Hefazi, Mehrdad

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) efficacy is complicated by graft-versus-host disease (GVHD), a leading cause of morbidity and mortality. Regulatory T cells (Tregs) have shown efficacy in preventing GVHD. However, high Treg doses are often required, necessitating substantial ex vivo or in vivo expansion that may diminish suppressor function. To enhance in vivo suppressor function, murine Tregs were transduced to express an anti–human CD19 chimeric antigen receptor (hCAR19) and infused into lethally irradiated, hCD19-transgenic recipients for allo-HSCT. Compared with recipients receiving control transduced Tregs, those receiving hCAR19 Tregs had a marked decrease in acute GVHD lethality. Recipient hCD19 B cells and murine hCD19 TBL12-luciferase (TBL12 luc ) lymphoma cells were both cleared by allogeneic hCAR19 Tregs, which was indicative of graft-versus-tumor (GVT) maintenance and potentiation. Mechanistically, hCAR19 Tregs killed syngeneic hCD19 + but not hCD19 – murine TBL12 luc cells in vitro in a perforin-dependent, granzyme B–independent manner. Importantly, cyclophosphamide-treated, hCD19-transgenic mice given hCAR19 cytotoxic T lymphocytes without allo-HSCT experienced rapid lethality due to systemic toxicity that has been associated with proinflammatory cytokine release; in contrast, hCAR19 Treg suppressor function enabled avoidance of this severe complication. In conclusion, hCAR19 Tregs are a potentially novel and effective strategy to suppress GVHD without loss of GVT responses.

Abstract Pivotal clinical trials of B-cell maturation antigen-targeted chimeric antigen receptor T (CART)-cell therapy in patients with relapsed/refractory multiple myeloma (MM) resulted in remarkable initial responses, which led to a recent US Food and Drug Administration approval. Despite the success of this therapy, durable remissions continue to be low, and the predominant mechanism of resistance is loss of CART cells and inhibition by the tumor microenvironment (TME). MM is characterized by an immunosuppressive TME with an abundance of cancer-associated fibroblasts (CAFs). Using MM models, we studied the impact of CAFs on CART-cell efficacy and developed strategies to overcome CART-cell inhibition. We showed that CAFs inhibit CART-cell antitumor activity and promote MM progression. CAFs express molecules such as fibroblast activation protein and signaling lymphocyte activation molecule family-7, which are attractive immunotherapy targets. To overcome CAF-induced CART-cell inhibition, CART cells were generated targeting both MM cells and CAFs. This dual-targeting CART-cell strategy significantly improved the effector functions of CART cells. We show for the first time that dual targeting of both malignant plasma cells and the CAFs within the TME is a novel strategy to overcome resistance to CART-cell therapy in MM.

Abstract With the development of various strategies of anti-CD19 immunotherapy for treatment of B cell malignancies, it remains to be elucidated whether CD19 targeting with a monoclonal antibody impairs subsequent CD19 targeted chimeric antigen receptor T cell (CART19) therapy. In this study, we evaluated the potential interference between the CD19 targeting monoclonal antibody tafasitamab and CART19 treatment (using a construct similar to the FDA approved therapy, FMC63-41BBζ, tisagenlecleucel) in preclinical lymphoma and leukemia models. For in vitro assays, the CD19 + cell lines JeKo-1 (mantle cell lymphoma), Nalm6 (acute lymphoblastic leukemia), and OCI-Ly7 (diffuse large B cell lymphoma) were used. When CART19 were co-cultured with CD19 + tumor cells in the presence of increasing concentrations of tafasitamab (10-400 µg/mL) or the isotype control, an impairment of CART19 functions at the highest concentration of tafasitamab was observed. However, when tafasitamab was cocultured with tumor cells overnight and removed from the culture by washing, there was no impairment of CART19 anti-tumor activity or effector functions compared to a pre-culture with isotype control. We then aimed to validate these findings in xenograft mouse models by determining whether prior tafasitamab treatment impairs subsequent CART cell function. First, a two-step JeKo-1 xenograft model was created through inoculation of 1x10 6 luciferase + JeKo-1 cells to immunocompromised NOD-SCID-γ -/- (NSG) mice via tail vein injection. One week later, tumor burden was assessed by bioluminescence imaging (BLI) and mice were randomized to receive either 1) PBS (Group 1) or 2) 10 mg/kg of tafasitamab (Group 2) (Fig. 1A). Treatment was performed until the mice reached the endpoint due to the high tumor load. When reaching the endpoint, mice were euthanized and spleen was harvested. Splenic cells were then injected to naïve NSG mice. Two weeks after the inoculation of splenic cells (PBS or tafasitamab treated), mice were imaged with BLI and randomized according to their tumor burden to receive 2x10 6 of 1) untransduced T cells (UTD) or 2) CART19 (i.v.) (Fig. 1B). Mice, treated with CART19 demonstrated potent anti-tumor effects and showed prolonged survival compared to UTD control mice. Interestingly, CART19-treated mice inoculated with Group 2 splenic cells (pretreated with tafasitamab) showed a significantly better tumor control and overall survival compared with mice engrafted Group 1 splenic cells (PBS pretreatment) (Fig. 1C). This finding led us to further investigate the effects of prior tafasitamab therapy on subsequent CART19 treatment. In this context, we studied the sequential treatment with tafasitamab and CART19 in an additional JeKo-1 xenograft model (Fig. 1D). In brief, NSG mice were inoculated with luciferase + JeKo-1 on day -14. On day -8, mice were imaged and randomized to the PBS (Group I) or tafasitamab (10 mg/kg) group. On day -1 tafasitamab treated mice were imaged again, randomized to the tafasitamab discontinuation (Group II) or continuation (Group III). Treatment with PBS was continued in Group I. All groups were administered with 1x10 6 of CART19 on day 0. Cotreatment of JeKo-1 xenografts with tafasitamab and CART19 led to impaired anti-tumor activity presumably as a result of CD19 binding competition between tafasitamab and CART19 (Group III). Mice treated with tafasitamab followed by CART19 had improved tumor control compared to mice treated with PBS followed by CART19 (Fig. 1E-F). Serial peripheral blood analysis demonstrated that mice from Group I showed the highest CART19 expansion on day 10. Strikingly, Group II exhibited weak CART19 expansion at day 10, but strongest expansion on day 23 (Fig. 1G). We therefore hypothesized that prior treatment with tafasitamab results in modulation of CART19 cell activation. To test this hypothesis, spleens of satellite mice were harvested 24 hours after CART19 administration. Flow cytometric analysis revealed that the mice from Group II showed significantly lower CD69 and HLA-DR expression on CART19 cells compared with Group I, and reduced apoptosis as measured by the Annexin assay (Fig. 1H-I). In summary, concomitant treatment of tafasitamab and CART19 led to impaired anti-tumor activity while, in contrast, sequential treatment of tafasitamab and CART19 did not inhibit CART19 anti-tumor activity but rather promoted anti-tumor effects in xenograft models. Figure 1 Figure 1. Disclosures Sakemura: Humanigen: Patents & Royalties. Augsberger: MorphoSys AG: Current Employment. Schanzer: MorphoSys AG: Current Employment. Patra-Kneuer: MorphoSys AG: Current Employment. Heitmüller: MorphoSys AG: Current Employment. Steidl: MorphoSys AG: Current Employment. Endell: MorphoSys AG: Current Employment. Ding: DTRM: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees. Parikh: Pharmacyclics, MorphoSys, Janssen, AstraZeneca, TG Therapeutics, Bristol Myers Squibb, Merck, AbbVie, and Ascentage Pharma: Research Funding; Pharmacyclics, AstraZeneca, Genentech, Gilead, GlaxoSmithKline, Verastem Oncology, and AbbVie: Membership on an entity's Board of Directors or advisory committees. Kay: Acerta Pharma: Research Funding; Targeted Oncology: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Research Funding; Behring: Membership on an entity's Board of Directors or advisory committees; CytomX Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero Pharmaceuticals: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Genentech: Research Funding; Sunesis: Research Funding. Nowakowski: Celgene, NanoString Technologies, MorphoSys: Research Funding; Celgene, MorphoSys, Genentech, Selvita, Debiopharm Group, Kite/Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees. Cox: Humanigen: Patents & Royalties. Kenderian: Humanigen, Inc.: Consultancy, Honoraria, Research Funding.

Abstract CD19-targeted chimeric antigen receptor T cell (CART19) therapy has been remarkably successful in treating a subset of patients with hematological malignancies. However, it is associated with significant toxicities, including cytokine release syndrome (CRS) and neurotoxicity (NT). Furthermore, the rate of durable responses after CART19 therapy is low, and most patients develop resistance to the therapy. One of the predominant mechanisms for CART cell resistance is intrinsic T cell dysfunction which leads to a suboptimal CART product. The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has been shown to favorably modulate CART phenotype and function through downregulation of inhibitory receptors and enhancement of CART cell efficacy in preclinical models and early clinical studies. However, ibrutinib is an irreversible receptor tyrosine kinase (RTK) inhibitor, and thus there are concerns that it inhibits CART cell proliferation and expansion. In contrast to ibrutinib and other BTK inhibitors, vecabrutinib has recently emerged as a potent, reversible, non-covalent BTK inhibitor that can block the activity of both wild type and C481 mutant BTK as well as interleukin-2-inducible T-cell kinase (ITK). We hypothesized that treatment with vecabrutinib improves CART19 antitumor efficacy through the reversible inhibition of BTK/ITK, independent of its antitumor effect. To test this hypothesis, we assessed whether vecabrutinib potentiates CART19 cytotoxicity against CD19 + tumor cells. Killing of the CD19 + mantle cell lymphoma cell line, JeKo-1, was significantly increased when CART19 were combined with vecabrutinib (Fig. 1A). Of note, vecabrutinib did not induce antitumor activity in this model as a single agent (Fig. 1A). In addition, vecabrutinib (10 µM) treatment of CART19 resulted in enhanced antigen-specific proliferation (Fig. 1B). Next, we aimed to study the direct impact of vecabrutinib on CART19 cytokine production. We measured the levels of multiple pro-inflammatory cytokines in the supernatant of CART19 co-cultured for 3 days with JeKo-1 cells and vecabrutinib. Vecabrutinib did not impair CART19 degranulation, as measured by flow cytometric assessment of CD107a expression (Fig. 1C). Cytokines known to play a crucial part in the development of CRS, such as IL-6, IL-10, and MIP-1β were significantly downregulated in cocultures with vecabrutinib (Fig. 1D-F). We then investigated whether the addition of vecabrutinib to CART19 caused a synergistic antitumor effect in vivo. We used our established JeKo-1 lymphoma xenograft models. Here NSG mice were engrafted with CD19 + luciferase + JeKo-1 cells. Mice were then imaged for engraftment and randomized to treatment with 1) untransduced control T cells, 2) CART19 with vehicle, or 3) CART19 with vecabrutinib 50 mg/kg BID administered via oral gavage over a period of 4 weeks. Vecabrutinib combination with CART19 treatment led to sustained antitumor activity (Fig.1G) and increased CART19 proliferation (Fig.1H). Encouragingly, mice treated with vecabrutinib better maintained their body weight following CART19 infusion, suggesting possibly dampened toxicity (Fig.1I). We also measured serum cytokine levels from patients on a phase 1 clinical trial (NCT03037645) testing vecabrutinib in B cell malignancies at baseline and four weeks after treatment. We found that vecabrutinib significantly reduces the levels of multiple pro-inflammatory cytokines linked to CART cell toxicity, including MIP-1β, IP-10, and TNF-a (Fig. 1J-L), further validating our preclinical findings. Finally, we interrogated the transcriptome of CART19 co-cultured with JeKo-1 with or without vecabrutinib. Total RNA sequencing of activated CART19 highlighted a significant enhanced expression of multiple genes involved in the PI3K/AKT and Th1 pathways compared to antigen stimulated CART19 alone (Fig.1M, N). These results could explain the enhanced proliferation and cytotoxicity induced by vecabrutinib. In summary, we demonstrate for the first time that using the reversible BTK inhibitor, vecabrutinib, is a potent and a novel strategy to favorably modulate CART19 function by increasing their efficacy and decreasing toxicity. Further studies are currently underway to compare the effect of reversible versus irreversible BTK/ITK inhibition on CART functions and to further validate the mechanisms responsible for the observed effects. Figure 1 Figure 1. Disclosures Sakemura: Humanigen: Patents & Royalties. Cox: Humanigen: Patents & Royalties. Fox: Sunesis Pharmaceuticals: Current Employment. Ding: Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; DTRM: Research Funding. Parikh: Pharmacyclics, MorphoSys, Janssen, AstraZeneca, TG Therapeutics, Bristol Myers Squibb, Merck, AbbVie, and Ascentage Pharma: Research Funding; Pharmacyclics, AstraZeneca, Genentech, Gilead, GlaxoSmithKline, Verastem Oncology, and AbbVie: Membership on an entity's Board of Directors or advisory committees. Kay: MEI Pharma: Research Funding; Sunesis: Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; CytomX Therapeutics: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Research Funding; Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Behring: Membership on an entity's Board of Directors or advisory committees; Genentech: Research Funding; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Targeted Oncology: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Research Funding; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Acerta Pharma: Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncotracker: Membership on an entity's Board of Directors or advisory committees. Kenderian: Humanigen, Inc.: Consultancy, Honoraria, Research Funding.