Engineered Tumor-Targeted T Cells Mediate Enhanced Anti-Tumor Efficacy Both Directly and through Activation of the Endogenous Immune System.

Chimeric antigen receptor (CAR) T cell therapy has proven clinically beneficial against B cell acute lymphoblastic leukemia and non-Hodgkin's lymphoma. However, suboptimal clinical outcomes have been associated with decreased expansion and persistence of adoptively transferred CAR T cells, antigen-negative relapses, and impairment by an immunosuppressive tumor microenvironment. Improvements in CAR T cell design are required to enhance clinical efficacy, as well as broaden the applicability of this technology. Here, we demonstrate that interleukin-18 (IL-18)-secreting CAR T cells exhibit enhanced in vivo expansion and persistence and significantly increase long-term survival in syngeneic mouse models of both hematological and solid malignancies. In addition, we demonstrate that IL-18-secreting CAR T cells are capable of modulating the tumor microenvironment, as well as enhancing an effective endogenous anti-tumor immune response. IL-18-secreting CAR T cells represent a promising strategy to enhance the clinical outcomes of adoptive T cell therapy

Chimeric Antigen Receptor-Induced Antigen Loss Protects CD5.CART Cells From Fratricide Without Compromising On-Target Cytotoxicity

Chimeric antigen receptor T cells (CART) targeting lymphocyte antigens can induce T cell fratricide and require additional engineering to mitigate self-damage. We demonstrate that the expression of a chimeric antigen receptor (CAR) targeting CD5, a prominent pan-T cell antigen, induces rapid internalization and complete loss of the CD5 protein on T cells, protecting them from self-targeting. Notably, exposure of healthy and malignant T cells to CD5.CART cells induces similar internalization of CD5 on target cells, transiently shielding them from cytotoxicity. However, this protection is short-lived, as sustained activity of CD5.CART cells in patients with T cell malignancies results in full ablation of CD5+ T cells while sparing healthy T cells naturally lacking CD5. These results indicate that continuous downmodulation of the target antigen in CD5.CART cells produces effective fratricide resistance without undermining their on-target cytotoxicity

Feasibility and Preclinical Efficacy of CD7-Unedited CD7 Car T Cells for T Cell Malignancies

Chimeric antigen receptor (CAR)-mediated targeting of T lineage antigens for the therapy of blood malignancies is frequently complicated by self-targeting of CAR T cells or their excessive differentiation driven by constant CAR signaling. Expression of CARs targeting CD7, a pan-T cell antigen highly expressed in T cell malignancies and some myeloid leukemias, produces robust fratricide and often requires additional mitigation strategies, such as CD7 gene editing. In this study, we show fratricide of CD7 CAR T cells can be fully prevented using ibrutinib and dasatinib, the pharmacologic inhibitors of key CAR/CD3ζ signaling kinases. Supplementation with ibrutinib and dasatinib rescued the ex vivo expansion of unedited CD7 CAR T cells and allowed regaining full CAR-mediated cytotoxicity in vitro and in vivo on withdrawal of the inhibitors. The unedited CD7 CAR T cells persisted long term and mediated sustained anti-leukemic activity in two mouse xenograft models of human T cell acute lymphoblastic leukemia (T-ALL) by self-selecting for CD7−, fratricide-resistant CD7 CAR T cells that were transcriptionally similar to control CD7-edited CD7 CAR T cells. Finally, we showed feasibility of cGMP manufacturing of unedited autologous CD7 CAR T cells for patients with CD7+ malignancies and initiated a phase I clinical trial (ClinicalTrials.gov: NCT03690011) using this approach. These results indicate pharmacologic inhibition of CAR signaling enables generating functional CD7 CAR T cells without additional engineering

IL-18 Secreting CAR T Cells Enhance Cell Persistence, Induce Prolonged B Cell Aplasia and Eradicate CD19+ Tumor Cells without Need for Prior Conditioning

Abstract Chimeric antigen receptor (CAR) T cell therapy has consistently shown significant results against acute lymphoblastic leukemia (ALL) in clinical trials1. However, results with other hematological or solid malignancies have been far more modest2. These disparate outcomes could be partially due to an inhibitory tumor microenvironment that suppresses CAR T cell function3. Thus, in order to expand the anti-tumor CAR T cell applications, a novel strategy in which these cells are capable of overcoming the hostile tumor microenvironment is needed. The cytokine interleukin-18 (IL-18) induces IFN-γ secretion, enhances the Th1 immune response and activates natural killer and cytotoxic T cells4. Early phase clinical trials that utilized systemic administration of recombinant IL-18 for the treatment of both solid and hematological malignancies have demonstrated the safety of this therapy5. We hypothesize that CAR T cells that constitutively secrete IL-18 could enhance CAR T cell survival and anti-tumor activity, and also activate cells from the endogenous immune system. To generate CAR T cells that constitutively secrete IL-18, we modified SFG-1928z and SFG-19m28mz CAR T cell constructs and engineered bicistronic human and murine vectors with a P2A element to actively secrete the IL-18 protein (1928z-P2A-hIL18 and 19m28mz-P2A-mIL18, respectively). Human and mouse T cells were transduced with these constructs and in vitro CAR T cell function was validated by coculturing the CAR T cells with CD19+ tumor cells and collecting supernatant for cytokine analysis. Both human and mouse CAR T cells secreted increased levels of IL-18, IFN-γ and IL-2. Proliferation and anti-tumor cytotoxic experiments were conducted with human T cells by coculturing CAR T cells with hCD19+ expressing tumor cells. 1928z-P2A-hIL18 CAR T cells had enhanced proliferation over 7 days and enhanced anti-tumor cytotoxicity over 72 hours when compared to 1928z CAR T cells (p=0.03 and 0.01, respectively) Next, the in vivo anti-tumor efficacy of the IL-18 secreting CAR T cell was tested in xenograft and syngeneic mouse models. Experiments were conducted without any prior lympho-depleting regimen. In the human CAR T cell experiments, Scid-Beige mice were injected with 1x106 NALM-6 tumor cells on day 0 and 5x106 CAR T cells on day 1. Survival curves showed a significant improvement in mouse survival with the 1928z-P2A-hIL18 CAR T cell treatment when compared to 1928z CAR T cell (p=0.006). Subsequently, to determine if IL-18 secreting CAR T cells could also improve anti-tumor efficacy in immunocompetent mice, we tested the murine 19m28mz-P2A-mIL18 CAR T cells in a syngeneic mouse model. The C57BL/6 hCD19+/- mCD19+/- mouse model was utilized and injected with 1x106 EL4 hCD19+ tumor cells on day 0 and 2.5 x106 CAR T cells on day 1. Mice treated with 19m28mz-P2A-mIL18 CAR T cells had 100% long-term survival, when compared to 19m28mz (p&lt;0.0001). 19m28mz-P2A-mIL18 CAR T cells were detected in peripheral blood for up to 30 days after injection, whereas the 19m28mz CAR T cells were not detectable at any time point. In addition, 19m28mz-P2A-mIL18 CAR T cells were capable of inducing B cell aplasia for greater than 70 days, whereas 19m28mz treatment was not capable of inducing B cell aplasia. In vivo serum cytokine analysis demonstrated that 19m28mz-P2A-mIL18 CAR T cells, as compared to 19m28mz, significantly increased the levels of IFN-γ and TNF-α in the peripheral blood for up to 14 days after injection (p&lt;0.0001 and 0.01, respectively). Despite the increase in IFN-γ and TNF-α cytokines, there was no increase in IL-6 levels. Our findings demonstrate that anti-CD19 CAR T cells that constitutively secrete IL-18 significantly increase serum cytokine secretion, enhance CAR T cell persistence, induce long-term B cell aplasia and improve mouse survival, even without any prior preconditioning. To our knowledge, this is the first description of an anti-CD19 CAR T cell that constitutively secretes IL-18 and that induces such high levels of T cell proliferation, persistence and anti-tumor cytotoxicity. We are currently investigating other mechanisms by which this novel CAR T cell functions, its interactions with the endogenous immune system, as well as testing its applicability in other tumor types. We anticipate that the advances presented by this new technology will expand the applicability of CAR T cells to a wider array of malignancies. Disclosures Brentjens: Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. </jats:sec

Enhancing CAR T Cell Anti-Tumor Efficacy through Secreted Single Chain Variable Fragment (scFv) Immune Checkpoint Blockade

Abstract T cell therapies have had valuable clinical responses in patients with cancer. Chimeric antigen receptor (CAR) T cells can be genetically engineered to recognize tumor cells and CAR T cell therapy has shown impressive results in the setting of B cell acute lymphoblastic leukemia but has been less effective in treating other types of hematologic and solid tumors. The inhibitory tumor microenvironment (TME), including expression of ligands that bind inhibitory receptors on T cells, e.g. programmed death receptor 1 (PD-1), can dampen CAR T cell responses. Separately, immune checkpoint blockade therapy involving the disruption of PD-1 and programmed death receptor ligand1 (PD-L1) interaction allows for re-activation of tumor-infiltrating lymphocytes (TIL) to have anti-tumor function. This approach has shown clinical responses in a range of malignancies, but has been less efficacious in poorly immunogenic tumors. To prevent PD-1-mediated dampening of CAR T cell function, we have co-modified CAR T cells to secrete PD-1 blocking single chain variable fragments (scFv). We first designed mouse constructs with which we could investigate the scFv-secreting CAR T cells in the context of a syngeneic immune-competent intact TME. CAR constructs were engineered directed against either human CD19 or MUC-16 (ecto) with mouse signaling domains and a anti-mouse PD-1 scFv. Mouse T cells transduced with these constructs expressed the CAR on the surface and secreted detectable amounts of scFv that bound to mouse PD-1. The scFv-secreting CAR T cells were cytotoxic and produced IFN-g when co-cultured with PD-L1 expressing tumors in vitro . We utilized a syngeneic mouse model to study scFv secreting CAR T cells in a model with an intact TME. In tumor-bearing mice treated with CAR T cells, scFv-secreting CAR T cells enhanced survival as compared to second generation CAR T cells. The survival benefit achieved with scFv-secreting CAR T cells was comparable to that achieved with systemic infusion of PD-1 blocking antibody, but with localized delivery of PD-1 blockade. Mice treated with scFv-secreting CAR T cells had detectable scFv in vivo in the TME. Lastly, long term surviving mice had detectable CAR T cells in the bone marrow by PCR, demonstrating persistence and suggesting an immunological memory. We next aimed to translate PD-1 blocking scFv CAR T cells to a clinically relevant human model utilizing a novel anti-human PD-1 blocking scFv. CAR constructs were engineered with recognition domains directed against human CD19 or MUC-16 (ecto) and human signaling domains. Human T cells modified with the CAR constructs express the CAR on the surface and secrete detectable amounts of PD-1 blocking scFv. The scFv binds to human PD-1 and scFv-secreting CAR T cells are cytotoxic to PD-L1 expressing tumors. Expression of PD-1-blocking scFv enhances CAR T cell function against PD-L1 expressing tumors in xenograft models of hematological and solid tumors by enhancing survival in tumor-bearing mice as compared to second generation CAR T cells. Furthermore, scFv-secreting CAR T cells exhibit in vivo bystander T cell enhancement of function, suggesting scFv-secreting CAR T cells can reactivate endogenous TILs in the TME. These data support the novel concept that localized delivery of scFv by CAR T cells can successfully block PD-1 binding to PD-L1 and work in an autocrine manner to prevent dampening of CAR T cell responses as well as a paracrine manner to activate endogenous tumor infiltrating lymphocytes to enhance the overall anti-tumor efficacy of CAR T cell therapy. Disclosures Curran: Juno Therapeutics: Research Funding; Novartis: Consultancy. Yan: Eureka Therapeutics Inc: Employment. Wang: Eureka Therapeutics Inc.: Employment, Equity Ownership. Xiang: Eureka Therapeutics Inc.: Employment. Liu: Eureka Therpeutics Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties. Brentjens: Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties, Research Funding. </jats:sec