Despite the remarkable activity of CD19 directed chimeric antigen receptor T cell (CART19) therapy in the treatment of B cell malignancies, the therapy is limited by the development of severe life-threatening complications such as neurotoxicity (NT) and cytokine release syndrome (CRS). Additionally, durable efficacy following CART19 therapy is not optimal. Emerging literature suggests that inhibitory myeloid cells and their cytokines play an important role in inducing CAR-T cell toxicities and also contribute to the inhibition of their effector functions. Specifically, GM-CSF was identified as a critical cytokine in the development of NT and CRS after CART19 therapy. Neutralization of GM-CSF in preclinical models has been shown to prevent CRS and enhance CART anti-tumor activity through modulation of myeloid cell behavior, resulting in reduction of tumor associated macrophages. In addition to the predominant effect of GM-CSF on myeloid cells, there appears to be a direct effect on CART19 cells. In this study, we aimed to evaluate the direct effect of GM-CSF on CART cells. Our initial finding of enhanced anti-tumor activity of CART19 cells after GM-CSF inhibition suggested a direct effect of GM-CSF on CART cells (Sterner et al. 2019, Blood). In these experiments, a guide RNA (gRNA) targeting exon 3 of GM-CSF in a CRISPR-Cas9 lentiviral vector was used to knock out GM-CSF during CART cell manufacturing. This resulted in a disruption efficiency of approximately 70% of the GM-CSF gene. Using a high tumor burden xenograft model for relapsed acute lymphoblastic leukemia established through the engraftment of the CD19+ luciferase+ NALM6 cell line (1x106 cells intravenously) in immunocompromised NOD-SCID-γ-/- mice, treatment with low doses of GM-CSFk/o CART19 resulted in improved anti-tumor activity and overall survival compared to GM-CSFwt CART19. The lack of myeloid cells in this model pointed to an intrinsic effect of GM-CSF on CAR-T cells. To ensure that this was not related to an off-target effect of the gRNA, whole exome sequencing (WES) of the modified cells was performed. There was no difference in the single nucleotide variants or indel counts between GM-CSFk/o CART19 and GM-CSFwt CART19 (Figure 1A). WES was significant for only two alterations in the exon 3 targeted by the gRNA (Figure 1B). The high efficiency and accuracy of targeting exon 3 of GM-CSF indicated that the improvement in CART function is unlikely related to an off-target effect of the gRNA and suggested the possibility of a direct interaction between GM-CSF and CART cells as a potential mechanism behind the improved anti-tumor activity. To investigate this interaction, we first assessed the expression of GM-CSF receptors on CART cells. While resting CART cells do not express any GM-CSF receptors, our analysis robustly indicates that activated CART cells significantly upregulate both α and β subunits of the GM-CSF receptor. This finding was significant both when CART cells are activated through their T cell receptor with CD3/CD28 beads (Figure 1C) or through the CAR with irradiated NALM6 cells (Figure 1D). Additionally, activated GM-CSFk/o CART19 cells also upregulated GM-CSF receptors, indicating this upregulation is induced by T cell stimulation. These results suggest a direct interaction between GM-CSF and upregulated GM-CSFR on activated CART cells. Having demonstrated that 1) GM-CSF depletion enhances CART19 efficacy in xenograft models in the absence of monocytes and 2) T cell activation increases GM-CSF receptor expression, we sought to uncover the downstream changes resulting from this effect. Transcriptome interrogation of GM-CSFk/o CART19 revealed a distinct signature including a significant inhibition of the Fas death pathway, a known critical pathway in inducing CART cell apoptosis. This suggests a potential mechanism for enhanced CART19 activity following GM-CSF depletion (Figure 1E). In summary, our results strongly indicate that CART cells increase expression of GM-CSF receptor subunits when activated, resulting in modulation of CART cell functions. Furthermore, GM-CSFk/o CART19 revealed a distinct transcriptome signature compared to GM-CSFwt CART19. These results illuminate a novel mechanism for a direct modulatory effect of GM-CSF on activated CART cells. Disclosures Cox: Humanigen: Patents & Royalties. Sterner:Humanigen: Patents & Royalties. Sakemura:Humanigen: Patents & Royalties. Ahmed:Humanigen: Employment. Chappell:Humanigen: Employment. Durrant:Humanigen: Employment. Parikh:Acerta Pharma: Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Genentech: Honoraria; Janssen: Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; Ascentage Pharma: Research Funding. Kay:MorphoSys: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Agios: Other: DSMB. Kenderian:Novartis: Patents & Royalties, Research Funding; Tolero: Research Funding; Lentigen: Research Funding; Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Kite/Gilead: Research Funding; Morphosys: Research Funding.

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Chimeric-antigen receptor (CAR) T-cell immunotherapy employs autologous-T cells modified with an antigen-specific CAR. Current CAR-T manufacturing processes tend to yield products dominated by effector T cells and relatively small proportions of long-lived memory T cells. Those few cells are a so-called stem cell memory T (TSCM) subset, which express naïve T-cell markers and are capable of self-renewal and oligopotent differentiation into effector phenotypes. Increasing the proportion of this subset may lead to more effective therapies by improving CAR-T persistence; however, there is currently no standardized protocol for the effective generation of CAR-TSCM cells. Here we present a simplified protocol enabling efficient derivation of gene-modified TSCM cells: Stimulation of naïve CD8+ T cells with only soluble anti-CD3 antibody and culture with IL-7 and IL-15 was sufficient for derivation of CD8+ T cells harboring TSCM phenotypes and oligopotent capabilities. These in-vitro expanded TSCM cells were engineered with CARs targeting the HIV-1 envelope protein as well as the CD19 molecule and demonstrated effector activity both in vitro and in a xenograft mouse model. This simple protocol for the derivation of CAR-TSCM cells may facilitate improved adoptive immunotherapy.

CD19 directed chimeric antigen receptor T cell (CART) therapy has shown remarkable activity in B cell lymphoma and acute lymphoblastic leukemia leading to the approval of two CART therapies. With the emergence of therapeutic anti-CD19 antibodies for the treatment of B cell malignancies, it remains to be elucidated whether such antibodies would interfere with the ability of CD19 targeting CARTs to exert their anti-tumor effect in a subsequent therapy. To address a part of this question, we investigated the potential for functional interference between the monoclonal anti-CD19 antibody tafasitamab (MOR208) and CD19 directed CART cells (CART19). CART19 cells were generated through lentiviral transduction of healthy donor T cells with a second generation CD19 CAR construct (FMC63-CD8h-CD8TM-41BBζ) which is similar to the construct used for the FDA-approved CART tisagenlecleucel. Tafasitamab, is an Fc-enhanced humanized monoclonal antibody which mediates antibody-dependent cellular toxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and direct cytotoxicity. It is currently being studied in phase 2 and 3 clinical trials in diffuse large B-cell lymphoma (DLBCL) in combination with the immunomodulatory agent lenalidomide (L-MIND) and the chemotherapeutic drug bendamustine (B-MIND). As a first step we confirmed the relevance of the tested CD19-positive target cell lines, JEKO (mantel cell lymphoma), Ly7 (DLBCL) and NALM-6 (ALL) based on functional activity of tafasitamab and CART19. In a 24 hours ADCC (tafasitamab titration plus natural killer (NK) cells; Figure 1A) and T cell cytotoxicity assays (CART19, E:T titrations; data not shown) distinct activity was observed for both therapies on all tested cell lines. Secondly, we studied whether the observed CART19 activity may be influenced by tafasitamab in case of a direct CD19 binding competition between tafasitamab and the CAR. To test for such binding competition we incubated the CD19+ cell lines NALM6 or JEKO with up to 100 µg/ml tafasitamab, to saturate the receptors. Subsequent flow cytometry analysis using the FMC63 antibody (carrying the same CD19 binding domain as contained in CART19) failed to detect CD19 expression, indicating a direct binding competition between FMC63 and tafasitamab (Figure 1B). Next, to investigate the potential impact of such binding competition on CART19 cell effector functions, we co-cultured tafasitamab CD19+ JEKO cell line at increasing concentrations of up to 100µg/ml, and then added CART19 cells at different effector to target ratios to the cell culture. The presence of tafasitamab, binding to the CD19 antigen, did not affect important CART cell effector functions such as antigen specific killing (Figure 1C), degranulation (Figure 1D), cytokine production or proliferation of CART19 (Figure 1E). In summary, our studies indicate that CART19 continue to exhibit potent antigen specific effector functions despite presence of tafasitamab and the related competition for CD19 binding. Besides the presented in vitro work the questions of therapeutic sequencing of tafasitamab and CART19 is being studied in xenograft models and will be presented at the meeting. Disclosures Sakemura: Humanigen: Patents & Royalties. Cox:Humanigen: Patents & Royalties. Schanzer:MorphoSys AG: Employment. Endell:MorphoSys AG: Employment, Patents & Royalties. Nowakowski:Selvita: Membership on an entity's Board of Directors or advisory committees; NanoString: Research Funding; MorphoSys: Consultancy, Research Funding; Genentech, Inc.: Research Funding; F. Hoffmann-La Roche Ltd: Research Funding; Curis: Research Funding; Bayer: Consultancy, Research Funding; Celgene: Consultancy, Research Funding. Kay:MorphoSys: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Agios: Other: DSMB. Kenderian:Novartis: Patents & Royalties, Research Funding; Tolero: Research Funding; Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Lentigen: Research Funding; Morphosys: Research Funding; Kite/Gilead: Research Funding.