Background: B cell maturation antigen (BCMA) is a B-lineage antigen that is retained on malignant plasma cells in multiple myeloma (MM), and is under investigation as a target antigen for humoral and cellular immunotherapy. Targeting BCMA with chimeric antigen receptor (CAR) T-cells, T-cell engaging antibodies and antibody-drug conjugates has resulted in high rates of clinical responses however, the depth and durability of these responses is still not satisfactory and most patients ultimately relapse. This has been attributed at least in part to low or non-uniform BCMA expression on MM cells, as well as MM cell escape after BCMA down-regulation or even loss. Here, we show that epigenetic modulation with all-trans retinoic acid (ATRA) augments BCMA expression at the gene (and protein) level and leads to enhanced BCMA molecule density on the surface of MM cells that translates into increased anti-MM potency of BCMA CAR T-cells. Methods: Primary MM cells and myeloma cell lines were treated with titrated doses of ATRA (25, 50, 100 nM), alone and in combination with the g-secretase inhibitor crenigacestat (10 nM). BCMA expression was analyzed by flow cytometry, RT-qPCR and direct stochastic optical reconstruction microscopy (dSTORM). BCMA CAR T-cells were derived from healthy donors and MM patients (n>6) and their anti-MM function analyzed in vitro and in the NSG/MM.1S murine xenograft model in vivo. Results: By RT-qPCR, we observed a 1.8-fold (MM.1S) and 2.1-fold (OPM-2) increase in BCMA gene expression after treatment with 50 nM ATRA for 72 hours. By flow-cytometry, we confirmed increased BCMA protein expression, with 1.9-fold (MM.1S and OPM-2) increase in mean fluorescence intensity relative to isotype control staining. Super-resolution dSTORM microscopy on MM.1S cells confirmed the increase in BCMA protein expression and showed a homogenous distribution pattern of BCMA molecules across the cell surface without an increase in cluster formation. These data were confirmed with primary MM cells from patients with newly diagnosed (n=7) and relapsed/refractory (n=11) MM. The increase in MFI for BCMA expression on primary MM cells after ATRA treatment was 1.2-fold - 2.2-fold (mean: 1.6-fold; p=.01 at 50 nM ATRA). By ELISA, we did not detect increased levels of soluble BCMA protein in supernatant of MM.1S cells after ATRA treatment. Accordingly, we found superior cytolytic activity, cytokine secretion and proliferation of CD8+ and CD4+BCMA CAR T-cells in response to ATRA-treated vs. non-treated primary MM cells and MM cell lines. In the NSG/MM.1S xenograft model, we confirmed increased BCMA expression on MM.1S after systemic treatment with ATRA, and superior anti-MM activity after adoptive transfer of BCMA CAR T-cells. Further, we confirmed that epigenetic modulation of BCMA-expression with ATRA works synergistically with g-secretase inhibitor treatment that has recently been shown to prevent cleavage of BCMA molecules from the surface of MM cells (Pont Blood 2019). Combination treatment with ATRA and the g-secretase inhibitor crenigacestat led to higher BCMA density on primary MM cells (and cell lines) than each single-agent treatment alone, resulting in maximum reactivity of by BCMA CAR T-cells in vitro and in vivo. Conclusions: Taken together, the data show that BCMA expression on MM cells can be increased by epigenetic modulation with ATRA. After ATRA treatment, MM cells have increased susceptibility to BCMA CAR T-cell treatment in pre-clinical models vitro and in vivo, that can be increased even further by combination treatment of ATRA and g-secretase inhibitors. These data suggest the potential to improve responses (depth and durability) of immunotherapies directed against BCMA. Disclosures Einsele: Takeda: Consultancy, Honoraria, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau; GlaxoSmithKline: Honoraria, Research Funding, Speakers Bureau.
Background: FMS-like tyrosine kinase 3 (FLT3) is a transmembrane protein uniformly expressed on leukemic blasts in acute myeloid leukemia (AML), and driver of leukemia-genesis in FLT3-ITD+ (Internal tandem duplication) AML. There is an increasing body of pre-clinical and clinical data suggesting that FLT3-ITD+ AML blasts respond to FLT3 inhibitor treatment by augmenting FLT3 expression in order to sustain the survival signal provided by this mutation. Here, we analyzed FLT3 expression on FLT3 wild type and FLT3-ITD+ AML cells after treatment with the FLT3 inhibitors midostaurin, quizartinib and crenolanib, and determined the antileukemia efficacy of combination treatment with FLT3 inhibitors and FLT3 CAR T cells in vitro and in vivo. Methods: MOLM-13 and MV4;11 AML cells (both FLT3-ITD+) were cultured in the presence of IC50 doses of midostaurin, quizartinib and crenolanib, respectively to induce resistance (MOLM-13R/MV4;11R). A FLT3-CAR comprised of BV10 scFv binding domain, CD28-CD3ζ signal module and EGFRt marker was encoded in a lentiviral vector and expressed in CD8+ and CD4+ T cells of healthy donors and patients (n=6). T cell mediated cytolytic activity was evaluated in luminescence-based assay, cytokine production analyzed by ELISA and proliferation assessed by CFSE dye dilution. NSG mice (n= 4-6 per group) were engrafted with MOLM-13/ffLuc AML cells and treated with 5x106 CAR T cells alone or in combination with FLT3 inhibitors. Results: We detected a significant increase in FLT3 expression on both MV4;11 and MOLM-13 AML cells after treatment with each of the inhibitors as assessed by mean fluorescence intensity (quizartinib > crenolanib > midostaurin). The increase in FLT3 expression occurred specifically on these FLT3-ITD+ AML cell lines and was not observed on FLT3 wt AML (THP-1), acute lymphoblastic leukemia (NALM-16), mixed lineage leukemia (KOPN-8 and SEM) cell lines and normal hematopoietic stem cells. We applied single molecule sensitive super-resolution microscopy to demonstrate that the average number of FLT3 molecules (per micrometer sq.) on MV4;11 AML cells had increased from 0.80 (untreated) to 10.7 (quizartinib), 4.7 (crenolanib), and 3.3 (midostaurin) (p<.05). Of interest, midostaurin induced clustering of FLT3, while FLT3 was still present as monomers after quizartinib and crenolanib treatment. Intriguingly, the higher FLT3 density after FLT3 inhibitor treatment translated into superior antileukemia reactivity of FLT3 CAR T cells against AML cell lines and primary AML cells in vitro and in vivo. We observed the strongest increase in cytolytic activity, cytokine production and proliferation by CD8+ and CD4+ FLT3 CAR T cells after treatment with crenolanib and quizartinib, followed by midostaurin (p<.05). We confirmed that upregulation of FLT3 occurred on MOLM-13 cells during FLT3 inhibitor therapy in NSG mice in vivo, and observed synergistic antileukemia efficacy of FLT3 CAR T cells in combination with each of the compounds. The mean frequency of FLT3 CAR T cells in mice that received FLT3 CAR T cells and an FLT3 inhibitor was 2-4 fold higher compared to mice had received FLT3 CAR T cells alone (p<.05) and was the highest in the cohort of mice that had received FLT3 CAR T cells in combination with crenolanib. FLT3 CAR T cells alone and each of the combination treatments of FLT3 CAR T & FLT3 inhibitor achieved 100% response rate which compares favorably to untreated or FLT3 inhibitor alone (0%). However, the mean fold reduction in leukemia burden (b/w day 7 and 10) was greater in all three combination treatment compare to only CAR treatment (p<.05). The most potent combination was FLT3 CAR T cells & crenolanib that accomplished the strongest reduction in leukemia burden as assessed by bioluminescence imaging and flow cytometry. Conclusion: Collectively, the data show that FLT3 inhibitors augment cell surface expression of FLT3 in FLT3-ITD+ AML cells which leads to enhanced recognition and elimination by FLT3 CAR T cells. This is, to our knowledge, the first demonstration that small molecule inhibitors and CAR T cell immunotherapy can be used synergistically to treat a hematologic malignancy. We confirmed this principle with each of the FLT3 inhibitors in our panel, and observed the strongest antileukemia activity of FLT3 CAR T cells in combination with crenolanib. Our data encourage the clinical evaluation of this combination treatment in high risk patients with FLT3-ITD+ AML. Disclosures Jetani: University hospital wuerzburg: Employment, Patents & Royalties: H.J. and M.H are co-inventors on a patent related to the use of FLT3-CAR T-cells to treat AML filed by the University of Wuerzburg, Wuerzburg, Germany. Bonig:Kiadis Pharma: Consultancy.
Introduction Acute Myeloid Leukemia (AML) is a genetically heterogeneous disease characterized by clonal expansion of immature myeloid progenitor cells in the bone marrow (BM). Mutations of the FMS-like tyrosine kinase 3 (FLT3) gene occur in approximately 30% of AML cases, with Internal Tandem Duplications (ITD) being the most common type of mutation. Several FLT3 specific inhibitors (TKI) have been developed such as quizartinib, crenolanib and midostaurin (recently approved for clinical use). Nevertheless FLT3-ITD is associated with unfavorable prognosis and patients develop drug resistance with the underlying mechanisms remaining largely unexplained. Recently, changes within the actin cytoskeleton were associated with drug resistance development in various cancers. FLT3-ITD mutations are associated with RAC1 activation. RAC1 belongs to the family of RHO GTPases and enhances the actin polymerization by inducing the expression of N-WASP or WAVE2 and ARP2/3 complex. Therefore, we investigated actin cytoskeleton rearrangements through RAC1 activation as a potential mechanism contributing to Midostaurin resistance in AML. Material and methods First, we developed two Midostaurin resistant AML cell lines (MID-RES, MV4-11 and MOLM-13). Single cell measurements of Cell Stiffnes, cell adhesion forces between tumor and HS5 stroma cells and Actin filaments were performed by Atomic Force Microscopy (FluidFM®) and SIM microscopy, respectively. RAC1 activation was measured by RAC1 activation kit provided by Cytoskeleton. FLT3 surface and intracellular expression was measured by Flow cytometry and western blot, respectively. Cell death was analyzed by Annexin/PI staining in flow cytometry. Results The MID-RES cell lines MV4-11/MOLM-13 showed higher FLT3 surface and intracellular expression compared to their MID sensitive parental cells. In line with our expectations, we observed RAC1 activation, as well as an up-regulation of actin polymerization positive regulators such as N-WASP, WAVE2, PFN1 and ARP2/3 complex and the inhibition of actin polymerization negative regulator P-ser3 CFL1 in MID-RES cells. FLT3 receptor knock down by siRNAs reversed the MID resistance and reduced RAC1 activation and actin polymerization inducers expression. Likewise, bioinformatic analysis from publicly available microarray expression data (E-MTAB-3444), confirmed positive correlation between actin polymerization inducers and FLT3 signaling expression in 178 FLT3-ITD (r=0,67) and 461 FLT3 WT(r= 0,57) de novoAML patients. RAC1 induced Actin polymerization positively correlates with actin filaments growth and cell stiffness, which was observed in our MID-RES cells, higher load of actin filaments and increased cell stiffness. The combination between RAC1 specific inhibitor, EHT1864 and Midostaurin synergistically induces cell death in MID-RES cells by arresting cell cycle in G0/G1 phase and activating apoptosis. Beside, this combination reduced the adhesion forces to stroma cells, decreased the expression of PFN1/N-WASP/ARP2 and consequently reduced drastically the number of actin filaments and cell stiffness in MID-RES cells. EHT1864 and Midostaurin (alone and in combination) were not toxic in PBMCs obtained from healthy donors. Interestingly, this combination increase >45 % cell death in cells obtained from refractory FLT3-mutated AML patient (this patient was relapsed (≥ 50% residual blasts in the bone marrow)under Chemotherapy+Midostaurin combination).The specific knock down of PFN1/N-WASP/ARP2 with siRNAs equally reversed the resistance to Midostaurin. Of note, RAC1 regulates the anti-apoptotic BCL2. Indeed, EHT1864 in combination with Midostaurin reduced anti-apoptotic family BCL2/MCL1 expression and increases the pro-apoptotic proteins BAX/PUMA. As expected, our MID-RES cells showed higher sensitivity to BCL2 inhibitor Venetoclax, than their parental cells. The combinations EHT1864+venetoclax, venetoclax+midostaurin and venetoclax+Midostaurin+EHT1864 synergistically induced cell death in MID-RES cells. Conclusion Actin polymerization inducers N-WASP, ARP2/3 complex and PFN1 may provide a valuable approach to overcome Midostaurin resistance in AML. Our data further suggest that the addition of BCL2 inhibition through EHT1864 and venetoclax could represent an interesting strategy to potentiate the activity of Midostaurin in FLT3 mutated AML. Disclosures Duell: Regeneron Pharmaceuticals, Inc.: Research Funding. Rosenwald:MorphoSys: Consultancy.
Objective: We investigated blood samples from fully SARS-CoV2-vaccinated subjects and from patients up to one year after infection with SARS-CoV2, and compared short and long term T cell and antibody responses, with a special focus on the recently emerged delta variant (B.1.617.2). Methods and Results: In 23 vaccinated subjects, we documented high anti-SARS-CoV2 spike protein receptor binding domain (RBD) antibody titers. Average virus neutralization by antibodies, assessed as inhibition of ACE2 binding to RBD, was 2.2-fold reduced for delta mutant vs. wt RBD. Specific CD4+ T cell responses as measured using stimulation with peptides representing wt or alpha, beta, gamma and delta variant SARS-CoV2 entire spike proteins by flow cytometric intracellular cytokine staining, did not differ significantly between vaccinees. One year post infection in one of the earliest SARS-CoV2 populations in the Western world, mean specific antibody titers were lower than in vaccinees; ACE2 binding to delta mutant vs. wt RBD was 1.65-fold reduced. T cell responses, especially interferon gamma expression, to mutant SARS-CoV2 spike were significantly reduced compared to those in vaccinees. Conclusion: Strong T cell responses occurred for wildtype and mutant SARS-CoV2 variants, including delta (B.1.617.2), in fully vaccinated individuals, whereas they were partly reduced 1 year after natural infection. Antibody neutralisation of delta mutant was reduced compared to wt, as assessed in a novel inhibition assay with a finger stick drop of blood. Hence, immune responses after vaccination are stronger compared to those after naturally occurring infection, pointing out the need of the vaccine to overcome the pandemic.
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