Porter, David

Background: Chimeric antigen receptor (CAR) T cells have repeatedly demonstrated capacity to induce high rates of response and durable remissions for B cell cancers. In contrast, CAR T cells for the treatment of acute myeloid leukemia (AML) have not yet achieved this impact. We previously demonstrated that CD123 is expressed on AML blasts at high frequency, thus making it an attractive target antigen for AML-directed CAR T cells. Methods and Results: We conducted a pilot study of CD123-directed CAR T cells (CART-123) in adults with relapsed or refractory AML. The primary objective was safety with a secondary objective of anti-leukemia efficacy. Twenty-two subjects were screened, and 20 were eligible for the trial. Fludarabine and cyclophosphamide were used for lymphodepletion (LD). Twelve subjects were infused with CART-123. There were two treatment-related deaths due to cytokine release syndrome (CRS) and infection. Four subjects (33%) achieved complete response with incomplete count recovery (CRi), three subjects had progressive disease, and five had stable disease. Of the four who achieved CRi, one subject remains alive in remission after a relapse and subsequent consolidative hematopoietic stem cell transplant (HSCT). One other subject is alive after HSCT following CART-123 failure. There was evidence of CAR T cell expansion and ten subjects (83%) experienced clinical CRS ranging from Grade I to Grade V. There was no neurotoxicity. To address the apparent paradox of high rates of CART-123 bioactivity (clinical CRS and in vivo expansion) co-occurring with low rates of response, we hypothesized that cytokines produced during CRS may undermine the anti-AML effect of CART cells. We obtained serum drawn from trial subjects at baseline (prior to LD chemotherapy) or during CRS. Serum drawn during CRS promotes primary AML survival in cell culture, while matched baseline serum from the same subjects does not. Individual cytokines secreted during CRS that signal through receptors expressed on myeloid progenitors (such as GM-CSF and Flt3 ligand) reproduced this survival advantage in primary AML blasts when tested at physiologically relevant levels. In contrast, these cytokines did not produce a pro-survival effect in primary patient B-ALL blasts. Crucially, candidate myeloid-acting cytokines, as well as serum drawn during the peak of clinical CRS, promoted resistance of AML to killing by CART-123 in vitro. Using scRNAseq of bone marrow aspirates from subjects on this clinical trial, we found that GM-CSF and FLT3L were most frequently expressed by CD4+ T cells. Exposure of primary AML samples to CRS cytokines led to upregulation of signaling (e.g. JAK/STAT5) and pro-survival pathways. In previous work in B-ALL, we showed that relative resistance to killing can translate to a functional deficit in CART-19 due to development of exhaustion in the CART cells. We similarly find that that exposure to GM-CSF reduces AML blast sensitivity to CART-123, and that the persistence of antigen leads to exhaustion in CART cells (Figure 1). Specifically, co-incubation of CART-123 cells and cytokine-exposed primary AML blasts leads to increased populations of PD-1+/CD39+/CTLA4+/LAG3+ CAR T cells, consistent with an exhaustion phenotype. Finally, we found that anti-apoptotic effects of cytokines can be prevented, and CART-123 killing of AML can be restored, via ruxolitinib blockade of JAK/STAT signaling in both in vitro and in vivo settings. Significance: Our work reveals CRS as active in undermining CART-123 lethality first by promoting survival of AML, and subsequently by exhausting CAR T cells. This is in stark contrast to its role in lymphoid malignancies and underscores biological differences in myeloid versus lymphoid neoplasms. These results highlight a need to tailor therapy to limit cytokine effects on AML blasts to improve outcomes with AML-directed CAR T cell therapy.

Abstract Background: Synthetic lethality (SL) is characterized by the chemical inhibition of multiple aberrant genes to differentially kill malignant cells. We investigated small-molecule combinations for SL through in vitro and in vivo screening and optimization studies (as in the granted patents in USA and China) and demonstrated the synergistic potential of best-in-class combinations of targeted agents, drug repositioning, immune modulation and low-dose combinations. We hypothesize that BTK and mTOR inhibition coupled with an IMID will target multiple key signaling pathways, improve differential apoptosis and target acquired drug resistance. DTRM-555, an optimized mechanism-based combination, consists of the clinically differentiated BTK inhibitor DTRMWXHS-12 (or DTRM-12), everolimus (EV) and pomalidomide (POM). In xenograft tumor models, DTRM-555 administered orally has demonstrated superior efficacy over monotherapy at very low dose combinations of the three agents (1/18 of DTRM-12, 1/6 of EV, and 1/6 of POM). DTRM-12 monotherapy has been safe and well tolerated in simultaneous phase Ia trials (US and China) with preliminary efficacy across 50 mg through 400 mg once daily (QD). Here we report the US clinical results of the oral combination therapies DTRM-505 (DTRM-12 plus EV) and DTRM-555 (DTRM-12 plus EV plus POM) for the first time (NCT02900716). Methods: The safety and anti-tumor activity of the oral doublet therapy DTRM-505 and the oral triplet therapy DTRM-555 are evaluated using a "3+3" study design. Enrollment was conducted concurrently with a phase Ia study of DTRM-12 monotherapy; with DTRM-505 opened for accrual when monotherapy DTRM-12 cleared the 200-mg dose level. Eligible patients are at least 18 years old, having ECOG performance status less than 2 with relapsed/refractory chronic lymphocytic leukemia (CLL), diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL) or classical Hodgkin lymphoma (cHL). Study treatment is administered daily for 21 consecutive days over a 28-day cycle. The dose-limiting toxicity (DLT) period was defined during the first cycle of therapy, and treatment was continued until disease progression or unacceptable toxicity. Patients underwent tumor evaluations every 2 months and response was recorded using revised response criteria (IWCLL and Cheson 2014). Results: Between 9/2016 and 7/2018, 19 patients have been treated with DTRM-12 monotherapy in the USA (6 pts, 50-300 mg) and China (13 pts, 50-400 mg). Patients have tolerated up to 23+ cycles of DTRM-12 monotherapy. Seven patients, including DLBCL, CLL, MCL, FL and cHL, have been treated with oral doublet therapy DTRM-505 for 2 to 12 cycles. Three patients (2 DLBCL, 1 FL) have been treated with oral triplet therapy DTRM-555, including one patient transitioned from the doublet DTRM-505 under an exploratory arm. No patient has discontinued due to an adverse event (AE) and no DLT has occurred. Recurring AEs are mostly grade 1 or 2 and responses have been seen with DTRM-505 and DTRM-555 across diverse lymphoma histologies (See Tables). All patients evaluable for response demonstrated partial response to DTRM-505 and DTRM-555, with 2/7 (doublet) and 3/3 (triplet) patients remaining on therapy. Conclusion: The maximum tolerated dose has not been reached for DTRM-12 monotherapy and for the current fixed dose doublet and triplet combinations. Toxicities have been mild and repeated cycles of study treatment have been well tolerated without delays of subsequent treatment cycles. Enrollment continues for refractory lymphoma patients to demonstrate the safety and efficacy for the first-in-class oral triplet therapy DTRM-555. Disclosures Huntington: Janssen: Consultancy; Bayer: Consultancy; Celgene: Consultancy. Schuster:Merck: Consultancy, Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; OncLive: Honoraria; Dava Oncology: Consultancy, Honoraria; Physician's Education Source, LLC: Honoraria; Nordic Nanovector: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees. He:Zhejiang DTRM Biopharma Co. Ltd.: Other: Founder and CEO; Zhejiang DTRM Biopharma LLC: Other: Founder and CEO. Shen:Zhejiang DTRM Biopharma LLC: Other: Clinical Operations Director. Kennard:AbbVie, Gilead, Verastem: Consultancy. Dwivedy Nasta:Incyte: Research Funding; Takeda/Millenium: Research Funding; Debiopharm: Research Funding; Pharmacyclics: Research Funding; Rafael/WF: Research Funding; Aileron: Research Funding; Roche: Research Funding; Merck: Other: DSMC; Celgene: Membership on an entity's Board of Directors or advisory committees. Landsburg:Takeda: Consultancy; Curis: Consultancy, Research Funding. Porter:Novartis: Other: Advisory board, Patents & Royalties, Research Funding; Genentech: Other: Spouse employment; Kite Pharma: Other: Advisory board. Svoboda:Bristol-Myers Squibb: Consultancy, Research Funding; TG Therapeutics: Research Funding; Kyowa: Consultancy; Regeneron: Research Funding; Merck: Research Funding; Seattle Genetics: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; KITE: Consultancy. Song:Peking University Cancer Hospital (Beijing Cancer Hospital): Employment. Zhu:Beijing Cancer Hospital: Employment. Brander:Pharmacyclics, an AbbVie Company: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; TG Therapeutics: Consultancy, Honoraria, Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; Novartis: Consultancy, Other: DSMB; Genentech: Consultancy, Honoraria, Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; Teva: Consultancy, Honoraria; Acerta: Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; DTRM: Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; BeiGene: Other: Institutional research funding for non investigator initiated clinical trial, Research Funding. Ding:Merck: Research Funding. Mato:Pharmacyclics, an AbbVie Company: Consultancy, Research Funding; AstraZeneca: Consultancy; Regeneron: Research Funding; Celgene: Consultancy; Acerta: Research Funding; TG Therapeutics: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Portola: Research Funding; Johnson & Johnson: Consultancy; Prime Oncology: Honoraria; Medscape: Honoraria.

Abstract Introduction: Anti-CD19 chimeric antigen receptor T cells (CART19) and bi-specific anti-CD19/CD3 antibodies (blinatumomab) generate unprecedented complete response rates of 45-90% in relapsing/refractory acute lymphoblastic leukemia (r/r B-ALL). However, a subset of patients treated with these targeted approaches will relapse and a significant portion of these relapses are characterized by the loss of detectable CD19 (about 30% of relapses after blinatumomab and up to 50% after CART19), a clear manifestation of immunoediting. Hence, novel effective strategies are needed in order to be able to treat those patients and ideally prevent antigen-loss. CD123, the interleukin-3 receptor alpha, is involved in hematopoiesis and has been shown to be expressed in several hematologic neoplasms, including acute myeloid leukemia (AML) and more recently also B-ALL. Targeting CD123 with chimeric antigen receptor T cells (CART123) was shown to lead to deep and long-term responses in human primary AML xenografts. The goal of this study was to pre-clinically evaluate the impact of targeting CD123 and CD19 with chimeric antigen receptor T cells for the treatment and prevention of CD19-negative leukemia relapses occurring after CD19-directed therapies. Results: We analyzed the expression of CD123 in 42 r/r B-ALL samples and found that CD123 is highly and homogeneously expressed on the surface of most B-ALL blasts (81.75%, range: 5.1-99.6), making it a promising candidate for targeted therapy in B-ALL. Moreover, CD123 was also found to be expressed in the putative leukemia stem cells (LSC), identified as CD34-pos CD38-neg. Notably, in some Ph+ B-ALL samples we found CD19-ve CD123+ve cells with a BCR-ABL translocation by FISH, suggesting that these cells too are malignant. The expression of CD123 was detected in all (n=6) CD19-negative B-ALL blasts analyzed after relapse from CART19 treatment (Figure 1). These findings indicate that CD123 represents an ideal marker to target CD19-neg ALL blasts occurring after CART19 or blinatumomab. Therefore, we generated anti-CD123 chimeric antigen receptor T cells costimulated with 4-1-BB using a lentiviral vector (CART123). We then evaluated the CART123 anti-B-ALL efficacy both in vitro and in vivo against primary B-ALL and cell line (NALM-6). CART123 showed intense anti-B-ALL ex vivo activity, as defined by specific CD107a degranulation, cytokine production, cytotoxicity and proliferation, not statistically different from that of CART19. In order to test the role of CART123 to target CD19-negative relapses we developed a novel in vivo model, engrafting immunodeficient NSG mice with blasts obtained from a patient relapsing with CD19-ve disease after CART19 treatment. At day 14 mice were randomized to receive CART19, CART123 or control T cells (untransduced, UTD). CART19 and control T cell treated mice had no anti-tumor activity, while CART123 led to complete eradication of the disease and long-term survival. We next developed a murine model to test the hypothesis that a combined approach simultaneously targeting CD123 and CD19 could treat and prevent CD19-negative relapses. NSG mice were injected with a mixture of primary CD19-neg and CD19-pos blasts from the same patient that were labeled with different click beetle luciferases (red or green) in order to be able to track the respective clones in vivo. Mice were then randomized to receive UTD, CART19 or the combination of CART19 and CART123 (same total dose of T cells). As shown in Figure 2, mice treated with UTD had progression of both leukemic clones (CD19-pos and CD19-neg) while CART19 showed rapid progression mostly of the CD19-neg disease (red luciferase); on the contrary only mice treated with the combination of CART123 and CART19 showed rapid clearance of the disease, with improved overall survival (64 days for CART19, not reached for CART19+CART123). As a final strategy, we expressed both CAR19 and CAR123 in the same T cells and showed potent anti-leukemia activity (CD107a degranulation 81.7%). Conclusions: Here we demonstrate that CD123 is expressed in CD19-negative B-ALL relapses occurring after CD19-directed therapies, and that combining CART123 cells with CART19 cells is an effective therapy for the treatment and prevention of antigen-loss relapses in B-ALL murine xenografts. Disclosures Ruella: Novartis: Patents & Royalties, Research Funding. Kenderian:Novartis: Patents & Royalties, Research Funding. Scholler:Novartis: Patents & Royalties. Lacey:Novartis: Patents & Royalties, Research Funding. Melenhorst:Novartis: Patents & Royalties, Research Funding. Hunter:Surface Oncology - SAB: Membership on an entity's Board of Directors or advisory committees, Research Funding. Porter:Novartis: Patents & Royalties, Research Funding. June:University of Pennsylvania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight; Novartis: Research Funding. Grupp:Novartis: Consultancy, Research Funding. Gill:Novartis: Patents & Royalties, Research Funding.

Key Points Tac/Sir prophylaxis provides equivalent GVHD-free survival when compared with Tac/Mtx in MRD transplantation. Tac/Sir is associated with more rapid engraftment and reduced oropharyngeal mucositis after MRD transplantation.