Non-transduced Research Articles

Abstract P65: Single Cell Profiling Reveals Unique TCR Repertoire and Transcriptomic Signatures of Epstein-Barr Virus-Specific T cells (EBVSTs) Armoured with Chimeric Antigen Receptors (CARs)

Abstract Allogeneic Epstein-Barr Virus-specific T cells (EBVSTs) have thus far been a safe immunotherapy product in the clinic, with little graft-versus-host-disease reported in over 300 recipients to date. Consequently, EBVSTs armoured with chimeric antigen receptors (CARs) have emerged as a promising platform for allogeneic T cell therapies. Believing that a deep characterisation of CAR EBVST cell products can enhance insights into their clinical safety, we performed single cell T cell receptor (TCR) sequencing on CAR EBVSTs, taking CD30.CAR EBVSTs as a prototype. Non-transduced (NT) EBVSTs and CD30.CAR EBVSTs generated from three healthy donors exhibited a narrower, more focused TCR repertoire by diversity indices, compared to resting T cells and CD30.CAR-transduced activated T cells (ATCs) from the same donors. EBVSTs harboured a lower frequency of unique TCR clonotypes and a higher proportion of expanded clones, indicating a tendency toward clonal dominance. Narrower TCR repertoires corresponded to greater IFNγ and TNFα secretion in response to stimulation with EBV peptides, with no cross-reactivity toward other common viruses, and were associated with lower allogeneic reactivity in mixed lymphocyte reactions. To investigate whether the EBVST nature of the product might contribute to better CAR activity, we concurrently performed single cell transcriptomic sequencing on NT EBVSTs, CD30.CAR EBVSTs, and CD30.CAR ATCs. Transcriptomic signatures revealed that each sample type contained two major, distinct populations of CD4+ T cells: proliferative and activated. While the proliferative subsets appeared to be closely related, the activated populations had distinct transcriptional profiles. In particular, CD30.CAR EBVSTs appeared to be more polyfunctional and metabolically poised than CD30.CAR ATCs. The restricted TCR repertoire and apparent functional superiority of CD30.CAR EBVSTs may underlie their safety and efficacy observed in early-stage clinical trials and preclinical models, with the larger implication that these qualities extend to CAR EBVSTs targeting antigens beyond CD30. Citation Format: Lindsay Kua, Ting Wei Lim, Fiona Wong, Pei Yun Teo, Lionel Low, Ivan Horak, Jinmiao Chen, Kar Wai Tan. Single Cell Profiling Reveals Unique TCR Repertoire and Transcriptomic Signatures of Epstein-Barr Virus-Specific T cells (EBVSTs) Armoured with Chimeric Antigen Receptors (CARs) [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P65.

Preclinical Evaluation of Off-The-Shelf PD-L1+ Human Natural Killer Cells Secreting IL15 to Treat Non-Small Cell Lung Cancer.

We described previously a human natural killer (NK) cell population that upregulates PD-L1 expression upon recognizing and reacting to tumor cells or exposure to a combination of IL12, IL18, and IL15. Here, to investigate the safety and efficacy of tumor-reactive and cytokine-activated (TRACK) NK cells, human NK cells from umbilical cord blood were expanded, transduced with a retroviral vector encoding soluble (s) IL15, and further cytokine activated to induce PD-L1 expression. Our results show cryopreserved and thawed sIL15_TRACK NK cells had significantly improved cytotoxicity against non-small cell lung cancer (NSCLC) in vitro when compared with non-transduced (NT) NK cells, PD-L1+ NK cells lacking sIL15 expression (NT_TRACK NK), or NK cells expressing sIL15 without further cytokine activation (sIL15 NK cells). Intravenous injection of sIL15_TRACK NK cells into immunodeficient mice with NSCLC significantly slowed tumor growth and improved survival when compared with NT NK and sIL15 NK cells. The addition of the anti-PD-L1 atezolizumab further improved control of NSCLC growth by sIL15_TRACK NK cells in vivo. Moreover, a dose-dependent efficacy was assessed for sIL15_TRACK NK cells without observed toxicity. These experiments indicate that the administration of frozen, off-the-shelf allogeneic sIL15_TRACK NK cells is safe in preclinical models of human NSCLC and has potent antitumor activity without and with the administration of atezolizumab. A phase I clinical trial modeled after this preclinical study using sIL15_TRACK NK cells alone or with atezolizumab for relapsed or refractory NSCLC is currently underway (NCT05334329).

Development of optimized cytotoxicity assays for assessing the antitumor potential of CAR-T cells

CAR-T cells are T cells expressing a chimeric antigen receptor (CAR) rendering them capable of killing tumor cells after recognition of a target antigen. CD19 CAR-T cells have revolutionized the treatment of hematological malignancies. Their function is typically assessed by cytotoxicity assays using human allogeneic cell lines expressing the target antigen CD19 such as Nalm-6. However, an alloreactive reaction is observed with these cells, leading to a CD19-independent killing. To address this issue, we developed a fluorescence microscopy-based potency assay using murine target cells to provide an optimized cytotoxicity assay with enhanced specificity towards CD19.Murine NIH/3T3 (3T3) fibroblast-derived cell line and EL4 T-cell lymphoma-derived cell line were used as targets (no xenoreactivity was observed after coculture with human T cells). 3T3 and EL4 cells were engineered to express eGFP (enhanced Green Fluorescent Protein) and CD19 or CD22 using retroviral vectors. CD19 CAR-T cells and non-transduced (NT) control T cells were produced from several donors. After 4 h or 24 h, alloreactive cytotoxicity against CD19+ Nalm-6-GFP cells and CD19− Jurkat-GFP cells was observed with NT or CAR-T cells. In the same conditions, CAR-T but not NT cells specifically killed CD19+ but not CD19− 3T3-GFP or EL4-GFP cells. Both microscope- and flow cytometry-based assays revealed as sensitive as impedance-based assay. Using flow cytometry, we could further determine that CAR-T cells had mostly a stem cell-like memory phenotype after contact with EL4 target cells.Therefore, CD19+ 3T3-GFP or EL4-GFP cells and fluorescence microscopy- or flow cytometry-based assays provide convenient, sensitive and specific tools to evaluate CAR-T cell function with no alloreactivity.

Disruption of SOCS1 Promotes the Antitumor Activity of CD19-Specific CAR NK Cells Coexpressing IL-15

Background Chimeric antigen receptor (CAR) expressing NK cells exhibit robust antitumor responses in patients with hematologic malignancies but have limited persistent without cytokines supplement. Interleukin-15 (IL-15) promotes NK cells expansion and survival, while IL-15 signaling is often inhibited by the suppressor of cytokine signaling (SOCS) proteins (CIS, SOCS1-7), which is rapidly induced in response to IL-15 and negatively regulates cytokine signaling through the JAK/STAT pathway in NK cells. We hypothesized that SOCS1 would be a logical checkpoint to target in human NK cells to increase their persistence and thereby enhance their antitumor potency. Here, we devised a strategy that couples targeting the SOCS1 protein, with CD19-specific human NK cells that constitutively express IL-15. Method Firstly, we evaluated the SOCS1 mRNA expression levels on NK cells after cultured with or without IL-15 for 6 or 24 hours by reverse transcription polymerase chain reaction (RT-PCR). The retroviral vector encoding CAR19.4-1BB-zeta-2A-IL-15 (named BBz.IL-15) and the production of transient retroviral supernatants have been previously described. Human peripheral blood NK cells isolated from healthy volunteer donors were stimulated with CD16/CD137 mAb and expanded with 500 IU/ml IL-2 (PeproTech) for 5 days before transduced with CAR retrovirus in plates coated with RetroNectin. CAR expression on NK cells was determined by flow cytometry, and IL-15 secretion was measured by ELISA. Knockout (KO) of SOCS1 by CRISPR/Cas9 using the Neon transfection system was performed on day 3 post transduction in both non-transduced NK cells (NT-NK) and CAR-transduced NK cells (CAR-NK). To assess KO efficiency, we used polymerase chain reaction (PCR) gel electrophoresis, western blot, and Sanger sequencing. The degranulation, cytokine production, and cytotoxicity of CAR NK cells were analyzed on day 7 post electroporation. Results The expression of SOCS1 on NK cells was significantly induced within 6 hours of IL-15 treatment. Our approach for combined retroviral transduction with the anti-CD19 CAR and RNP-mediated gene editing of SOCS1 showed that surface expression of the CAR was maintained on NK cells in culture, with construct containing BBz.IL15 (37.18 ± 11.71%) demonstrating comparable transduction efficiency compared with the BBz construct (45.48 ± 10.54%) (P＞0.05), and the efficiency of SOCS1 KO was high in both the nontransduced (NT) control and CAR-expressing NK cells by TIDE and remained stable over time. When tested against CD19-postive Raji cells, SOCS1 KO BBz.IL-15 NK cells produced more IFN-γ and TFN-α, and displayed greater degranulation (CD107a) against their targets than did their BBz.IL-15 NK controls (BBz.IL-15 SOCS1 KO vs BBz.IL-15 control: 15.75±2.05% vs 7.93±0.47% for IFN-γ, P=0.032; 6.27±1.27% vs 1.88±0.26% for TFN-α, P=0.04; 23.25±2.33% vs 12.35±2.33% for CD107a, P=0.042). However, there was no significant difference between the SOCS1 KO BBz. or NT NK cells and their respective BBz. and NT controls regarding IFN-γ and TFN-α production and degranulation (BBz.SOCS1 KO vs BBz.control: 16.95±5.30% vs 16.85±8.70% for IFN-γ, P＞0.05; 6.83±2.98% vs 4.06±2.47% for TFN-α, P＞0.05; 26.65±7.14% vs 25.8±2.12% for CD107a, P＞0.05) (NT SOCS1 KO vs NT control: 21.75±6.58% vs 7.5±2.18% for IFN-γ, P＞0.05; 7.23±3.46% vs 1.58±0.03% for TFN-α, P＞0.05; 15.05±3.46% vs 6.84±2.15% for CD107a, P＞0.05). To confirm this observation, NK cells were cocultured with Raji cells at various E:T ratios for 6h. SOCS1 KO NK cells could kill the tumors more efficiently in comparison to the control. Proliferation data showed the SOCS1 KO NK cells can grow faster than the control group. Conclusion In conclusion, the results demonstrate that SOCS1 might be a potential target for genetic modification to improve activity of CAR- NK cells.

CD70 CAR NK Cells in the Treatment of Multiple Myeloma

Despite the high response rates of chimeric antigen receptor (CAR) - T cells targeting the B-cell maturation antigen (BCMA) for multiple myeloma (MM), patients eventually still relapse. At this time there is no clear subsequent therapy. Furthermore, CAR T-cell therapy is associated with significant toxicities such as cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome. CD70 is a normally transiently expressed on activated T-cells, B-cells, or dendritic cells, but have been found to be upregulated on many hematologic and solid cancer cells such as acute myeloid leukemia, triple negative breast cancer, and renal cell carcinoma. However, CD70 as a potential target for treatment of multiple myeloma, including patients who have progressed on BCMA targeted therapies, has not been fully investigated. We assessed bone marrow aspirates from MM patients by flow cytometry and demonstrated that CD70 was expressed on the majority of myeloma cells, and on average, relapsed myeloma patients had higher expression of CD70. In addition, using immunohistological staining, we found that CD70 was expressed on 10 of 10 patients who had progressed on BCMA targeted therapies (seven who had progressed on belantamab mafodotin, an antibody drug conjugate, three who had progressed on idecabtagene vicleucel, a BCMA CAR-T), suggesting that CD70 could be a viable target in patients who have failed BCMA targeted therapy. As natural killer (NK) cells are a promising off-the-shelf treatment with minimal toxicity as compared to CAR-T cells, we generated CD70-targeting CAR NK cells for the treatment of MM. We found in the chromium cytotoxicity assay that CD70 CAR NK cells had higher killing as compared to non-transduced (NT) NK cells at multiple effector-to-target ratios against MM1s, a myeloma cell line confirmed to express CD70. There was no difference in killing when co-cultured with H929, a myeloma cell line lacking CD70 expression, demonstrating the specificity of the CD70 CAR NK cell. The enhanced killing was also confirmed in an incucyte assay in which the CD70 CAR NK cells cleared tumor faster than NT NK, even in MM1s in which BCMA was knocked out using CRISPR (simulating BCMA antigen loss). When tested in a myeloma mouse model, the CD70 CAR NK exhibited greater tumor control as shown in Figure 1, when compared to NT NK, which lead to increased survival of the mice. Similar results were seen in a mouse model using BCMA KO myeloma cells. In summary, we were able to demonstrate that CD70 can be a feasible target for the treatment of multiple myeloma, including in patients who have failed BCMA targeted therapy. Based on these results, we have initiated a Phase I/II clinical trial (NCT05092451) that is currently recruiting.

Targeting T-Cell Lymphoma Using CD70-Directed Cord Blood-Derived CAR-NK Cells

T-cell lymphomas (TCL) are a heterogenous group of non-Hodgkin lymphoma characterized by resistance to conventional therapies posing an urgent need for safe and effective therapies. CAR-NK cells have emerged as a promising “off-the-shelf” cancer immunotherapy and are especially attractive for the treatment of T-cell malignancies. This is especially the case given the challenge of targeting T cell malignancies with CAR T cells due to shared antigens across the therapeutic, normal and malignant T cells posing the risk of CAR T cell fratricide, T-cell aplasia, and contamination of the CAR T products with malignant T cells. We have developed an FDA-approved system for ex vivo NK cell expansion, which reliably generates clinically relevant doses of GMP-grade NK cells from a cord blood (CB) unit. We have used this platform to engineer NK cells to express a CAR targeting CD19 using GMP-grade retroviral vectors and demonstrated the safety and efficacy of this platform in the clinic. We now propose to expand this approach to target TCL. CD70, a surface ligand of CD27, is overexpressed in a variety of cancers, including TCL. Signaling mediated by CD70-CD27 is thought to induce proliferation of TCL cells via activation of the NF-κB pathway. Thus, CD70 is an attractive target for CAR NK-cell therapy against TCL. We analyzed the publicly available primary TCL dataset (GSE19069) including anaplastic large cell lymphoma (30 samples), angioimmunoblastic TCL (37 samples), peripheral TCL (PTCL; 50 samples), and adult T cell leukemia/lymphoma (13 samples) for CD70 expression. The majority of the samples expressed CD70 transcript with a variable level of expression ( Figure 1). Using flow cytometry, we screened a panel of TCL cell lines with diverse histologic, cytogenetic and molecular properties for the expression of CD70. We demonstrated positive CD70 expression on most of the TCL cell lines profiled encompassing anaplastic large cell lymphoma (ALK+: KAR299; ALK-: MAC2A; fusion gene NPM-ALK+: SU-DHL1 and SUP-M2), hepatosplenic gamma-delta TCL (DERL-7), Sezary syndrome (SS; H9 and HuT78), and PTCL NOS (OCI-LY12). The CD70 intensity was highest on MAC2A, HuT78, KAR299 and SUP-M2, intermediate to low on DERL-7 and H9, and negative on SU-DHL1 and OCI-LY12. We designed a novel retroviral vector targeting CD70 that: 1) encodes the CD70-targeting CAR gene based on the CD27 extracellular domain; 2) ectopically produces interleukin (IL)-15 to support NK cell proliferation; and 3) expresses the suicide gene inducible caspase 9 ( iC9) that can be pharmacologically activated to eliminate transduced cells in the event of toxicity. The construct will be referred to as CAR.70/IL-15. Non-transduced (NT) NK cells and NK cells transduced with a construct that leads to IL-15 production without a CAR (IL-15 NK cells) were used as controls. We demonstrated that CAR.70/IL-15 NK cells had a significantly enhanced cytotoxicity compared to NT- and IL-15 NK cells against the CD70+ cell lines while there was no difference in cytotoxicity between the NK cell conditions in the negative cell lines pointing to a CD70-targeted fashion of anti-TCL NK cell cytotoxicity. Moreover, CAR.70/IL-15 NK cells exhibited greater cytotoxicity against CD70+ cell lines in long-term cytotoxicity assays ( Figure 2). In a tumor rechallenge assay against KAR299 where fresh tumor cells were added every 3-4 days, CAR.70/IL-15 NK cells showed superior potency in continuous tumor control. In addition, CAR.70/IL-15 NK cells exhibited higher levels of degranulation (CD107a) and cytokine production (TNFα and INF-γ) when cocultured with CD70+ TCL cell lines, compared to NT and IL-15 NK cells. Furthermore, these CAR NK cells targeting CD70 outperformed CAR NK cells engineered to target CD19, which is an irrelevant target for TCL, further confirming the specific CD70-targeted activity of CAR.70/IL-15 NK cells. CyTOF immunophenotyping revealed that CAR.70/IL-15 NK cells are characterized by the increased expression of markers of cytotoxicity such as granzyme-b, perforin, TRAIL; and activating coreceptors/proliferation markers such as DNAM, CD25 and Ki67. Taken together, our findings validate CD70 as a promising target for TCL treatment using CAR NK cells. Our approach provides a solid foundation for the clinical translation of CD70-targeting CAR NK-cell therapy for patients with TCL.

Preclinical testing of FOLR1-CAR T cells against osteosarcoma (OS).

2555 Background: Survival rates for OS are poor. Prior literature suggests folate receptor-a (FOLR1) overexpression in OS, making it a target for CAR T development. Methods: OS transcriptome data from TCGA, PCGP and NCI PIVOT programs were interrogated for FOLR1 gene expression. A 2nd generation FOLR1-CAR T cell was created by fusing the ScFv from farletuzumab (anti-FOLR1 monoclonal antibody) with a 4-1BB costimulatory and CD3z cytotoxicity domain. Non-transduced (NT) T cells served as controls. In vitro functional analysis was performed against U2OS cell line using flow cytometry (FC) and Incucyte. U2OS xenograft in NSG mice was generated via intra-femoral inoculation for local tumor and via tail vein (TV) tumor injection for pulmonary disease. Tumor engraftment was confirmed by in vivo imaging system (IVIS). Disease progression was monitored by IVIS and CT imaging. 5 million T cells were administered via TV. Circulating human T cells (HTC) collected from retro-orbital peripheral blood (PB) were detected via FC. Results: FOLR1 transcript was expressed in >75% of OS in TCGA, PCGP and PIVOT. In vitro evaluation of FOLR1-CAR T demonstrated robust antitumor activity (E:T ratio 1:1) with cytotoxicity of 76.5% and 95.5% at 6 and 24 hrs. Disease progression was observed in NT T cell treated mice, with an average fold increase in IVIS radiance of 35.1 in local (n=2) and 142.8 in pulmonary disease (n=3) over 11 weeks (wks) versus complete resolution of local (n=2) and pulmonary disease (n=3) in FOLR1-CAR T treated mice (see table). NT T treated mice with local disease demonstrated significant bone destruction; no local tumors were found in FOLR1-CAR T treated mice. HTC (CD45+/CD3+) expansion was detected in PB one wk post-treatment (PT); in mice with metastasis 0.190% and 0.136% of live circulating cells were human T cells in FOLR1-CAR T and NT T treated mice, respectively. Improved overall survival was observed in FOLR1-CAR T treated mice (>100 vs 87.5 days). Conclusions: FOLR1-CAR T cells appear to have in vivo activity against U2OS cell line and support further testing of safety and feasibility in an early phase clinical trial for relapsed/refractory OS. [Table: see text]

Asymmetrical Expression of PD1 and CD25 in T-cells Post-Initial Activation

Abstract After TCR engagement, T cells induce expression of CD25, which forms the IL2 high affinity receptor complex, concurrently with PD1, which plays a down-regulatory role in activation. Cessation of TCR stimulation leads to eventual contraction of T cell responses. Chimeric antigen receptor (CAR) T cell therapy has seen increased use in the past decade. CAR T-cells are activated and transduced ex vivo and expanded with low amounts of cytokines prior to administration. The expression of CD25 and PD1 on CAR T cells at different stages of the process has been incompletely characterized. Purified T cells were activated in vitro for 3 days followed by expansion using low dose IL2. We demonstrate that both CD25 and PD-1 are strongly upregulated during initial activation, but by day 8, under expansion conditions, PD1 is downregulated to negligible levels. Despite absence of TCR signaling, CD25 expression remains moderate-to-high explaining the ability to maintain proliferation. This discrepancy was higher in transduced CAR T-cells compared to activated non-transduced (NT) T cells indicating the possibility of CAR tonic signaling. Furthermore, restimulation of both NT and CAR-T cells after this expansion phase resulted in further increased CD25 expression whereas PD-1 expression remained low. Our studies suggest that during initial activation, TCR engagement is necessary to maintain PD-1, but not CD25 expression. However, upon restimulation, TCR engagement is capable of further upregulating CD25, but not PD-1 expression. These results indicate that TCR signaling induces asymmetrical expression of PD-1 and CD25, a dynamic interplay that may regulate T-cell function and memory response and that PD1 may not be a limiting factor in the process.

Abstract 913: Engineering allogeneic off-the-shelf CD19-directed CAR-iNKT cells without additional genetic manipulations for the treatment of hematological malignancies

Abstract Despite the approval of three autologous CAR-T cell products, B-cell malignancies still represent an unmet need as several of the relapsed/refractory patients are ineligible for autologous CART cell therapies. Invariant natural killer T (iNKT) cells are a unique subset of immune cells that display properties of both T cells and natural killer cells and bridge the innate and adaptive immune responses upon antigen recognition. Unlike conventional β T cells, iNKT cells express an invariant T cell receptor (TCR) that recognize glycolipids presented in the context of the monomorphic, MHC-class I related molecule, CD1d. iNKT cells are ideal cell types for allogeneic, off the shelf approach as they can target cancers without the risk of graft-versus-host disease (GvHD), circumventing the need to delete or knock out the endogenous TCR. Here, we report the engineering of ‘off-the-shelf’ iNKT cells directed against CD19 and their robust in vitro and in vivo functional profile. Briefly, peripheral blood derived iNKT cells were isolated from healthy donors and were engineered to express a CD19 CAR using a 3rd generation lentiviral vector. To demonstrate the CAR-dependent and independent anti-tumor activity, CAR19-iNKT cells were compared in vitro against non-transduced (NT) iNKT cells in cytotoxicity assays and intracellular cytokine staining assays. Allogeneic CAR19-iNKT cells upregulated activation markers, secreted cytokines, and lysed cancer cell lines and primary tumor cells from chronic lymphocytic leukemia and marginal zone lymphoma patients in an antigen-specific manner. Excellent post thaw recovery and viability was noted in cryopreserved CAR19-iNKT cells. Finally, the anti-tumor activity of cryopreserved CAR19-iNKT cells were evaluated in an established aggressive NSG mice model of SEM-luc, a B cell lymphoblastic leukemia cell line expressing luciferase. Robust antitumor activity by CAR19-iNKT cells was observed without any obvious adverse effects compared to controls (untreated and NT iNKT cells). The persistence of CAR19-iNKT cells in this model is currently being evaluated. These data demonstrate a unique, off the shelf CAR19-iNKT cell platform for targeting hematological malignancies that combines the specificity of CD19 targeting CAR with the innate features of iNKT cells. The data indicates that iNKT cells are a potent cell type for generating allogeneic off the shelf products for treating a broad spectrum of hematological malignancies and solid tumors by targeting different receptors on the respective tumors. Citation Format: Kanagaraju Ponnusamy, Janani Kanagaraju, Yingxin Wang, Bryan Lye, Hongwei Ren, Sandhya Buchanan, Michael J. Baker, Mini Bharathan, Anastasios Karadimitris. Engineering allogeneic off-the-shelf CD19-directed CAR-iNKT cells without additional genetic manipulations for the treatment of hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 913.

Pre-Clinical Evaluation of Allogeneic 'Off-the-Shelf' CAR-iNKT Cells Targeting CD19 (ALA-101)-Expressing Hematological Malignancies

Background Arovella Therapeutics is a preclinical stage company developing allogeneic 'off-the-shelf' human invariant natural killer T (iNKT) cell-based therapies for hematological malignancies and solid tumors. iNKT cells are a unique and rare subset of lymphocytes that express an invariant T cell receptor (iTCR) and are restricted by the glycolipid-presenting, non-polymorphic CD1d. Unlike allogeneic conventional T cells that can cause acute graft versus host disease (aGVHD) following administration, iNKT cells exert a protective effect against aGVHD after allogeneic stem cell transplantation in humans. Early clinical studies suggest that non-engineered and CAR-engineered allogeneic iNKT cells are safe in humans and represent a promising therapeutic approach, bypassing the requirement to delete the endogenous TCR. Their inherent biological properties enable iNKT cells to mount a swift and robust antitumor immune response when engineered with a Chimeric Antigen Receptor (CAR). The use of iNKT cells from healthy donors has many advantages over allogeneic T cells and NK cells, including recognition of CD1d+ on tumor cells through the iTCR in addition to the CAR mediated recognition of a tumor specific antigen. Here, we report results from early preclinical studies assessing the functional profile of iNKT cells engineered to express an optimized CAR targeting CD19 (ALA-101) for allogeneic use. Methods iNKT cells were purified from the peripheral blood of healthy donors followed by lentiviral vector transduction (development construct) to express a CD19-targeting CAR. After transduction, cells were expanded to generate ALA-101. The CD19 CAR functionality of ALA-101 was compared against non-transduced (NT) iNKT cells or with CAR19-T cells transduced with the same lentiviral vector. Flow cytometry was used to assess the expression of the CAR and ALA-101 phenotypes and to determine CD19 and CD1d expression levels in the primary patient samples; marginal zone lymphoma (MZL) cells and two chronic lymphocytic leukemia (CLL) samples. In vitro cytotoxicity assays and intracellular cytokine staining were used to evaluate the CAR-dependent and -independent anti-tumor activities using B cell lymphoblastic leukemia (B-ALL) SEM cell line and primary tumor cells from patients. In vivo efficacy testing of ALA-101 is ongoing in a disseminated NSG mouse model of SEM cells expressing luciferase. Results The purity of iNKT cells following selection from peripheral blood mononuclear cells of healthy donors was greater than 99%. ALA-101 was optimized for CD19 CAR functionality using a pre-clinical lentiviral vector. Using multiple independent donors, we confirmed that iNKT cells engineered with lentiviral vector encoding CD19 CAR (ALA-101) maintained the surface CAR expression over 35 days and expressed variable frequencies of CD27, CD62L and CCR7. Compared to day 11 CAR19-T cells, day 17 and day 35 ALA-101 cells demonstrated higher cytotoxicity against the CD19+CD1d- B-ALL SEM cells; similarly, ALA-101 demonstrated higher cytotoxic activity against CD19+CD1d+ malignant B cells from patients with CLL and MZL. Of note, when tested at different time points, ALA-101 cells expanded for 17 and 35 days showed similar cytotoxicity against primary patient samples and SEM cells. Intracellular cytokine staining from tumor cell co-cultures demonstrated that the majority of ALA-101 expressed TNFα, Perforin and Granzyme B. Conclusions Here we report the preclinical activity of ALA-101, a novel allogeneic CAR19-iNKT cell product for the treatment of CD19+ B-cell malignancies. Our data shows the successful generation of polyfunctional allogeneic iNKT cells in terms of cytotoxicity and cytokine profile when engineered to express a CD19-targeting CAR. We have initiated the manufacturing of ALA-101 with the intention to enter clinic trials in CD19 positive malignancies. Taken together, the data indicates that iNKT cells are a potent allogeneic, 'off-the-shelf' cell therapy to treat a broad spectrum of hematological malignancies and solid tumors.

Abstract LB211: Tumor-reactive and anti-PD-L1 co-stimulated killer cells (TRACK-NK) for immunotherapy of non-small cell lung cancer

Abstract Non-small cell lung cancer (NSCLC) remains one of the major causes of cancer related mortality worldwide, yet clinical advances with checkpoint inhibitors suggest that an anti-tumor immune response can be harnessed to prolong survival in NSCLC patients. We recently described the identification of a tumor reactive natural killer (NK) cell population by its in vivo upregulation of PD-L1 in the presence of tumor, referred to here as TRACK-NK cells. PD-L1(+) NK cells are more potent than PD-L1(-) NK cells against tumor cell targets. Further, the administration of anti-PD-L1 antibody (atezolizumab) functions as an immune agonist on TRACK-NK cells resulting in significantly enhanced degranulation (P < 0.001) and secretion of IFN-gamma (P < 0.001) in the presence of tumor. Here we investigated the functional activity and product safety of allogeneic, off-the-shelf TRACK-NK cells retrovirally transduced to express soluble (s) IL-15 to treat NSCLC without or with the addition of atezolizumab. TRACK-NK cells derived from umbilical cord blood NK cells were expanded over 1,000-fold ex-vivo in 17 days with ~50% of the final product engineered to express sIL15 and cytokine-activated to express PD-L1. The phenotype of the final TRACK-NK cell product showed a significant increase in the expression of PD-L1, CD25 and CD69 when compared to non-transduced (P < 0.01) or NK cells transduced to express sIL15 without cytokine activation (s15 NK cells) (P < 0.01). Following cryopreservation, our thawed TRACK-NK cell product demonstrated high recovery (>85%) and viability (>80%). In a Real Time Cell Analysis (RTCA) in vitro cytotoxicity assay against a human NSCLC cell line (A549), TRACK-NK cells were 10 times more potent than non-transduced (NT) NK cells (P < 0.001) and 2 times more potent than s15 NK cells (P < 0.01). To assess the efficacy of our frozen, allogeneic off-the-shelf TRACK-NK cells in vivo, we utilized immunodeficient mice bearing human A549 metastatic NSCLC. Intravenous infusion of TRACK-NK cells resulted in significantly more suppression of luciferase-A549 NSCLC growth in mice compared to mice treated with NT NK cells (P < 0.05) or s15 NK cells (P < 0.01). In terms of dose response, 1 × 107 TRACK-NK cells per dose delivered every other day for a total of 4 doses showed significantly better tumor control compared to a dose of 5 × 106 TRACK-NK cells (P < 0.001) but similar control compared to 2 × 107 TRACK-NK cells. The combination of TRACK-NK cells plus atezolizumab showed better control of NSCLC growth in vivo compared to TRACK-NK cells alone (P < 0.01). Finally, we evaluated the safety of our TRACK-NK product in immunodeficient mice without tumor engraftment and did not observe any significant change in body weight, body temperature, liver enzymes, kidney function, or blood counts compared to mice treated with vehicle control. No cytokine release syndrome was observed. In summary, our studies using human allogeneic off-the-shelf TRACK-NK cells proved to be safe and non-toxic, demonstrated enhanced cytotoxicity against NSCLC in vitro and enhanced control of tumor growth in vivo, further improved with atezolizumab. As the intravenous infusion of activated NK cells naturally traffic to lung, our TRACK-NK platform will move forward to the clinic for the treatment of relapsed and refractory NSCLC patients. Citation Format: Ting Lu, Anthony G. Mansour, Rui Ma, Christian Bustillos, Zhiyao Li, Shoubao Ma, Zhenlong Li, Hanyu Chen, Kun-Yu Teng, Jianying Zhang, Michael A. Caligiuri, Jianhua Yu. Tumor-reactive and anti-PD-L1 co-stimulated killer cells (TRACK-NK) for immunotherapy of non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB211.

Antitumor activity of T cells expressing a novel anti-folate receptor alpha (FOLR1) costimulatory antigen receptor (CoStAR) in a human xenograft murine solid tumor model and implications for in-human studies.

2535 Background: ITIL-306 is an autologous tumor-infiltrating lymphocyte (TIL) therapy that integrates T-cell receptor (TCR)-specific antigen recognition (Signal 1) with robust costimulation via the novel CoStAR transgene upon engagement with FOLR1 (Signal 2; Sukumaran, et al. JITC. 2021;9:198). Here, we assessed IL-2 independent effector function by anti-FOLR1 CoStAR T cells in vitro and evaluated activity in a novel, more representative murine solid tumor model. Methods: For in vitro studies, healthy donor T cells were manufactured to express anti-FOLR1 CoStAR or left nontransduced (NTD). Product cells were stimulated with a single (Day [D]0; ±IL-2) or serial (D0, 7, 14, 21; no IL-2) addition of target cells expressing membrane-anchored OKT3 (Signal 1) and FOLR1 (Signal 2). T-cell activation and proliferation were measured. For in vivo studies, the carcinoembryonic antigen (CEA)-positive (Signal 1) H508 cell line was engineered to express FOLR1 (Signal 2) and injected into 6 mice/group (D–21). NTD or healthy donor T cells expressing HLA-A*02-restricted anti-CEA TCR only, anti-FOLR1 CoStAR only, or dual TCR+CoStAR were given intravenously (D0), and compared with PBS control. Tumor growth, survival, and T-cell expansion were assessed in 2 donors to D96. Results: After single and serial stimulation, only anti-FOLR1 CoStAR T cells showed sustained proliferation without exogenous IL-2. PD-1 positivity was low ( < 10%) on anti-FOLR1 T cells up to D24. In vivo, T-cell persistence on D14 was increased in dual TCR+CoStAR vs anti-CEA TCR only mice ( P<.001) and all other groups ( P<.01-.001). Tumor growth up to D58 was significantly lower in dual TCR+CoStAR ( <.5 cm3) vs all other groups (each > 2 cm3; P<.0001). Survival was significantly longer in dual TCR+CoStAR ( P<.01) vs all other groups; only dual TCR+CoStAR mice were alive at D58, with 5/6 alive at D96. The second donor showed similar tumor control irrespective of exogenous IL-2 in responder mice, with > 50% alive at D96, and robust T-cell expansion in dual TCR+CoStAR mice. Conclusions: When combined with TCR-specific binding, CoStAR significantly enhanced T-cell proliferation, persistence, and antitumor activity in vivo vs TCR alone, resulting in tumor control and prolonged survival. Effects were not observed with CoStAR alone, underscoring that signaling through CoStAR alone does not induce T-cell effector function. The sustained proliferation of anti-FOLR1 CoStAR T cells without exogenous IL-2 support in vitro and in vivo supports a clinical TIL regimen free of high-dose IL-2. These results suggest that CoStAR may improve the clinical performance and benefit/risk profile of TILs, whereby fewer toxicities are expected with the removal of post-TIL IL-2 support. This will be explored in an upcoming first-in-human clinical study with ITIL-306.

Anti-CCR9 CAR-T Cells for T Acute Lymphoblastic Leukemia

The prognosis of relapsed/refractory T acute lymphoblastic leukemia (T-ALL) is poor with a dire lack of new treatment options which impart meaningful survival benefits. This is in stark contrast with B-ALL, where CD19-directed CAR-T cell therapy has revolutionized the treatment of relapsed/ refractory (r/r) disease. However, CAR-T for T-ALL is challenging since target antigens described to date are expressed on normal T cells. This leads to two main problems: loss of essential normal T cells and self-kill ‘fratricide’ of CAR-T.To avoid these issues, we sought to identify potential immunotherapy targets for T-ALL which are not expressed on normal T cells or other essential cell types. We analysed the collated gene expression profiles of 35 normal tissues (n=172 samples) as compared to MOLT-4, a T-ALL cell line. Using subtractive transcriptomics, we identified 12 transcripts uniquely expressed in MOLT-4 but not in normal mature tissues. Of these, CCR9 (C-C Motif Chemokine Receptor 9) was the most attractive, being cell-surface resident and thus potentially amenable to immunotherapy. Further, publicly available RNA-seq data confirmed CCR9 was expressed in >70% of cases of paediatric T-ALL.CCR9 is a G-protein coupled receptor for the natural ligand CCL25, and is expressed in gut intraepithelial γδ T cells, some plasmacytoid dendritic cells and double-positive thymocytes, but in less than 5% of normal circulating T and B cells. CCR9 is not expressed in hematopoietic stem cells (HSCs) or myeloid cells.Using flow cytometry, 74/102 cases (73%) of primary T-ALL expressed CCR9, with expression enriched in cases of relapsed/ refractory disease - 64% diagnostic v 85% relapsed v 86% primary refractory (Figure 2a). The median antigens/ cell was 1732 and expression was preserved or increased upon relapse (1320 diagnostic v 1889 relapsed v 2175 refractory). On normal blood cells, expression was limited to ~9% of B cells and <3% of T cells, at low density (<500 antigens/cell).We developed a novel rat-derived anti-CCR9 scFv, and generated a second-generation CAR with 4-1BB-CD3ζ endodomain, expressed in a gamma-retroviral vector. T cells transduced with anti-CCR9 CAR (CARCCR9) expanded similarly to control anti-CD19 CAR (CAR19), with no evidence of fratricide. No CCR9+ cells were detected following transduction, suggesting ‘purging’ of CCR9+ T cells.We co-cultured CARCCR9 or control CAR19 for 48hrs with multiple T-ALL cell lines, including CCR9-negative variants generated using CRISPR-Cas9. We showed specific cytotoxicity, cytokine secretion (interferon-gamma and IL-2) and proliferation of CARCCR9 against CCR9+ cell lines, including at low target density of ~400 copies/ cell. In addition, in 72hr co-cultures at a 1:1 ratio, CARCCR9 but not NT or CAR19 T cells secreted interferon-gamma and lysed primary blasts from 3 patients with T-ALL.We tested CARCCR9 in vivo. We intravenously (IV) injected NSG mice with 3 x 10^6 MOLT-4 cells, engineered to express luciferase. Nine days later (D+9), mice received 8 x 10^5 non-transduced (NT), CAR19 or CARCCR9 cells IV. While untreated mice and NT or CAR19 recipients experienced disease progression, weight loss and death, mice receiving CARCCR9 had disease regression, continued weight gain and prolonged survival beyond day 80 (median OS NT 16 days, CAR19 16 days, CARCCR9 NR, p = 0.003). Further, these mice were re-injected with 1 x 10^6 MOLT4-Fluc on D+49, and in 3/4 (75%), no increasing BLI signal was detected, suggesting continued anti-leukemic immunosurveillance.We also tested CARCCR9 in 2 patient-derived xenograft (PDX) models of T-ALL. NSG mice were injected with 1 x 10^6 primary blasts, then received 8 x 10^5 NT, CAR19 or CARCCR9 cells IV on D+20. All recipients of NT or CAR19 displayed increasing ALL burden in peripheral blood over time, with weight loss, splenomegaly and eventual leukaemic death. By contrast, all CARCCR9 recipients had undetectable leukemia and long-term disease-free survival (median OS NT 42 days, CAR19 42 days, CARCCR9 NR, p = 0.003), with no detectable blasts in marrow or spleen at necropsy.Thus, we have demonstrated potent anti-leukemic function of anti-CCR9 CAR-T cells both i n vitro and in vivo. Further, efficacy was not associated with loss of essential normal T cells or with CAR-T fratricide. We propose that anti-CCR9 CAR-T cells could be a safe and effective treatment strategy for T-ALL, and potentially a major advance in a neglected clinical area. DisclosuresMaciocia: Autolus: Current equity holder in publicly-traded company, Research Funding. Maciocia: Autolus: Current equity holder in publicly-traded company. Leon: BenevolentAI: Current Employment. Pule: Autolus: Current Employment, Current equity holder in publicly-traded company. Mansour: Astellas: Consultancy, Honoraria; Janssen: Consultancy.

Anti-CD117 CAR T Cells Incorporating a Safety Switch Eradicate Acute Myeloid Leukemia and Deplete Human Hematopoietic Stem Cells

Anti-CD117 CAR T Cells Incorporating a Safety Switch Eradicate Acute Myeloid Leukemia and Deplete Human Hematopoietic Stem Cells

Development of CD19 CAR Engineered Human Placental CD34 +-Derived Natural Killer Cells (CAR19-CYNK) As an Allogeneic Cancer Immunotherapy

Development of CD19 CAR Engineered Human Placental CD34 +-Derived Natural Killer Cells (CAR19-CYNK) As an Allogeneic Cancer Immunotherapy

Selectively targeting myeloid-derived suppressor cells through TRAIL receptor 2 to enhance the efficacy of CAR T cell therapy for treatment of breast cancer

BackgroundSuccessful targeting of solid tumors such as breast cancer (BC) using chimeric antigen receptor (CAR) T cells has proven challenging, largely attributed to the immunosuppressive tumor microenvironment (TME). Myeloid-derived suppressor...

CS1-Specific Chimeric Antigen Receptor Drives Cord Blood NK Cell Maturation and Expansion In Vivo

Chimeric antigen receptors (CAR) have been used extensively to redirect the specificity of T cells against leukemic cells, with remarkable clinical responses in patients with lymphoid malignancies. CAR-transduced natural killer (NK) cells might serve as an alternative to CAR T cells as they lack the potential to cause GVHD, and could be made available as an off-the-shelf allogeneic product for immediate clinical use. Moreover, unlike CAR-T cells, CAR NK cells retain their intrinsic capacity to recognize and target tumor cells through their native receptors, in theory making disease escape through downregulation of the CAR target antigen less likely. We have identified cord blood (CB) as a readily available source of allogeneic NK cells.Thus, in this study, we reprogrammed CB-NK cells to recognize multiple myeloma (MM) using a retroviral construct encoding a CAR against CS1, a coreceptor expressed on MM cells, IL-15 to enhance their in vivo proliferation and survival, and inducible caspase-9, a suicide gene that can be activated as needed. We consistently achieved a median CAR positivity of >90%, 14 days post transduction and viability of >95% with only limited CS1-mediated fratricide (since CS1 is also expressed on NK cells) during long-term ex vivo culture. CS1-CAR NK cells killed the CS1-expressing MM.1S cell line significantly better than ex vivo expanded non-transduced (NT) CB-NK cells at all effector:target ratios tested (p<0.001). We next tested the in vivo trafficking and antitumor activity of iC9/CAR.CS1/IL-15+ CB-NK cells in a xenograft mouse model of MM. Mice engrafted with MM1.S cells received saline (control) or 1 x107 ex vivo expanded CB-NK cells that were either unmodified or transduced with iC9/CAR.CS1/IL-15; iC9/CAR.CS1/IL15+ CB-NK cells controlled tumor expansion and prolonged survival of mice significantly better than NT CB-NK cells.This was associated withsignificantly higher frequencies of circulating NK cells in the CAR+ CB-NK cell treated mice (median=19%, range 13.6-31.1%) compared to the NT CB-NK group (median=1.26%, range 0.98-2.49%, p=0.0003). We next used mass cytometry (CyTOF) to interrogate the features that shape NK cell diversity, their maturation and their trafficking in vivo. The expression of CAR.CS1 on NK cells infiltrating tumor sites remained stable over time in vivo (median 86.7%, range 81-89.8% compared to the infused product). This was associated with significantly less tumor infiltration in liver (0.013% vs 33.8%), spleen (0.01% vs 23.4%) and BM (4.3% vs 79.8%) harvested on day 21 post-infusion, compared to mice treated with NT NK cells. Interestingly, the residual MM cells in the BM of iC9/CAR.CS1/IL-15 treated mice lost CS1 expression on their surface, suggesting that target antigen downregulation may also play a part in escape from CAR-mediated immune recognition in MM, as also described with CD19 CAR T cell therapy.Using a panel of 40 monoclonal antibodies, we profiled the global signature of NK cells harvested from organs of animals sacrificed on day 21 after infusion and compared them to CAR+ CB-NK cells prior to infusion. The diversity of iC9/CAR.CS1.IL-15-transduced CB-NK cells, measured using the inverse Simpson index, was similar to that of ex vivo expanded NT CB-NK cells (167 vs 114). Additionally, using t-SNE maps to visualize the high-dimensional data generated by CyTOF, we observed complete overlap of clusters for ex vivo expanded NT CB-NK cells and CS1-CAR-transduced CB-NK cells, indicating, that any potential CS1-related fratricide does not significantly impact on the repertoire of the CAR-transduced CB-NK cells (Figure 1).Interestingly, there was a profound and significant increase in the diversity of the infiltrating CS1-CAR CB-NK cells in the liver (830), spleen (843) and BM (965) on day 21 after infusion, suggesting in vivo maturation and expansion of NK subpopulations in response to the tumor. t-SNE analysis revealed a clear separation between iC9/CAR.CS1.IL-15 transduced CB-NK cells prior to infusion and tissue-infiltrating CAR+ CB-NK cells, indicating significant shifts in NK subpopulations, with an increase in diversity and maturation after adoptive transfer in vivo (Figure 1). The data from this analysis will have important implications for future studies as we seek to generate CAR+ CB-NK cells with enhanced antitumor potential. [Display omitted] DisclosuresNeelapu:Karus: Research Funding; Poseida Therapeutics, Inc: Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Cellectis Inc.: Research Funding; Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Orlowski:BioTheryX: Consultancy, Membership on an entity's Board of Directors or advisory committees.

MyD88/CD40 Enhanced CD19-Specific CAR-T Cells Maintain Therapeutic Efficacy Following Resolution of Cytokine-Related Toxicity Using Inducible Caspase-9

Introduction: The efficacy of chimeric antigen receptor-modified T cells (CAR-T) targeting CD19+ leukemias and lymphomas is dependent on their in vivo expansion following adoptive transfer. Additional genetic augmentations to improve CAR-T expansion may improve therapeutic efficacy but risks increasing CAR-T toxicity. Here, we demonstrate that a highly active CD19-specific CAR-T cell constitutively expressing a MyD88/CD40 (MC) costimulatory fusion protein and the inducible caspase-9 (iC9) safety switch, is effective at eliminating tumors but induces acute cytokine-related toxicity in animal models. This toxicity, however, can be resolved by administration of rimiducid to induce partial CAR-T apoptosis preserving long-term anti-tumor effects.Methods: T cells were activated with anti-CD3/CD28 antibodies and transduced with a retrovirus encoding iC9, a first-generation CAR (with CD3ζ) targeting CD19, and a constitutively active MC costimulatory protein (SFG-iC9-CD19.ζ-MC). In vitro coculture assays were used to measure specific killing of CD19+ tumor cells (Daudi and Raji) and cytokine production. To assess anti-tumor efficacy, immunodeficient NSG mice were injected i.v. with 5x105 Raji-EGFPluciferase (EGFPluc) or 1x106 Daudi-EGFPluc tumor cells followed by i.v. administration of variable doses (1x106 to 1x107) non-transduced (NT) or iC9-CD19.ζ-MC-modified T cells on days 4 to 10 after tumor engraftment. Tumor growth was measured by in vivo imaging. CAR-T cell toxicity was assessed by weight loss and physical appearance. In initial experiments, CAR-T toxicity was resolved by i.p. administration of 0.5 mg/kg rimiducid after 15% weight loss. Serum cytokines were measured before and after rimiducid treatment. In addition, mice were treated with neutralizing antibodies to hIL-6, hIFN-γ and hTNF-α (100 ug i.p. twice weekly). Further, iC9-CD19.ζ-MC-modified T cells were sorted into CD4+ and CD8+ T cell fractions to assess the impact of cytokine production from CAR-T cells on animal health. Mice treated with iC9-CD19.ζ-MC-modified T cells that also received rimiducid to resolve toxicity were subsequently rechallenged with tumors to measure persistence of residual CAR-T cells, and at the end of the experiment were sacrificed and the spleens analyzed for the presence of human T cells and functional CAR-T activity using in vitro assays.Results: T cells were efficiently transduced (40-85%) with the iC9-CD19.ζ-MC retrovirus. In vitro assays showed that these CAR-T cells eliminated CD19+ Raji and Daudi tumor cells at low (1:20) effector-to-target (E:T) ratios in 7 day coculture assays, and produced high levels of IL-2 (669±324 pg/ml), IFN-γ (6715±2541 pg/ml), IL-6 (753±398 pg/ml) and TNF-α (2668±1867 pg/ml). NSG mice engrafted with CD19+ EGFPluc-expressing Raji or Daudi tumor cells were treated with variable doses of iC9-CD19.ζ-MC-modified T cells which were capable of eliminating tumor by day 21. However, acute toxicity (>15% body weight reduction) was observed in a CAR-T cell dose dependent manner occuring day 7 to 14 post-CAR-T injection. Systemic administration of 0.5 mg/kg rimiducid rapidly resolved toxicity and mice regained weight within 3-5 days. Serum cytokine concentrations of hIL-6 and hIFN-γ decreased to background levels within 24 hours post-rimiducid treatment. All mice treated with iC9-CD19.ζ-MC-modified T cells demonstrated long-term (>100 days) control of tumor growth and rejected CD19+ when rechallenged at day 150. Use of neutralizing antibodies against hIL-6, hIFN-γ and hTNF-α showed that blockade of hTNF-α resulted a significant reduction in weight loss. Further, iC9-CD19.ζ-MC-modified T cells were fractionated into CD4+ and CD8+ and measured for cytokine production, showing that CD4+ CAR-T cells produced higher levels of IL-2 (60-fold), IFN-γ (4-fold), IL-6 (37-fold) and TNF-α (10-fold) compared to CD8+ CAR-T cells. Furthermore, treatment of tumor-bearing animals with CD8+ selected iC9-CD19.ζ-MC-modified T cells reduced toxicity and the requirement of iC9 activation, while maintaining anti-tumor efficacy.Conclusions: Enhanced CAR-T cell activity can raise the risk of T cell toxicity. Constitutively active MC costimulation provides robust CAR-T cell proliferation and anti-tumor activity, but also increase cytokine-related toxicity. iC9 can be used to resolve toxicity without affecting long-term efficacy of CAR-T treatment in animals. DisclosuresFoster:Bellicum Pharmaceuticals: Employment, Other: stockholders . Lin:Bellicum Pharmaceuticals: Employment. Lu:Bellicum Pharmaceuticals: Employment. Chang:Bellicum Pharmaceuticals: Employment. Shinners:Bellicum Pharmaceuticals: Employment. Khalil:Bellicum Pharmaceuticals: Employment. Crisostomo:Bellicum Pharmaceuticals: Employment. Mahendravada:Bellicum Pharmaceuticals: Employment. Slawin:Bellicum Pharmaceuticals: Consultancy, Equity Ownership. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership, Other: stockholders .

Overcoming the breast tumor microenvironment by targeting MDSCs through CAR-T cell therapy.

1032 Background: Successful targeting of solid tumors such as breast cancer (BC) using CAR T cells (CARTs) has proven challenging, largely due to the immune suppressive tumor microenvironment (TME). Myeloid derived suppressor cells (MDSCs) inhibit CART’s function and persistence within the breast TME. We generated CAR T cells targeting tumor-expressed mucin 1 (MUC1) (Bajgain P et al, 2018) for BC. To potentiate expansion and persistence of MUC1 CARTs and modulate the suppressive TME, we developed a novel chimeric co-stimulatory receptor, TR2.4-1BB, encoding a ScFv derived from a TNF-related apoptosis-inducing ligand receptor 2 (TR2) mAb followed by a 4-1BB endodomain. We hypothesize that engagement with TR2 expressed on TME-resident MDSCs, will lead to both MDSC apoptosis and CART co-stimulation, promoting T cell persistence and expansion at tumor site. Methods: Function of the novel TR2.4-1BB receptor, was assessed by exposing non-transduced (NT) and TR2.4-1BB transduced T cells to recombinant TR2 and nuclear translocation of NFκB was measured by ELISA. Functionality of in vitro generated MDSCs was determined by the suppression assay. In vitro CART/costimulatory receptor T cell function was measured by cytotoxicity assays using MUC1+ tumor targets in presence or absence of MDSCs. In vivo anti-tumor activity was assessed using MDSC enriched tumor-bearing mice using calipers to assess tumor volume and bioluminescence imaging to track T cells. Results: Nuclear translocation of NFκB was detected only in TR2.4-1BB T cells. MDSCs significantly attenuated T cell proliferation by 50±5% and IFNγ production by half compared with T cells cultured alone. Additionally, presence of MDSCs, diminished cytotoxic potential of MUC1 CARTs against MUC1+ BC cell lines by 25%. However, TR2.4-1BB expression on CAR.MUC1 T cells induced MDSC apoptosis thereby restoring the cytotoxic activity of CAR.MUC1 against MUC1+ BC lines in presence of TR2.4-1BB (67±8.5%). There was an approximate two-fold increase in tumor growth due enhanced angiogenesis and fibroblast accumulation in mice receiving tumors + MDSCs compared to tumors alone. Treatment of these MDSC-enriched tumors with MUC1.TR2.4-1BB CARTs led to superior tumor cell killing and significant reduction in tumor growth (24.54±8.55 mm3) compared to CAR.MUC1 (469.79.9±81.46mm3) or TR2.4-1BB (434.86±64.25 mm3) T cells alone (Day 28 after T cell injection). The treatment also improved T cell proliferation and persistence at the tumor site. Thereby, leading to negligible metastasis demonstrating ability of CARTs to eliminate tumor and prevent dissemination. We observed similar results using HER2.TR2.4-1BB CARTs in a HER2+ BC model. Conclusions: Our findings demonstrate that CARTs co-expressing our novel TR2.4-1BB receptor have higher anti-tumor potential against BC tumors and infiltrating MDSCs, resulting in TME remodeling and improved T cell proliferation at the tumor site.

GMP-Compliant Universal Antigen Presenting Cells (uAPC) Promote the Metabolic Fitness and Antitumor Activity of Armored Cord Blood CAR-NK Cells.

Natural killer (NK) cells are innate lymphocytes recognized for their important role against tumor cells. NK cells expressing chimeric antigen receptors (CARs) have enhanced effector function against various type of cancer and are attractive contenders for the next generation of cancer immunotherapies. However, a number of factors have hindered the application of NK cells for cellular therapy, including their poor in vitro growth kinetics and relatively low starting percentages within the mononuclear cell fraction of peripheral blood or cord blood (CB). To overcome these limitations, we genetically-engineered human leukocyte antigen (HLA)-A− and HLA-B− K562 cells to enforce the expression of CD48, 4-1BBL, and membrane-bound IL-21 (mbIL21), creating a universal antigen presenting cell (uAPC) capable of stimulating their cognate receptors on NK cells. We have shown that uAPC can drive the expansion of both non-transduced (NT) and CAR-transduced CB derived NK cells by >900-fold in 2 weeks of co-culture with excellent purity (>99.9%) and without indications of senescence/exhaustion. We confirmed that uAPC-expanded research- and clinical-grade NT and CAR-transduced NK cells have higher metabolic fitness and display enhanced effector function against tumor targets compared to the corresponding cell fractions cultured without uAPCs. This novel approach allowed the expansion of highly pure GMP-grade CAR NK cells at optimal cell numbers to be used for adoptive CAR NK cell-based cancer immunotherapy.