Chimeric antigen receptor (CAR)-expressing T cells induce durable remissions in patients with relapsed/refractory B cell malignancies. CARs are synthetic constructs that, when introduced into mature T cells, confer a second, non-major histocompatibility complex-restricted specificity in addition to the endogenous T cell receptor (TCR). The implications of TCR activation on CAR T cell efficacy has not been well defined. Using an immunocompetent, syngeneic murine model of CD19-targeted CAR T cell therapy for pre-B cell acute lymphoblastic leukemia in which the CAR is introduced into T cells with known TCR specificity, we demonstrate loss of CD8 CAR T cell efficacy associated with T cell exhaustion and apoptosis when TCR antigen is present. CD4 CAR T cells demonstrate equivalent cytotoxicity to CD8 CAR T cells and, in contrast, retain in vivo efficacy despite TCR stimulation. Gene expression profiles confirm increased exhaustion and apoptosis of CD8 CAR T cells upon dual receptor stimulation compared to CD4 CAR T cells and indicate inherent differences between CD4 and CD8 CAR T cells in the use of T cell-associated signaling pathways. These results provide insights into important aspects of CAR T cell immune biology and indicate opportunities to rationally design CAR constructs to optimize clinical efficacy.

Background: Outcomes for adults and children with acute myeloid leukemia (AML) are dismal with 20-40% and 60% 5-year event-free survival, respectively. Alternative therapeutic strategies for AML are thus needed to improve outcomes. Chimeric antigen receptor (CAR) T cell immunotherapy has induced remarkable clinical responses in multiple phase 1 clinical trials for patients with relapsed or chemorefractory B cell leukemias, encouraging great interest in developing similar approaches for AML. Prior studies have demonstrated efficacy of CD33 or CD123-redirected CAR T cells in AML models, although the genetic heterogeneity of AML will likely require identification of additional therapeutic targets. In the current studies, we report preliminary in vitro and in vivo efficacy of new CAR T cells targeting the FMS-like tyrosine kinase 3 (FLT3) in human AML. FLT3 mutations via internal tandem duplication or kinase domain point mutations occur in approximately 25% of AML and result in FLT3 surface protein overexpression, suggesting potential efficacy of FLT3-targeting therapies. Both types of FLT3 alterations induce ligand-independent activation of FLT3 signaling, further demonstrating a critical role of FLT3 in AML pathogenesis. Hypothesis: FLT3 is a promising target for CAR T cell immunotherapy based treatment of AML. Results: Quantitative flow cytometric analysis of human AML cell lines demonstrated FLT3 surface expression ranging from 1338 (MOLM-13), 2594 (MOLM-14), and 2710 (MV4;11) receptors/cell versus 623 receptors/cell on negative control U937 cells. We first generated FLT3-redirected CAR construct consisting of a single chain variable fragment (scFv) derived from a well-characterized anti-human FLT3 antibody coupled to T cell 4-1BB (CD137) costimulatory and CD3-zeta activation domains. CD33 CAR T cells based on Gemtuzumab created by identical methodologies were also used as AML CAR T cell controls. In vitro studies verified that human T cells transduced with the FLT3 CAR construct induced interferon-gamma and interleukin-2 production after co-culture with AML cell lines MOLM-13, MOLM-14, and MV4;11. One dose of FLT3 CAR T cells inhibited leukemia proliferation in vivo in NOD-SCID-IL2Rγc-/- (NSG) mice engrafted with FLT3-mutant MOLM-13 or MOLM14 cell lines. These first data demonstrate potent preclinical activity of FLT3 CAR T cells and warrant further study in additional AML models. However, on target/off tumor toxicities can occur with AML antigen-targeted immunotherapies, as previously reported in studies of CD33 and CD123 CAR T cells. Normal expression of FLT3 has been mainly described on CD34+ hematopoietic progenitor stem cell populations, and FLT3-targeted therapies have potential to induce aplastic anemia. To address this question of hematologic toxicity of FLT3 CAR T cells, we created normal human hematopoiesis xenograft models in NOD scid gamma Il3-GM-SF (NSGS) mice engrafted with CD34+ cord blood cells for treatment with anti-AML CAR T cells. No difference in human granulocyte numbers was observed in marrows of engrafted mice treated with FLT3 CAR T cells, CD33 CAR T cells, or non-transduced T cells. A significant reduction in monocytes was observed in FLT3 CAR T cell-treated animals, however (p<0.05 by t test). To determine potential for increased hematologic toxicity in the presence of greater target antigen levels, we injected MOLM-14 into CD34+ cell-engrafted mice, then treated animals with control or anti-AML CAR T cells. We surprisingly found no decrement in defined hematopoietic stem cell (HSC) or granulocyte macrophage progenitor (GMP) populations, but did observe increased multipotent and common myeloid progenitor (MPP, CMP) cell numbers and an increase in total human cell engraftment 5 days after FLT3 CAR treatment in comparison to non-transduced T cell-treated animals. Relative to CD33 CAR T cells, FLT3 CAR T cells induced less toxicity to HSCs and MPPs and equivalent toxicity to CMPs and GMPs, indicating lower hematologic toxicity with FLT3 targeting. Conclusions: Taken together, these initial data demonstrate potent in vitro and in vivo anti-AML activity with limited hematopoietic toxicity of FLT3 CAR T cell immunotherapy. Future studies are focused on testing the effectiveness on other AML cell lines with varying expression of FLT3. Disclosures No relevant conflicts of interest to declare.

Chimeric Antigen Receptor T-cell (CART) immunotherapy led to unprecedented responses in patients with refractory/relapsed B-cell non-Hodgkin lymphoma (NHL); nevertheless, two-thirds of patients fail this treatment. Resistance to apoptosis is a key feature of cancer cells that associates with treatment failure. In 87 NHL patients treated with anti-CD19 CART, we found that chromosomal alteration of BCL-2, a critical anti-apoptotic regulator, in lymphoma cells was associated with reduced survival. Therefore, we combined CART19 with the FDA-approved BCL-2-inhibitor, venetoclax, and demonstrated in vivo synergy in venetoclax-sensitive NHL. However, higher venetoclax doses for venetoclax-resistant lymphomas resulted in CART toxicity. To overcome this limitation, we developed venetoclax-resistant CART by overexpressing mutated BCL-2(F104L) which is not recognized by venetoclax. Notably, BCL-2(F104L)-CART19 synergized with venetoclax in multiple lymphoma xenograft models. Furthermore, we uncovered that BCL-2 overexpression in T cells per se enhanced CART anti-tumor activity in preclinical models and in patients by prolonging CART persistence.

Utilizing a clinically relevant haploidentical (HI) murine transplant model, lethally irradiated B6D2F1 (H2K b/d ) mice were transplanted with T cell-depleted (TCD) BM from B6CBAF1 (H2K b/k ) mice. We found that administration of IL-15 significantly increases the numbers of CD8 þ T and natural killer (NK) cells in spleen and BM after transplantion without GVHD. Graft-versus-tumor (GVT) potency of the graft was evaluated upon tumor challenge using P815 tumor cells (H2 d ). IL-15 administration without T-cell infusion did not result in any survival improvement. However, IL-15 in combination with very low-dose T-cell infusion (1 Â 10 4 ) significantly increased GVT activity and improved survival in recipients of HI hematopoietic SCT (HSCT). This effect was observed when IL-15 was given at a later time point, rather than immediately following transplantation. IL-15 administration also specifically increased slow-proliferative CD8 þ T-cell proliferation and IFN-g secretion in CD8 þ T cells in recipients of CFSE (carboxyfluorescein succinimidyl ester)-labeled HI T-cell infusion, whereas there was no effect on CD4 þ T-cell proliferation, suggesting the critical effect of IL-15 on CD8 þ T-cell homeostasis in HI host. We conclude that IL-15 can be used for enhancing antileukemia effect of HI-HSCT, which requires presence of donor-derived T cells.

Interleukin-15 (IL-15) is a potent cytokine that increases CD8+ T and NK cell numbers and function in experimental models. However, obstacles remain in using IL-15 therapeutically, specifically its low potency and short in vivo half-life. To help overcome this, a new IL-15 superagonist complex comprised of an IL-15N72D mutation and IL-15RαSu/Fc fusion (IL-15SA, also known as ALT-803) was developed. IL-15SA exhibits a significantly longer serum half-life and increased in vivo activity against various tumors. Herein, we evaluated the effects of IL-15SA in recipients of allogeneic hematopoietic stem cell transplantation. Weekly administration of IL-15SA to transplant recipients significantly increased the number of CD8+ T cells (specifically CD44+ memory/activated phenotype) and NK cells. Intracellular IFN-γ and TNF-α secretion by CD8+ T cells increased in the IL-15SA-treated group. IL-15SA also upregulated NKG2D expression on CD8+ T cells. Moreover, IL-15SA enhanced proliferation and cytokine secretion of adoptively transferred CFSE-labeled T cells in syngeneic and allogeneic models by specifically stimulating the slowly proliferative and nonproliferative cells into actively proliferating cells.We then evaluated IL-15SA's effects on anti-tumor activity against murine mastocytoma (P815) and murine B cell lymphoma (A20). IL-15SA enhanced graft-versus-tumor (GVT) activity in these tumors following T cell infusion. Interestingly, IL-15 SA administration provided GVT activity against A20 lymphoma cells in the murine donor leukocyte infusion (DLI) model without increasing graft versus host disease. In conclusion, IL-15SA could be a highly potent T- cell lymphoid growth factor and novel immunotherapeutic agent to complement stem cell transplantation and adoptive immunotherapy.

Immunotherapy has revolutionized the treatment of cancer. In particular, immune checkpoint blockade, bispecific antibodies, and adoptive T-cell transfer have yielded unprecedented clinical results in hematological malignancies and solid cancers. While T cell-based immunotherapies have multiple mechanisms of action, their ultimate goal is achieving apoptosis of cancer cells. Unsurprisingly, apoptosis evasion is a key feature of cancer biology. Therefore, enhancing cancer cells’ sensitivity to apoptosis represents a key strategy to improve clinical outcomes in cancer immunotherapy. Indeed, cancer cells are characterized by several intrinsic mechanisms to resist apoptosis, in addition to features to promote apoptosis in T cells and evade therapy. However, apoptosis is double-faced: when it occurs in T cells, it represents a critical mechanism of failure for immunotherapies. This review will summarize the recent efforts to enhance T cell-based immunotherapies by increasing apoptosis susceptibility in cancer cells and discuss the role of apoptosis in modulating the survival of cytotoxic T lymphocytes in the tumor microenvironment and potential strategies to overcome this issue.

Chimeric Antigen Receptor (CAR)-modified T cells are a class of immunotherapy, most known for producing durable remissions in B-cell malignancies. To date, On-Target/Off-Tumor effects, systemic cytokine syndromes, and neurotoxicity are some types of toxicities encountered in early CAR T cell therapy trials. Initially, we sought to investigate potential toxicity by a novel, FLT3-targeting CAR T cell (FLT3 CAR). As a tumor-associated antigen, FLT3 is expressed on AML, ALL and MLL, as well as non-malignant hematopoietic subsets, warranting evaluation for On-Target toxicity. To examine this, human peripheral mobilized CD34+ stem cells were engrafted into either NSG or NSGSÑan NSG-derived knock-in expressing human interleukin-3, GM-CSF, and stem cell factorÑimmunodeficient murine stains. After establishing hematopoietic xenografts and allowing for reconstitution of circulating human myeloid cells, we treated mice with either FLT3 CARs or mock-transduced T cells derived from a marrow-autologous donor [Figure 1A]. In the presence of FLT3 CARs, we observed loss of circulating mature monocytes compared to mock T cell treated controls. To elucidate if myeloid loss originated from an On-Target toxicity towards a FLT3 expressing progenitor or by some other mechanism, we treated marrow-humanized mice with either FLT3 CAR, a lymphoid restricted CD22-targeting CAR, a mature myeloid restricted CD33-targeting CAR or mock transduced T cells. Two weeks following treatment, we analyzed progenitor subsets including human hematopoietic stem cells (HSC), multipotent progenitors (MPP), common myeloid progenitors (CMP), granulocyte-macrophage progenitors (GMP), maturing marrow granulocytes, common lymphoid progenitors (CLP), and megakaryocyte erythroid progenitors (MEP). Surprisingly, we observed significant marrow loss of CMPs, GMPs, MEPs and CLPs across all mice receiving CARs compared to mice treated with mock transduced T cells alone [Figure 1B]. Absolute numbers of HSCs, MPPs, and total humans cells were equivalent across treatment groups. As the CD19-targeting CAR therapy is most well characterized, we repeated this experiment using CD19 CARs with consistent outcomes [Figure 1C]. Flow cytometric characterization of these progenitor populations confirms that loss still occurs in the absence of target-antigen expression. Taken together, these results suggest that CAR T cell therapy may exert a deleterious effect on specific marrow progenitors through a target antigen-independent mechanism. Furthermore, while a FLT3-CAR On-Target toxicity towards FLT3 expressing progenitors (CLP, GMP, and CMP) can not be fully excluded, an exacerbated progenitor loss compared to target antigen-null subset toxicity in the CD33 CAR and CD22 CAR treated groups was not observed. Interestingly, prolonged cytopenias are anecdotally observed in Phase I CAR trials, often attributed to heavy pretreatment of enrollees. Local inflammatory cytokine milieu, particularly IFNg, is implicated in marrow suppression in other settingsÑchronic viral infections, donor lymphocyte infusion and disseminated non-tuberculous mycobacterial infections. We hypothesize that prolonged CAR T cell cytokine secretion may exert similar marrow suppression. This could be due to either continuous CAR-engagement with reconstituting antigen-positive progenitors or an intrinsic supraphysiologic basal level of cytokine secretion by CAR T cells. We are currently analyzing cytokine profiles in our humanized murine model to further evaluate this hypothesis. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

correlated with Progression free survival (PFS) and Overall Survival (OS). CR30 was calculated according to original method (Qian Shi et al. JCO, 2017). To align the endpoints, POD24 and CR30 are labeled as fail (F) for progression within 24 months, no CR30 and achieve (A) for no POD24 and achieve CR30, respectively.Results: Median follow-up was 84 months (range 1-119). One hundred and forty-one patients (28%) failed POD24, and 235 (47%) failed the CR30 endpoint. Five-year OS was 79% and 95% for patients failing and achieving POD24 (p < 0.001) and was 82% and 98% for patients failing to achieve and achieving CR30 (p < 0.001).Prognostic role of POD24 and CR30 was observed for all three study arms (p < 0.001). Both indexes retained their prognostic role for OS after adjustment by FLIPI (p < 0.001) and FLIPI2 was better than POD24 to identify low risk patients, while POD24 was more accurate for the identification of high-risk subjects.POD24 and CR30 showed a comparable behavior and a satisfying correlation with PFS and can be used for the early assessment of patient outcome.