Lymph node resident memory t cells for treatment of cancer

Abstract

Disclosed herein are compositions comprising CD8+ CD69+ CD103+ CD62L- tissue-resident memory T (Trm) cells derived from tissue- or tumor-draining lymph nodes, and methods of their use for treating cancers. The Trm cells are isolated from a CD8+ T cell population and expanded using a specific cytokine composition. Kits comprising antibodies and cytokines for preparing the Trm cells are also described.

Description

Atty. Dkt. No. 178981.00044LYMPH NODE RESIDENT MEMORY T CELLS FOR TREATMENT OF CANCERCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No.63 / 777,912 filed on March 26, 2025, the content of which is incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under R01 CA225028 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] An obstacle to current tumor-infiltrating lymphocyte (TIL) therapies is that TILs are often exhausted, dysfunctional, or limited in their clonal expansion capacity. Prior published studies have used tumor-draining lymph nodes as a source of T cells for adoptive immunotherapy. However, a limitation is that an overwhelming majority of CD8 T cells from lymph nodes lack tumor antigen specificity. Other studies have used total tumor-infiltrating lymphocytes (TILs) for therapy. However, a limitation to this approach is that most TILs are already exhausted by their ongoing exposure to factors in the tumor microenvironment. Recent studies have identified resident memory CD8 T cells that reside in tumor- and regional tissue-draining lymph nodes. However, these cells have not been isolated or studied for their anti-tumor properties. Therefore, new and improved strategies for developing and maintaining tumor antigen specific T cell populations for therapeutic use are needed.SUMMARY

[0004] In an aspect, provided herein is a composition comprising a cell population comprising tissue-resident memory T (Trm) cells expressing CD8, CD69 and CD103. The Trm cells may express CD45RO. The Trm cells may be negative for CD62L. The Trm cells may be derived from tissue-draining lymph nodes or tumor-draining lymph nodes.

[0005] The composition may further comprise a pharmaceutically acceptable delivery vehicle.Atty. Dkt. No. 178981.00044

[0006] The cell population may be prepared by: isolating T cells from tissue-draining lymph nodes or tumor-draining lymph nodes; isolating CD8+ CD69+ and CD103+ Trm cells from the T cells; and expanding the Trm cells in a T cell media comprising one or more cytokines selected from IL-2, IL-7, IL-15, TGFp, and IFNa. The one or more cytokines may consist of at least one of IL-7 and IL-15. The one or more cytokines may consist of IL-2. The IL-7 may be at a concentration of between about 5 and about 100 ng / mL. The IL- 15 may be at a concentration of between about 5 and about 200 ng / mL. The IL-2 may be at a concentration of between about 1 and about 100 ng / mL. The TGF0 may be at a concentration of between about 0.5 and about 20 ng / mL. The IFNa may be at a concentration of between about 0.001 and about 2 ng / mL.

[0007] The step of expanding the Trm cells may comprise: seeding the Trm cells in the T cell media, wherein the T cell media comprises an anti-CD3 / CD28 agent; culturing the cells for between about 5 and about 21 days; refreshing the T cell media with the anti-CD3 / CD28 agent; and supplementing the cells with about 50% fresh T cell media comprising an about 2X concentration of the one or more cytokines. The anti-CD3 / CD28 agent may comprise an anti-CD3 / CD28 bead; wherein the bead is at a concentration of about three beads per cell. The step of culturing the cells may be done for about seven days. The step of isolating the Trm cells from the CD8+ T cells may comprise: incubating the CD8+ T cells in an antibody mix comprising a CD8 antibody, a CD69 antibody, and a CD 103 antibody; wherein each antibody is conjugated to a dye; and wherein each dye is different; and sorting the cells to identify CD8+ CD69+ CD 103+ Trm cells.

[0008] The antibody mix may further comprise a CD62L antibody and a CD45RO antibody; wherein each antibody is conjugated to a dye; and wherein the step of sorting the cells further comprises identifying CD62L- and CD45RO+.

[0009] The step of purifying the CD8+ T cells from the tissue-draining lymph nodes or tumordraining lymph nodes comprises: incubating a sample comprising the lymph nodes, collagenase IV, and HBSS medium; neutralizing the sample with sterile PBS and 2% FBS; filtering the sample; spinning the sample and discarding a supernatant; and suspending the cell in sterile PBS and 2% FBS.

[0010] The Trm cells may be derived from a human subject. The Trm cells may be derived from a subject that has a cancer. The Trm cells may exhibit at least 10% clonal overlap with tumorinfiltrating lymphocytes (TILs) from at least one tumor in the subject.Atty. Dkt. No. 178981.00044

[0011] After the step of isolating CD8+ CD69+ and CD103+ Trm cells from the T cells, the Trm cells may be at least 90% pure. The step of expanding the Trm cells may expand the Trm cells at least three-fold. After step of expanding the Trm cells, at least 25% of the Trm cells may be CD103+CD69+. After the step of expanding the Trm cells, at least 25% of the Trm cells may produce Granzyme B and IFN-y when contacted with a mitogen or an anti-CD3 agent.

[0012] In another aspect, a method for treating a subj ect in need thereof with therapeutic Trm cells, the method comprising administering to the subject an effective amount of the composition described herein.

[0013] The subject may have a cancer. The cancer may be a melanoma.

[0014] The composition may comprise between about 106and about 109Trm cells.

[0015] The method may further comprise administering to the subject at least one of an IL-2 / anti-IL-2 complex therapy, an IL-15, and an IL-7 therapy. The method may further comprise administering an additional anti-cancer therapy to the subject.

[0016] In another aspect, provided herein is a method for preparing therapeutic tissue-resident memory T cells (Trm), the method comprising: isolating T cells from lymph nodes, isolating CD8+ CD69+ CD103+ CD62L- Trm cells from the T cells; andexpanding the Trm cells in a T cell media comprising one or more cytokines selected from IL-2, IL-7, IL- 15, TGF0, and IFNa.

[0017] The IL-7 may be at a concentration of between about 5 and about 100 ng / mL. The IL-15 may be at a concentration of between about 5 and about 200 ng / mL. The IL-2 may be at a concentration of between about 1 and about 100 ng / mL. The TGFP may be at a concentration of between about 0.5 and about 20 ng / mL. The IFNa may be at a concentration of between about 0.001 and about 2 ng / mL. The one or more cytokines may consist of at least one of IL-7 and IL-15. The one or more cytokines may consist of IL-2.

[0018] The step of expanding the Trm cells may comprise: seeding the cells in the T cell media, wherein the T cell media comprises an anti-CD3 / CD28 agent; culturing the cells for between about 5 and about 21 days; refreshing the T cell media with the anti-CD3 / CD28 agent; andsupplementing the cells with about 50% fresh T cell media comprising an about 2X concentration of the cytokines.

[0019] The anti-CD3 / CD28 agent may comprise an anti-CD3 / CD28 bead; wherein the bead is at a concentration of about three beads per cell.

[0020] The step of culturing the cells may be done for about seven days.Atty. Dkt. No. 178981.00044

[0021] The step of isolating the Trm cells from the CD8+ T cells may comprise: incubating the CD8+ T cells in an antibody mix comprising a CD8 antibody, a CD69 antibody, and a CD 103 antibody; wherein each antibody is conjugated to a dye; and wherein each dye is different; and sorting the cells to identify CD8+ CD69+ CD103+ Trm cells.

[0022] The antibody mix may further comprise a CD62L antibody and a CD45RO antibody; wherein each antibody is conjugated to a dye; and wherein the step of sorting the cells further comprises identifying Trm cells that are additionally CD62L- and CD45RO+.

[0023] The step of isolating the CD8+ T cells from lymph nodes comprises: incubating a sample comprising the lymph nodes, collagenase IV, and HBSS medium; neutralizing the sample with sterile PBS and 2% FBS; fdtering the sample; spinning the sample and discarding a supernatant; and suspending the cell in sterile PBS and 2% FBS.

[0024] In another aspect, provided herein is a population of therapeutic Trm cells prepared by the method described herein.

[0025] In another aspect, provided herein is a kit comprising: a CD8 antibody; a CD69 antibody; a CD103 antibody; and at least one of IL-7; IL-15; IL-2; TGF0; and IFNa. The kit may further comprise at least one of a CD45RO antibody; and a CD62L antibody.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIGS. 1A-1B. Image of Trm cells in mouse tumor-draining lymph nodes (From A. Molodtsov and N.Khatwani et. al., Immunity, 2021): Immunofluorescence confocal microscopy of pmel cells in regional LNs of mice with that had previously been treated by neoadjuvant immunotherapy and surgery, resulting in melanoma associated vitiligo (MAV). (A) Colors indicate staining for anti-CD8 (red), anti-B220 (blue), anti-CD169 (green), and anti-Thyl.l+ (gplOO melanoma antigen-specific “pmel” CD8 T cells; white). Inset at right depicts pmel cells (yellow arrows) in proximity to CD 169+ cells in the subcapsular sinus. (B) Staining was performed as in (A) but including anti-CD103 (magenta) instead of B220, to identify CD103-expressing Trm cells. Yellow arrows indicate Thy 1.1+ pmel cells (middle), most of which coexpress CD 103 (right), both in the T cell zone and subcapsular sinus regions.

[0027] FIG. 2. Image of Trm cells in melanoma patient lymph nodes. Immunohistochemistry on patient melanoma-involved sentinel lymph node, stained with anti-CD8 (green) and anti-CD69 (red), with co-stained cells (black; arrows) detected throughout regions. Image of LN is from aAtty. Dkt. No. 178981.00044single patient, but with similar cells identified in a total of n = 4 patients. (Right) Immunofluorescence imaging of metastatic melanoma patient tumor-involved LN, with CD8+CD103+CD69+ triple-stained Trm cells appearing white. Image is representative of specimens from n = 2 patients analyzed, (from Molodtsov et al. 2021)

[0028] FIG.3. Mouse lymph nodes containing Trm cells are resistant to melanoma growth.Luciferase-expressing B16 melanoma cells were directly injected into regional lymph nodes (RLNs) draining the prior site of surgical tumor excision, and LN tumor burden was imaged 7 days later. As a control naive mice (no previous neoadjuvant treatment, surgery, or MAV) were treated the same way. (from Molodtsov et. al 2021)

[0029] FIGS. 4A-4B. Human and Mouse Trm cells from tumor-draining lymph nodes appear less exhausted than Trm cells from tumors. (A) CD8+ T cells were sorted from tumors and matched draining lymph nodes of twelve NSCLC patients and underwent scRNA sequencing and paired TCR sequencing to identify clonotypes that were matched between LN and tumor (total clonal numbers shown at bottom). Cells comprising these clonotypes that distributed to Trm clusters were compared between LN and tumor regarding transcriptional features of effector function, exhaustion and sternness (top). Red boxes indicate higher sternness and lower exhaustion features for LN Trm cells compared with tumor Trm cells (top). Unpublished data. (B) Trm cells in mouse LNs do not express high exhaustion markers. FACS of Trm cells taken from mice treated as in FIG. 1, showing minimal expression of exhaustion markers. Skin draining inguinal lymph nodes were harvested from mice and stained with anti-Tim3, anti-PD-1, anti-Slamf6 and anti- CD39 antibodies for flow cytometry-based phenotyping. Gating strategy: cells gated on live CD8+CD44+CD62L-CD69+CD 103+.

[0030] FIGS. 5A-5B. Fluorescent activated cell sorting (FACS) of Trm cells from mouse lymph nodes. Mice received neoadjuvant treatment and surgery as in FIG. 1 (A) Gating strategy is depicted. Dot plot at far right depicts the sorted CD69+CD103+ Trm population in the upper right quadrant (20% of antigen experienced CD44+CD62L-CD8+ T cells). (B) Within tumordraining LNs, CD103+CD69+ Trm cells contain the highest fraction of tumor-specific CD8 T cells. FACS plots depicting proportion of tumor specific CD8+T cells (CD8+CD44+Thyl.l+) in CD69+CD103+ resident memory (Trm) or CD44+CD62L+ central memory (Tcm) compartments.

[0031] FIGS. 6A-6F. (A) Trm cells expanded better in IL-15+IL-7 compared with IL-2. Sorted Trm cells were seeded in a 96 U bottom plate at a density of 10,000-30,000 cells / well in T cellAtty. Dkt. No. 178981.00044media supplemented with IL-7 (10 ng / mL) + IL-15 (20 ng / mL) or IL-2 (2ng / ml) and anti-CD3 / CD28 beads (Thermofisher) at a 3 Bead:l Tcell ratio. Cells were cultured at 37C and 5% CO2. 50% media was refreshed with a 2X cytokine concentration without disturbing the cells on Day 3. Solid and Dashed lines indicate independent experiments, and red arrows indicate no of TCR stimulations. (B) Highest ex-vivo expansion of mouse Trm cells is achieved with a single bead (anti-CD3 / CD28) restimulation in IL-7+IL-15 over 7-18 days. Graph depicting counts of mouse Trm cells as determined by Trypan Blue staining. Mouse Trm cells were purified from skin draining LNs and expanded with IL-7 and IL- 15 or IL-2 under the following restimulation conditions: Initial stim anti-CD3 / CD28 beads added on the day of seeding the cells (Day 0) Cont stim: anti-CD3 / CD28 beads added on Day 0, 4, 7, 14. (C) Trm cells expanded in IL-7+IL-15 have higher viability than those expanded in IL-2. FACS plots depicting viability staining of mouse Trm cells purified from skin draining lymph nodes and expanded ex vivo for 7 days in the presence of IL-7(10ng / mL) and IL- 15 (20ng / mL) or IL-2 (2ng / mL) with anti-CD3 / CD28 beads at 3 beads to IT cell ratio. (D) Highest mouse Trm cell viability is obtained by culture with IL-7 and IL- 15 using a single bead restimulation (anti-CD3 / CD28) over 7-15 days. Graph depicting viabilities of mouse Trm cells as determined by Trypan Blue staining. Mouse Trm cells were purified from skin draining LNs and expanded with IL-7 and IL- 15 or IL-2 under the following restimulation conditions: Initial stim anti-CD3 / CD28 beads added on the day of seeding the cells (Day 0) Cont stim: anti-CD3 / CD28 beads added on Day 0, 4, 7, 14. (E) Graph representing fold expansion of mouse Trm or Tcm cells from lymph nodes over 7 days of expansion with IL-7 and IL-15 and anti-CD3 / CD28 beads. Each point on the plot represents an independent experiment. (F) FACS plots depicting that the Trm cell product is enriched for tumor Ag-specific CD8 T cells (gpl0025-33 specific pmel cells) denoted by CD44+Thyl.l+. (G) Mouse Trm cells maintain a clear memory phenotype (CD127+SLAMF6+) and high CD 103 expression following ex-vivo reactivation (anti-CD3 / CD28 beads) and culture in IL-7 and IL-15 for 7 days. FACS plots depicting expression of CD44, CD62L (L-selectin), CD103(Itgae), CD127(IL-7R) and Slamf6 on Trm cells purified from skin / tumor draining lymph nodes of mice and expanded for 7 days in the presence of IL-7 and IL-15 and Anti-CD3 / CD28 beads.

[0032] FIG. 7. Cell therapy experimental outline. 6xl05B16-KVP tumor cells were inoculated subcutaneously on the right flanks of C57B6L / 6 mice and grown for 7 days prior to cell therapy. We followed a published protocol for cell therapy based on the use of in vitro cultured pmel TCRAtty. Dkt. No. 178981.00044Transgenic T cells (Muranski etal, Blood, 2008 and Nelson et. al. 2015 ). One day before therapy, tumor bearing mice were treated with sublethal total body irradiation of 5 Gy. Trm cells, PBS or positive control cells (as detailed in section 2.4) were intravenously injected on DO followed by IL-2 complex (IL-2 + anti-IL-2 potentiating antibody) on days 0, 2, and 4. Tumors were measure thrice weekly.

[0033] FIG.8. Study demonstrating significant tumor growth inhibition with 20,000 LN Trm cells. B16F10-KVP tumor bearing mice were treated with no cells, 20,000 LN Trm cells, 20,000 LN Tcm cells, or naive pmel cells (positive control) as described in Fig. 7, section 2.4 and 2.5.2. LN TRM demonstrate significant tumor growth inhibition and improved survival in mice bearing B16F10 KVP tumors, (top) Error bars depict SEM of N=5-7 mice per group. Red arrows indicate IL-2 complex treatment. Statistical significance was calculated by 2-way ANOVA. (bottom) Log-Rank analysis depicts overall survival.

[0034] FIGS. 9A-9D. Transferred Trm cells engraft in tumor and other tissues, persist as memory, and are largely tumor-antigen specific. Ex vivo cultured CD45.2+ LN Trm cells were adoptively transferred as described in FIG. 7, in CD45.1 congenic mice which allows for tracing of the transferred population. (A) Engraftment of transferred populations was assessed 13 days after adoptive transfer in subcutaneous tumors and draining LNs. Data shown are combined from two independent experiments. (B) In tumor, a high proportion of engrafted Trm cells are tumor antigen-specific as evidenced by co-staining with two Kb tetramers specific for the melanoma antigen Trp2(180-188); 13 days post-treatment; gated on CD45.2+ transferred Trm cells; flow plots depict data from 5 different mice in a single experiment. (C) LN Trm cells accumulate and persist in subcutaneous tumor and draining LNs of treated mice. Engraftment of transferred (CD45.2+) populations was assessed in TDLN (top) and tumor (bottom) of separate mice on Day 24 (left) and Day 32 (right), post adoptive transfer. (D) LN Trm cell products differentiate into multiple memory subsets in vivo. FACS plots depicting differentiation of transferred Trm cell products (CD8+CD45.2+) at Day 13 (top) and Day 32 (bottom) post cell therapy in tdLNs and tumors of recipient mice.

[0035] FIGS. 10A-10D. Lung-draining lymph nodes from patients with non-small cell lung cancer (NSCLC) contain Trm cells that are clonally matched to tumor-infiltrating CD8 T cells and are highly functional (A) Human NSCLC patient TdLNs were digested enzymatically as described and stimulated with PMA / Ionomycin for 3 hrs followed by flow cytometryAtty. Dkt. No. 178981.00044phenotypic characterization. Flow cytometry plots (left) depicting expression of effector molecules GzmB and IFN-y in CD103-CD69-, CD103-CD69+, and CD103+CD69+ (Trm) subsets in the tdLNs of three NSCLC patients. Graph(right) shows combined data from 6 NSCLC patients indicate that the CD69+CD103+ Trm subset is the most functional. (B-D) CD8+ T cells were obtained from NSCLC patient tumor-draining lymph nodes, excised during routine standard-of-care surgery. Sorted CD8+ T cells underwent single cell RNA sequencing and paired TCR sequencing. (B) UMAP plot depicting transcriptional clustering, with two Trm-like clusters circled (top) and Dotplot indicating the transcriptional profiles of two prominent Trm clusters (bottom; CIO in purple and C5 in green) with high levels of Trm- and function-associated transcripts, and low expression of circulation- and exhaustion-associated transcripts. (C) UMAP plot depicting transcriptional clustering. Contours depict the overall level of TCR clonal expansion of each cluster, with the Trm cluster (C5) highlighted in green. (D) Enrichment scores indicating level of TCR clonal match between lymph node T cells (across each transcriptional cluster; on x-axis) and patient-matched tumors; The Trm cluster (C5) is significantly more clonally matched to tumor than other clusters.

[0036] FIGS. 11A-11C. Human LN Trm cells can be sorted and grown ex-vivo, and expand better in IL-7 + IL-15 vs IL-2. (A) Gating strategy depicting FACS of Trm cells from human lymph nodes. Dot plot at far right depicts the sorted CD69+CD103+ Trm population in the upper right quadrant (24% of antigen experienced CD45RO+CD62L-CD8+ T cells). (B-C) Sorted Trm cells were seeded in a 96 U bottom plate at a density of 10,000-30,000 cells / well in T cell media supplemented with IL-7 (10 ng / mL) + IL-15 (20 ng / mL). at a 3 Bead:l Tcell ratio. Cells were cultured at 37C and 5% CO2. (B) 7 day LN Trm cell expansion from five individual NSCLC patients including two patients (in red) that had previously received neoadjuvant chemo / immunotherapy treatment. (C) Cells were cultured in IL-7 (10 ng / mL) + IL-15 (20 ng / mL) or IL-2 (Ing / ml). 50% media was refreshed with a 2X cytokine concentration without disturbing the cells on Day 7 and Day 14. Solid and Dashed lines indicate samples from two patients and red arrows indicate number of TCR stimulations.

[0037] FIG. 12. Human Trm cells maintain a CD103+CD69+ phenotype following ex vivo bead (anti-CD3 / CD28) reactivation and 7 days of culture in IL-7+IL-15. Sorted Trm cells were seeded in a 96 U bottom plate at a density ofl 0,000-30,000 cells / well in T cell media supplemented with IL-7 (lOng / mL) + IL-15 (20 ng / mL) and anti-CD3 / CD28 beads(Thermofisher). Cells wereAtty. Dkt. No. 178981.00044cultured at 37C and5% C02. 50% media was refreshed with a 2X cytokine concentration without disturbing the cells on Day 4. Flow cytometry-based phenotyping was done on Day 7.

[0038] FIG. 13. Trm cell culture medium (IL-15+IL-7) can additionally include TGF-beta or TGF-beta + IFN-alpha, to enhance maintenance of the Trm phenotypic state ex vivo. FACS plots of mouse LN derived Trm cells expanded ex vivo for 5 days in the presence if IL-7(10ng / mL),IL-15(20ng / mL), TGF-beta(10ng / mL) with or without IFN-alpha (Ing / mL) and anti-CD3 / CD28 beads.

[0039] FIGS. 14A-14C. (A) Human LN Trm still produce high level of Gzmb after 7 days of ex vivo expansion. Sorted Trm cells were seeded in a 96 U bottom and expanded for 7 days as described. On Day 7 cells were restimulated with anti-CD3 antibody and Monensin overnight at 5%CO2 and 37C followed by flow cytometry based phenotypic characterization. (B) Cultured human LN Trm cells are functional in producing IFN-y and GZMB. Sorted human Trm cells (from NSCLC patient TDLNs) were cultured as described in section 3.3 and restimulated with PMA / Ionomycin on Day 21 to assess production of IFN-y and GZMB. (C) FACS plots depicting expression of effector molecules IFN-gamma, TNF-alpha and GzmB (Granzyme B) on Trm cells expanded as described. On Day 6, cells were restimulated with anti-CD3 antibody at 5ug / mL overnight followed by flow cytometry analysis for phenotypic characterization.DETAILED DESCRIPTION

[0040] The inventors previously discovered a population of resident memory CD8 T (Trm) cells that reside durably in tumor draining lymph nodes where they provide protection against metastatic disease (Molodtsov and Khatwani et. al. Immunity 2021). They identified these populations in mice and melanoma patients, and showed that these lymph node (LN) Trm cells express cellsurface markers CD 103 and CD69, and have a high level of tumor antigen specificity. The inventors demonstrate herein that these cells can be successfully sorted, grown, and re-transferred therapeutically, where they significantly restrain the growth of established melanoma tumors.

[0041] In a first aspect, provided herein is a composition comprising a cell population comprising tissue-resident memory T (Trm) cells expressing CD8, CD69 and CD 103 (CD8+ CD69+ CD103+). In embodiments, the Trm cells express CD45RO. The composition is a purified Trm cell population isolated from lymph node that has been reactivated ex vivo.

[0042] Tissue-resident memory T cells (Trm cells) are a subset of memory T cells that occupyAtty. Dkt. No. 178981.00044epithelial, mucosal and other tissues (skin, mucosa, lung, brain, pancreas, gastrointestinal tract) without recirculating. Trm cells develop from circulating CD8+effector T cells upon exposure to antigen. Most Trm cells express cluster of differentiation 69 (CD69) and CD 103 markers. CD69 is a transmembrane C-Type lectin protein, expressed in T cells, and implicated in T cell differentiation and lymphocyte retention in lymphoid organs. CD103, also known as integrin, alpha E (ITGAE), is an integrin protein that binds integrin beta 7 (ITGB7) to form a heterodimeric integrin molecule aEp7, a ligand for E-cadherin. CD8 is a transmembrane glycoprotein that serves as a co-receptor for the T cell receptor (TCR). Together, CD8 and TCR play a role in T cell signaling and aiding with cytotoxic T cell-antigen interactions.

[0043] In embodiments, the Trm cells are negative for CD62L. CD62L, also referred to as L-selectin, is a cell surface protein expressed by T cells. CD45RO is an isoform of CD45 lymphocyte common antigen. It is essential for T cell antigen signaling, promoting survival, and modulating immune responses. CD45RO serves as a marker for memory T cells.

[0044] The Trm cells may be derived from tissue-draining lymph nodes or tumor-draining lymph nodes. Tissue-draining lymph nodes are lymph nodes regionally located in the body, and serve to receive lymph fluid and filter it for bacteria and cell debris. The tissue-draining lymph nodes may be located in the neck or throat, armpit, skin, groin, abdomen, chest, etc. Tumor-draining lymph nodes receive lymphatic draining from a tumor, and prime, activate, and expand tumor-specific T cells (e.g. CD8+ T cells).

[0045] The cell population may be prepared by isolating T cells from tissue-draining lymph nodes or tumor-draining lymph nodes; isolating CD8+ CD69+ and CD103+ Trm cells from the T cells; and expanding the Trm cells in a T cell media comprising one or more cytokines selected from IL-2, IL-7, IL-15, TGF0, and IFNa. After isolating CD8+ CD69+ and CD 103+ Trm cells from the T cells, the Trm cells may be at least 90% pure, comprising no more than 10% cells other than Trm cells.

[0046] T cell media includes essential nutrients and growth factors for T cell survival, proliferation, and function. In embodiments, the T cell media is an RPMI media with glutamine (500mL) including: 10% FBS: 50mL, 1%NEAA: 5ml non-essential amino acids, l%NaP: 5ml sodium pyruvate, 1% HEPES: 5mL, 1% P / S: 5mL (Penicillin / Streptomycin), and bME: 500ul (b-mercaptoethanol).

[0047] Cytokines are small proteins involved in cell signaling as immunomodulating agents. ManyAtty. Dkt. No. 178981.00044T cell media formulations include IL-2. However, as shown in the Examples, while lymph nodederived Trm cells grow in IL -2, IL-7 and IL- 15 LN yielded higher expansion. Therefore, in embodiments, the T cell media includes IL-7 and IL- 15.. In other embodiments, the T cell media includes IL-2. In embodiments, the T cell media includes IL-7, IL- 15, and IL-2. When the media is supplemented with IL-7, the IL-7 may be at a concentration of between about 5 and about 100 ng / mL. The IL-7 may be at a concentration of about 10 ng / mL. When the media is supplemented with IL-15, the IL-15 may be at a concentration of between about 5 and about 200 ng / ml. The IL-15 may be at a concentration of about 20 ng / mL. When the media is supplemented with IL-2, the IL-2 may be at a concentration of between about 1 and about 100 ng / mL. The IL-2 may be at a concentration of about 2 ng / mL. When the media is supplemented with TGFp, the TGFp may be at a concentration of between about 0.5 and about 20 ng / mL. The TGFp may be at a concentration of about lOng / mL. When the media is supplemented with IFNathelFNamay be at a concentration of between about 0.001 and about 2 ng / mL. The IFNa may be at a concentration of about 1 ng / mL.

[0048] In embodiments, the Trm cells are expanded in the presence of membrane-bound cytokines.

[0049] T cell expansion is the process of T cell activation and proliferation which occurs naturally in vivo when the human body is actively fighting an infection or an immune system attack. In vitro, clonal T cell expansion happens when a naive T cell recognizes and binds to a specific antigen, activating the T cell to proliferate rapidly — or “expand”. Expanding the cells may comprise seeding the cells in the T cell media, wherein the T cell media comprises an anti-CD3 or anti-CD3 / CD28 agent; culturing the cells for between about 5 and about 21 days; refreshing the T cell media with the anti-CD3 / CD28 agent; and supplementing the cells with 50% fresh T cell media comprising an about 2X concentration of the cytokines. The anti-CD3 / CD28 agent may be a bead, such as a Dynabead, wherein the bead is provided at a concentration of about three beads per cell. The anti-CD3 / CD28 agent may be a bispecific anti-CD3 / CD28 antibody. The agent may be a commercially available membrane cell lysate expressing surface anti-CD3 and anti-CD28, together with membrane-bound IL- 15 and IL-7. In exemplary embodiments, the step of culturing the cells is done for about seven days. Expanding the cells may comprise transferring the cells to a larger well or vessel than they were seeded in, continually, as the cells proliferate.

[0050] The step of expanding the Trm cells may expand the Trm cells at least three-fold. In embodiments, the step of expanding the cells, at least 25% of the Trm cells are CD103+CD69+. After the step of expanding the cells, at least 25% of the Trm cells produce Granzyme B and IFN-Atty. Dkt. No. 178981.00044Y when contacted wtih with a mitogen (e.g. Phorbol 12-myristate 13-acetate (PMA)) or an anti-CD3 agent.

[0051] Isolating the Trm cells from the CD8+ T cells may comprise incubating the CD8+ T cells in an antibody mix comprising a CD8 antibody, a CD69 antibody, and a CD103 antibody; wherein each antibody is conjugated to a dye; and wherein each dye is different; and sorting the cells to identify CD8+ CD69+ CD 103+ cells. Sorting the cells may be performed by fluorescence-activated cell sorting (FACS). The antibody mix may further comprise a CD62L antibody and a CD45RO antibody; wherein each antibody is conjugated to a dye; wherein all antibodies in the antibody mix is conjugated to a different dye; and wherein sorting the cells further comprises identifying CD62L negative (CD62L-) and CD45RO+ cells. The Trm cells may be CD8+ CD69+ CD 103+ CD62L-. The dyes may be fluorophores or any labels that may be used to distinguish the antibodies.

[0052] Purifying the CD8+ T cells from lymph nodes may comprise: incubating a sample comprising the lymph nodes, collagenase IV, and Hanks’ Balanced Salt Solution (HBSS) medium; neutralizing the sample with sterile phosphate buffered saline (PBS) and 2% fetal bovine serum (FBS); filtering the sample; spinning the sample and discarding a supernatant; and suspending the cell in sterile PBS and 2% FBS.

[0053] In embodiments, the Trm cells are derived from tissue-draining lymph nodes or tumordraining lymph nodes from a human subject. The Trm cells are derived from tissue-draining lymph nodes or tumor-draining lymph nodes from a subject that has a cancer. The Trm cells may exhibit at least 10% clonal overlap with tumor-infiltrating lymphocytes (TILs) from at least one tumor in the subject.

[0054] The composition may further comprise a pharmaceutically acceptable delivery vehicle. Delivery vehicles may include sterile, pharmaceutical-grade infusion buffer, which may contain human serum albumin or dextran.

[0055] The term “culture” refers to a population of cells grown under controlled conditions in vitro.

[0056] “Contacting” a cell or a cell culture with an agent refers to adding the agent to a medium or buffer comprising the cell culture.

[0057] In a second aspect, provided is a method for treating a subject in need, the method comprising administering to the subject an effective amount of any of the compositions described herein comprising the cell population consisting of the Trm cells. The subject may have a cancer.Atty. Dkt. No. 178981.00044The cancer may be melanoma. The Trm cells may be derived from the subject’s lymph nodes. The method may further comprise administering an additional anti-cancer therapy.

[0058] As used herein, the terms “treat”, “treatment”, and “treating” refer to reducing the amount or severity of a particular condition, disease state, or symptoms thereof, in a subject presently experiencing or afflicted with the condition or disease state. The terms do not necessarily indicate complete treatment (e.g., total elimination of the condition, disease, or symptoms thereof). “Treatment,” encompasses any administration or application of a therapeutic or technique for a disease (e.g., in a mammal, including a human), and includes inhibiting the disease, arresting its development, relieving the disease, causing regression, or restoring or repairing a lost, missing, or defective function; or stimulating an inefficient process. “Treating” includes the reducing, repressing, delaying or preventing a cancer (e.g. melanoma) growth, reduction of tumor volume, and / or preventing, repressing, delaying or reducing metastasis of the tumor. Treating may also include reducing the number of tumor cells within the subject. The term “treatment” can be characterized by at least one of the following: (a) reducing, slowing or inhibiting growth of cancer and cancer cells, including slowing or inhibiting the growth of metastatic cancer cells; (b) preventing further growth of tumors; (c) reducing or preventing metastasis of cancer cells within a subject; (d) reducing or ameliorating at least one symptom of cancer; and (e) extending the survival of the subject. In some embodiments, the optimum effective amount can be readily determined by one skilled in the art using routine experimentation.

[0059] As used herein, “preventing”, “prevent”, and “preventative” refer to partially or completely delaying or precluding the onset or recurrence of a disorder or conditions and / or one or more of its attendant symptoms or barring a subject from acquiring or reacquiring a disorder or condition or reducing a subject’s risk of acquiring or reacquiring a disorder or condition or one or more of its attendant symptoms.

[0060] As used herein, the term “administering” an agent, such as a therapeutic, to a subject is intended to refer to contacting, dispensing, delivering or applying the substance to the subject. Administering is done by any suitable route for delivery of the therapeutic agent to the desired location, including delivery by either the parenteral or oral route, intramuscular injection, subcutaneous / intradermal injection, intravenous injection, intrathecal administration, buccal administration, transdermal delivery, and administration by the intranasal or respiratory tract route. In embodiments, the Trm cells are administered intravenously.Atty. Dkt. No. 178981.00044

[0061] The terms “effective amount” or “therapeutically effective amount” refer to an amount sufficient to effect beneficial or desirable biological and / or clinical results. The amount of the Trm cells that is therapeutically effective may vary depending on the particular disease or the condition of the subject. The Trm cells may be administered at a dose of between about 106and about 109cells. The Trm cells may be administered at a dose of at least about 106cells.

[0062] The terms “subject” and “patient” are used herein interchangeably to refer to a mammal to be treated by the methods and compositions described herein. “Mammals” means any member of the class Mammalia including, but not limited to, humans and non-human animals, non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, and swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice, and guinea pigs; and the like.

[0063] Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. Cancer includes solid tumors and hematological malignancies. Cancers that may be treated using the compositions and methods described herein include, but are not limited to melanomas, B cell cancers, myeloid leukemias, T cell cancers, breast cancers, pancreatic cancers, colorectal cancers, brain cancers, head and neck cancers, etc.

[0064] T cell transfer therapy is a type of immunotherapy that makes one’s own immune cells better able to attack cancer. Tumor-infiltrating lymphocytes (TIL) therapy is a type of T cell transfer therapy involving collecting a subject’s own immune cells (from tumors), growing large numbers of these cells in the lab, and then transferring the cells back to the subject. In contrast to TIL therapy, the methods described herein utilize T cells derived from the lymph node.

[0065] The method may further comprise administering to the subject an IL-2 or an IL-2 / anti-IL-2 complex therapy, or IL-15 or IL-7 therapy. Interleukin-2 (IL-2) has been used clinically for boosting effector immune response for the treatment of metastatic melanoma and other cancer types. Use of IL-2 at high doses is limited by its high toxicity. Complexing IL-2 with an anti-IL-2 monoclonal antibody (IL-2 / anti-IL-2 complex) have been shown to enhance the therapeutic efficacy of IL-2. IL-15 and IL-7 therapies include, without limitation, cytokine therapies and IL-15 and IL-7 receptor agonists. For example, Anktiva® (nogapendekin alfa inbakicept-pmln) is an IL- 15 agonist.

[0066] “Additional anti-cancer therapies” as described herein include, but are not limited to, chemotherapy, radiation, bone marrow transplant, surgery and immunotherapy.Atty. Dkt. No. 178981.00044

[0067] In a third aspect, provided herein is a method for preparing therapeutic tissue-resident memory T cells (Trm), the method comprising: isolating T cells from lymph nodes, isolating CD8+ CD69+ CD 103+ CD62L- Trm cells from the T cells; and expanding the Trm cells in a T cell media comprising one or more cytokines selected from IL-2, IL-7, IL- 15, TGFp, and IFNa. The steps of isolating T cells from lymph nodes, isolating the Trm cells from the T cells, and expanding the Trm cells are described above. The cells may further be CD45RO+.

[0068] In a fourth aspect, provided herein is a population or culture of therapeutic Trm cells prepared by any of the methods described herein.

[0069] In a fifth aspect, provided herein is a kit comprising a CD8 antibody; a CD69 antibody; a CD103 antibody; and at least one of IL-7; IL-15; IL-2; TGF0, and IFNa.

[0070] The kit may further comprise a CD45RO antibody; and a CD62L antibody.

[0071] Miscellaneous

[0072] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.”

[0073] As used herein, “about,” “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of these terms which are not clear to persons of ordinary skill in the art given the context in which they are used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

[0074] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising” in that these latter terms are “open” transitional terms that do not limit claims only to the recited elements succeeding these transitional terms. The term “consisting of,” while encompassed by the term “comprising,” should be interpreted as a “closed” transitional term that limits claims only to the recited elements succeeding this transitional term. The term “consisting essentially of,” while encompassed by the term “comprising,” should be interpreted as a “partially closed” transitional term which permits additional elements succeeding this transitional term, but only if those additional elements do not materially affect the basic and novel characteristics of the claim. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of’ and “consisting of’ those certain elements.Atty. Dkt. No. 178981.00044

[0075] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

[0076] In those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0077] " Substantial identity" of amino acid sequences means that a polynucleotide or polypeptide comprises a sequence that has at least 85% sequence identity to a reference sequence (SEQ ID NO) using a sequence alignment program; preferably BLAST using standard parameters. A preferred percent identity of polynucleotides and polypeptides can be any integer from 85% to 100%. A preferred percent identity may be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to a reference sequence.

[0078] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any ofAtty. Dkt. No. 178981.00044these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or description found in the cited references.

[0079] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0080] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0081] EXAMPLES

[0082] The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.

[0083] Example 1

[0084] Section 1. Background

[0085] CD8+ tissue-resident memory (Trm) cells are essential to the immune responses against cancer and are conventionally identified by surface expression of CD69 and CD 103 proteins. These memory T cells have properties of cytotoxic effector cells (they express IFNy and GZMB) while possessing stem like properties that enable their longevity and plasticity. Our laboratory’s published work identified a new population of Trm cells that reside stably in tumor draining lymphAtty. Dkt. No. 178981.00044nodes (TDLNs) where they prevent the growth of melanoma metastasis.

[0001] Compared with other CD8 T cells in TDLNs, we showed that Trm cells are also more likely to have specificity for tumor-expressed antigens and to be clonally matched to tumors. [1] Despite their tumor antigen specificity and their important role in mediating anti-tumor immunity, the field has yet to harness the power of LN-derived Trm cells for the immunotherapy of cancer. Prior published studies have used tumor-draining lymph nodes as a source of T cells for adoptive immunotherapy. However, a limitation is that an overwhelming majority of CD8 T cells from lymph nodes lack tumor antigen specificity. Other studies have used total tumor-infiltrating lymphocytes (TILs) for therapy. However, a limitation to this approach is that most TILs are already exhausted by their ongoing exposure to factors in the tumor microenvironment. Our ability to sort LN Trm cells using cellsurface markers enables us to selectively capture and expand this functional, tumor antigen-specific population for therapeutic applications. Our preliminary studies demonstrate ex vivo culture of purified CD8 Trm cells from mouse and human tumor- draining lymph nodes in cytokines (IL-7 + IL- 15) with T cell receptor (TCR) stimulation. Additionally, by employing the B16F10-KVP mouse melanoma model, we show that these cells can be transferred therapeutically and that very small numbers (20,000 per mouse) significantly restrain growth of established melanoma tumors in mice.

[0086] 1.1. Generation of LN Trm cells in mice. In mice bearing progressive B16 melanoma, we have shown that neoadjuvant treatment with anti-CD4 monoclonal antibody to deplete regulatory T cells (Tregs), followed by surgical excision of the primary tumor, leaves mice with robust long-lived memory CD8 T cell responses, that are protective against melanoma rechallenge [2-4], To track tumor-specific CD8+T-cell responses over time, congenic Thyl.l+T-cells expressing a transgenic T cell receptor (TCR) specific for the shared melanoma / melanocyte antigen gplOO, called “pmel cells” were used. Briefly, 104naive pmel cells were transferred into recipient C57BL / 6 micefollowed by intradermal inoculation of B16F10 melanoma cells on the right flank.4 and 10 days later, these mice were injected intraperitoneally with anti-CD4 antibody. 12 days after tumor inoculation, surgery was performed to remove tumors and initiate effector T-cell contraction and memory formation. Memory T cell populations were studied 30 days later.

[0087] 1.2 Tumor-specific Trm cells in LNs are characterized by expression of CD69 and CD 103. As shown in FIG. 1, we identified LN Trm cells in tumor draining LNs of mice 30 days after primary melanoma excision. These cells localized to the subcapsular sinus and T cell zones andAtty. Dkt. No. 178981.00044expressed the cell surface markers CD 103 and CD69. We also evaluated melanoma patient lymph nodes and identified a similar population of CD103+CD69+ Trm cells (FIG. 2).

[0088] 1.3 Trm cells in lymph nodes provide regional tumor protection [1], To determine if LN Trm cells are protective against metastatic-like disease, we rechallenged mice 30 days after surgery with B16 melanoma cells directly injected into lymph nodes that had drained the primary tumor. Importantly, these mice did not contain transgenic pmel cells, enabling us to study endogenous Trm cell-mediated protection. Imaging of luminescent tumor cells revealed significant protection against melanoma seeding and growth in lymph nodes (FIG. 3; [1]). Parabiosis studies were also conducted to demonstrate that tumor protection in lymph nodes was mediated by Trm cells as opposed to circulating memory T cells [1],

[0089] 1.4 Trm cells in human non-small cell lung cancer (NSCLC) -draining LNs are less exhausted than their clonal counterparts in tumors. We harvested tumor and matched draining lymph nodes from 12 non-small cell lung cancer patients and sorted CD8 T cells for single cell RNA sequencing and paired single-cell TCR sequencing. A total of 1657 clonotypes were shared between tumor and lymph node across all of the patients, and we found that the gene expression profile of these cells was overall more stem-like and less exhausted when cells were localized to LNs versus tumors (FIG. 4). These data support the idea that Trm cells from lymph nodes are more functional than Trm cells from tumors, making them attractive candidates for cell therapy.

[0090] Section 2. Experimental methodologies for harvesting, sorting and expanding LN Trm cells for adoptive therapy. Mice were treated with neoadjuvant Treg depletion (anti-CD4 monoclonal antibody) followed by surgery as described in section 1.1. Eight to ten mice were euthanized and lymph nodes were harvested and combined.

[0091] 2.1 Mouse TN digestion. Lymph nodes were defatted and enzymatically digested using 400U / mL collagenase IV (Worthington Biochem#LS004189) and HBSS medium (Corning#21-020-CV). In a 6 well plate, 3mL of the collagenase IV+HBSS digestion buffer was added and kept on ice. LNs from each mouse were put in 1 well of a 6 well plate with the digestion buffer. Using 0.5ml syringes, LNs were dissociated at 4C and then digested at 37C for 30 mins followed by neutralization with sterile PBS containing 20 mM EDTA (3 mL neutralization buffer per well). The plate was incubated at 37C for 5 mins followed by filtration through sterile 40-micron filter into 50 mL falcon tubes. Residual tissue pieces were mashed through the filter using a syringe plunger. Falcon tubes containing digested LN suspension were spun at 1500RPM for 5 mins at 4CAtty. Dkt. No. 178981.00044and supernatant was discarded. Pellets were resuspended in sterile flow buffer (PBS with 2% FBS). CD8 T cells were isolated using a mouse CD8 T cell negative isolation kit from Stemcell technologies.

[0092] 2.2 Mouse Trm cell sorting. Purified CD8 T cells from combined LNs were spun down and resuspended in a 200ul master mix of antibodies in flow buffer (see Table 1). Cells were stained for 30 mins on ice in the dark. Cells were spun down and washed with flow buffer and resuspended in 500ul of flow buffer in 5mL polystyrene tubes. Cells were sorted on a SONY fluorescence activated cell sorter (FACS) according to the following gating strategy: singlets, CD8+, CD44+CD62L-, CD69+CD103+ (FIG. 5). These sorted cells were cultured and used for adoptive cell therapy as described below. These cells also contained a small sentinel population of pmel cells for tracking.

[0093] Table 1. Antibodies and dilutions used for mouse Trm cell sorting.Antibodies Dilution (1 to)CD8 APC 100CD44- BV711 100CD 103 BV421 40CD62L- BV510 80CD69-PeCy7 20Thy 1.1 PE 200Fc block 100

[0094] 2.3 Ex vivo expansion of mouse LN Trm cells with CD3 / CD28 beads and cytokines for use in adoptive cell therapy. Sorted Trm cells were seeded in a 96 U bottom plate at a density of 10,000-30,000 cells / well in T cell media supplemented with IL-7 (10 ng / mL) + IL-15 (20 ng / mL) or IL-2 (2ng / ml) and anti-CD3 / CD28 beads (Thermofisher) at a 3 Bead:l Tcell ratio. Cells were cultured at 37C and 5% CO2. 50% media was refreshed with a 2X cytokine concentration without disturbing the cells on Day 3. Anti-CD3 / CD28 beads were refreshed as indicated with red arrows (FIG. 6). For adoptive cell therapy studies, cells were left undisturbed in the 96 U bottom plate after Day 0 until they were harvested for adoptive therapy. Analysis of cell expansion indicated that Trm cells preferred IL-7 + IL- 15 to IL-2 (FIG. 6).

[0095] 2.4 Adoptive Cell Therapy (ACT) experimental design. FACS-sorted LN Trm cells wereAtty. Dkt. No. 178981.00044cultured for either 7 or 14 days prior to their use in adoptive therapy. Cells were cultured in IL-7 + IL-15 with anti-CD3 / CD28 bead stimulation as described in section 2.3. To generate tumors, 6xl05B 16-KVP tumor cells were inoculated subcutaneously on the right flanks of C57B6L / 6 mice and grown for 7 days prior to cell therapy. We followed a published protocol for cell therapy based on the use of pure pmel TCR transgenic T cells. [5, 6] One day before therapy, tumor bearing mice were treated with sublethal total body irradiation of 5 Gy. Unless otherwise noted, in each study, PBS injection was used as negative control and purified pmel T cells were used as a positive control as they function aggressively against B 16 tumors expressing the cognate KVP antigen. On the day of therapy, cultured Trm cells (or Tcm cells) were transferred in mice viatail vein injections. As published, mice were also given human IL-2 / anti-human IL-2 complex therapy (prepared prior to the day of adoptive transfers as 1.5 ug IL2 + 7.5ug anti-IL-2, stored at -80C). IL-2 complex was injected in 200 ul volume i.p. on Day 0, 2 and 4 post cell transfer. Tumor growth was measured every other day starting on day 0 with digital calipers. An experimental outline is shown in FIG.7. Length and width of tumors was reported in tumor area (mm2). Overall survival was calculated using Kaplan Meier Analysis.

[0096] 2.5 Results of adoptive cell therapy studies

[0097] 2.5.1 Results for Study 1 (RIG. 8). After 14 days of culture in IL-7 + IL-15 with anti-CD3 / CD28 beads, as described in section 2.4, Trm cells were harvested and administered i.v. via tail vein, at a dose of 20,000 cells per mouse. Significant tumor growth retardation was observed in mice receiving Trm cells as compared with PBS treated mice. Mice receiving 1.5 million pure Pmel cells (cultured with IL-7+IL-15 and TGF-[3) also demonstrated significant tumor retardation as a positive control. This pilot study demonstrates that ex vivo cultured LN Trm cells significantly retard established B 16 tumor growth at a dose of 20,000 cells / mouse. FIG. 13 shows that addition of TGF-beta with or without IFN-alpha during ex-vivo reactivation / culture further promotes the Trm phenotype.

[0098] 2.5.2 Results for Study 2 (FIGS. 8 and 9). Trm cells were sorted and cultured as described for Study 1, but instead of 14 days, cells were cultured for 7 days. LN-derived “Tcm” cells (that had been sorted on a CD69' CD103" CD62L111phenotype) were used as a comparison to determine if Trm cells are more effective than CD62LhlT cells. Pure naive pmel cells were used as a positive control. Each cell product was administered at a dose of 20,000 cells / mouse, and mice were treated according to the schedule shown in FIG. 7. Indeed, similar to Study 1, just 20,000 LN Trm cellsAtty. Dkt. No. 178981.00044resulted in significant growth inhibition of established Bl 6 tumors (FIG. 8, upper). Mice treated with LN Trm cells also led to a significant increase in overall survival (FIG. 8, lower). By comparison LN Tcm cells failed to result in significant tumor growth inhibition or extended survival (FIG. 9).

[0099] Also in Study 2, the transferred T cell populations had been labeled with a CD45.2 congenic marker to enable their tracking in recipient mice (which express CD45.1). Despite transferring only 20,000 cells, we detected LN Trm cells in tumors and draining lymph nodes of treated mice over 1 -month post-transfer, indicating their long-term persistence as memory (FIG.9C). FIGS. 9A-9B illustrate that Transferred Trm cells engraft in tumor, lymph nodes, and spleen, and a high proportion are tumor antigen-specific.

[0100] Section 3. LN Trm cells from patients with non-small cell lung cancer (NSCLC): Sorting, growth, and in vitro function

[0101] To extend the above findings to humans, we obtained tumor-draining lymph nodes from two patients who were undergoing standard of care surgery for NSCLC. These patients had both been treated with anti-PD-1 therapies in the neoadjuvant setting. Thoracic lymph nodes were isolated in close proximity to the tumor mass and processed as fresh tissues (same day) in the laboratory.

[0102] 3.1. Human LN Enzymatic digestion. Lymph nodes were defatted as much as possible and enzymatically digested using 725 units / mL of collagenase IV (Worthington Biochem#LS004189) and HBSS medium (Coming#21-020-CV). In a 15mL falcon tube, 3 mL of the collagenase IV+HBSS digestion buffer was added per LN and samples were digested at 37C for 15 mins in the stand rotator followed by neutralization with ice cold sterile PBS+ 2% FBS. Digested LNs were filtered through sterile 70-micron filters into 50 mL falcon tubes. Residual tissue pieces were mashed through a filter using a syringe plunger. Falcon tubes containing digested LN suspensions were spun at 1500 RPM for 5 min at 4C and supernatants were discarded. Pellets were resuspended in sterile flow buffer (PBS with 2% FBS).

[0103] 3.2 Human LN Trm cell Fluorescence Activated Cell Sorting (FACS). Purified CD8 T cells from combined LNs were spun down and resuspended in a 200ul master mix of antibodies in flow buffer (see Table 2). Cells were stained for 30 mins on ice in dark. Cells were spun down and washed with flow buffer and resuspended in 500ul of flow buffer in 5mL polystyrene tube. Cells were sorted on a SONY fluorescence activated cell sorter (FACS) according to the followingAtty. Dkt. No. 178981.00044gating strategy: singlets, CD8+, CD45RO+CD62L-CD69+CD103+ (FIG. 12A). These sorted cells were cultured and assessed for effector functions as described below.

[0104] Table 2. Antibodies and dilutions used for human Trm cell sorting.Antibodies Dilution (1 to)CD8 AF488 100CD69 PE 20CD45RO-APC 20CD103-BV711 50CD103 Pe-Cy7 20CD62L- BV421 20Fc block 100

[0105] FIG. 10A demonstrates that within human non-small cell lung cancer (NSCLC) regional / tumor-draining lymph nodes (TDLNs), Trm cells are the highest GzmB and IFN-g dualproducers. FIG. 10B illustrates Trm populations identified in NSCLC patient TDLNs through single cell RNA sequencing. FIG. 10C shows the IFN-gamma-high CD8+ Trm cell cluster in NSCLC patient TDLNs is most highly clonally matched to tumor, indicating high tumor-antigen specificity. FIG. 12B illustrates that purified Trm cells can be expanded from lymph nodes of NSCLC patients regardless of prior chemo / immunotherapy treatment status.

[0106] 3.3 Assessment of human LN Trm cytokine preference for growth over 14 days with repeated TCR stimulation. Sorted human LN Trm cells were seeded in 96 U bottom plate with human anti-CD3 / CD28 Dynabeads (3 beads to 1 T cell). The cells were either supplemented with T cell media and IL-2 (1 ng / mL) or T cell media with IL-7 (10 ng / mL) and IL- 15 (20 ng / mL). Dynabeads were refreshed on Day 7 keeping the ratio same. On Day 7, cells were moved to 48 well plate and supplemented with 50% fresh media containing 2X cytokine concentration. FIG. 12 depicts two independent experiments involving LNs from two lung cancer patients. These data suggest that, similar to our findings in mice, human LN Trm cells are more effectively cultured with IL-7 + IL- 15 compared with IL-2.

[0107] 3.4 Assessment of human Trm cell effector functions. After twenty-one days of culture according to the methodology described in section 3.3, LN Trm cells were restimulated with PMA-Atty. Dkt. No. 178981.00044ionomycin in the presence of brefeldin-A for 4 hours and then stained with antibodies to TFN-g and Granzyme-B, to assess function. We found that LN Trm cells were highly functional, with over half of the cells producing GzmB in both patients, and 25-35% of the cells making both IFN-y and GzmB. These effector molecules are known to promote cytotoxic function of Trm cells, and we show that in both patients, LN Trm cells remain capable of making effector molecules even after culture for 3 weeks (see FIG. 14A). FIG. 14C shows that ex-vivo expanded mouse Trm cells produce high levels of GranzymeB. FIG. 14A shows that human LN Trms still produce high level of Gzmb after 7 days of ex vivo expansion.

[0108] Summary of Novel Findings

[0109] 1. Tumor-draining lymph nodes provide a niche for Trm cells that persist as long-lived, functional, stem-like, tumor-specific T cells.

[0110] 2. This cell population can be isolated from both mouse and human tumor-draining lymph nodes, and cultured in IL-15 and IL-7.

[0111] 3. Human LN Trm cells retain robust effector function even following prolonged culture and restimulation.

[0112] 4. Cultured mouse LN Trm cells can be employed for potent cell therapy against established melanoma tumors in vivo.

[0113] 5. Mouse LN Trm cells expand in the host and persist for >1 month following therapeutic transfer, likely owing to their stem-like properties.

[0114] References1. Molodtsov, A.K.,etal., Residentmemory CD8(+)Tcellsinregionallymphnodesmediateimmunity to metastatic melanoma. Immunity, 2021.54(9): p.2117-2132 e7.2. Byrne, K.T.,etal.,AutoimmunevitiligodoesnotrequiretheongoingprimingofnaiveCD8Tcellsfor diseaseprogressionorassociatedprotectionagainstmelanoma. JImmunol, 2014.192(4):p.1433- 9.3. Malik, B.T.,etal., Resident memory Tcellsintheskinmediatedurableimmunitytomelanoma. Sci Immunol, 2017.2(10).4. Zhang, P., et al., Induction of postsurgical tumor immunity and T-cell memory by a poorly immunogenic tumor. Cancer Res, 2007.67(13): p.6468-76.5. Nelson, M.H., et al., The inducible costimulator augments Tcl7 cell responses to self and tumor tissue. JImmunol, 2015. 194(4): p. 1737-47.6. Muranski, P., et al., Tumor-specific Thl7-polarized cells eradicate large establi shedmelanoma.Atty. Dkt. No. 178981.00044Blood, 2008. 112(2): p. 362-73.

[0115] Embodiments

[0116] Embodiment 1. A composition comprising a cell population comprising tissue-resident memory T (Trm) cells expressing CD8, CD69 and CD 103.

[0117] Embodiment 2. The composition of embodiment 1, wherein the Trm cells express CD45RO.

[0118] Embodiment 3. The composition of embodiment 1 or 2, wherein the Trm cells are negative for CD62L.

[0119] Embodiment 4. The composition of any one of the preceding embodiments, wherein the Trm cells are derived from tissue-draining lymph nodes or tumor-draining lymph nodes.

[0120] Embodiment 5. The composition of any one of the preceding embodiments, further comprising a pharmaceutically acceptable delivery vehicle.

[0121] Embodiment 6. The composition of any one of the preceding embodiments, wherein the cell population is prepared by: isolating T cells from tissue-draining lymph nodes or tumordraining lymph nodes; isolating CD8+ CD69+ and CD 103+ Trm cells from the T cells; and expanding the Trm cells in a T cell media comprising one or more cytokines selected from IL-2, IL-7, IL- 15, TGFP, and IFNa.

[0122] Embodiment 7. The composition of embodiment 6, wherein the one or more cytokines consist of at least one of IL-7 and IL-15.

[0123] Embodiment 8. The composition of embodiment 6, wherein the one or more cytokines consist of IL-2.

[0124] Embodiment 9. The composition of embodiment 6 or 7, wherein the IL-7 is at a concentration of between about 5 and about 100 ng / mL.

[0125] Embodiment 10. The composition of embodiment 6 or 7, wherein the IL-15 is at a concentration of between about 5 and about 200 ng / mL.

[0126] Embodiment 11. The composition of embodiment 6 or 8, wherein the IL-2 is at a concentration of between about 1 and about 100 ng / mL.

[0127] Embodiment 12. The composition of embodiment 6, wherein the TGF0 is at a concentration of between about 0.5 and about 20 ng / mL.

[0128] Embodiment 13. The composition of embodiment 6, wherein the IFNa is at a concentration of between about 0.001 and about 2 ng / mL.Atty. Dkt. No. 178981.00044

[0129] Embodiment 14. The composition of any one of embodiments 6-13, wherein the step of expanding the Trm cells comprises: seeding the Trm cells in the T cell media, wherein the T cell media comprises an anti-CD3 / CD28 agent; culturing the cells for between about 5 and about 21 days; refreshing the T cell media with the anti-CD3 / CD28 agent; and supplementing the cells with about 50% fresh T cell media comprising an about 2X concentration of the one or more cytokines.

[0130] Embodiment 15. The composition of embodiment 14, wherein the anti-CD3 / CD28 agent comprises an anti-CD3 / CD28 bead; wherein the bead is at a concentration of about three beads per cell.

[0131] Embodiment 16. The composition of embodiment 14 or 15, wherein the step of culturing the cells is done for about seven days.

[0132] Embodiment 17. The composition of any one of embodiments 6-16, wherein the step of isolating the Trm cells from the CD8+ T cells comprises:

[0133] incubating the CD8+ T cells in an antibody mix comprising a CD8 antibody, a CD69 antibody, and a CD 103 antibody; wherein each antibody is conjugated to a dye; and wherein each dye is different; and sorting the cells to identify CD8+ CD69+ CD 103+ Trm cells.

[0134] Embodiment 18. The composition of embodiment 17, wherein the antibody mix further comprises a CD62L antibody and a CD45RO antibody; wherein each antibody is conjugated to a dye; and wherein the step of sorting the cells further comprises identifying CD62L- and CD45RO+.

[0135] Embodiment 19. The composition of any one of embodiments 16-18, wherein the step of purifying the CD8+ T cells from the tissue-draining lymph nodes or tumor-draining lymph nodes comprises: incubating a sample comprising the lymph nodes, collagenase IV, and HBSS medium; neutralizing the sample with sterile PBS and 2% FBS; filtering the sample; spinning the sample and discarding a supernatant; and suspending the cell in sterile PBS and 2% FBS.

[0136] Embodiment 20. The composition of any one of embodiments 1-19, wherein the Trm cells are derived from a human subject.

[0137] Embodiment 21. The composition of any one of embodiments 1-20, wherein the Trm cells are derived from a subject that has a cancer.

[0138] Embodiment 22. The composition of embodiment 20 or 21, wherein the Trm cells exhibit at least 10% clonal overlap with tumor-infdtrating lymphocytes (TILs) from at least one tumor in the subject.Atty. Dkt. No. 178981.00044

[0139] Embodiment 23. The composition of any one of embodiments 6-22, wherein after the step of isolating CD8+ CD69+ and CD 103+ Trm cells from the T cells, the Trm cells are at least 90% pure.

[0140] Embodiment 24. The composition of any one of embodiments 6-23, wherein the step of expanding the Trm cells expands the Trm cells at least three-fold.

[0141] Embodiment 25. The composition of any one of embodiments 6-24, wherein after the step of expanding the Trm cells, at least 25% of the Trm cells are CD103+CD69+.

[0142] Embodiment 26. The composition of any one of embodiments 6-25, wherein after the step of expanding the Trm cells, at least 25% of the Trm cells produce Granzyme B and IFN-y when contacted with a mitogen or an anti-CD3 agent.

[0143] Embodiment 27. A method for treating a subject in need thereof with therapeutic Trm cells, the method comprising administering to the subject an effective amount of the composition of any one of embodiments 1-26.

[0144] Embodiment 28. The method of embodiment 27, wherein the subject has a cancer.

[0145] Embodiment 29. The method of embodiment 28, wherein the cancer is melanoma.

[0146] Embodiment 30. The method of any one of embodiments 27-29, wherein the composition comprises between about 106and about 109Trm cells.

[0147] Embodiment 31. The method of any one of embodiments 27-30, further comprising administering to the subject at least one of an IL-2 / anti-IL-2 complex therapy, an IL- 15, and an IL-7 therapy.

[0148] Embodiment 32. The method of any one of embodiments 27-30, further comprising administering an additional anti-cancer therapy to the subject.

[0149] Embodiment 33. A method for preparing therapeutic tissue-resident memory T cells (Trm), the method comprising: isolating T cells from lymph nodes, isolating CD8+ CD69+ CD103+ CD62L- Trm cells from the T cells; and expanding the Trm cells in a T cell media comprising one or more cytokines selected from IL-2, IL-7, IL-15, TGFp, and IFNa.

[0150] Embodiment 34. The method of embodiment 33, wherein the IL-7 is at a concentration of between about 5 and about 100 ng / mL.

[0151] Embodiment 35. The method of embodiment 33 or 34, wherein the IL-15 is at a concentration of between about 5 and about 200 ng / mL.Atty. Dkt. No. 178981.00044

[0152] Embodiment 36. The method of any one of embodiments 33-35, wherein the IL-2 is at a concentration of between about 1 and about 100 ng / mL.

[0153] Embodiment 37. The method of any one of embodiments 33-36, wherein the TGF0 is at a concentration of between about 0.5 and about 20 ng / mL.

[0154] Embodiment 38. The method of any one of embodiments 33-37, wherein the IFNa is at a concentration of between about 0.001 and about 2 ng / mL.

[0155] Embodiment 39. The method of any one of embodiments 33-35, wherein the one or more cytokines consist of at least one of IL-7 and IL-15.

[0156] Embodiment 40. The method of embodiment 33 or 36, wherein the one or more cytokines consist of IL-2.

[0157] Embodiment 41. The method of any one of embodiments 33-40, wherein the step of expanding the Trm cells comprises: seeding the cells in the T cell media, wherein the T cell media comprises an anti-CD3 / CD28 agent; culturing the cells for between about 5 and about 21 days; refreshing the T cell media with the anti-CD3 / CD28 agent; and supplementing the cells with about 50% fresh T cell media comprising an about 2X concentration of the cytokines.

[0158] Embodiment 42. The method of embodiment 41, wherein the anti-CD3 / CD28 agent comprises an anti-CD3 / CD28 bead; wherein the bead is at a concentration of about three beads per cell.

[0159] Embodiment 43. The method of any one of embodiments 33-42, wherein the step of culturing the cells is done for about seven days.

[0160] Embodiment 44. The method of any one of embodiments 33-43, wherein the step of isolating the Trm cells from the CD8+ T cells comprises:

[0161] incubating the CD8+ T cells in an antibody mix comprising a CD8 antibody, a CD69 antibody, and a CD103 antibody; wherein each antibody is conjugated to a dye; and wherein each dye is different; and sorting the cells to identify CD8+ CD69+ CD 103+ Trm cells.

[0162] Embodiment 45. The method of embodiment 44, wherein the antibody mix further comprises a CD62L antibody and a CD45RO antibody; wherein each antibody is conjugated to a dye; and wherein the step of sorting the cells further comprises identifying Trm cells that are additionally CD62L- and CD45RO+.

[0163] Embodiment 46. The method of any one of embodiments 33-45, wherein the step of isolating the CD8+ T cells from lymph nodes comprises: incubating a sample comprising theAtty. Dkt. No. 178981.00044lymph nodes, collagenase IV, and HBSS medium; neutralizing the sample with sterile PBS and 2% FBS; filtering the sample; spinning the sample and discarding a supernatant; and suspending the cell in sterile PBS and 2% FBS.

[0164] Embodiment 47. A population of therapeutic Trm cells prepared by the method of any one of embodiments 33-46.

[0165] Embodiment 48. A kit comprising: a CD8 antibody; a CD69 antibody;

[0166] a CD103 antibody; and at least one of IL-7; IL-15; IL-2; TGF[3; and IFNa.

[0167] Embodiment 49. The kit of embodiment 48, further comprising: a CD45RO antibody; and a CD62L antibody.

Claims

Atty. Dkt. No. 178981.00044CLAIMSWe claim:

1. A composition comprising a cell population comprising tissue-resident memory T (Trm) cells expressing CD8, CD69 and CD103.

2. The composition of claim 1, wherein the Trm cells express CD45RO.

3. The composition of claim 1, wherein the Trm cells are negative for CD62L.

4. The composition of claim 1, wherein the Trm cells are derived from tissue-draining lymph nodes or tumor-draining lymph nodes.

5. The composition of claim 1, further comprising a pharmaceutically acceptable delivery vehicle.

6. The composition of claim 1, wherein the cell population is prepared by:isolating T cells from tissue-draining lymph nodes or tumor-draining lymph nodes; isolating CD8+ CD69+ and CD 103+ Trm cells from the T cells; andexpanding the Trm cells in a T cell media comprising one or more cytokines selected from IL-2, IL-7, IL- 15, TGF0, and IFNa.

7. The composition of claim 6, wherein the one or more cytokines consist of at least one of IL-7 and IL- 15.

8. The composition of claim 6, wherein the one or more cytokines consist of IL-2.

9. The composition of claim 6, wherein the IL-7 is at a concentration of between about 5 and about 100 ng / mL.

10. The composition of claim 6, wherein the IL-15 is at a concentration of between about 5 and about 200 ng / mL.Atty. Dkt. No. 178981.0004411. The composition of claim 6, wherein the IL-2 is at a concentration of between about 1 and about 100 ng / mL.

12. The composition of claim 6, wherein the TGF0 is at a concentration of between about 0.5 and about 20 ng / mL.

13. The composition of claim 6, wherein the IFNa is at a concentration of between about 0.001 and about 2 ng / mL.

14. The composition of claim 6, wherein the step of expanding the Trm cells comprises: seeding the Trm cells in the T cell media, wherein the T cell media comprises an anti- CD3 / CD28 agent;culturing the cells for between about 5 and about 21 days;refreshing the T cell media with the anti-CD3 / CD28 agent; andsupplementing the cells with about 50% fresh T cell media comprising an about 2X concentration of the one or more cytokines.

15. The composition of claim 14, wherein the anti-CD3 / CD28 agent comprises an anti-CD3 / CD28 bead; wherein the bead is at a concentration of about three beads per cell.

16. The composition of claim 14, wherein the step of culturing the cells is done for about seven days.

17. The composition of claim 6, wherein the step of isolating the Trm cells from the CD8+ T cells comprises:incubating the CD8+ T cells in an antibody mix comprising a CD8 antibody, a CD69 antibody, and a CD 103 antibody; wherein each antibody is conjugated to a dye; and wherein each dye is different; andsorting the cells to identify CD8+ CD69+ CD 103+ Trm cells.Atty. Dkt. No. 178981.0004418. The composition of claim 17, wherein the antibody mix further comprises a CD62L antibody and a CD45RO antibody; wherein each antibody is conjugated to a dye; and wherein the step of sorting the cells further comprises identifying CD62L- and CD45RO+.

19. The composition of claim 16, wherein the step of purifying the CD8+ T cells from the tissue-draining lymph nodes or tumor-draining lymph nodes comprises:incubating a sample comprising the lymph nodes, collagenase IV, and HBSS medium; neutralizing the sample with sterile PBS and 2% FBS;filtering the sample;spinning the sample and discarding a supernatant; andsuspending the cell in sterile PBS and 2% FBS.

20. The composition of claim 1, wherein the Trm cells are derived from a human subject.

21. The composition of claim 1, wherein the Trm cells are derived from a subject that has a cancer.

22. The composition of claim 20, wherein the Trm cells exhibit at least 10% clonal overlap with tumor-infiltrating lymphocytes (TILs) from at least one tumor in the subject.

23. The composition of claim 6, wherein after the step of isolating CD8+ CD69+ and CD 103+ Trm cells from the T cells, the Trm cells are at least 90% pure.

24. The composition of claim 6, wherein the step of expanding the Trm cells expands the Trm cells at least three-fold.

25. The composition of claim 6, wherein after the step of expanding the Trm cells, at leastAtty. Dkt. No. 178981.0004426. The composition of claim 6, wherein after the step of expanding the Trm cells, at least 25% of the Trm cells produce Granzyme B and IFN-y when contacted with a mitogen or an anti-CD3 agent.

27. A method for treating a subject in need thereof with therapeutic Trm cells, the method comprising administering to the subject an effective amount of the composition of claim 1.

28. The method of claim 27, wherein the subject has a cancer.

29. The method of claim 28, wherein the cancer is melanoma.

30. The method of claim 27, wherein the composition comprises between about 106and about 109Trm cells.

31. The method of claim 27, further comprising administering to the subject at least one of an IL-2 / anti-IL-2 complex therapy, an IL-15, and an IL-7 therapy.

32. The method of claim 27, further comprising administering an additional anti-cancer therapy to the subject.

33. A method for preparing therapeutic tissue-resident memory T cells (Trm), the method comprising:isolating T cells from lymph nodes,isolating CD8+ CD69+ CD 103+ CD62L- Trm cells from the T cells; andexpanding the Trm cells in a T cell media comprising one or more cytokines selected from IL-2, IL-7, IL-15, TGF0, and IFNa.

34. The method of claim 33, wherein the IL-7 is at a concentration of between about 5 and about 100 ng / mL.Atty. Dkt. No. 178981.0004435. The method of claim 33, wherein the IL-15 is at a concentration of between about 5 and about 200 ng / mL.

36. The method of claim 33, wherein the IL-2 is at a concentration of between about 1 and about 100 ng / mL.

37. The method of claim 33, wherein the TGFp is at a concentration of between about 0.5 and about 20 ng / mL.

38. The method of claim 33, wherein the IFNa is at a concentration of between about 0.001 and about 2 ng / mL.

39. The method of claim 33, wherein the one or more cytokines consist of at least one of IL-7 and IL-15.

40. The method of claim 33, wherein the one or more cytokines consist of IL-2.

41. The method of claim 33, wherein the step of expanding the Trm cells comprises:seeding the cells in the T cell media, wherein the T cell media comprises an anti- CD3 / CD28 agent;culturing the cells for between about 5 and about 21 days;refreshing the T cell media with the anti-CD3 / CD28 agent; andsupplementing the cells with about 50% fresh T cell media comprising an about 2X concentration of the cytokines.

42. The method of claim 41, wherein the anti-CD3 / CD28 agent comprises an anti-CD3 / CD28 bead; wherein the bead is at a concentration of about three beads per cell.

43. The method of claim 33, wherein the step of culturing the cells is done for about seven days.Atty. Dkt. No. 178981.0004444. The method of claim 33, wherein the step of isolating the Trm cells from the CD8+ T cells comprises:incubating the CD8+ T cells in an antibody mix comprising a CD8 antibody, a CD69 antibody, and a CD 103 antibody; wherein each antibody is conjugated to a dye; and wherein each dye is different; andsorting the cells to identify CD8+ CD69+ CD103+ Trm cells.

45. The method of claim 44, wherein the antibody mix further comprises a CD62L antibody and a CD45RO antibody; wherein each antibody is conjugated to a dye; and wherein the step of sorting the cells further comprises identifying Trm cells that are additionally CD62L- and CD45RO+.

46. The method of claim 33, wherein the step of isolating the CD8+ T cells from lymph nodes comprises:incubating a sample comprising the lymph nodes, collagenase IV, and HBSS medium; neutralizing the sample with sterile PBS and 2% FBS;filtering the sample;spinning the sample and discarding a supernatant; andsuspending the cell in sterile PBS and 2% FBS.

47. A population of therapeutic Trm cells prepared by the method of claim 33.

48. A kit comprising:a CD 8 antibody;a CD69 antibody;a CD 103 antibody; andat least one of IL-7; IL- 15; IL-2; TGF0; and IFNa.

49. The kit of claim 48, further comprising:a CD45RO antibody; anda CD62L antibody.

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