Adoptive T cell transfer in lymphocyte-replete hosts is mediated by transient STAT5B activation during engraftment

Abstract

The present invention relates to a method for promoting functional engraftment of engineered T cells that circumvents the need for lymphodepletion. The present invention relates to a method for functional engraftment of cells in the absence of lymphodepletion, comprising the step of transient activation of Stat5 signaling by transfection of T cells with a plasmid or RNA encoding a constitutively active Stat5 molecule or a constitutively active cytokine receptor that activates STAT5 signaling, Stat3, Stat5a, Stat5b, interleukin-7 receptor, interleukin-2 receptor, interleukin-9 receptor, interleukin-15 receptor, and engineered orthogonal receptors, prior to adoptive transfer.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention generally relates to methods for promoting functional engraftment of engineered T cells that avoid the need for lymphocyte depletion. All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes, and are incorporated by reference to the same extent as if each individual publication, patent, patent application, or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. The citation of a reference herein should not be construed as an admission that it is prior art to the present invention.

[0002] Priority to related applications This application claims the benefit of U.S. Provisional Application No. 63 / 440,728, filed January 24, 2023. The entire contents of the above-identified application are incorporated herein by reference in their entirety.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made in part with government support under NCI P30CA138313. The government has certain rights in this invention. [Background technology]

[0004] Background of the Invention The success of adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TILs), chimeric antigen receptors (CARs), or T cell receptor (TCR)-engineered T cells relies on preconditioning regimens designed to lymphodeplete patients prior to infusion (1, 2). Lymphodepleting regimens, such as chemotherapy and total body irradiation (TBI), eliminate endogenous cytokine-responsive cells that act as cytokine sinks (3). Furthermore, lymphodepletion eradicates suppressor cell populations within the host immune system by eliminating immunosuppressive cell types, including regulatory T cells (4, 5) and myeloid-derived suppressor cells (MDSCs) (6). Patients who undergo lymphodepleting regimens prior to ACT enjoy better response rates and improved survival and antitumor immunity (1, 7). However, although these regimens are important for maximizing clinical benefit, the agents used, such as fludarabine and cyclophosphamide, are largely nonspecific agents with significant toxicity profiles ( 8 , 9 ).

[0005] Lymphocyte-depleting regimens can induce various side effects, including leukopenia, pulmonary veno-occlusive disease, and reactive myelopoiesis (10). These side effects contribute to patient exclusion and have hindered the widespread adoption of clinical trials utilizing engineered T cell-based therapeutics. In addition to significant side effects, physiological responses to lymphodepletion can adversely affect the efficacy of ACT in eligible patients (11). Furthermore, management of these toxicities often requires prolonged inpatient support and hospitalization, causing extreme financial burdens for both patients and healthcare providers. Despite the fact that lymphodepleting regimens have many drawbacks, it is clear that they promote the functional engraftment and persistence of transferred T cells (12, 13), while ACT without prior lymphodepletion inhibits the engraftment of adoptively transferred T cells (14), resulting in reduced clinical benefit. There is a need in the art for a cell-autonomous method to promote functional engraftment of engineered T cells that eliminates the need for lymphodepletion. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Dudley ME et al., J Immunother. (2002)25(3):243~51 [Non-patent document 2] Dudley ME et al., Science (2002) 298(5594):850-4 [Non-patent document 3] Ramos CA et al. Molecular Therapy (2018) 26(12):2727~37 Summary of the Invention [Means for solving the problem]

[0007] Summary of the Invention The present invention relates to a method for functional engraftment of cells in the absence of lymphodepletion, comprising the step of transient activation of Stat5 signaling by transfection of T cells with a plasmid or RNA encoding a constitutively active Stat5 molecule or a constitutively active cytokine receptor that activates STAT5 signaling, Stat3, Stat5a, Stat5b, interleukin-7 receptor, interleukin-2 receptor, interleukin-9 receptor, interleukin-15 receptor, and engineered orthogonal receptors, prior to adoptive transfer.

[0008] The present invention also relates to a method for reducing cytokine release syndrome (CRS) in a patient undergoing cancer treatment, comprising administering, prior to adoptive transfer, T cells transiently transfected with RNA or DNA encoding an activated form of Stat5 or a cytokine receptor.

[0009] The present invention further relates to a method for increasing cell engraftment efficiency, which comprises the step of transient activation of Stat5 signaling by transfection of DNA or RNA encoding activated Stat5 or a cytokine receptor prior to adoptive transfer.

[0010] Furthermore, the present invention relates to a method for reducing IL-6 production in a patient in need thereof, comprising administering to the patient, prior to adoptive transfer, transient activation of Stat5 signaling by transfection of DNA or RNA encoding activated Stat5 or a cytokine receptor. [Brief explanation of the drawings]

[0011] [Figure 1] Figure 1 shows that transient expression of Stat5b* promotes CD8+ T cell engraftment in the absence of lymphodepletion. a) Experimental schematic showing three groups of animals undergoing adoptive transfer (ACT) using either conventional 5Gy lymphodepletion, no lymphodepletion followed by ACT, or no lymphodepletion followed by ACT with cells transfected with a plasmid encoding STAT5* immediately before ACT. b) Representative ventral images of ffLuc-Thy1.1-manipulated CD8+ T cells 3 days after ACT. c) Animals were measured for 29 days after ACT, and the intensity of the signal emitted by ffLuc-Thy1.1+ T cells was expressed as luminescence. Quantification of total (dorsal + ventral) luminescence is plotted (n = 10 for LD and LD pT-CMV-mStat5b*, n = 5 for no LD and Stat5b* mRNA). ACT using Stat5* results in significantly more efficient engraftment when compared to conventional methods of lymphodepletion before ACT. d) Peripheral cytokine levels 24 hours before and 4 days after ACT. Cytokines thought to be involved in ACT-related toxicity in humans are shown. *p<0.05 by unpaired Student's t-test (d). Error bars indicate SEM.

[0012] [Figure 2]Figure 2 shows that Stat5 drives superior recall responses in the absence of lymphodepletion. a) Experimental scheme of b-c. b) Representative images of ffLuc-Thy1.1 engineered CD8+ T cells 40 days after ACT and 24 hours after rechallenge with LLC-ova cells. c) Animals were measured 24 and 72 hours after LLC-ova challenge, and the intensity of the signal emitted by ffLuc-Thy1.1 transfected T cells was expressed as luminescence. d) Tumor growth kinetics and e) survival of mice challenged with LLC-ova.

[0013] [Figure 3] Figure 3 shows that CD62L re-expression is reduced by transient expression of Stat5*. A) Schematic of the experiment. Mice were either transferred using conventional lymphodepletion or with transient expression of Stat5*. B and C) Representative plots and graph summary of flow cytometry data comparing CD62L expression, and F) Schematic of a competition study examining whether the effect of lymphodepletion on CD62L expression outweighs the effect of transient Stat5* expression during engraftment. G and E) Representative plots and summary of data comparing the phenotype of GFP+ and thy1.1+ (Stat5*-transfected) cells from the same spleen.

[0014] [Figure 4] Figure 4 shows that ATC without lymphodepletion using transient Stat5* induces a unique phenotype of tumor-infiltrating T cells. A) Schematic of the experiment: Subcutaneous tumors were initiated prior to ACT. Tumors were harvested 6 days after adoptive transfer, and the phenotype of engrafted cells was assessed by flow cytometry for markers associated with T cell exhaustion. B), C) PD1 levels are suppressed, but PD1+Tim3+ double-positive cells remain unchanged due to the strong induction of Tim3 in Stat5*-engrafted cells, despite no change in Lag-3 expression.

[0015] [Figure 5]Figure 5 shows that adoptive transfer using transient Stat5b* without lymphodepletion results in functional tumor control. a) Experimental scheme of b-e. b) Tumor growth kinetics and c) survival of mice bearing LLC-ova tumors injected with OT1+ T cells engrafted via conventional lymphodepletion (blue) or Stat5b* (red) (n = 5 mice per group). d) Tumor growth kinetics and e) survival of mice bearing B16-F10 tumors injected with PMEL+ T cells engrafted via conventional lymphodepletion (blue) or Stat5b* (red) (n = 5 mice per group). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by linear regression of tumor growth and log-rank of survival, Mantel-Cox test (c, e). Error bars indicate SEM.

[0016] [Figure 6] FIG. 6 shows that cryopreserved human T cell products retain and express transfected mRNA after long-term cryopreservation.

[0017] [Figure 7] FIG. 7 shows that cryopreserved Stat5b* mRNA-modified T cells are highly functional and engraft efficiently in lymphocyte-replete mice. DETAILED DESCRIPTION OF THE INVENTION

[0018] Detailed Description of the Invention It should be understood that the description of the present invention is simplified to illustrate elements relevant to a clear understanding of the invention and, for clarity, excludes many other elements found in typical pharmaceutical compositions. Those skilled in the art will recognize that other elements and / or steps are desirable and / or required in practicing the present invention. However, because such elements and steps are well known in the art and do not facilitate a better understanding of the present invention, a discussion of such elements and steps is not provided herein. The disclosure herein relates to all such variations and modifications to such elements and methods known to those skilled in the art. Furthermore, the embodiments specified and illustrated herein are for illustrative purposes only and are not intended to be exclusive or limiting in describing the present invention.

[0019] Functional engraftment is characterized by the expansion and persistence of T lymphocytes and antitumor function. Specific mechanisms contributing to effective engraftment are thought to be related to homeostatic T cell proliferation. The increased availability of homeostatic cytokines that occurs after lymphodepletion is associated with the proliferation of γ-antigens, including interleukin-2 (IL-2), IL-7, and IL-15. c This results in enhanced T cell proliferation, which is thought to be regulated via cytokines. These cytokines play a pivotal role in regulating T cell homeostasis and inflammatory responses through key signaling pathways. Current lymphodepletion regimens eliminate cellular cytokine sinks, thus increasing the availability of homeostatic cytokines to support the efficacy of adoptively transferred T cells (3).

[0020] Traditionally, the cytokines IL-2, IL-7, and IL-15 signal through the JAK-STAT pathway, primarily via activation of JAK1 / 3 and STAT5, known mediators of T cell antitumor immunity (15, 16). In response to cytokine stimulation, the C-terminal tyrosines (mY699, hY694) of Stat5b are phosphorylated, mediating Stat5b activation and dimerization, thus promoting nuclear translocation and DNA binding (17, 18). We chose to utilize a form of Stat5b* that mimics the structural aspects of phosphorylated Stat5b independently of tyrosine phosphorylation by substituting histidine 298 for arginine, referred to in this manuscript as Sta5b*.

[0021] We believe that transient Stat5b activation during the early phase of adoptive transfer eliminates the need for prior lymphodepletion. We demonstrate that transient expression of an active form of Stat5 during the early phase of adoptive transfer results in functional T cell engraftment and eliminates the spike in IL-6 typically observed during the engraftment period, a driving factor for cytokine release syndrome (CRS). We observed that cells engrafted in lymphocyte-replete mice using Stat5b* had superior recall responses and improved tumor control in multiple immunocompetent tumor models. These results indicate that this novel engraftment strategy, which removes lymphodepletion regimens from ACT protocols and instead relies on transient Stat5b* expression during the engraftment phase, may also offer an opportunity to improve T cell function after ACT.

[0022] Thus, we discovered that transient expression of constitutively active STAT5 (STAT5-CA) during adoptive transfer obviates the need for prior lymphodepletion, results in long-term functional engraftment, and improves tumor control in immunocompetent cancer models.

[0023] In one embodiment, the inventors discovered that transient transfection of CAR-T cells with constitutively active STAT5 immediately prior to adoptive transfer allows functional engraftment of the cells in the absence of lymphodepletion.

[0024] In one embodiment of the present invention, we have found that transient expression (via DNA, mRNA, or cirRNA) of constitutively active receptors that activate signaling pathways such as Jak / Stat and other common gamma-chain cytokine receptors promotes T cell engraftment and function. This is based on the demonstration that cell-autonomous adoptive transfer of functional T cells uses methods that do not require genetic modification of T cells or lymphodepletion of the host. This reasonably extends to other constitutively active receptors that can be transiently delivered to T cells to safely enhance adoptive transfer without the risk of transformation. This also extends to the use of this transient expression methodology to enhance adoptive transfer or T cell function in situations of mild lymphodepletion, reduced lymphodepletion, or conventional lymphodepletion.

[0025] These constitutively active molecules may include, but are not limited to, constitutively active forms of Stat3, Stat5a, Stat5b, interleukin 7 receptor, interleukin 2 receptor, interleukin 9 receptor, interleukin 15 receptor, and engineered orthogonal receptors, In another embodiment of the present invention, the inventors discovered that adoptive transfer of T cells without prior lymphodepletion results in reduced production of several cytokines, including IL6, which has been characteristically identified as antagonizing therapeutic benefit by driving cytokine release syndrome (CRS) and contributing to T cell dysfunction [2].

[0026] In a further embodiment of the present invention, the inventors have discovered that transient transfection of T cells with STAT5-CA prior to adoptive transfer increases engraftment efficiency, such that a much lower dose of cells can be administered, thereby shortening product manufacturing time and further reducing the risk of side effects.

[0027] Another embodiment of the present invention provides a cryopreserved T cell product that is ready for infusion without host lymphodepletion. The T cell manufacturing process requires collection of T cells from the patient by leukapheresis, activation and lentiviral transduction of the cells with a CAR-expressing construct, and expansion of the transduced cells for 2-3 weeks. After production, the cells are cryopreserved and transported to the point-of-care where they are thawed and infused.

[0028] The success of adoptive cell therapy using tumor-infiltrating lymphocytes (TILs), CARs, or T cell receptor (TCR)-engineered T cells relies on efficient engraftment of the cellular product. Historically, preconditioning regimens designed to lymphodeplete patients prior to infusion have been used to promote engraftment of adoptively transferred cells. Lymphodepletion (LD) is now considered essential for effective CAR-T therapy and is a universal component of adoptive T cell transfer protocols. However, while these regimens have historically been considered critical for maximizing clinical benefit, the drugs used (fludarabine and cyclophosphamide) are largely nonspecific agents with significant toxicity profiles. Replacing, simplifying, or improving the LD process has been a major priority for pharmaceutical companies over the past decade. We developed an approach that relies on transfection of manufactured T cells with mRNA encoding a constitutively active mutein of Stat5b (Stat5b*) prior to adoptive transfer. We observe that this transient activation of the Stat5b pathway "trick" T cells into thinking they are entering a lymphodepleted host, resulting in efficient functional engraftment of the T cell product, which proves superior to traditional lymphodepletion-based protocols. This technique potentially eliminates the need for LDs and revolutionizes T cell therapy. [Example]

[0029] The following examples further describe and demonstrate specific embodiments within the scope of the present invention. Techniques and formulations can generally be found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA). The present disclosure is further illustrated by the following examples, which should not be construed as limiting the scope or spirit of the disclosure to the specific procedures described herein. It should be understood that the examples are provided to illustrate particular embodiments, and no limitation on the scope of the disclosure is thereby intended. It should be understood that various other embodiments, modifications, and equivalents thereof, which may suggest themselves to those skilled in the art, may be employed without departing from the spirit of the present disclosure and / or the scope of the appended claims.

[0030] General Materials and Methods molecular biology Transfection-grade plasmids were prepared using endotoxin-free buffer and the ZymoPURE II Plasmid Maxiprep Kit (ZYMO Research, USA) according to the manufacturer's protocol. Plasmid vectors pT-effluc-thy1.1 20 and pRP--CAG>hyPBase 29, pTPB-CMV-GFP, pCMV-mStat5b*, pT7-mStat5b*, and pT7-hStat5b* were synthesized by Vector Builder Biosciences (Chicago, IL). All plasmid vectors were verified by DNA sequencing. Transfection-grade plasmids were prepared using endotoxin-free buffer and the ZymoPURE II Plasmid Maxiprep Kit (ZYMO Research, USA) according to the manufacturer's protocol.

[0031] mouse OT1 (C57BL / 6-Tg(TcraTcrb)1100Mjb / J), PMEL (B6.Cg-Thy1a / Cy Tg(TcraTcrb)8Rest / J), and B6 albino (B6(Cg)-Tyrc-2J / J) mice were obtained from the Jackson Laboratory. Luciferase (β-actin-luc) was obtained from Taconic Bioscience. All animal experiments were approved by the Medical University of South Carolina (MUSC) Institutional Animal Care and Use Committee, and the MUSC Laboratory Animal Resources Department maintained all mice.

[0032] cell culture B16-F10 and LLC-OVA tumor lines, as well as HEK293T cells, were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS. All cell lines were determined to be mycoplasma-free in December 2021. OT-1 T cells, human CD4+ T cells, and human CD8+ T cells were maintained in RPMI supplemented with 10% FBS, 300 mg / L L-glutamine, 100 units / mL penicillin, 100 μg / mL streptomycin, 1 mM sodium pyruvate, 100 μM NEAA, 1 mM HEPES, and 55 μM 2-mercaptoethanol. For activation and expansion of OT-1 and PMEL T cells, whole splenocytes from OT1 or PMEL mice were activated with 1 μg / mL OVA 257-264 peptide or 1 μg / mL glycoprotein 100 (gp100). For Luc+ T cell isolation, CD8+ lymphocytes were purified from the spleen using a MACS Mouse CD8a+ T Cell Isolation Kit (Miltenyi Biotec, Auburn, CA). Purified CD8+ lymphocytes were then activated with anti-CD3e 2.5 μg / ml (BD Bioscience, San Jose, CA) and anti-CD28 2 μg / ml (BD Bioscience, San Jose, CA). All T cells were expanded for 3 days using 200 U / mL rhIL-2 (NCI). T cells were split on day 3 and expanded in rhIL-2 or IL-15 (50 ng / mL) until day 7.

[0033] In vitro transcription mRNA was transcribed using the mMessage mMachine T7 ULTRA Transcription Kit (Ambion, Life Technologies, Grand Island, NY) using the protocol provided by the manufacturer. mRNA was resuspended in nuclease-free water and stored at -80°C prior to transfection.

[0034] Adoption model After in vitro expansion, lymphocytes were briefly washed in PBS by centrifugation and transfected with the Neon (Life Technologies, Grand Island, NY) transfection system according to the manufacturer's instructions for mouse T cells. In vitro-expanded OT-1 T cells were prepared for adoptive transfer by transfection with 5 μg pRP--CAG>hyPBase and 10 μg pCMV-EL-Thy1.1 or 10 μg pT-GFP and 10 μg pCMV-mStat5b*. For mRNA transfection, OT-1 T cells were transfected with the indicated concentrations of GFP mRNA or 20 μg Stat5b* mRNA using the previously described setup 30. Transfected T cells were then placed in complete T cell medium for 1 hour, after which the cells were resuspended in PBS and directly transferred into recipient mice. Luc+ T cells were transfected with 20 μg Stat5b* mRNA. Prior to adoptive transfer, mice were preconditioned by exposure to 5 Gy of lymphodepleting radiation using a cesium irradiator or with cyclophosphamide (Thermo Scientific, Waltham, MA) in PBS administered intraperitoneally at a dose of 200 mg / kg, unless otherwise indicated. T cells were transfected as described above, allowed to recover in complete TCM for 1 hour, and then adoptively transferred via IP injection. For ex vivo analysis of ELThy1.1- or GFP-transferred T cells, mice were sacrificed 3 days after transfer, and organs were processed into single-cell suspensions for FACS analysis.

[0035] Tumor cell challenge For the primary challenge, mice were injected subcutaneously (sc) with 2.5 x 105 LLC-ova tumor cells 40 days after the initial adoptive transfer of OT-1+ T cells.

[0036] Tumor model For adoptive cell therapy experiments, LLC-ova adenocarcinoma or B16-F10 melanoma was established subcutaneously by injecting 2.5 x 10 cells into the right flank of male B6(Cg)-Tyrc-2J / J mice. Tumor-bearing hosts were irradiated with 5 Gy 24 h prior to T cell transfer. After 7 days of tumor growth, mice were transfected with 1 x 10 OT1 or PMEL T cells using the Neon transfection system before being injected via IP injection in 100 μL of phosphate-buffered saline. Tumor growth was measured every other day with a caliper, and survival was monitored at the experimental endpoint of tumor growth >400 mm2. For ex vivo analysis of ff-Luc-Thy1.1-transfected T cells, mice were sacrificed 5 days after transfer, and organs were processed into single-cell suspensions for FACS analysis.

[0037] Bioluminescence imaging Mice were anesthetized using isoflurane and IP injected with luciferin substrate (Perkin Elmer, Waltham, MA) at a standard concentration of 150 mg / kg in PBS. Approximately 10 minutes after luciferase injection, mice were imaged on an AMI-HT (Spectral Imaging, Tucson, AZ). All data shown represent the average luminescence observed by summing dorsal and ventral measurements obtained from the same region of interest drawn over the trunk and head of each individual mouse.

[0038] Serum cytokine assessment Blood was collected by retro-orbital vein bleeding into Microvette CD300 EDTA potassium collection tubes (Sarstedt, Newton, NC), and serum was measured using the Mouse Cytokine Pro-inflammatory Focused 10-Plex Discovery Assay (MDF10) (Eve Biotechnologies, Calgary, AB, Canada).

[0039] Flow cytometry Prior to extracellular staining, cells were stained with the Live / Dead Fixable Aqua Dead Cell Stain Kit (Invitrogen). Fluorescent dye-conjugated antibodies were purchased from Biolegend (San Diego, CA), eBioscience (San Diego, CA), or BD Pharmigen (Mountain View, CA). Cells were stained extracellularly with CD90.1 (HIS51)-PE, CD8a (53-6.7)-PerCP-eFluor 710, CD44 (IM7)-PECy7, CD62L (MEL-14)-BV421, PD-1 (29F.1A12)-APC, Tim-3 (RMT3-23)-FITC, Lag-3 (C9B7W)-APC-eFluor 780, CD3 (145-2C11)-FITC, (GK1.5)-APC-eFluor 780, and CD25 (PC61.5)-PECy7, or isotype controls, followed by cell surface antibody staining in PBS containing 2% FBS. Antibody-stained cells were run directly on a CytoFLEX flow cytometer (Beckman Coulter Life Sciences, Indianapolis, IN), and analysis was performed with FlowJo software.

[0040] Nanostring gene expression analysis RNA isolation: Cells were immediately pelleted by centrifugation at 4°C and resuspended in the homogenization buffer containing thioglycerol provided in the purification kit. RNA was extracted with a Promega Maxwell RSC 16 using the Maxwell RSC simplyRNA Cells kit (catalog no. AS1390) according to the manufacturer's instructions, and RNA concentration was measured using a NanoDrop 8000.

[0041] Gene quantification: Direct quantification of selected transcripts was performed using 100 ng of total RNA with the Nanostring nCounter system using markers in the NS_MM_EXHAUSTION codeset. RCC files were imported into nSolver 4.0 software and analyzed using the default analysis pipeline for normalization, differential expression, and agglomerative clustering.

[0042] statistical analysis Using GraphPad Prism, p values ​​were calculated by one-way analysis of variance (ANOVA) with Dunnett's multiple comparison test, unpaired Student's t-test, or paired Student's t-test, as indicated in the figure legends. Tumor growth curves were analyzed by simple linear regression. A value of p<0.05 was considered significant. Values ​​of p<0.05 were ranked as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0043] Example 1 Stat5b* regulates CD8+ cells in the absence of lymphocyte depletion + Supports T cell engraftment material and method vector The plasmid vectors pT-effluc-thy1.1 (19) and pCMV-m7pB (20) have been previously described. pT-SB-Stat5b was synthesized by Vector Builder Biosciences (Chicago, IL). All plasmid vectors were verified by DNA sequencing. Transfection-grade plasmids were prepared using endotoxin-free buffer and the ZymoPURE II Plasmid Maxiprep Kit (ZYMO Research, USA) according to the manufacturer's protocol.

[0044] mouse OT1 (C57BL / 6-Tg(TcraTcrb)1100Mjb / J) mice and B6 albino (B6(Cg)-Tyr c-2J(J / J) mice were obtained from the Jackson Laboratory. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Medical University of South Carolina (MUSC), and the MUSC Department of Laboratory Animal Resources maintained all mice.

[0045] cell culture B16-F10 and LLC-OVA tumor lines were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS. All tumor lines were determined to be mycoplasma-free in December 2021. OT1 T cells were maintained in RPMI supplemented with 10% FBS, 300 mg / L L-glutamine, 100 units / mL penicillin, 100 μg / mL streptomycin, 1 mM sodium pyruvate, 100 μM NEAA, 1 mM HEPES, and 55 μM 2-mercaptoethanol. For activation and expansion of OT1 and PMEL T cells, whole splenocytes from OT1 or PMEL mice were activated with 1 μg / mL OVA 257-264 peptide or 1 μg / mL glycoprotein 100 (gp100) and expanded with 200 U / mL rhIL-2 (NCI) for 3 days. T cells were split on day 3 and expanded in rhIL-2 or IL-15 (50 ng / mL) until day 7. Human PBMCs were activated with CD3 / CD28 activators and expanded in 500 U of rhIL-2 (NCI), and cultures were maintained in RPMI supplemented with 10% FBS, 300 mg / L L-glutamine, 2 mM GlutaMAX, 100 units / mL penicillin, 100 μg / mL streptomycin, 50 μg / mL gentamicin, 25 mM HEPES, and 55 μM 2-mercaptoethanol.

[0046] Adoption model After in vitro expansion, lymphocytes were briefly washed in PBS by centrifugation and transfected with the Neon (Life Technologies, Grand Island, NY) transfection system according to the manufacturer's instructions for mouse T cells, as previously described (19). In vitro-expanded OT-1 T cells were prepared for adoptive transfer by transfection with 5 μg of pCMV-M7PB and 10 μg of pT-effluc-thy1.1 or 10 μg of pT-GFP and 10 μg of pSB-mSTAT5. Prior to adoptive transfer, mice were preconditioned by exposure to 5 Gy of lymphodepleting radiation using a cesium irradiator, unless otherwise indicated. T cells were transfected as described above, allowed to recover in complete TCM for 1 h, and then adoptively transferred via IP injection. For ex vivo analysis of ff-Luc-Thy1.1- or GFP-transferred T cells, mice were sacrificed 3 days after transfer, and organs were processed into single-cell suspensions for FACS analysis.

[0047] Antigen challenge In the primary challenge, mice received 2.5 × 10 5 LLC-ova tumor cells were injected subcutaneously (sc).

[0048] Tumor model For adoptive cell therapy experiments, male B6(Cg)-Tyr c-2J 2.5 x 10 5 LLC-ova adenocarcinoma or B16-F10 melanoma were established subcutaneously by injecting 1 × 10 cells, and tumor-bearing hosts were irradiated with 5 Gy 24 h prior to T cell transfer. After 7 days of tumor growth, 1 × 10 cells were transfected using the Neon transfection system before being injected into mice in 100 μL of phosphate-buffered saline via IP injection. 7 OT1 or PMEL T cells were transfected. Tumor growth was measured every other day with a caliper, and survival was defined as tumor growth ≥ 400 mm 2For ex vivo analysis of ff-Luc-Thy1.1 transferred T cells, mice were sacrificed 5 days after transfer and organs were processed into single cell suspensions for FACS analysis.

[0049] Bioluminescence imaging Mice were anesthetized using isoflurane and IP injected with luciferin substrate (Perkin Elmer, Waltham, MA) at a standard concentration of 150 mg / kg in PBS. Approximately 10 minutes after luciferase injection, mice were imaged on the AMI-HT. All data shown represent the average luminescence observed by summing dorsal and ventral measurements obtained from the same region of interest drawn over the trunk and head of each individual mouse.

[0050] Serum evaluation Blood was collected by retro-orbital vein bleeding into Microvette CD300 EDTA potassium collection tubes (Sarstedt, Newton, NC), and serum was measured using the Mouse Cytokine Pro-inflammatory Focused 10-Plex Discovery Assay (MDF10) (Eve Biotechnologies, Calgary, AB, Canada).

[0051] Flow cytometry Fluorochrome-conjugated antibodies with the following specificities were purchased from Biolegend (San Diego, CA), eBioscience (San Diego, CA), or BD Pharmigen (Mountain View, CA): CD90.1, CD8a, CD44, CD62L, PD-1, Tim-3, and Lag-3. Cell surface antibody staining was performed in PBS containing 2% FBS. Antibody-stained cells were analyzed using a CytoFLEX flow cytometer (Beckman Coulter Life Sciences, Indianapolis, IN), and analysis was performed using FlowJo software.

[0052] statistical analysis Using GraphPad Prism, p values ​​were calculated using one-way analysis of variance (ANOVA) with Dunnett's multiple comparison test, unpaired Student's t-test, or paired Student's t-test, as indicated in the figure legends. A value of p<0.05 was considered significant. Values ​​of p<0.05 were ranked as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0053] To assess the ability of T cells to engraft, we first modified murine OT-1 T cells with the previously described pT-effluc-thy1.1 transposon (21). The luciferase modification allowed us to track T cells in vivo and quantify engraftment and persistence by measuring luciferase activity over time. Using this model system, we transiently transfected our OT-1 T lymphocytes with a plasmid encoding a constitutively active version of murine Stat5b (pCMV-mStat5b*) prior to adoptive transfer (Figure 1a). We hypothesized that this novel engraftment strategy would activate pathways downstream of the γc cytokine receptor to promote T cell engraftment without prior lymphodepletion. We observed engraftment of adoptively transferred luciferase-positive cells into lymphoid tissues in lymphocyte-depleted recipients as well as in lymphocyte-replete animals transferred with T cells expressing constitutively active Stat5b* plasmid or mRNA (Figure 1b). The luciferase signal peaked at 24 hours and then decayed over time, following the kinetics expected in a host lacking OVA antigen expression in any tissue (Figure 1c). Interestingly, we noted that cells transfected with pCMV-mStat5b* or Stat5b* mRNA engrafted more efficiently than cells transferred into lymphocyte-depleted hosts. This suggests that Stat5b* expression during ACT may result in more efficient engraftment and provide an opportunity to reduce the number of cells required to effectively treat patients.

[0054] T-cell engraftment without prior lymphodepletion eliminates preclinical correlates of CRS One of the most frequent and severe adverse effects of these T cell ACT-based therapies is called cytokine release syndrome (CRS). CRS is associated with the overproduction of IL-6 during T cell engraftment. This excess IL-6 is not produced by the engrafting CAR-T cells, but rather by recipient bone marrow cells as a result of reactive myelopoiesis induced by lymphodepletion regimens in response to the production of cytokines such as IL1b by proliferating T cells. To examine differences in cytokine levels before and after ACT in the context of transient Stat5* expression, serum concentrations were assessed using the Mouse Cytokine Proinflammatory Focused 10-Plex Discovery Assay (Eve Technologies) for mice in each treatment group. Consistent with previous observations regarding CRS and cytokine levels described above, IL-6 was the only cytokine observed to increase after ACT. Notably, the increase in serum IL-6 was lost in mice that did not undergo lymphodepletion (11). Taken together, this suggests that ACT of CAR-T cells without prior lymphodepletion may represent an opportunity to reduce or eliminate the risk of CRS in patients.

[0055] Adoptively transferred T cells maintain functional surveillance of OVA-expressing tumor cells long after engraftment.

[0056] In addition to tracking T cell persistence in vivo, luciferase modification allowed us to assess their ability to respond to antigen challenge. To this end, when initial luminescence had decayed, we subcutaneously injected LLC-ova lung cancer into the flank of each mouse. We observed antigen-specific memory CD8 T cells, as indicated by an increase in bioluminescence at 24 hours in both lymphocyte-depleted and lymphocyte-replete cohorts engrafted with constitutively active Stat5b* plasmid or mRNA. +We observed a rapid T cell response (Fig. 2b, c, supplemental RNA data, adds RNA data to figure).

[0057] Example 2 T cell assessment To further evaluate the functional fitness of T cells engrafted using transient Stat5*, we next assessed the effector function of engrafted antigen-specific T cells. As expected, engrafted T cells resulted in significant tumor control in the lymphodepleted group, but this group produced a lower antitumor effect than Stat5b* plasmid or mRNA or their counterparts. Surprisingly, many of the mice engrafted by ACT using Stat5* without lymphodepletion provided complete tumor control and significantly enhanced overall survival in the host mice (Figure 2d, e), suggesting that this engraftment method yields T cells with a functionally superior phenotype. This prompted us to investigate the phenotype of cells generated by each engraftment mode.

[0058] Stat5b* promotes the development of non-canonical effector memory cell phenotypes.

[0059] We examined the phenotype of adoptively transferred cells under each condition by collecting cells from the spleen, lymph nodes, or tumor. Interestingly, we observed that cells engrafted using the transient Stat5* protocol failed to upregulate CD62L after adoptive transfer, as observed using conventional lymphodepletion regimens (Figure 3). To further investigate whether lymphodepletion or Stat5* has a dominant effect in suppressing CD62L expression, we lymphodepleted mice and then performed a competition assay in which Stat5*-transfected thy1.1+ cells and mock-transfected GFP+ cells engrafted. Comparison of CD62L expression between Stat5*-positive and Stat5*-negative cells further supports the model that Stat5* drives a dominant change in cell phenotype as defined by CD62L.

[0060] Transient expression of Stat5* during engraftment reduces PD1+Tim3+ expression in solid tumor models. To determine whether antitumor function would be altered in Stat5*-engrafted mice, we conducted studies to examine the phenotype of adoptively transferred T cells in an immunosuppressive tumor model (Figure 4). Cells were adoptively transferred using conventional lymphodepletion or by transiently expressing Stat5* during engraftment. Tumors were harvested 6 days after ACT, and cell phenotype was characterized by flow cytometry. We observed a unique cell phenotype in the Stat5* group, in which T cells showed a significant decrease in PD1 expression compared to cells engrafted using conventional lymphodepletion. Consistent with previous observations, Tim3 expression was increased as a result of transient Stat5b* expression compared to tumor-infiltrating cells engrafted using conventional lymphodepletion. Although Tim3 is often identified as a marker of exhaustion when combined with PD1 expression, we observe independent regulation of the two genes in response to Stat5b*, which reduces the number of canonically exhausted cells based on the expression of both Tim3+ and PD1+ cells in tumors.

[0061] Example 3 Transient expression of Stat5b* during engraftment promotes tumor control We evaluated the functional consequences of Stat5b* activation on tumor control in lymphocyte-replete hosts. Control luciferase-labeled T cells or Stat5b* luciferase-labeled T cells were injected into LLC-ova (Figure 5b, c) or B16-F10 (Figure 5d, e) tumor-bearing mice. Surprisingly, Stat5b* T cells exhibited potent and durable tumor control, similar to the effects of their T cell counterparts engrafted by standard lymphocyte-depletion regimens (Figure 5b, d). As expected, treatment of lymphocyte-replete hosts with control T cells abrogated tumor control in both tumor models, reducing survival and supporting the previously described role of lymphocyte depletion in maintaining antitumor immunity of adoptively transferred T cells. Consistent with these data, survival was substantially enhanced in lymphodepleted mice infused with antigen-specific T cells and in lymphocyte-replete hosts with Stat5b*-conditioned T cells compared with the respective control T cells in lymphocyte-replete hosts. Together, these data demonstrate that Stat5b* is sufficient for T cell-mediated tumor control in lymphocyte-replete hosts.

[0062] Example 4 Cryopreserved CAR-T cell products To facilitate processing at the manufacturing site, a novel cryopreserved CAR-T cell product containing highly stable mRNA encoding Stat5* is provided, ready for thawing and infusion into patients, including lymphocyte-replete hosts. Figure 6 demonstrates the stability and efficient expression of mRNA in human CAR-T cells after thawing. Figure 6 shows an experimental scheme for the isolation, expansion, and transfection of human CD8+ T cells from peripheral blood. After transfection, cells were cultured for 24 hours or cryopreserved and thawed 44 days later before flow cytometry analysis. Figure 6 also shows summary data showing (b) the percentage of viable transfected cells expressing GFP, or (c) GFP intensity as measured by FITC MFI, and (d) representative flow cytometry plots of GFP+ cells 1 day after transfection when thawed and analyzed 45 days after production compared to cryopreserved cells.

[0063] Figure 7 further demonstrates the stability and function of our Stat5b* mRNA in mediating engraftment in lymphocyte-replete animals after thawing cryopreserved cells. Panel a) shows an experimental schematic illustrating three groups of animals in which cells were transfected with control mRNA or Stat5b* mRNA, thawed, and immediately cryopreserved for 10 days before being transferred into animals and then subjected to adoptive cell therapy (ACT). The experimental groups underwent ACT without lymphodepletion, ACT after conventional 5 Gy-irradiated lymphodepletion, or ACT with Stat5b* mRNA-transfected cells without lymphodepletion. Panel b) shows representative ventral images of luciferase-engineered CD8+ T cells 5 days after ACT. Panel c) shows that animals were measured 8 days after ACT (ongoing study), and the intensity of the signal emitted by luciferase-engineered T cells was expressed as luminescence. Quantification of total (dorsal + ventral) luminescence is plotted (n=5 for all groups).

[0064] Collectively, these data support the invention that Stat5b* can be transfected into CAR-T cells after manufacturing, immediately cryopreserved, and then transported to the point of care where the cells are thawed and infused into lymphocyte-replete patients, eliminating the need for lymphodepletion before adoptive transfer.

[0065] Example 5 Stat5b*-transfected mouse CD8 + Gene expression changes in T cells We believe that adoptive cell therapy may be improved by RNA transfection of constitutively active Stat5 transcripts prior to adoptive transfer. To evaluate this process, we utilized Stat5b* plasmid DNA or mRNA to transfect OT1 cells. + CD8 + T cells were transfected and gene expression was measured using the nanoString™ (Seattle, WA) platform with the NS_MM_EXHAUSTION CSO code set. We first evaluated the overall survival and transfection efficiency of T cells after RNA and DNA electroporation and found that RNA transfection was associated with lower cytotoxicity (Figure S2a, b) and higher transfection efficiency. We then harvested RNA from the cells and analyzed them using a pre-made nanoString™ (Seattle, WA) Immune Exhaustion Panel (NS_MM_EXHAUSTION panel: Catalog No. PSTD-M-EXHAUST-12). The results of this analysis showed that CD8 + We clearly show that in T cells, Stat5b*RNA transfection increases the expression of genes normally associated with Stat5b activation (Socs genes, Grzmb, Ccr5) while decreasing the expression of genes known to be downregulated by Stat5b activation (Il7r, Jak3, Ccr7). The invention is further described in the following numbered paragraphs:

[0066] 1. A method for functional engraftment of cells in the absence of lymphodepletion, comprising the step of transient transfection of T cells with constitutively active STAT5 prior to adoptive transfer.

[0067] 2. A method for reducing cytokine release syndrome (CRS) in a patient undergoing cancer treatment, comprising administering to the patient T cells transiently transfected with constitutively active STAT5 prior to adoptive transfer.

[0068] 3. A method for increasing cell engraftment efficiency, which involves a step of transient transfection of T cells with STAT5 prior to adoptive transfer.

[0069] 4. A method for reducing IL-6 production in a patient in need thereof, comprising administering to the patient T cells transiently transfected with constitutively active STAT5 prior to adoptive transfer.

[0070] 5. A method for producing a cryopreserved T cell product, comprising: transiently transfecting T cells with constitutively active STAT5 prior to adoptive transfer to produce transfected T cells; cryopreserving the transfected T cells; A method comprising:

[0071] 6. A point-of-use product comprising the cryopreserved, transfected T cells of paragraph 5.

[0072] 7. A method for reducing cytokine release syndrome (CRS) in a patient undergoing cancer treatment, comprising administering to said patient, after thawing, the cryopreserved, transfected T cells of paragraph 6.

[0073] 8. A method for reducing the production of IL-6 in a patient in need thereof, comprising administering to said patient, after thawing, the cryopreserved, transfected T cells of paragraph 6. References

[0074] [ka] [ka] [ka] [ka]

[0075] It is to be understood that the invention is not limited to the particular embodiments of the invention described above, and variations of the particular embodiments may be made and still fall within the scope of the appended claims.

Claims

1. 1. A method for functional engraftment of cells in the absence of lymphodepletion, comprising the step of transient transfection of T cells with constitutively active STAT5 prior to adoptive transfer.

2. 1. A method for reducing cytokine release syndrome (CRS) in a patient undergoing cancer treatment, comprising administering to the patient T cells transiently transfected with constitutively active STAT5 prior to adoptive transfer.

3. A method for increasing cell engraftment efficiency comprising the step of transient transfection of T cells with STAT5 prior to adoptive transfer.

4. 1. A method for reducing IL-6 production in a patient in need thereof, comprising administering to said patient T cells transiently transfected with constitutively active STAT5 prior to adoptive transfer.

5. 1. A method for producing a cryopreserved T cell product, comprising: transiently transfecting T cells with constitutively active STAT5 prior to adoptive transfer to produce transfected T cells; cryopreserving the transfected T cells; A method comprising:

6. 6. A point-of-use product comprising the cryopreserved, transfected T cells of claim 5.

7. 10. A method for reducing cytokine release syndrome (CRS) in a patient undergoing cancer treatment, comprising administering to said patient the cryopreserved, transfected T cells of claim 6 after thawing.

8. 10. A method for reducing IL-6 production in a patient in need thereof, comprising administering to said patient the cryopreserved, transfected T cells of claim 6 after thawing.

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