Horvei, Paulina

Abstract In the development of various strategies of anti-CD19 immunotherapy for the treatment of B-cell malignancies, it remains unclear whether CD19 monoclonal antibody therapy impairs subsequent CD19-targeted chimeric antigen receptor T-cell (CART19) therapy. We evaluated the potential interference between the CD19-targeting monoclonal antibody tafasitamab and CART19 treatment in preclinical models. Concomitant treatment with tafasitamab and CART19 showed major CD19 binding competition, which led to CART19 functional impairment. However, when CD19+ cell lines were pretreated with tafasitamab overnight and the unbound antibody was subsequently removed from the culture, CART19 function was not affected. In preclinical in vivo models, tafasitamab pretreatment demonstrated reduced incidence and severity of cytokine release syndrome and exhibited superior antitumor effects and overall survival compared with CART19 alone. This was associated with transient CD19 occupancy with tafasitamab, which in turn resulted in the inhibition of CART19 overactivation, leading to diminished CAR T apoptosis and pyroptosis of tumor cells.

Abstract The receptor tyrosine kinase AXL is a member of the TYRO3, AXL, and proto-oncogene tyrosine-protein kinase MER family and plays pleiotropic roles in cancer progression. AXL is expressed in immunosuppressive cells, which contributes to decreased efficacy of immunotherapy. Therefore, we hypothesized that AXL inhibition could serve as a strategy to overcome resistance to chimeric antigen receptor T (CAR T)–cell therapy. To test this, we determined the impact of AXL inhibition on CD19-targeted CAR T (CART19)–cell functions. Our results demonstrate that T cells and CAR T cells express high levels of AXL. Specifically, higher levels of AXL on activated Th2 CAR T cells and M2-polarized macrophages were observed. AXL inhibition with small molecules or via genetic disruption in T cells demonstrated selective inhibition of Th2 CAR T cells, reduction of Th2 cytokines, reversal of CAR T-cell inhibition, and promotion of CAR T-cell effector functions. AXL inhibition is a novel strategy to enhance CAR T-cell functions through two independent, but complementary, mechanisms: targeting Th2 cells and reversing myeloid-induced CAR T-cell inhibition through selective targeting of M2-polarized macrophages.

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 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.

Abstract BACKGROUND Chimeric antigen receptor T cell (CART) therapy has revolutionized the treatment landscape for hematological malignancies, its efficacy remains limited in solid tumors. EGFRvIII is a truncated version of the wild type EGFR in which deletion of exons 2–7 of the extracellular domain leads the variant (EGFRvIII) that is strongly antigenic and is mostly expressed on tumor cells. EGFR amplification (tEGFR) almost uniformly precedes the presence of EGFRvIII on tumor cells. The heterogeneity of surface receptor expression and immunosuppressive stromal microenvironment underscore the need to develop CART strategies to target multiple tumor antigens simultaneously. METHODS we generated tEGFR/EGFRvIII directed CAR construct by cloning a clinically relevant tEGFR and EGFRvIII specific scFv into a second generation CAR construct (41BB stimulated) in a third generation lentivirus backbone. This was used to transfect 293T cells and the generated lentivirus particles were used to transduce T cells and generate EGFRvIII/tEGFR CART cells. GBM primary patient derived cell lines were used in these experiments. These cells were passaged and maintained in patient derived xenograft models. RESULTS EGFRvIII/tEGFR directed CART cells exhibited potent antitumor activity against EGFRvIII/tEGFR + GBM cell lines: with 100% killing at 1.25:1, 2.5:1, 5:1 and 10:1 E:T ratio on multiple PDX cell lines with EGFRvIII expression and EGFR over-expression (greater than five copies of EGFR gene) at 24 hours of incubation. Conclusion: We demonstrate that targeting EGFRvIII and over-expressed EGFR with CART cells is feasible, efficacious and represents a promising therapeutic strategy to target GMB. Data from in vivo and combinatorial CART experiments will be reported at the meeting.

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.

Recent successes and FDA approvals of CD19 redirected chimeric antigen receptor T cell (CART19) therapy have been achieved in pivotal clinical trials in patients with B cell malignancies. However, durable response rates in non-Hodgkin B cell lymphoma (B-NHL) and chronic lymphocytic leukemia (CLL) are low. Tumor-associated macrophages have emerged as key mediators of tumor-induced CART cell immunosuppression. Axl receptor tyrosine kinase (Axl-RTK) is overexpressed in various human cancers including B-NHL or CLL and correlates with poor overall survival. Thus, several Axl-RTK inhibitors are currently being investigated in clinical trials. We have previously reported that the Axl-RTK inhibitor TP-0903 modulates CART19 function by Th1 polarization. Moreover, we observed a significant reduction in myeloid-derived cytokines in preclinical models. This led us to hypothesize that Axl-RTK inhibition also modulates monocytes which in turn affects CART function. To test our hypothesis, we interrogated the interactions between Axl-RTK inhibition, monocytes, and CART. First, we isolated CD14+ monocytes from peripheral mononuclear cells (PBMCs) from healthy donors by magnetic negative selection. Flow cytometric analysis revealed a robust Axl expression on monocytes (Fig.1A). Monocytes were maintained in ImmunoCult Macrophage Medium (STEMCELL Technologies) and differentiated with lipopolysaccharide and interferon-γ for M1-type and interleukin (IL)-4 for M2-type macrophages. After confirming the macrophage phenotypes, cells were assessed for Axl expression. Flow cytometric analysis demonstrated that M2-type macrophages expressed higher Axl compared to M1 macrophages (Fig.1B). Then, we treated PBMCs from healthy donors with TP-0903 for 24 hours to determine the specific cell types affected by Axl-RTK inhibition. Flow cytometric analysis revealed that monocytes were significantly inhibited by TP-0903 compared to B cells or T cells (Fig.1C). Having demonstrated that TP-0903 strongly suppresses monocytes and that M2-type macrophages express Axl on their surface, we then performed a cytotoxicity assay against CD19+ mantle cell lymphoma cell line, JeKo-1, to study the impact of Axl-RTK inhibition on monocyte-induced CART suppression. We used CART19 generated in our laboratory (FMC63-41BBζ). CART19, JeKo-1, and M2-type macrophages were co-cultured at 1:2:1 ratio and incubated for 3 days. The data revealed that M2-type macrophages strongly suppress CART19 killing (Fig.1D) which could be overcome with TP-0903 (Fig.1E). Next we studied monocyte-induced CART19 suppression with antigen-specific proliferation. CART19, JeKo-1, and monocytes were co-cultured at 1:5:1 ratio and incubated for 5 days. Flow cytometric analysis revealed that in the presence of monocytes, antigen-specific proliferation of CART19 was significantly suppressed, which was reversed in the presence of TP-0903 (Fig.1F). To assess how Axl inhibition modulates myeloid cell-derived cytokines/chemokines, we performed Multiplex (Millipore-Sigma) with the supernatants obtained from these co-culture assays. The presence of monocytes resulted in a significant increase in IL-17a, IL-6, IL-1 receptor agonist, IL-1β, and soluble CD40 ligand. However, Axl-RTK inhibition resulted in selective reduction of those cytokines/chemokines (Fig.1G) while preserving other effector cytokines. Finally, we tested whether Axl-RTK inhibition with TP-0903 could overcome the negative effects of monocytes in vivo. Here, immunocompromised NSG mice were injected with 1x106 luciferase+ JeKo-1 cells intravenously. Ten days later, mice were injected with freshly isolated monocytes derived from a healthy donor. Two weeks after JeKo-1 inoculation, tumor burden was assessed with bioluminescence imaging. Mice were then randomized according to the tumor burden to receive 0.5x106 CART19 as a monotherapy or in combination with 20 mg/kg/day of oral TP-0903. The combination therapy resulted in superior anti-tumor effects and overall survival compared to CART19 monotherapy (Fig.1H-I). In summary, Axl-RTK inhibition with TP-0903 demonstrates a direct suppression of inhibitory monocytes, which in turn enhances CART cell function. Our findings support the strategy of further refining and testing the novel and potent combination of Axl-RTK inhibition and CART cell therapy for the treatment of B cell malignancies. Figure 1 Disclosures Sakemura: Humanigen: Patents & Royalties. Cox:Humanigen: Patents & Royalties. Mouritsen:Sumitomo Dainippon Pharma Oncology, Inc.: Current Employment. Foulks:Tolero Pharmaceuticals, Inc.: Current Employment. Warner:Tolero Pharmaceuticals, Inc.: Current Employment. Ding:Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; DTRM: Research Funding; Astra Zeneca: Research Funding; Abbvie: Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; alexion: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees. Parikh:GlaxoSmithKline: Honoraria; Verastem Oncology: Honoraria; Genentech: Honoraria; Ascentage Pharma: Research Funding; AbbVie: Honoraria, Research Funding; Merck: Research Funding; TG Therapeutics: Research Funding; AstraZeneca: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; MorphoSys: Research Funding; Pharmacyclics: Honoraria, Research Funding. Kay:Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; MEI Pharma: Research Funding; Abbvie: Research Funding; Tolero Pharmaceuticals: 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; Acerta Pharma: Research Funding; Sunesis: Research Funding; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; 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; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees. Kenderian:Mettaforge: Patents & Royalties; Lentigen: Research Funding; 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.