In parallel, 6 days before T cell transfer, OVA-expressing B78ChOva melanoma cells were implanted into the flanks of recipient mice that are congenically marked with CD45.1.1. The congenically-marked CD45.2.2 control and FMNL1-overexpressing OT-I T cells were mixed 1:1 and co-transferred into the CD45.1.1 B78ChOva tumor bearing mice. Tumors from 3 recipient mice / timepoint were harvested at 3-6 days post T cell transfer. The number of transferred T cells (marked by CD45.2.2 expression) present at the tumor site were analyzed by flow cytometry (as in FIG.9). Control and FMNL1-overexpressing T cells were distinguished using CFP vs. GFP expression from the transduction vectors. In addition, the phenotype of the transferred control and FMNL1-overexpressing T cells will be analyzed by staining for markers of activation (e.g., CD69, CD44, CD49d, Granzyme B), exhaustion (e.g., PD-1, CTLA-4, LAG-3, TIGIT, TIM-3), proliferation (Ki67), and apoptosis (Annexin-V and active Caspase 3 / 7). Lymph nodes (tumor- draining and non-draining) and spleen from the recipient mice were analyzed to determine the 24 94952371.1number and phenotype of control and FMNL1-overexpressing T cells in these secondary lymphoid organs. The experiments confirmed that FMNL1 overexpression enhances T cell trafficking and accumulation at the tumor site (Fig.9B). By analyzing the number of transferred T cells in a time-course manner, FMNL1 overexpression was identified to improve not only the initial trafficking (T cell numbers at early timepoints, e.g., day 2-3) but also the persistence of T cells at the tumor site (day 6 post-transfer) (Fig.9A). Furthermore, staining of Ki67 assists in determining if differences in T cell proliferation contribute to increased accumulation of FMNL1-overexpressing T cells. By analyzing proliferation, exhaustion, and apoptosis marker expression, it will be determined that FMNL1 overexpression results in less T cell exhaustion and promotes T cell survival, persistence and / or expansion at the tumor site.

[0074] These data support that FMNL1 overexpression does not interfere with the ability of T cells to stop and kill tumor cells, and to remain at the tumor site. FMNL1 overexpression improves the accumulation, localization, and persistence of T cells in the tumor over time, and this data supports a role for FMNL1 in this process (FIGS.9 and 10). To minimize mouse-to- mouse tumor environmental variability, control and FMNL1 overexpressing T cells were co- transferred in the same recipient mice. Transduced T cells with higher levels of fluorescent marker correlate with higher FMNL1 overexpression, which were confirmed by Western blot.

[0075] One limitation of anti-tumor T cell-based therapies is that in many cases insufficient T cells reach the tumor site and / or infiltrate the tumor mass. These experiments determined that FMNL1 overexpression improves the ability of transferred tumor-specific T cells to control tumor growth. In other experiments related to solid tumors, several days before T cell transfer, B78ChOva melanoma cells will be implanted into the flanks of recipient mice. OT-I TIL cells will be activated, transduced with control or FMNL1 vectors, and then sorted as indicated herein similar to verified T cell overexpression. The B78ChOva tumor bearing mice will receive either control or FMNL1-overexpressing OT-I TIL cells (OT-I T cells), or no T cell transfer as a negative control. Tumor growth will be analyzed daily by measuring the tumor volume. Tumor volumes will be calculated using the ellipsoid volume formula: volume = 4 / 3π x length x width x height. Mice will be scored for up to 60 days or until sacrificed. Cohorts of 10 mice for each group (no T cells, control T cells, or FMNL1-overexpressing T cells) will be used. FMNL1 overexpression will be verified to enhance T cell accumulation and infiltration at the tumor site 25 94952371.1and thus reduce the rate of tumor growth compared to recipient mice that receive transfer of control T cells.

[0076] In another example, it will be determined that T cells engineered to overexpress FMNL1 have increased anti-tumor activity and improve the efficacy of checkpoint inhibitor therapy. For these experiments, immune checkpoint inhibitor therapies will be used in combination with Formin family overexpressing T cells disclosed herein. Immune checkpoint inhibitor therapies have shown promise in the treatment of solid tumors but are still ineffective in a majority of cases. This lack of efficacy can in part be due to insufficient T cells at the tumor site or lack of infiltration of T cells into the tumor mass. It is noted that the B16 melanoma- derived model is poorly immunogenic but partially responsive to immunotherapy, including checkpoint inhibitors, and provides a system to test checkpoint inhibitor therapy in combination with an enhancement of T cell migration approach. Therefore, it will be tested whether FMNL1 overexpression in transferred tumor-specific T cells can increase the efficacy of immune checkpoint inhibitor treatment.

[0077] For these exemplary methods, recipient mice will be implanted with B78ChOva tumor cells and then, 6 days later, control or FMNL1-overexpressing OT-I T cells transferred into the tumor bearing mice. Starting 48 hours after the T cell transfer, the tumor bearing mice will be receiving anti-PD-L1 and anti-CTLA-4 antibodies every 3 days. As controls, mouse cohorts that receive immune checkpoint antibodies but no T cell transfer, and cohorts with T cell transfer but no antibody treatment will be included. Tumor growth will be analyzed daily by measuring tumor volumes for up to about 30 to about 60 days or more. It will be determined that combinations of immune checkpoint inhibitor treatment and enhanced accumulation and infiltration into the tumor mass of T cells overexpressing FMNL1 (or its active mutants) interact to further increase anti-tumor activity and slow tumor growth, and potentially fully eradicate the tumor.

[0078] In other studies, these systems were tested using CAR-T cells by overexpressing FMNL1 or other Formin family protein in CAR-T cells directed against solid tumor models (Fig. 10, 13, and 14). A strength of this approach is that it can both increase the ability of T cells to extravasate at the tumor site and enhance the capacity of T cells to migrate through restrictive tissue barriers to infiltrate the tumor mass. One advantage of this platform is that FMNL1 overexpression can be applied to CAR-T cells expressing any kind of CAR receptor regardless of 26 94952371.1composition or antigen specificity. Another advantage is that FMNL1 or other Formin family protein overexpression can be applied to different adoptive transfer T cell-based therapies, such as TIL T cells. Additionally, the T cell tumor infiltration enhancement provided by FMNL1 is not limited to a specific tumor antigen and is not MHC restricted, broadening its applicability.

[0079] In another example, these overexpression systems will be tested using regulatory T cell data in a mouse type-1 diabetes model setting by overexpressing FMNL1 or other Formin family protein in regulatory T cell of use to test its effect on type-1 diabetes or other autoimmune model. In one example, regulatory T cells were purified from donor mice and then expanded in vitro and transduced with FMNL1-expressing or control vectors and then sorted. Control and FMNL1 overexpressing regulatory T cells were transferred at a 1:1 ratio in type 1 diabetes model mice and 6 days later the pancreatic islets and other tissues were harvested for flow cytometry quantification of the transferred regulatory T cells. Increased accumulation of FMNL1 overexpressing regulatory T cells compared to control regulatory T cells was found in the pancreatic islets of mice that are models of type 1 diabetes (FIGS.15A and 15B). It is expected that these engineered T cells will improve outcomes of autoimmune disorders by overcoming the reduced accumulation of the regulatory T cells in affected subjects and treating the autoimmune condition. Example 4

[0080] In another Example, and further to Examples 1 to 3, above, T cells were transduced with a retroviral construct expressing FMNL1 (see e.g., Materials and Methods referenced in Example 2) and then sorted based a fluorescent marker co-expressed in the plasmid. Cell lysates for Western blotting were then made from control-transduced and FMNL1-transduced T cells. Overexpression of FMNL1 was confirmed with densitometry analysis of Western blots. Densitometry values were normalized to the relative protein loading measured by GAPDH. The results are illustrated in FIG.3 and illustrate that cells “overexpressing” FMNL expressed about 55% more FMNL compared to control. The transduced T cells overexpressing FMNL1 were observed to overexpress FMNL1 at levels typically about 30% to about 90% higher than controls.

[0081] In another example, it was tested whether low or high overexpressing FMNL1 cells demonstrate increased accumulation at the tumor site and whether a threshold of expression was required to see effects. In this example, WT T cells were transduced with an anti-human CD19 27 94952371.1CAR construct and with a FMNL1 plasmid with an IRES / GFP reporter and sorted using FACS into low FMNL1 overexpressing cells (in green gate) and high overexpressing FMNL1 cells (in yellow gate) (FIG.8A). Cells were then co-transferred with control CAR-T cells and accumulation at tumor sites was quantified. It was found that CAR-T cells with lower FMNL1 overexpression did not enhance overall accumulation at tumor site compared to control CAR-T cells, while high FMNL1 overexpressing CAR T cells had increased accumulation (FIG.8B). Example 5

[0082] In another Example and further to Example 3 above, wildtype ovalbumin-specific T cells (i.e., tumor infiltrating lymphocytes (TILs) designed to target B78ChOva melanoma cells) were activated and transduced with either an FMNL1-expressing (FMNL1 O.E) or a control retroviral construct. The transduced T cells were then sorted and co-transferred at a 1:1 ratio into mic bearing ovalbumin-expressing melanoma tumors (as described in Example 3). Tumors and other tissues were harvested 3 to 6 days post T-cell transfer for quantification of transferred T cells. FIG.9A illustrates total TIL accumulation at the tumor site normalized to control T cells and FIG.9B illustrates the ratio of FMNL1 T cell: control T cells in various tissues at day 6. It was found that FMNL1 overexpression enhances TIL accumulation specifically at the tumor site and not in other tissues. Example 6

[0083] In another exemplary method and further to Example 3 above, WT T cells were transduced with an anti-human CD19 CAR construct and either a FMNL1-expressing or a control construct. Sorted FMNL1-overexpressing CAR-T cells and control CAR-T cells were co- transferred at a 1:1 ratio into mice bearing human CD19-expressing melanoma tumors. The tumors were then harvested 3- or 6-days post CAR-T transfer for quantification of transferred CAR-T cells by flow cytometry. FIG.10A illustrates control CAR-T cell and FMNL1 overexpressing CAR-T cell accumulation at the tumor site 3- and 6-days post-transfer (normalized to control) and FIG.10B illustrates FMNL1 CAR-T cell: control CAR-T cell ratios in various tissues at day 6. These data demonstrate that FMNL1 overexpression enhances CAR-T cell accumulation specifically at the tumor site and not in other tissues.

[0084] In another example, it was tested whether FMNL1 overexpression and associated accumulation at tumor sites leads to improved therapeutic outcomes. To this end, in one exemplary method, T cells were transduced with an anti-human CD19 CAR construct and either 28 94952371.1a FMNL1-expressing or a control construct. Mice bearing human CD19-expressing melanoma tumors were intravenously injected with either control CAR-T cells or with FMNL1 overexpressing CAR-T cells. Mice were monitored for tumor progression with FIG.11A illustrating tumor size 0 to 60 days post injection in mice receiving control CAR-T cells (black) and mice injected with FMNL-1 overexpressing CAR-T cells (red). FIG.11B illustrates average survival curves for the mice injected with control CAR-T cells (black) and FMNL-1 overexpressing CAR-T cells (red). It was found that in one mouse injected with the FMNL-1 CAR cells that tumor progression was halted completely (i.e., the mouse was “cured”) and in the other mice tumor progression was slowed significantly, resulting in improved survival. Therefore, these data suggest that the improved tumor infiltration of the FMNL-1 CAR-T cells can directly translate to improved therapeutic outcome. Example 7

[0085] In another Example, and further to Example 1 above, it was tested whether other Formin mutants or variants or other Formin family proteins could also improve tumor infiltration. Specifically, in one exemplary method, an active mutant of Formin (FMNL1-mut) containing a Leucine 1062 to Aspartic Acid substitution or equivalent amino acid thereof (described further in Example 1, above) was tested. T cells were transduced with an anti-human CD19 CAR construct and either an active mutant FMNL1 (FMNL1 mut)-expressing or a control construct. Sorted mutant FMNL1-expressing CAR-T cells or control CAR-T cells were co- transferred at a 1:1 ratio into mice bearing human CD19-expressing melanoma tumors. The tumors were then harvested 6 days post CAR-T cell transfer for quantification of transferred CAR-T cells by flow cytometry. FIG.12A illustrates active mutant FMNL1 (FMNL1-mut)- expressing CAR-T cells accumulation at the tumor site relative to control and FIG.12B illustrates mutant FMNL1 CAR-T cell:control CAR-T cell ratios in various tissues at day 6. These data illustrate that a mutant constitutively active FMNL1 also can enhance CAR-T cell accumulation specifically at a tumor site and not at other tissues.

[0086] In another exemplary method, another Formin protein (mDia1) was tested. In this exemplary method, T cells were transduced with an anti-human CD19 CAR construct and either an mDia1-expressing or a control construct. Sorted mDia1-overexpressing CAR-T cells or control CAR-T cells were then co-transferred at a 1:1 ratio into mice bearing human CD19- expressing melanoma tumors. The tumors were harvested 6 days post CAR-T transfer for 29 94952371.1quantification of transferred CAR-T cells by flow cytometry. FIG.13A illustrates mDia1 overexpressing CAR-T accumulation at the tumor site relative to control and FIG.13B illustrates mDia-1 CAR-T cell:control CAR-T cell ratios in various tissues at day 6. This data illustrates that CAR-T cells overexpressing the related Formin protein (mDia1) also had enhanced accumulation at the tumor sites specifically and not at other tissue sites. Example 8

[0087] In another Example, and further to Example 3 above, whether FMNL1 can improve tumor infiltration in other cancer models was tested. Specifically, T cells were transduced with an anti-human CD19 CAR construct and either a FMNL1-expressing or a control construct. Sorted FMNL1-overexpressing CAR-T cells or control CAR-T cells were co-transferred at a 1:1 ratio into mice bearing human CD19-expressing lung carcinoma tumors (LL2). The tumors were harvested 3- or 6-days post CAR-T transfer for quantification of transferred CAR-T cells by flow cytometry. FIG.14A illustrates control CAR-T cell and FMNL1 overexpressing CAR-T cell accumulation at the LL2 lung carcinoma tumor site and FIG.14B illustrates FMNL1 CAR-T cell:control CAR-T cell ratios in various tissues at day 6. These data, together with FIG.10, established that FMNL1 overexpression again enhances CAR-T infiltration into different solid tumor types (i.e., melanoma and lung carcinoma) in support that these technologies are applicable to treating any solid tumor by an engineered T-cell therapy disclosed herein. **************************************************** All the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods have been described in terms of embodiments, it is apparent to those of skill in the art that variations maybe applied to the compositions and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope herein. More specifically, certain agents that 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 as defined by the appended claims. 30 94952371.1

Claims

WHAT IS CLAIMED IS:

1. A composition comprising: isolated engineered T cells or other isolated engineered immune-related cells over-expressing at least one Formin family member protein wherein over- expression of the at least one Formin family member protein comprises at least about a 25 percent (%) or more increase in the level of expression of the at least one Formin family member protein in the engineered isolated T cell population compared to a control T cell population not over- expressing the at least one Formin family member protein; and a medium.

2. The composition according to claim 1, wherein the at least one Formin family member protein comprises at least one of Formin-like-1 (FMNL1 or FRL1), Diaphanous homolog-1 (mDia1 or Dia1 or Diaph1 or Drf1), mDia2, mDia3, FMNL2, FMNL3, Daam1, Daam2, FHOD1, FHOD3, FMN1, FMN2, INF1, INF2, Delphilin, or variant thereof, or mutant thereof, or the like or combination thereof.

3. The composition according to claim 1 or 2, wherein the isolated engineered T cells comprise at least one of CD3+ CD8+ T cells, CD3+ CD4+ T cells, CD3+ CD8+ CD4+ T cells, CD4+ CD25+ T cells, or a combination thereof.

4. The composition according to any one of claims 1-3, wherein the isolated engineered T cells comprise chimeric antigen receptor (CAR)-T cells or T cells with receptors specific for one or more tumor- antigens, or regulatory T cells.

5. The composition according to any of the previous claims, further comprising at least one marker or agent for at least one of selecting and tracking the isolated engineered T cells over- expressing at least one Formin family member protein.

6. The composition according to claim 5, wherein the marker comprises a fluorescent marker, a surface protein or other selection marker. 31 94952371.

17. The composition according to any of the preceding claims, wherein migration of the isolated engineered T cell population is characterized in that the isolated engineered T cell population comprises enhanced migration capabilities or enhanced accumulation at a targeted disease site compared to a control T cell population not overexpressing the at least one Formin family member protein.

8. The composition according to claim 1, wherein the other isolated engineered immune- related cells comprise isolated engineered natural killer (NK) cells.

9. The composition according to claim 8, wherein migration of the isolated engineered NK cell population is characterized in that the isolated engineered NK cell population comprises enhanced migration capabilities or enhanced accumulation at a targeted disease site compared to a control NK cell population not overexpressing the at least one Formin family member protein.

10. The composition according to any of the preceding claims, wherein the composition comprises a pharmaceutical composition and further comprises a pharmaceutically acceptable excipient.

11. The composition according to any one of claims 1-10, wherein the at least one Formin family member protein comprises at least one human Formin family member protein or other mammalian derived Formin family member protein.

12. A method for reducing onset of, or treating a health condition in a subject comprising, upregulating, or inducing expression or overexpression of at least one Formin family member protein in a population of T cells or other immune-related cell population to create an engineered population of T cells or an engineered population of other immune-related cells composition; and administering the composition of engineered population of T cells or engineered population of other immune-related cells to the subject and reducing the onset of, or treating the health condition.

13. The method according to claim 12, wherein cell population comprises T cells and the health condition comprises cancer or an autoimmune condition. 32 94952371.

114. The method according to claim 13, wherein the cancer comprises a solid tumor.

15. The method according to any one of claims 12-14, further comprising treating the subject with the health condition comprising cancer with one additional treatment comprising a standard anti-cancer treatment to reduce or eliminate the cancer in the subject.

16. The method according to claim 15, wherein the other standard anti-cancer treatment comprises at least one of immune checkpoint inhibitor treatment, radiation, chemotherapy, surgical excision, or other surgical procedure.

17. The method according to any one of claims 13-16, wherein the cancer comprises one or more of brain, lung, heart, stomach, liver, kidney, pancreatic, skin (e.g., melanoma), intestinal, lymph, esophageal, ovarian, prostate, spleen, bone, breast, or other cancer having solid tumors.

18. The method according to any one of claims 13-16, wherein the autoimmune condition comprises one or more of multiple sclerosis, type 1 diabetes, lupus, rheumatoid arthritis, scleroderma, Crohn's disease, psoriasis, inflammatory bowel disease (IBD or IBS), ulcerative colitis, Hashimoto’s thyroiditis, and alopecia.

19. The method according to any one of claims 12-18, wherein the subject comprises a human subject.

20. An in vitro method for generating an engineered T cell population or other immune-related cell population with enhanced migration comprising, obtaining T cells or other immune-related cells and upregulating, or inducing expression or overexpression of at least one Formin family member protein compared to a control population in a plurality of the obtained T cells or other immune-related cells to create an engineered population of T cells or engineered population of other immune-related cells. 33 94952371.

121. The method according to claim 20, wherein the T cells or other immune-related cells are expanded by at least one of prior to, or after upregulating or inducing expression or overexpression of the at least one Formin family member protein in the T cells or other immune-related cells.

22. The method according to claim 20 or 21, further comprising selecting the engineered population of T cells or engineered population of other immune-related cells having at least about a 25% upregulation, induced expression or overexpression of the at least one Formin family member protein compared to a control cell population.

23. The method according to any one of claims 20-22, wherein the at least one Formin family member protein comprises at least one of Formin-like-1 (FMNL1 or FRL1), Diaphanous homolog- 1 (mDia1 or Dia1 or Diaph1 or Drf1), mDia2, mDia3, FMNL2, FMNL3, Daam1, Daam2, FHOD1, FHOD3, FMN1, FMN2, INF1, INF2, Delphilin, or the like, or active mutant or active truncated molecule thereof, or combination thereof.

24. The method according to any one of claims 20-23, further comprising expanding the engineered population of T cells or engineered population of other-immune related cells with upregulated, induced expression or overexpression of the at least one Formin family member protein prior to introduction to a subject to treat a health condition.

25. The method according to any one of claims 20-24, further comprising harvesting the engineered population of T cells or engineered population of other-immune related cells with upregulated, induced expression or overexpression of the at least one Formin family member protein; and preparing a pharmaceutical composition for administration to a subject.

26. The method according to any one of claims 20-25, further comprising storing the engineered population of T cells or engineered population of other-immune related cells with upregulated, induced expression or overexpression of the at least one Formin family member protein. 34 94952371.

127. The method according to claim 26, wherein storing comprises freezing in a medium the engineered population of T cells or engineered population of other-immune related cells with upregulated, induced expression or overexpression of the at least one Formin family member protein and storing for later use.

28. The method according to any one of claims 20-27, wherein the T cells comprise at least one of CD3+ CD8+ T cells, CD3+ CD4+ T cells, CD3+ CD8+ CD4+ T cells, CD4+ CD25+, or a combination thereof.

29. The method according to any one of claims 20-28, wherein the other immune-related cells comprise NK cells.

30. A kit comprising the composition according to any one of claims 1-11; and at least one container. 35 94952371.1

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