ALBERT

Description: 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

Description: Introduction: Venetoclax is an oral BCL2 inhibitor, effective in CLL and under investigation in multiple clinical trials to treat relapsed or refractory multiple myeloma (RRMM) in combination with a proteasome inhibitor (VPI) - either carfilzomib or bortezomib (Moreau P, 2017; Kumar S, 2017). VPI has shown promising initial results in a phase 2 trial with an overall response rate of 78% and a very good partial response or better (≥VGPR) rate of 56% (Costa L J, 2018; Terpos E, 2019). However, a separate phase 3 trial evaluating bortezomib given with venetoclax or placebo found improved response rates and progression free survival but a decrease in overall survival rate in the venetoclax arm of the study (data not published). The primary association with increased mortality was infection. Better characterizing infections in patients treated with VPI may give insight into the pathophysiology and suggest strategies for safe and effective use of this therapy in RRMM. Methods: We retrospectively reviewed charts of patients treated with VPI for infectious complications from initiation of treatment until one month after progression treated at the Hospital of the University of Pennsylvania. Infections were classified by site, pathogen and severity of infection. Additional data collected included demographics, blood cell counts, quantitative immunoglobulins, SPEP M-spike, light chain analysis, cytogenetics, prior lines of therapy, prophylactic antibiotics and IVIG use. We determined non-monoclonal IgG (NM-IgG) via the difference between serum M-spike and IgG for IgG patients. The two-sample Wilcoxon rank-sum was used to compare different subgroups within our study population. Results: We identified 18 patients treated with a VPI combination regimen of which 78% were male. The median age was 64.5 years (range 47-76) and a median of 3 (1-10) prior regimens. 14 were treated with carfilzomib and 4 with bortezomib combinations. 4 patients progressed by the time of data collection. 8 patients were positive for t(11;14). 11 patients experienced 35 discrete infectious episodes resulting in 4 hospitalizations. Respiratory infections predominated (29/35) with 24 upper respiratory infections or sinusitis. 4 were lower respiratory infections and comprised all the hospitalizations. Among respiratory infections, viruses were the only pathogens identified, including influenza, rhinovirus, coronavirus and RSV. Other sites of infection were gastrointestinal (including recurrent C. difficile) and urinary tract. No CNS, blood, or intra-abdominal infections were identified. Reductions in lymphocyte counts and non-monoclonal IgG were near universal but significant reductions in neutrophil counts were not observed. A greater proportional reduction of NM-IgG was noted in good-responders, defined as CR and VGPR (≥VGPR, n=9), with a mean of 78% decrease compared to 47% in poor-responders (PR/MR/SD/PD, n=4, p=0.045). Importantly, treatment of those with t(11;14) positive status demonstrated no significant difference in NM-IgG. Additionally, there was a relative risk ratio favoring a reduction in infections among those with t(11;14). Conclusion: Patients with RRMM treated with VPI experience frequent infectious complications, some severe, with sustained reductions in serum IgG and lymphocyte counts, but without neutropenia. Reduction in NM-IgG is associated with improved response depth. This phenomenon could explain the paradoxical improvement of response rate and progression free survival with concomitant reduction in overall survival due to infections noted by other reports. Interestingly, the presence of t(11;14) in this analysis was not associated with as great a drop in NM-IgG, as was seen in responders as whole, indicating a more differential effect. The absence of significant reduction in NM-IgG is a potential mechanism for relative reduction of infections in this subgroup. Suggestions of survival benefit among t(11;14) positive subgroups in other reports may be related to this preservation of NM-IgG. Replenishing NM-IgG with IVIG is a hypothetical mitigating strategy that could be of benefit in other subgroups treated with this combination of therapy. Disclosures Cohen: Poseida Therapeutics, Inc.: Research Funding. Garfall:Surface Oncology: Consultancy; Novartis: Patents & Royalties: inventor on patents related to tisagenlecleucel (CTL019) and CART-BCMA, Research Funding; Janssen: Research Funding; Amgen: Research Funding; Tmunity: Honoraria, Research Funding. Vogl:Karyopharm Therapeutics: Consultancy; Takeda: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Active Biotech: Consultancy. Mangan:janssen: Speakers Bureau; celgene: Speakers Bureau; takeda: Speakers Bureau; amgen: Speakers Bureau. Stadtmauer:Celgene: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Novartis: Consultancy, Research Funding; Tmunity: Research Funding; Abbvie: Research Funding. OffLabel Disclosure: venetoclax for myeloma

Description: 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.