Transient STAT5B activation mediated T cell adoptive metastasis in lymphocyte sufficient hosts during implantation

Abstract

The present invention relates to methods for promoting functional implantation of engineered T cells that will not require depletion of lymphocytes.

Description

Technical Field

[0001] The present invention generally relates to methods for promoting the functional implantation of engineered T cells, which methods do not require lymphocyte depletion. All disclosures, patents, patent applications and other references cited in this application are incorporated herein by reference in their entirety for all purposes, and the extent of their citation is as if specifically and individually indicating that each individual disclosure, patent, patent application or other reference is incorporated by reference in its entirety for all purposes. Citation of references herein should not be construed as an admission that the references are prior art of the present invention.

[0002] Priority of related applications

[0003] 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 applications are hereby incorporated by reference in their entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made in part with government support under NCIP 30CA138313. The government has certain rights in this invention. Background Art

[0006] The success of adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL), chimeric antigen receptors (CAR) or T cell receptor (TCR) engineered T cells relies on a pretreatment regimen designed to deplete the patient's lymphocytes before infusion (1, 2). Lymphocyte depletion regimens, such as chemotherapy and total body irradiation (TBI), eliminate endogenous cytokine-responsive cells that act as cytokine sinks (3). Further, lymphocyte depletion eradicates suppressive cell populations in the host immune system by eliminating immunosuppressive cell types, including regulatory T cells (4, 5) and myeloid-derived suppressor cells (MDSC) (6). Patients who receive lymphodepletion regimens before ACT enjoy better response rates and improved survival and anti-tumor immunity (1, 7). However, although these regimens are crucial for maximizing clinical benefit, the agents used, such as fludarabine and cyclophosphamide, are largely nonspecific agents with significant toxicity characteristics (8, 9).

[0007] Lymphocyte depletion regimens can result in various side effects, including leukopenia, pulmonary veno-occlusive disease, and reactive myelopoiesis (10). These side effects have led to patient exclusion and have hindered the widespread adoption of clinical trials utilizing engineered T cell-based therapies. In addition to significant side effects, the physiological response to lymphocyte depletion can adversely affect the efficacy of ACT in eligible patients (11). Furthermore, management of these toxicities often requires long-term inpatient support and hospitalization, placing a significant economic burden on patients and healthcare providers. Despite the fact that lymphocyte depletion regimens have many disadvantages, it is clear that they promote functional engraftment and persistence of transferred T cells (12, 13), whereas ACT without prior lymphocyte depletion inhibits engraftment of adoptively transferred T cells (14) and reduces clinical benefit.

[0008] There is a need in the art for cell-autonomous methods for promoting functional engraftment of engineered T cells that would not require lymphocyte depletion. Summary of the Invention

[0009] The present invention relates to a method for functionally engrafting cells without lymphocyte depletion, the method comprising the step of transiently activating Stat5 signaling by transfecting T cells prior to adoptive transfer 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.

[0010] The present invention also relates to a method of reducing cytokine release syndrome (CRS) in a patient undergoing cancer therapy, comprising the step of administering T cells transiently transfected with RNA or DNA encoding an activated form of Stat5 or a cytokine receptor prior to adoptive transfer.

[0011] The present invention also relates to a method for improving the engraftment efficiency of cells, comprising the step of transiently activating Stat5 signaling by transfecting DNA or RNA encoding activated Stat5 or cytokine receptors prior to adoptive transfer.

[0012] Additionally, the present invention relates to a method of reducing IL-6 production in a patient in need thereof, comprising the step of administering to said patient transient activation of Stat5 signaling by transfection of DNA or RNA encoding activated Stat5 or a cytokine receptor prior to adoptive transfer. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Transient expression of Stat5b* was shown to promote CD8 + T cell implantation. a) Schematic diagram of the experiment showing three groups of animals undergoing adoptive transfer (ACT) using conventional 5gy lymphocyte depletion, ACT without lymphocyte depletion, and ACT without lymphocyte depletion (where cells were transfected with a plasmid encoding STAT5* immediately before ACT). b) ffLuc-Thy1.1 engineered CD8 + Representative ventral images of T cells. c) Animals were measured 29 days after ACT, and the signal intensity emitted by ffLuc-Thy1.1+ T cells is 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 with Stat5* produced significantly more effective engraftment compared to the conventional method of lymphocyte depletion before ACT. d) Peripheral cytokine levels 24 hours before ACT and 4 days after ACT. Cytokines thought to be associated with ACT-related toxicity in humans are shown. By unpaired Student's t-test, * p<0.05(d). Error bars indicate SEM.

[0014] Figure 2 Stat5 drives a robust recall response without lymphocyte depletion. a) Experimental schemes for b and c. b) Representative images of ffLuc-Thy1.1-engineered CD8+ T cells 40 days after ACT and 24 hours after re-challenge with LLC-ova cells. c) Animals were measured 24 and 72 hours after LLC-ova challenge, and the signal intensity emitted by ffLuc-Thy1.1-transfected T cells is expressed as luminescence. d) Tumor growth kinetics and e) Survival of mice challenged with LLC-ova.

[0015] Figure 3 Transient expression of Stat5*A reduces CD62L re-expression. A) Schematic diagram of experiments using conventional lymphocyte depletion or transfer of mice using transient expression of Stat5*. B and C) Representative plots and graphical summary of flow cytometry data comparing CD62L expression, and F) is a schematic diagram of a competition study testing whether the effect of lymphocyte depletion on CD62L expression is more pronounced than the effect of transient Stat5* expression during implantation. G and E) Representative plots and data summary comparing the phenotypes of GFP+ and thy1.1+ (Stat5* transfected) cells from the same spleen.

[0016] Figure 4Demonstrating that adoptive transfer (ATC) using transient Stat5* without lymphocyte depletion generates a unique phenotype of tumor-infiltrating T cells. A) Experimental schematic: Subcutaneous tumors were established prior to ACT. Tumors were harvested 6 days after adoptive transfer, and the phenotype of the implanted cells was assessed by flow cytometry for markers associated with T cell exhaustion. B) and C) PD1 levels were suppressed, while PD1+Tim3+ double-positive cells remained unchanged, due to the robust induction of Tim3 in Stat5*-implanted cells despite unchanged Lag-3 expression.

[0017] Figure 5 Figure 3. Adoptive transfer of transient Stat5b* without lymphodepletion results in functional control of tumors. a) Experimental scheme. b) Tumor growth kinetics and c) OT1 cells infused with either conventional lymphodepletion (blue) or Stat5b* (red). + Survival of LLC-ova tumor-bearing mice (n = 5 mice per group) after infusion of T cells (d) tumor growth kinetics and e) PMELs implanted with conventional lymphodepletion (blue) or stat5b* (red). + Survival rate of mice bearing B16-F10 tumors of T cells (n=5 mice per group). By linear regression and log-rank of tumor growth, Mantel-Cox survival ratio test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (c, e). Error bars indicate SEM.

[0018] Figure 6 Cryopreserved human T cell products are shown to retain and express transfected mRNA after long-term frozen storage.

[0019] Figure 7 It was shown that cryopreserved Stat5b* mRNA-modified T cells were highly functional and efficiently engrafted in lymphocyte-replete mice. DETAILED DESCRIPTION

[0020] Should be understood that, for the sake of clarity, the description in the present invention has been simplified to show the elements relevant to a clear understanding of the present invention, while eliminating many other elements found in typical pharmaceutical compositions. Those of ordinary skill in the art will recognize that other elements and / or steps are desirable and / or required for implementing the present invention. However, because such elements and steps are well known in the art, and because such elements are unfavorable for a better understanding of the present invention, the discussion of such elements and steps is not provided herein. Disclosure herein relates to all such changes and modifications to such elements and methods known to those skilled in the art. In addition, the embodiments identified and shown herein are only for exemplary purposes, and are not intended to be exclusive or to limit aspect its description of the present invention.

[0021] Functional engraftment is characterized by the expansion and persistence of T lymphocytes and their antitumor function. Specific mechanisms contributing to effective engraftment are thought to be related to homeostatic T cell proliferation. Following lymphocyte depletion, the increased availability of homeostatic cytokines leads to an enhanced T cell proliferative state, which is thought to be mediated by γ c The immune system is regulated by a number of cytokines, including interleukin-2 (IL-2), IL-7, and IL-15. These cytokines play a key role in regulating T cell homeostasis and inflammatory responses through key signaling pathways. Current lymphocyte depletion protocols eliminate the cellular cytokine pools, thereby increasing the availability of homeostatic cytokines to support the effectiveness of adoptively transferred T cells (3).

[0022] Typically, cytokines IL-2, IL-7, and IL-15 signal through the JAK-STAT pathway, primarily by activating JAK1 / 3 and STAT5, known mediators of T cell anti-tumor immunity (15, 16). In response to cytokine stimulation, Stat5b's C-terminal tyrosine (mY699, hY694) is phosphorylated, mediating Stat5b activation and dimerization, thereby promoting nuclear translocation and DNA binding (17, 18). The present inventors have chosen to utilize a form of Stat5b* that mimics the structural aspects of phosphorylated Stat5b independent of tyrosine phosphorylation by replacing arginine with histidine 298 (referred to as Sta5b* in this manuscript).

[0023] The inventors of the present application believe that during the initial period of adoptive transfer, transient Stat5b activation eliminates the need for prior lymphocyte depletion. The inventors of the present application describe that transient expression of the active form of Stat5 during the initial period of adoptive transfer leads to functional T cell implantation and eliminates the peak of IL-6 usually observed during implantation, which is a driving factor for cytokine release syndrome (CRS). The inventors of the present application observed that in a variety of immune-competent tumor models, cells implanted into lymphocyte-rich mice using Stat5b* had excellent recall responses and improved tumor control. These results suggest that this novel implantation strategy may represent an opportunity to remove lymphocyte depletion protocols from ACT protocols, rather than relying on transient Stat5b* expression during the implantation period, and also improves T cell function after ACT.

[0024] Thus, the present inventors discovered that transient expression of constitutively active STAT5 (STAT5-CA) during adoptive transfer eliminates the need for prior lymphocyte depletion, results in long-term functional engraftment, and improves tumor control in immunocompetent cancer models.

[0025] In one embodiment, the present inventors discovered that transient transfection of CAR-T cells with constitutively active STAT5 immediately prior to adoptive transfer allows functional engraftment of cells without lymphodepletion.

[0026] In one embodiment of the present invention, the present inventors have found that transient expression (via DNA, mRNA, cirRNA) of constitutively active receptors and other common gamma chain cytokine receptors that activate signal transduction pathways such as Jak / Stat promotes T cell implantation and function. This is based on such proof that the cell-autonomous adoptive transfer of functional T cells uses a method that does not require genetic modification of T cells or lymphocyte depletion 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 method to enhance adoptive transfer or T cell function in the context of mild, reduced or conventional lymphocyte depletion.

[0027] These constitutively active molecules can 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.

[0028] In another embodiment of the present invention, the present inventors have found that adoptive transfer of T cells without prior lymphocyte depletion leads to reduced production of several cytokines, including IL-6. IL-6 production has been characterized as an antagonist to therapeutic efficacy by driving cytokine release syndrome (CRS) and leading to T cell dysfunction [2].

[0029] In another embodiment of the present invention, the present inventors found that transient transfection of T cells with STAT5-CA prior to adoptive transfer improved engraftment efficiency, allowing for administration of much lower cell doses, thereby reducing product preparation time and further reducing the risk of side effects.

[0030] In another embodiment of the present invention, a cryopreserved T cell product is provided that is ready for infusion without host lymphocyte depletion. The T cell preparation process requires collecting T cells from the patient through leukapheresis, activation of the cells with a construct expressing CAR, lentiviral transduction, and expansion of the transduced cells for 2-3 weeks. After preparation, the cells are cryopreserved and transported to the point of care, where they are thawed and infused.

[0031] The success of adoptive cell therapy using tumor infiltrating lymphocytes (TIL), CAR or T cell receptor (TCR) engineered T cells depends on the effective implantation of cell products. Historically, pretreatment protocols designed to deplete lymphocytes in patients before infusion have been used to promote the implantation of adoptively transferred cells. Lymphocyte depletion (LD) is now considered to be necessary for effective CAR-T therapy and is a common component of adoptive T cell transfer protocols. However, although these protocols have historically been considered to be crucial for maximizing clinical benefit, the agents used (fludarabine and cyclophosphamide) are largely non-specific agents with significant toxicity characteristics. Over the past decade, replacing, simplifying or improving the LD step has been an important priority for pharmaceutical companies. The inventors of the present application have developed a method that relies on T cells prepared by transfection of mRNA encoding a constitutively active mutant protein (Stat5b*) of Stat5b before adoptive transfer. The inventors observed that this transient activation of the Stat5b pathway "tricked" T cells into thinking they were entering a lymphocyte-depleted host and resulted in efficient functional engraftment of T cell products, which was shown to be superior to conventional lymphocyte-depletion-based protocols. This technique could eliminate the need for LD and revolutionize T cell therapy.

[0032] Example

[0033] The following examples further describe and illustrate specific embodiments within the scope of the present invention. Techniques and formulations can generally be found in Remington's Pharmaceutical Sciences (Mike Publishing Company of Easton, Pennsylvania). The present disclosure is further illustrated by the following examples, which should not be construed as limiting the scope or spirit of the present disclosure to the specific procedures described herein. It should be understood that the examples are provided to illustrate certain embodiments and are not intended to limit the scope of the present disclosure thereby. It should be further understood that various other embodiments, modifications, and equivalents thereof that can be imagined by those skilled in the art may be resorted to without departing from the spirit of the present disclosure and / or the scope of the appended claims.

[0034] General Materials and Methods

[0035] Molecular Biology

[0036] Transfection grade plasmids were prepared according to the manufacturer's protocol using endotoxin-free buffer and ZymoPURE II plasmid Maxiprep kit (ZYMO Research, USA, USA). Plasmid vectors pT-effluc-thy1.120 and pRP--CAG>hyPBase 29, pTPB-CMV-GFP, pCMV-mStat5b*, pT7-mStat5b*, and pT7-hStat5b* were synthesized by Vector Builder Biosciences (Chicago, IL, USA). All plasmid vectors were confirmed by DNA sequencing. Transfection grade plasmids were prepared according to the manufacturer's protocol using endotoxin-free buffer and ZymoPURE II plasmid Maxiprep kit (ZYMO Research, USA, USA). Plasmid vectors were synthesized using pT-effluc-thy1.120 and pRP--CAG>hyPBase 29, pTPB-CMV-GFP, pCMV-mStat5b*, pT7-mStat5b*, and pT7-hStat5b*.

[0037] mice

[0038] 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 Jackson Laboratories. Luciferase (β-actin-luc) was obtained from Taconic Bioscience. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Medical University of South Carolina (MUSC), and all mice were maintained by the Laboratory Animal Resources Department of MUSC.

[0039] Cell culture

[0040] 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 OT-1 and PMEL T cell activation and expansion, 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 the MACS mouse CD8a+ T cell isolation kit (Miltenyi Biotec, Auburn, CA). The 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 with 200 U / mL rhIL-2 (NCI). T cells were split on day 3 and expanded to day 7 in rhIL-2 or IL-15 (50 ng / mL).

[0041] In vitro transcription

[0042] mRNA was transcribed using the mMessage mMachine T7 ULTRA Transcription Kit (Ambion, Life Sciences, Grand Island, NY) using the manufacturer's protocol. mRNA was resuspended in nuclease-free water and stored at -80°C until transfection.

[0043] Adoptive transfer model

[0044] 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. OT-1 T cells expanded in vitro 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 specified concentrations of GFP mRNA or 20 μg Stat5b* mRNA using the previously described setup 30. The transfected T cells were allowed to rest in complete T cell culture medium for 1 hour, then resuspended in PBS and transferred directly into recipient mice. Luc+ T cells were transfected with 20 μg Stat5b* mRNA. Prior to adoptive transfer, mice were exposed to 5 Gy of lymphocyte depletion radiation using a cesium irradiator or cyclophosphamide (Thermo Scientific, Waltham, MA) dissolved in PBS at a dose of 200 mg / kg by intraperitoneal injection, unless otherwise stated. T cells were transfected as described above, recovered in complete TCM for 1 hour, and then adoptively transferred by IP injection. For in vitro analysis of T cells transferred by ELThy1.1 or GFP, mice were sacrificed 3 days after transfer, and organs were processed into single cell suspensions for FACS analysis.

[0045] Tumor cell attack

[0046] For the primary challenge, mice were injected subcutaneously (sc) with 2.5×10 5 LLC-ova tumor cells on day 40 after the primary adoptive transfer of OT-1 + T cells.

[0047] Tumor models

[0048] For adoptive cell therapy experiments, LLC-ova adenocarcinoma or B16-F10 melanoma was established subcutaneously by injecting 2.5×105 cells into the right flank of male B6(Cg)-Tyrc-2J / J mice, and the tumor-bearing hosts were irradiated with 5Gy 24 hours before T cell transfer. After 7 days of tumor growth, 1×107 OT1 or PMEL T cells were transfected using the Neon transfection system and then infused with 100 μL of phosphate-buffered saline by IP injection into the mice. Tumor growth was measured with a caliper every other day, and survival was monitored with an experimental endpoint of tumor growth >400 mm2. For in vitro 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

[0050] Mice were anesthetized using isoflurane and injected IP 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, Tuscon AZ). All data shown represent the average luminescence observed by summing dorsal and ventral measurements obtained from the same region of interest mapped from the trunk and head of each individual mouse.

[0051] Serum cytokine assessment

[0052] Blood was collected by retro-orbital venipuncture in Microvette CD300 potassium EDTA collection tubes (Sarstedt, Newton, NC). Serum was measured using the Mouse Cytokine Proinflammatory Focused 10-Plex Discovery Assay (MDF10) (Eve Biotechnologies, Calgary AB, Canada).

[0053] Flow cytometry

[0054] Prior to extracellular staining, cells were stained with a Live / Dead Fixable Aqua dead stain cell kit (Invitrogen). Fluorescent dye-conjugated antibodies were purchased from Biogen (San Diego, CA), eBioscience (San Diego, CA), or BD Pharmigen (Mountain View, CA). The cells were then extracellularly stained 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 an isotype control. Cell surface antibody staining was performed in PBS containing 2% FBS. Antibody-stained cells were run directly on a CytoFLEX flow cytometer (Beckman Coulter Life Sciences, Indianapolis, IN) and analyzed with FlowJo software.

[0055] Nanostring gene expression analysis

[0056] RNA Isolation: Cells were immediately pelleted by centrifugation at 4°C and resuspended in homogenization buffer containing monothioglycerol provided in the purification kit. RNA was extracted using a Promega Maxwell RSC 16 using the Maxwell RSC simply RNA Cell Kit (Cat. No. AS1390) according to the manufacturer's instructions, and RNA concentration was measured using a NanoDrop 8000.

[0057] Gene quantification: Selected transcripts were directly quantified using 100 ng of total RNA on a Nanostring nCounter system using markers from the NS_MM_EXHAUSTION code set. RCC files were imported into nSolver 4.0 software and analyzed using the default analysis pipeline for normalization, differential expression, and agglomerative clustering.

[0058] Statistical analysis

[0059] GraphPad Prism was used to calculate p values ​​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. Tumor growth curves were analyzed by simple linear regression. Values ​​of p < 0.05 were considered significant. Values ​​of p < 0.05 were ranked as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0060] Example 1

[0061] Stat5b* supports CD8+ T cell engraftment without lymphodepletion

[0062] Materials and methods

[0063] carrier

[0064] Plasmid vectors pT-effluc-thy1.1 (19) and pCMV-m7pB (20) were previously described. pT-SB-Stat5b was synthesized by Vector Builder Biosciences (Chicago, IL). All plasmid vectors were confirmed by DNA sequencing. Transfection-grade plasmids were prepared according to the manufacturer's protocol using endotoxin-free buffer and the ZymoPURE II Plasmid Maxiprep Kit (ZYMO Research, USA).

[0065] mice

[0066] OT1 (C57BL / 6-Tg(TcraTcrb)1100Mjb / J) and B6 albino (B6(Cg)-Tyr c-2J All animal experiments were approved by the Institutional Animal Care and Use Committee of the Medical University of South Carolina (MUSC), and all mice were maintained by the Laboratory Animal Resources Department of MUSC.

[0067] Cell culture

[0068] 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 OT1 and PMEL T cell activation and expansion, whole splenocytes from OT1 or PMEL mice were activated with 1 μg / mL OVA257-264 peptide or 1 μg / mL glycoprotein 100 (gp100), and whole splenocytes were 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) to day 7. Human PBMCs were activated with CD3 / CD28 activator and expanded in 500 U 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.

[0069] Adoptive transfer model

[0070] 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 pCMV-M7PB and 10 μg pT-effluc-thy1.1 or 10 μg pT-GFP and 10 μg pSB-mSTAT5. Prior to adoptive transfer, mice were pretreated by exposure to 5 Gy of lymphocyte depletion radiation using a cesium irradiator, unless otherwise indicated. T cells were transfected as described above, allowed to recover in complete TCM for 1 hour, and then adoptively transferred by IP injection. For in vitro 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.

[0071] Antigen attack

[0072] For the first challenge, when the initial OT-1+ T cell luciferase luminescence subsided, 2.5×10 5LLC-ova tumor cells were injected subcutaneously (sc) into mice.

[0073] Tumor models

[0074] For adoptive cell therapy experiments, 2.5 × 10 5 cells were injected into male B6(Cg)-Tyr c-2J LLC-ova adenocarcinoma or B16-F10 melanoma was established subcutaneously in the right flank of 1 / J mice, and the tumor-bearing hosts were irradiated with 5 Gy 24 hours before T cell transfer. After 7 days of tumor growth, 1×10 7 OT1 or PMELT cells were then injected into mice via IP with 100 μL of phosphate-buffered saline. Tumor growth was measured with a caliper every other day, and tumors ≥400 mm were used for 2 Survival was monitored as the experimental endpoint. For in vitro 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.

[0075] Bioluminescence imaging

[0076] Mice were anesthetized using isoflurane and injected with luciferin substrate (PerkinElmer, Waltham, Massachusetts) at a standard concentration of 150 mg / kg in PBS. Approximately 10 minutes after the luciferase injection, mice were imaged on AMI-HT. All data shown represent the average luminescence observed by adding the dorsal and ventral measurements obtained from the trunk and head of each individual mouse.

[0077] Serum evaluation

[0078] Blood was collected by retro-orbital vena cava in Microvette CD300 potassium EDTA collection tubes (Shast, Newton, NC). Serum was measured using the Mouse Cytokine Pro-Inflammatory Focused 10-Plex Discovery Assay (MDF10) (Eve Biotech, Calgary, Alberta, Canada).

[0079] Flow cytometry

[0080] Fluorescent dye-conjugated antibodies were purchased from Biogen (San Diego, CA), eBioscience (San Diego, CA), or BD Pharmigen (Mountain View, CA) with the following specificities: 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 on a CytoFLEX flow cytometer (Beckman Coulter Life Sciences, Indianapolis, IN) and analyzed using FlowJo software.

[0081] Statistical analysis

[0082] GraphPad Prism was used to calculate p values ​​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. Values ​​of p < 0.05 were considered significant. Values ​​of p < 0.05 were ranked as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0083] To assess the ability of T cells to engraft, the present inventors first modified murine OT-1 T cells with the previously described pT-effluc-thy1.1 transposon (21). Luciferase modification allowed the present inventors to track T cells in vivo and quantify engraftment and persistence by longitudinally measuring luciferase. Using this model system, the present inventors transiently transfected a plasmid encoding a constitutively active form of murine Stat5b (pCMV-mStat5b*) into OT-1 T lymphocytes prior to adoptive transfer ( Figure 1 a). The inventors hypothesized that this novel implantation strategy would activate pathways downstream of the γc cytokine receptor to promote T cell engraftment without the need for prior lymphocyte depletion. The inventors observed that in lymphocyte-depleted recipients, as well as in lymphocyte-replete animals transferred with T cells expressing constitutively active Stat5b* plasmid or mRNA, adoptively transferred luciferase-positive cells engrafted into lymphoid tissues ( Figure 1 b). This luciferase signal peaked at 24 hours and then decayed over time according to the expected kinetics in a host without OVA antigen expression in any tissue ( Figure 1c). Interestingly, the inventors 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 enable more efficient engraftment and provide an opportunity to reduce the number of cells required to effectively treat patients.

[0084] Implantation of T cells without prior lymphodepletion eliminates preclinical correlates of CRS. One of the most common and severe side effects of these therapies based on T cell ACT is known as cytokine release syndrome (CRS). CRS is associated with excessive production of IL-6 during T cell implantation. This excess IL-6 is not produced by the implanted CAR-T cells, but rather by recipient bone marrow cells in response to cytokines such as IL1b produced by proliferating T cells and is a result of reactive myelopoiesis induced by the lymphodepletion regimen. To investigate the differences in cytokine levels before and after ACT in the context of transient Stat5*, serum concentrations were assessed in mice from each treatment group using the mouse cytokine proinflammatory focused 10-plex discovery assay (Eve Technologies). Consistent with previous observations on CRS and cytokine levels, the only cytokine observed to increase after ACT was IL-6. Notably, the increase in serum IL-6 disappeared in mice that did not receive lymphodepletion (11). Taken together, this suggests that CAR-T cell ACT without prior lymphodepletion may represent an opportunity to reduce or eliminate the risk of CRS in patients.

[0085] Long after implantation, adoptively transferred T cells maintained functional surveillance of OVA-expressing tumor cells.

[0086] In addition to tracking the persistence of T cells in vivo, luciferase modification allowed the inventors to assess their ability to respond to antigen challenge. To this end, as the initial luminescence subsided, the inventors injected LLC-ova lung cancer subcutaneously into the flank of each mouse. The inventors observed antigen-specific memory CD8 + The rapid response of T cells was indicated by the increase in 24-hour bioluminescence in the lymphocyte-depleted and lymphocyte-replete groups implanted with constitutively active Stat5b* plasmid or mRNA ( Figure 2 b, c supplement and add RNA data to the figure).

[0087] Example 2

[0088] T cell assessment

[0089] To further evaluate the functional fitness of T cells implanted with transient Stat5*, the inventors of the present application next evaluated the effector function of the implanted antigen-specific T cells. As expected, the implanted T cells provided significant tumor control in the lymphocyte-depleted group, although the anti-tumor effect produced by this group was worse than the anti-tumor effect of Stat5b* plasmid or mRNA or its counterpart. Remarkably, many mice implanted with ACT using Stat5* but without lymphocyte depletion provided complete tumor control and significantly improved the overall survival rate of the host mice ( Figure 2 d, e), indicating that this engraftment method produces T cells with a functionally superior phenotype. This facilitates the study of the phenotype of cells produced by the corresponding engraftment model.

[0090] Stat5b* promotes the development of an atypical effector memory cell phenotype.

[0091] The present inventors examined the phenotype of cells adopted under each condition by collecting cells from the spleen, lymph nodes, or tumors. Interestingly, the present inventors observed that cells implanted using the transient Stat5* protocol were unable to upregulate CD62L after adoptive transfer, as observed using the conventional lymphocyte depletion protocol. Figure 3 To further investigate whether lymphocyte depletion or Stat5* has a dominant effect in suppressing CD62L expression, the present inventors performed a competition assay in which mice were lymphocyte-depleted and then implanted with Stat5*-transfected thy1.1-positive cells and mock-transfected GFP+ cells. Comparison of CD62L expression between Stat5*-positive and -negative cells further supports a model in which Stat5* drives a dominant change in the cellular phenotype as defined by CD62L.

[0092] Transient expression of Stat5* during implantation reduced PD1+Tim3+ in solid tumor models. To determine whether anti-tumor function would be altered in mice implanted with Stat5*, the inventors conducted studies to examine the phenotype of adoptively transferred T cells in an immunosuppressive tumor model ( Figure 4). Cells were adoptively transferred using conventional lymphocyte depletion or by transient expression of Stat5* during implantation. Tumors were harvested 6 days after ACT, and the cell phenotype was characterized by flow cytometry. The inventors of the present application observed a unique cell phenotype in the Stat5* group, in which T cells showed a significant decrease in PD1 expression compared to cells implanted using conventional lymphocyte depletion. Consistent with previous observations, Tim3 expression was increased due to transient Stat5b* expression compared to tumor-infiltrating cells implanted using conventional lymphocyte depletion. When combined with PD1 expression, Tim3 is typically identified as a marker of exhaustion, however the inventors of the present application found that these two genes are independently regulated in response to Stat5b*. Based on the expression of both Tim3+ cells and PD1+ cells in the tumor, this resulted in a reduction in the number of typically exhausted cells.

[0093] Example 3

[0094] Transient expression of Stat5b* during implantation promotes tumor control

[0095] The present inventors evaluated the functional consequences of Stat5b* activation on tumor control in lymphocyte-rich hosts. Control luciferase-labeled T cells or Stat5b*-luciferase-labeled T cells were infused into LLC-ova ( Figure 5 b, c) or B16-F10( Figure 5 d, e) in tumor-bearing mice. Strikingly, Stat5b* T cells exhibited potent and durable tumor control, producing an effect similar to that of their T cell counterparts implanted with a standard lymphocyte depletion protocol ( Figure 5 b, d). As expected, treatment of lymphocyte-replete hosts with control T cells abolished tumor control and reduced survival in both tumor models, supporting the previously described role of lymphocyte depletion in maintaining anti-tumor immunity of adoptively transferred T cells. Consistent with these data, survival was significantly improved in lymphocyte-depleted mice infused with antigen-specific T cells and in lymphocyte-replete hosts conditioned with Stat5b* T cells compared to corresponding control T cells in lymphocyte-replete hosts. Taken together, these data demonstrate that Stat5b* is important for T cell-mediated tumor control in lymphocyte-replete hosts.

[0096] Example 4

[0097] Cryopreserved CAR-T cell products

[0098] To facilitate point-of-production processing, a novel cryopreserved CAR-T cell product containing ultrastable mRNA encoding Stat5* is provided, which can be easily thawed and infused into patients, including lymphocyte-replete hosts. Figure 6 Demonstrating the stability and efficient expression of mRNA in post-thaw human CAR-T cells. Figure 6 The experimental scheme for the isolation of human CD8+ T cells from peripheral blood, cell expansion and transfection is shown. After transfection, cells were cultured for 24 hours or frozen and thawed after 44 days before flow cytometric analysis. Figure 6 Also shown are summary data showing (b) the percentage of live transfected cells expressing GFP or (c) GFP intensity as measured by FITC MFI and (d) representative flow cytometry plots of GFP+ cells one day after transfection and after cryopreservation, thawed, and analyzed 45 days after preparation.

[0099] also, Figure 7 It is shown that Stat5b*mRNA mediates the stability and function of implantation in lymphocyte-rich animals after thawing the frozen preserved cells.Small figure a) shows an experimental schematic diagram, which shows that after the cells have been transfected with control mRNA or Stat5b*mRNA and immediately frozen for 10 days, before thawing and transferring into the animal body, three groups of animals receiving adoptive cell therapy (ACT) are received. The experimental group receives ACT after no lymphocyte depletion, conventional 5Gy irradiation lymphocyte depletion or no lymphocyte depletion, and then performs ACT with cells transfected with Stat5b*mRNA.Small figure b) shows a representative ventral image of CD8+T cells engineered by luciferase 5 days after ACT.Small figure c) shows that animals were measured (ongoing research) 8 days after ACT, and the intensity of the signal emitted by the T cells engineered by luciferase is expressed as luminescence. Quantification (all groups n=5) of total (dorsal+ventral) luminescence is drawn.

[0100] Together, these data support the present invention that Stat5b* can be transfected after CAR-T cell preparation, immediately cryopreserved, and shipped to the point of care, where the cells are thawed and infused into lymphocyte-replete patients, thereby obviating the need for lymphocyte depletion after adoptive transfer.

[0101] Example 5

[0102] Mouse CD8 transfected with Stat5b* + Changes in gene expression in T cells

[0103] The present inventors believe that adoptive cell therapy can be improved by transfecting constitutively active Stat5 transcripts with RNA prior to adoptive transfer. To evaluate this approach, the present inventors transfected OT1 cells with Stat5b* plasmid DNA or mRNA. + CD8 + T cells, and using nanoString TM(Seattle, WA) platform, using the NS_MM_EXHAUSTION CSO code set to measure gene expression. The inventors 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. Then, the inventors collected RNA from the cells and used the readily available nanoString TM They were analyzed using the NS_MM_EXHAUSTION Plate (Cat. No. PSTD-M-EXHAUST-12) (Seattle, WA). The results of this analysis clearly showed that in the CD8 + In T cells, Stat5b* RNA transfection increased the expression of genes commonly associated with Stat5b activation (Socs, Grzmb, Ccr5), while decreasing the expression of genes known to be downregulated by Stat5b activation (Il7r, Jak3, Ccr7).

[0104] The present invention is further described in the following numbered paragraphs:

[0105] 1. A method for functionally engrafting cells without lymphodepletion, said method comprising the step of transiently transfecting T cells with constitutively active STAT5 prior to adoptive transfer.

[0106] 2. A method of reducing cytokine release syndrome (CRS) in a patient receiving cancer therapy, the method comprising the step of administering to the patient T cells transiently transfected with constitutively active STAT5 prior to adoptive transfer.

[0107] 3. A method for improving cell engraftment efficiency, the method comprising the step of transiently transfecting T cells with STAT5 prior to adoptive transfer.

[0108] 4. A method of reducing IL-6 production in a patient in need thereof, said method comprising the step of administering to said patient T cells transiently transfected with constitutively active STAT5 prior to adoptive transfer.

[0109] 5. A method for producing a cryopreserved T cell product, the method comprising the steps of:

[0110] transiently transfecting T cells with constitutively active STAT5 prior to adoptive transfer to generate transfected T cells; and

[0111] - Cryopreserving the transfected T cells.

[0112] 6. A ready-to-use product comprising the cryopreserved transfected T cells according to paragraph 5.

[0113] 7. A method of reducing cytokine release syndrome (CRS) in a patient receiving cancer therapy, comprising the step of administering to the patient the thawed cryopreserved transfected T cells according to paragraph 6.

[0114] 8. A method of reducing IL-6 production in a patient in need thereof, comprising the step of administering to the patient the thawed cryopreserved transfected T cells according to paragraph 6.

[0115] References

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[0137] ***

[0138] It should be understood that the present invention is not limited to the specific embodiments of the invention described above, as variations may be made therein and still fall within the scope of the appended claims.

Claims

1. A method for functionally engrafting cells without lymphodepletion, said method comprising the step of transiently transfecting T cells with constitutively active STAT5 prior to adoptive transfer.

2. A method of reducing cytokine release syndrome (CRS) in a patient receiving cancer therapy, the method comprising the step of administering to the patient T cells transiently transfected with constitutively active STAT5 prior to adoptive transfer.

3. A method for improving cell engraftment efficiency, the method comprising the step of transiently transfecting T cells with STAT5 prior to adoptive transfer.

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

5. A method for producing a cryopreserved T cell product, the method comprising the steps of: T cells were transiently transfected with constitutively active STAT5 prior to adoptive transfer to generate transfected T cells; as well as - Cryopreserving the transfected T cells.

6. A ready-to-use product comprising the cryopreserved transfected T cells according to claim 5.

7. A method of reducing cytokine release syndrome (CRS) in a patient receiving cancer therapy, said method comprising the step of administering to said patient said cryopreserved transfected T cells after thawing according to claim 6.

8. A method of reducing IL-6 production in a patient in need thereof, comprising the step of administering to the patient the thawed cryopreserved transfected T cells according to claim 6.

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