Cox, Michelle J.

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.

Abstract Introduction: It has become increasingly apparent that chimeric antigen receptor T (CART) cell activation and differentiation level is an important determinant of CART cell fate and response to therapy. In this study, we aimed to 1) measure levels of activation-induced surface death receptors and ligands on CART cells; 2) investigate how CART cell activation could impact their fitness and clinical responses, and 3) identify cell-based targets to modulate CART cell activation, apoptosis, and cytotoxicity to improve anti-tumor activity. Methods: We performed flow cytometric studies on ex-vivo stimulated, clinically annotated CART products of patients with large B cell lymphoma from the pivotal ZUMA-1 clinical trial that led to FDA-approved Axicabtagene ciloleucel (Axi-Cel). We investigated possible correlations of a number of surface death receptors and ligands with T cell differentiation status and post-infusion CART cell expansion, utilizing samples from ZUMA-1 patients who achieved a complete response as a best outcome ('responders') vs patients who achieved stable or progressive disease('non-responders'). CART cell effector functions in vitro were measured, and CART apoptosis was assessed using Annexin V. For in vitro and in vivo functional studies, we used CART19 generated from healthy donors (HD CART19) as indicated in the specific experiment. CRISPR/Cas9 was employed during CART cell production to disrupt specific genes. A xenograft model of lymphoma was used to investigate the in vivo antitumor activity of CART19. Results: Following an ex vivo stimulation of Axi-Cel products with CD19 + target cells, we observed upregulation of death receptors and ligands in CART19 from non-responders, compared to responders. We also observed a possible association between such upregulated surface markers with CART cell differentiation as measured by CCR7 expression. In an extended in vitro co-culture assay, where HD CART19 cells were repeatedly stimulated through the CAR, we found that tumor necrosis factor α receptor 2 (TNFR2), unlike other death receptors and ligands, was persistently elevated, suggesting a possible role for TNFR2 in long-term antigen-dependent CART19 dysfunction (Figure 1A). We further found that HD CART19 upregulate TNFR2, but not TNFR1, upon CAR stimulation (Figure 1B). While non-specific TCR activation (CD3 stimulation) of HD CART19 cells protected them from activation-induced apoptosis, antigen-specific activation through the CAR resulted in significant initiation of apoptosis within 2 hours of stimulation (Figure 1C). Having identified a possible association between TNFR2 and CART19 dysfunction, we aimed to study the impact of TNFR2 knockout on HD CART19 functions. Using CRISPR/Cas9 during CART cell manufacturing, we generated TNFR2 k/o HD CART19 cells with a knockout efficiency of around 50%, where the expression levels of TNFR2 in activated CART19 cells were reduced, compared to control CART19 cells (with non-targeting gRNA CRISPR/Cas9, Figure 1D). TNFR2 k/o CART19 cells demonstrated reduced early activation surface markers compared to control CART19, as measured by CD25 and CD69 surface expression (Figure 1E), reduced apoptosis initiation as measured by the Annexin V assay (Figure 1F), and enhanced antigen-specific proliferation and cytotoxicity (Figure 1G). Finally, in an in vivo xenograft model of CD19 + lymphoma, TNFR2 k/o CART19 resulted in enhanced CART cell expansion and anti-tumor activity (Figure 1H). Conclusions: Our results indicate that TNFR2 plays a role in early activation and apoptosis initiation of CART19 following CAR stimulation with CD19 + target cells and present TNFR2 knockout as a strategy to enhance CART19 anti-tumor activity. Figure 1 Figure 1. Disclosures Cox: Humanigen: Patents & Royalties. Sakemura: Humanigen: Patents & Royalties. Ding: Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; DTRM: 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: Juno Therapeutics: 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; MEI Pharma: Research Funding; Celgene: 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; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Targeted Oncology: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Acerta Pharma: Research Funding; Genentech: 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; Sunesis: Research Funding; TG Therapeutics: Research Funding; Tolero Pharmaceuticals: Research Funding; 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; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees. Scholler: Kite: Current Employment. Bot: Kite, a Gilead Company: Current Employment; Gilead Sciences: Consultancy, Current equity holder in publicly-traded company, Other: Travel support. Mattie: Kite: Current Employment. Kim: Gilead Sciences: Current equity holder in publicly-traded company; Kite, a Gilead Company: Current Employment. Filosto: Kite, a Gilead Company: Current Employment; Tusk Therapeutics: Patents & Royalties: or other intellecular property; Gilead Sciences: Other: stock or other ownership . Kenderian: Humanigen, Inc.: Consultancy, Honoraria, Research Funding.

Abstract Anti-CD19 chimeric antigen receptor T (CART19) cell therapy has resulted in unprecedented outcomes in patients with relapsed/refractory B-cell malignancies, which led to the FDA approval for several indications. However, CART19 cell therapy is limited by the development of severe life-threatening toxicities, as well as by the limited rates of durable response. It has become apparent that myeloid cells contribute to the development of both CART cell toxicity and also to the inhibitory tumor microenvironment. We and others have identified that granulocyte-monocyte-colony-stimulating factor (GM-CSF) depletion results in decreased myeloid activation, reduced toxicities, and enhancement of CART19 cell therapy efficacy in pre-clinical models. Furthermore, we observed that GM-CSF knockout (GM-CSF k/o) in CART19 cells resulted in the improvement of their functions (in vitro and in vivo). These findings suggest that there is also a direct effect of GM-CSF on CART19 cells, which is independent of the GM-CSF impact on myeloid cell activation. To further evaluate this, we first examined GM-CSF receptor alpha (GM-CSFRα) expression by flow cytometry on resting and activated CART19 cells (using FMC63-41BBζ). When CART19 cells were stimulated with either anti-CD3/CD28 beads or lethally irradiated (120 Gy) CD19 + Nalm6 cells (B cell acute lymphoblastic leukemia cancer cell line), GM-CSFRα expression was upregulated upon both T cell receptor (TCR) (data not shown) and CAR stimulation (Figure 1A). Having demonstrated that GM-CSFRα is significantly upregulated on stimulated CART19 cells, we aimed to determine the impact of GM-CSF neutralization (clinical-grade anti-GM-CSF antibody, lenzilumab, 10 µg/mL) versus GM-CSFRα blockade (research-grade antibody, 10 µg/mL) on CART19 cell function and CART cell-monocyte interactions. An IgG isotype antibody was used as a control antibody. Neither the GM-CSF neutralizing antibody, nor GM-CSFRα blocking antibody, had any impact on CART19 cell antigen-specific killing against the CD19 + JeKo-1 cells (mantle cell lymphoma cancer cell line), in the presence or absence of CD14 + monocytes (ratio 1:1:1) isolated by magnetic beads from healthy donors (Figures 1B-C). Next, we compared the effects of GM-CSF neutralization versus GM-CSFRα blockade on CART19 cell antigen-specific proliferation. Here, CART19 cells were co-cultured with lethally irradiated CD19 + cell line JeKo-1 at 1:1 ratio in the presence of 10 µg/mL of the GM-CSF neutralizing antibody, increasing doses of the GM-CSFRα blocking antibody (10-100 µg/mL), or an IgG control. The absolute number of CART cells was measured by flow cytometry on day 5. GM-CSF neutralization did not affect CART19 cell proliferation, but GM-CSFRα blocking antibody significantly inhibited CART19 cell proliferation in a dose-dependent manner. Then, we assessed the effects of GM-CSF neutralizing antibody (20 µg/mL) versus GM-CSFRα blocking antibody (20 µg/mL) on CART19 cell antigen-specific proliferation in the presence of healthy donor monocytes (ratio 1:1:0.5) on day 3. Flow cytometric analysis revealed that GM-CSF neutralization, but not GM-CSFRα blockade, mitigated monocyte-suppression of CART19 antigen-specific proliferation (Figure 1E). In summary, our findings indicate significant differences on CART cell functions and CART cell-monocyte interactions when a specific cytokine, GM-CSF, is neutralized compared to blocking its receptor. Further mechanistic studies are ongoing to assess the functions of GM-CSFRα k/o and GM-CSF k/o CART cells. Figure 1 Figure 1. Disclosures Sakemura: Humanigen: Patents & Royalties. 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: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Research Funding; CytomX Therapeutics: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Research Funding; Dava Oncology: 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; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Research Funding; Acerta Pharma: Research Funding; Genentech: Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees; Behring: 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; Sunesis: Research Funding; Targeted Oncology: Membership on an entity's Board of Directors or advisory committees; 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; Janssen: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees. Durrant: Humanigen Inc.: Current Employment. Ahmed: Humanigen Inc.: Current Employment, Current equity holder in publicly-traded company. Chappell: Humanigen Inc.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Cox: Humanigen: Patents & Royalties. Kenderian: Humanigen, Inc.: Consultancy, Honoraria, Research Funding.

Abstract Although chimeric antigen receptor T (CART)–cell therapy has been successful in treating certain hematologic malignancies, wider adoption of CART-cell therapy is limited because of minimal activity in solid tumors and development of life-threatening toxicities, including cytokine release syndrome (CRS). There is a lack of a robust, clinically relevant imaging platform to monitor in vivo expansion and trafficking to tumor sites. To address this, we utilized the sodium iodide symporter (NIS) as a platform to image and track CART cells. We engineered CD19-directed and B-cell maturation antigen (BCMA)–directed CART cells to express NIS (NIS+CART19 and NIS+BCMA-CART, respectively) and tested the sensitivity of 18F-TFB-PET to detect trafficking and expansion in systemic and localized tumor models and in a CART-cell toxicity model. NIS+CART19 and NIS+BCMA-CART cells were generated through dual transduction with two vectors and demonstrated exclusive 125I uptake in vitro. 18F-TFB-PET detected NIS+CART cells in vivo to a sensitivity level of 40,000 cells. 18F-TFB-PET confirmed NIS+BCMA-CART-cell trafficking to the tumor sites in localized and systemic tumor models. In a xenograft model for CART-cell toxicity, 18F-TFB-PET revealed significant systemic uptake, correlating with CART-cell in vivo expansion, cytokine production, and development of CRS-associated clinical symptoms. NIS provides a sensitive, clinically applicable platform for CART-cell imaging with PET scan. 18F-TFB-PET detected CART-cell trafficking to tumor sites and in vivo expansion, correlating with the development of clinical and laboratory markers of CRS. These studies demonstrate a noninvasive, clinically relevant method to assess CART-cell functions in vivo.

Chimeric antigen receptor T (CART) cells are engineered with an artificial receptor which redirects T cells to recognize cancer cells expressing a particular surface antigen. CART cell therapy has been astonishingly successful at eradicating certain B cell malignancies, but relapse is common, and efficacy is lacking in many cancers. Gene editing of CART cells is being investigated to enhance efficacy and safety and to develop off-the-shelf products. Currently, genome engineering tools used to modify CART cells include zinc finger nucleases, transposons, TALENs, and CRISPR-Cas9. CRISPR-Cas9 uses a trans-activating (tracrRNA): CRISPR RNA (crRNA) duplex to trigger imprecise DNA repair through targeted double stranded breaks, causing indels and often resulting in loss of protein function. Gene-edited CART cells have entered the clinic to provide an allogeneic cell source (TALEN TCRα knockout), safer treatment (CRISPR-Cas9 GM-CSF knockout), and resistance to exhaustion (CRISPR-Cas9 PD-1 knockout). CRISPR-Cas9 PD-1 knockout (PD-1k/o) CART cells were well-tolerated in a first-in-human clinical trial. However, clinically tested CRISPR-Cas9-edited CART cells showed only modest loss of function (~25%) of PD-1 upon infusion. Additionally, off-target editing has been observed in the clinic and remains a concern. We hypothesized that using next-generation CRISPR-Cas12a systems will result in enhanced editing efficiency and precision. CRISPR-Cas12a has a smaller protein component than CRISPR-Cas9, uses a single crRNA without a tracrRNA for simplified delivery and leaves staggered 5' overhangs. These properties, along with lower intrinsic off-target activity than Cas9, render Cas12a a powerful gene editing tool. First, we compared the knockout efficiency of Cas9 and Cas12a in three therapeutically relevant genetic targets in T cells by delivering ribonucleoprotein complexes containing the crRNA and Cas protein of interest. We showed that Cas12a more effectively knocked out CD3, GM-CSF, and PD-1 expression compared to Cas9 (Figure 1A), demonstrating the potential of Cas12a in further genetically editing T cell therapies. We then used electroporation with Cas9 and Cas12a to generate PD-1k/o in lentivirally transduced CD19-targeted CART (CART19) cells with the aim of making exhaustion-resistant CART19 cells through CRISPR gene editing. CART19 and PD-1k/o CART19 cells were repeatedly stimulated with CD19+ NALM6 target cells for one week, and exhaustion marker expression was measured over time with flow cytometry. The expression of CTLA4, TIM3, and LAG3 were similar between CART19 groups, but PD-1 expression was lower in Cas9 PD-1k/o CART19 cells and almost completely eradicated in Cas12a PD-1k/o CART19 cells compared to wildtype or mock shocked CART19 cells (Figure 1B). We then compared the functionality of wildtype, mock shocked, and Cas9 or Cas12a PD-1k/o CART19 cells in vitro to ensure that neither the electroporation process nor PD-1 knockout impaired CART19 cell antitumor activity. Over a range of effector-to-target ratios and with repeated stimulation with target cells, cytotoxicity was comparable across all CART19 cell groups (Figure 1C). All CART19 cell groups demonstrated robust proliferation in response to both nonspecific and antigen-specific stimulation and over one week of repeated antigen stimulation with NALM6 target cells (Figure 1D). We also confirmed that all CART19 cell groups demonstrated strong degranulation and cytokine production in response to nonspecific and antigen-specific stimulation, regardless of electroporation or PD-1 knockout (Figure 1E). In summary, our data demonstrate that Cas12a can be used as a gene editing tool to efficiently knock out therapeutically relevant genes in CART19 cell therapy. Additionally, Cas12a demonstrated improved knockout efficiency over Cas9 in three different genomic targets. PD-1 knockout via Cas9 or Cas12a reduced PD-1 expression on the CART19 cell surface, and PD-1 expression was almost completely ablated with Cas12a gene editing. Electroporation and PD-1 knockout did not impact the effector functions of the CART19 cells, including cytotoxicity, degranulation, cytokine secretion, or proliferation. In vivo studies assessing the antitumor efficacy and CART19 cell persistence are ongoing. Overall, Cas12a is a promising, efficient method of gene knockout to enhance the safety and efficacy of CART cells. Disclosures Sakemura: Humanigen: Patents & Royalties. Cox:Humanigen: Patents & Royalties. Kenderian:MorphoSys: Research Funding; Sunesis: Research Funding; Tolero: Research Funding; BMS: Research Funding; Juno: Research Funding; Gilead: Research Funding; Kite: Research Funding; Novartis: Patents & Royalties, Research Funding; Torque: Consultancy; Humanigen: Consultancy, Patents & Royalties, Research Funding; Mettaforge: Patents & Royalties; Lentigen: Research Funding.

Although CD19-directed chimeric antigen receptor T cell (CART19) therapy is highly effective and was FDA approved for certain B-cell malignancies, most patients relapse after CART infusion within the first 1-2 years due to inadequate CART expansion in vivo. Vesicular stomatitis virus (VSV) has the ability to infect and lyse cancer cells. Clinical trials of VSV oncolytic therapy indicate that VSV efficiently infects cancer cells as well as innate immune cells. Therefore, we hypothesized that in patients who achieve suboptimal response to CART19, VSV engineered to express CD19 will augment anti-tumor activity through 1) direct lysis of cancer cells and 2) infecting cancer cells and innate immune cells with CD19 to further stimulate CART19. To test our hypothesis, human CD19 or GFP (control) was engineered between the glycoprotein and large-protein (Fig.1A) in a modified VSV backbone. A matrix inactivating mutation (M51R) rendered it incapable of suppressing anti-viral reactions of infected targets, potentially promoting its immunogenicity. First, we tested the anti-tumor activity of VSV-CD19 and VSV-GFP against the luciferase (luc)+CD19+ acute lymphoblastic leukemia cell line NALM6 and the luc+CD19- acute myeloid leukemia cell line MOLM13. VSV-CD19 and VSV-GFP successfully lysed NALM6 (Fig.1B) or MOLM13, both in vitro and in vivo (data not shown). Next, we investigated the efficiency of VSV-CD19 in infecting tumor and immune cells. 24 hours after exposure to VSV-CD19 or VSV-GFP, we analyzed the surface expression of CD19 on MOLM13 and revealed efficient CD19 delivery (Fig.1C). Next, we assessed VSV infection of peripheral blood mononuclear cells (PBMCs) from healthy donors (HDs). Freshly isolated HD PBMCs were infected with VSV-CD19 for 6 hours and subsequently assessed for CD19 expression. Consistent with findings from clinical trials, VSV-CD19 selectively infected and induced CD19 expression on monocytes while other cells were not affected (Fig.1D). To exclude potential toxicities against CART19, we co-cultured CART19 with VSV-CD19 or VSV-GFP using second-generation 4-1BB costimulated CART19. Both VSV-CD19 and VSV-GFP did not infect CART19 as evident by preservation of CART19 viability and lack of CD19 or GFP expression (Fig.1E). Having demonstrated that VSV-CD19 specifically delivered CD19 to monocytes, we next tested whether the infected monocytes stimulated CART19. VSV-CD19 infected monocytes induced potent antigen-specific proliferation of CART19 (Fig.1F) and resulted in enhanced anti-tumor activity against luc+NALM6 in vitro (Fig.1G). Next, we aimed to confirm these findings in vivo. We generated luc+CART19 to track CART19 expansion in vivo. Freshly isolated HD monocytes were infected with VSV-CD19 ex vivo. After 4 hours, VSV-CD19 was washed away and immunocompromised NSG mice were intravenously injected with VSV-CD19 infectedmonocytes. After 24 hours, 3.5x106 of luc+untransduced T cells (UTD) or luc+CART19 were injected intravenously. The T cell expansion was assessed by bioluminescence imaging (BLI). VSV-CD19 infected monocytes specifically stimulated and expanded CART19 (Fig.1H). Finally, we tested whether VSV-CD19 can stimulate and rescue suboptimal anti-tumor effects of CART19 in vivo using a NALM6 relapsed model. Here, 1x106 luc+NALM6 were injected intravenously into NSG mice on day -6. At day -1, mice were imaged and randomized according to tumor burden to receive 1x106 UTD or CART19 on day 0. Subsequently, at day 4, mice were re-imaged and randomized. At day 5, HD monocytes were injected intravenously. Three hours after administering monocytes, mice received 1x107 VSV-CD19 or VSV-GFP (Fig.1I). BLI revealed that CART19 plusVSV-CD19 showed better tumor control than CART19 monotherapy or CART19 plus VSV-GFP (Fig.1J-K). Furthermore, CART19 plus VSV-CD19 exhibited long-term survival (Fig.1L). In summary, VSV-CD19 not only demonstrated direct anti-tumor effects but also specifically delivered CD19 to monocytes and tumor cells, thereby re-stimulating and enhancing the anti-tumor activity of CART19. This work provides a rationale to study VSV-CD19 in patients who demonstrate only suboptimal response to CART19. This approach could also be applied to augment CART therapy in other tumors. Figure 1 Disclosures Sakemura: Humanigen: Patents & Royalties. Eckert:Genentech: Current Employment. Cox:Humanigen: Patents & Royalties. Parikh:Ascentage Pharma: Research Funding; GlaxoSmithKline: Honoraria; Verastem Oncology: Honoraria; MorphoSys: Research Funding; Genentech: Honoraria; Pharmacyclics: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Merck: Research Funding; Janssen: Honoraria, Research Funding; TG Therapeutics: Research Funding; AstraZeneca: Honoraria, Research Funding. Kay:Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Oncotracker: 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; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Research Funding; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding. Peng:Imanis: Other: Equity Ownership. Russell:Imanis: Other: Equity Ownership. Kenderian:Mettaforge: Patents & Royalties; Humanigen: Consultancy, Patents & Royalties, Research Funding; Lentigen: Research Funding; Torque: Consultancy; Novartis: Patents & Royalties, Research Funding; Kite: Research Funding; Gilead: Research Funding; Juno: Research Funding; BMS: Research Funding; Tolero: Research Funding; Sunesis: Research Funding; MorphoSys: Research Funding.