Fonkoua, Lionel Aurelien Kankeu

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.

AbstractThrough our involvement in KEYNOTE‐059, we unexpectedly observed durable responses in two patients with metastatic gastroesophageal adenocarcinoma (mGEA) who received ramucirumab (anti‐VEGFR‐2)/paclitaxel after immune checkpoint inhibition (ICI). To assess the reproducibility of this observation, we piloted an approach to administer ramucirumab/paclitaxel after ICI in more patients, and explored changes in the immune microenvironment. Nineteen consecutive patients with mGEA received ICI followed by ramucirumab/paclitaxel. Most (95%) did not respond to ICI, yet after irRECIST‐defined progression on ICI, all patients experienced tumor size reduction on ramucirumab/paclitaxel. The objective response rate (ORR) and progression‐free survival (PFS) on ramucirumab/paclitaxel after ICI were higher than on the last chemotherapy before ICI in the same group of patients (ORR, 58.8% vs 11.8%; PFS 12.2 vs 3.0 months; respectively). Paired tumor biopsies examined by imaging mass cytometry showed a median 5.5‐fold (range 4‐121) lower frequency of immunosuppressive forkhead box P3+ regulatory T cells with relatively preserved CD8+ T cells, post‐treatment versus pre‐treatment (n = 5 pairs). We then compared the outcomes of these 19 patients with a separate group who received ramucirumab/paclitaxel without preceding ICI (n = 68). Median overall survival on ramucirumab/paclitaxel was longer with (vs without) immediately preceding ICI (14.8 vs 7.4 months) including after multivariate analysis, as was PFS. In our small clinical series, outcomes appeared improved on anti‐VEGFR‐2/paclitaxel treatment when preceded by ICI, in association with alterations in the immune microenvironment. However, further investigation is needed to determine the generalizability of these data. Prospective clinical trials to evaluate sequential treatment with ICI followed by anti‐VEGF(R)/taxane are underway.

This series of nine articles (three original articles, six reviews) is presented by international leaders in biliary tract cancers (BTC) [...]

Introduction Immune-mediated hematologic disorders are relatively rare but potentially life-threatening immune-related adverse events (irAE) with immune checkpoint inhibitor (ICI) therapy (Shiuan, Beckermann et al 2017). The management strategy often relies on systemic corticosteroids and other immunosuppressant agents in steroid-refractory cases (Brahmer, Lacchetti et al 2018). We hereby describe our institution's experience in diagnosing and managing immune-related hematologic adverse events (ir-h-AE). Methods We retrospectively searched the electronic health record for all ir-h-AE diagnosed by treating providers and confirmed by study authors between 2015 and 2020. The search included patients on ICI and the following conditions: immune-related neutropenia (IRN), autoimmune hemolytic anemia (AIHA), cold agglutinin disease (CAD), immune thrombocytopenia (ITP), immune-related pancytopenia, aplastic anemia (AA), and pure red cell aplasia (PRCA). For AIHA, PRCA, and CAD, partial response (PR) was defined as hemoglobin greater than 10 g/dL but < 12 g/dL and 2 g/dL above the nadir without blood product transfusion. Complete response (CR) was defined as hemoglobin of ≥ 12 g/dL and 2 g/dL above the nadir without blood product transfusion (Peñalver, Alvarez-Larrán et al. 2010). For ITP, CR was defined as platelet count ≥ 100 × 109/L and absence of bleeding. PR was defined as platelet count ≥ 30 × 109/L and ≥ two-fold increase from the baseline count and absence of bleeding (Gómez-Almaguer, Tarín-Arzaga et al. 2013). For IRN, CR was defined as absolute neutrophil count (ANC) ≥ 1500/ mm3 on 2 consecutive measurements separated by 7 days; PR was defined as ANC ≥ 500/ mm3 sustained over 7 days. JMP® 14.1 was used for data analysis. Results Seventeen patients were identified, 9 (52.9%) of whom were female (table 1). Median age was 68 years (range 26-78). The most common pre-existing malignancies were melanoma (6 patients, 35.3%), non-small cell lung cancer (4 patients, 23.5%), and gastrointestinal cancer (3 patients, 17.6%). The ir-h-AE included 7 patients (41.2%) with warm AIHA, 4 (23.5%) with ITP (1 had immune-related pancytopenia that resolved with ITP persisting), 3 (17.6%) with IRN, 1 with AA, 1 with CAD, and 1 with PRCA and mild ITP. All patients had received an ICI dose within 60 days of diagnosis (range 3-52 days, median 18 days). Only 1 patient had a concomitant chronic autoimmune disorder (antinuclear antibody-positive arthritis). Four patients (23.5%) were receiving chemotherapy. Sixteen patients (94.1%) required treatment and received corticosteroids. Eight out of 15 evaluable patients (53.3%) responded to corticosteroids only. Another 4 responders (26.7%) required additional therapies including intravenous immunoglobulins (IVIG) (4, 23.5%) and/or rituximab (4, 23.5%). Overall response rate was 71.4% for AIHA and 100% for IRN. ITP was likely underrepresented in our cohort, and the response rate of 66.7% is likely lower than the response rate in clinical practice. This could be related to the fact that many mild thrombocytopenia cases are not further evaluated in clinical practice. None of our patients died from the sequelae of an ir-h-AE. Two patients (11.8%) died within 30 days of ir-h-AE diagnosis (disseminated intravascular coagulation likely due to the underlying malignancy and sepsis from bacterial pneumonia). At the time of ir-h-AE diagnosis, the malignancy was showing evidence of response in 9 patients (52.9%), progression in 6 patients (35.3%), and stability in 2 patients (11.8%). ICI therapy was permanently discontinued in 9 patients (52.9%) and temporarily held in 6 patients (35.3%). Three of the 5 patients (60%), who were rechallenged with ICI therapy, had recurrence of the same ir-h-AE (AIHA) with adequate response to therapy. Non-hematologic irAE (7 patients, 41.2%) included 3 patients with hepatitis, 2 with thyroiditis, 2 with pneumonitis, 1 with colitis, and 1 with dermatitis. Conclusion Our clinical experience with ir-h-AE highlights the importance of exercising vigilance in assessing the hematologic parameters of patients receiving ICI therapy. While relatively rare, ir-h-AE may impact the clinical course of patients with cancer and their eligibility for therapies that have the potential of offering durable control of the underlying malignancy. ir-h-AE respond to classical therapies including corticosteroids, IVIG, and rituximab. Disclosures Block: Merck: Research Funding; Bristol-Myers Squibb: Research Funding; Genentech: Research Funding; Pharmacyclics: Research Funding; Marker Therapeutics: Research Funding; Transgene: Research Funding; Immune Design: Research Funding. Kottschade:Bristol-Myers Squibb: Consultancy, 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.

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.

4541 Background: Anti-VEGFR2 therapy (ramucirumab/paclitaxel [RAM/TAX]) and ICI are approved as 2nd- and 3rd-line therapy (Tx), respectively, for pts with mGEA. We unexpectedly saw durable responses in 2 pts on RAM/TAX after progression on an ICI trial (KN-059; PMID 29674442). We performed a pilot to examine the clinical activity of ICI followed by RAM/TAX. Then we retrospectively compared the outcomes of pts who received this serial Tx to pts who received RAM/TAX without prior ICI. Methods: All pts with mGEA at Mayo Clinic who received RAM/TAX (2014-19) were included (N = 87). Outcomes were best objective response rate (ORR: complete [CR] or partial response) per RECIST1.1, progression-free survival (PFS), duration of response (DOR), and overall survival (OS). Chi square and multivariate (MV) logistic and Cox regression were used. Results: 15 consecutive pts with measurable mGEA received ICI immediately followed by RAM/TAX after irRECIST progression. Most pts (95%) did not respond to ICI. Yet on RAM/TAX, 100% (15/15) had tumor reduction (range -8% to -100%) with an ORR of 73% (11/15), including 3 CRs. In these pts (who received ICI followed by RAMTAX), PFS on RAMTAX was longer than on last chemotherapy before ICI (12.3 vs 3.0 m, P < .001). Outcomes on RAM/TAX in these pts were significantly better than in pts who received RAM/TAX alone (see Table). Associations were strengthened after adjusting for total lines of Tx, line of Tx of RAM/TAX, age, and ECOG PS. Exploratory analysis of paired tumor biopsies collected pre-ICI and on RAM/TAX in a small subset revealed that the frequency of intratumoral immunosuppressive FOXP3+ Tregs decreased on RAM/TAX, whereas the frequency of antitumor CD8+ T cells was preserved. Conclusions: RAM/TAX immediately preceded by ICI was associated with significantly higher OS, ORR, and DOR than RAM/TAX alone, suggesting ICI may enhance efficacy of subsequent anti-VEGFR/taxane therapy. This novel sequence of therapy will be tested prospectively in a new randomized phase 2 trial (NCT04069273). [Table: see text]