Ruella, Marco

CD19-directed CAR T-cells (CTL019) can produce durable remissions in chronic lymphocytic leukemia (CLL), but therapeutic success depends on whether autologous T-cells expand, persist, and retain cytotoxic function after manufacturing. Failure of CLL T-cells is often attributed to exhaustion, although many dysfunctional CLL T-cells retain inflammatory cytokine production. We tested whether this paradox reflects immunosenescence, an aging-like program defined by costimulatory loss, DNA damage, inflammatory secretion, repertoire restriction, and proliferative arrest. In response-linked preinfusion CTL019 products, nonresponders and short partial responders had higher senescence and senescence-associated secretory phenotype (SASP) programs than the functional-responder group, comprising complete responders and partial responders with transformed disease. These programs were detectable before infusion and became more prominent during manufacture in susceptible products. They tracked with weak in vivo CAR T-cell expansion and were associated with inferior survival. Serum proteomics revealed a circulating, CLL-associated SASP-like inflammatory milieu in nonresponders. Flow cytometry and T-cell receptor (TCR) profiling showed that poor responders carried CD27⁻CD28⁻ and KLRG1⁺ CD8 T-cells at apheresis, lower product CD27, and reduced product TCR diversity. Research-manufactured CAR T-cells from treatment-naïve CLL samples displayed the same state, most prominently in CD4 CAR T-cells, with SA-β-gal, p16, p53, DNA damage, depletion of less-differentiated states, and senescence-enriched transcriptomes. Under repeated CD19 stimulation, CLL-derived products reached a proliferative ceiling, and p53-high products lost cytotoxic reserve. Ibrutinib improved proliferative fitness, attenuated senescence-associated features and SASP output in paired patient and direct-exposure assays, and enhanced CAR T-cell expansion in an ibrutinib-resistant CLL model. Together, these data identify immunosenescence as a measurable and functionally consequential barrier to CAR T-cell efficacy in CLL and a candidate for therapeutic modulation.

Abstract Background: CD19-directed CART cell (CART19) therapy has transformed the treatment of B-cell malignancies but is limited by life-threatening toxicities, including cytokine release syndrome (CRS) and immune effector cell–associated neurotoxicity syndrome (ICANS), as well as low durable remission rates. Our understanding of the pathogenesis of CART-associated toxicities is limited, and although current treatments are effective, refractory cases remain challenging. Methods and Results: To investigate CRS/ICANS pathogenesis and identify new therapeutic targets, we analyzed plasma cytokine kinetics within the first month of CART19 cell therapy in 25 relapsed/refractory (r/r) large B-cell lymphoma (LBCL) and 14 r/r follicular lymphoma (FL) patients (NCT02030834). In this cohort, 22 (88%) patients developed any-grade CRS (73% LBCL, 27% FL) and 4 (16%) developed any-grade ICANS (50% LBCL, 50% FL). Across 30 analyzed cytokines, IL-5 emerged as the second most enriched cytokine based on the median peak-to-baseline (Day 0) ratio in patients who experienced CRS vs. those who did not. The IL-5 ratio was 6.6-fold higher (p<0.05) and 11.9-fold higher (p=0.093) in patients who experienced CRS or ICANS, respectively. Moreover, IL-5 was elevated in patients experiencing CRS on Days 1 and 2 (p<0.05). Prompted by our results, we evaluated the effect of IL-5 neutralization with a monoclonal antibody (mAb) on CART-associated toxicity in a patient-derived B-acute lymphoblastic leukemia (B-ALL) xenograft model. After engraftment (average of 110 human CD19+ cells/µL peripheral blood), NSG mice were randomized based on tumor burden and treated with 3.5×10⁶ CART19 plus either IL-5 mAb or IgG control (10 mg/kg, weekly), or no treatment. Mice receiving IL-5 mAb showed reduced weight loss (p<0.005, Days 13–16) and neuroscore (p<0.05, Day 15), as assessed by a daily, blinded 18-point assessment, vs. the IgG group. Additionally, while CRS-associated cytokines (GM-CSF, IFN-γ, MIP-1β) were significantly elevated in the IgG group (p<0.05) vs. the untreated group, they were not in the IL-5 mAb group. In this model, all mice treated with CART19 cells were able to clear the tumor burden. However, in a follow-up challenge model generated by engrafting NSG mice with 1x106 luciferase+ JeKo-1 cells, mice treated with a combination of 1x106CART19 cells + IL-5 mAb showed reduced weight loss (p<0.0001) and enhanced overall survival (p<0.01) vs. mice treated with CART19 + IgG. Next, we evaluated IL-5 neutralization in an immunocompetent toxicity model. BALB/c mice were treated with 10×10⁶ murine untransduced T cells, CART19 + IL-5 mAb, or CART19 + IgG (10 mg/kg, weekly). Mice receiving IL-5 mAb exhibited significantly reduced weight loss (p<0.05, Day 1) and lower neuroscore (p<0.05, Days 1, 6, and 8) vs. those receiving IgG control, with a consistent trend observed on other days, supporting a protective role for IL-5 blockade in mitigating CART-associated toxicities. To explore the direct effects of IL-5 on CART19 cells, we disrupted either IL-5 (IL-5KO-CART19) or its receptor (IL-5RαKO-CART19) using CRISPR-Cas9. In NSG mice engrafted with 1×10⁶ luciferase+NALM6 cells, mice receiving 1×10⁶ IL-5KO-CART19 vs. IL-5WT-CART19 showed a trend toward reduced weight loss (p=0.08), significantly increased antitumor activity by Day 20 (p<0.05), and improved overall survival (p<0.05), indicating that IL-5 production can significantly impair CART19 cell efficacy. We next evaluated changes in IL-5Rα gRNA representation in a published genome-wide CRISPR screen (PMID: 39266501). CART19 cells with IL-5Rα-targeting gRNAs were enriched following chronic stimulation, indicating a protective effect for IL-5 pathway interruption. In subsequent studies, IL-5RαKO-CART19 cells showed enhanced killing of JeKo-1 cells following 48 hours of co-culture (p<0.05) and reduced co-expression of multiple inhibitory markers (p<0.01) following chronic stimulation with JeKo-1 cells for one week vs. IL-5RαWT-CART19 cells, suggesting an improvement in CART19 cell activity and phenotype following disruption of IL-5Rα. Conclusion: Our data identify IL-5 as a previously unrecognized mediator of CART–associated toxicities and show significant improvement of CART19 activity with disruption of IL-5 signaling either indirectly with an IL-5 neutralizing antibody or directly with CRISPR gene editing. Thus, IL-5 could serve as a dual-purpose target to improve both safety and efficacy of CART cell therapy.

Cancer immunotherapy—including immune checkpoint inhibition (ICI) and adoptive cell therapy (ACT)—has become a standard, potentially curative treatment for a subset of advanced solid and liquid tumors. However, most patients with cancer do not benefit from the rapidly evolving improvements in the understanding of principal mechanisms determining cancer immune responsiveness (CIR); including patient-specific genetically determined and acquired factors, as well as intrinsic cancer cell biology. Though CIR is multifactorial, fundamental concepts are emerging that should be considered for the design of novel therapeutic strategies and related clinical studies. Recent advancements as well as novel approaches to address the limitations of current treatments are discussed here, with a specific focus on ICI and ACT.

Abstract Despite recent landmark advances in T-cell immunotherapy for the treatment of human cancer, metastatic solid tumors remain an intractable challenge. Macrophages are often the most abundant immune cell in the tumor microenvironment (TME), where they may convert into immunosuppressive (M2) tumor-associated macrophages (TAMs) and participate in disease progression. Currently, macrophage-orientated immunotherapeutic approaches under clinical development in oncology seek to reduce TAM infiltration (CSF-1 antagonists) or enhance TAM phagocytosis (CD47 antagonists). Transfer of autologous, activated, but nontargeted macrophages failed to demonstrate antitumor efficacy in past clinical trials. We hypothesized that genetically engineering human macrophages with CARs against tumor-associated antigens could redirect their phagocytic activity and lead to therapeutic efficacy with the potential for the induction of an antitumor T-cell response. We first demonstrate that CD3-zeta-based CARs are capable of inducing phagocytosis in human THP-1 macrophages, while truncated intracellular-domain deficient CARs were not. Targeted phagocytosis and clearance of CD19+, mesothelin +, and HER2+ cells by CARs targeted against each respective antigen was significantly superior to that by control untransduced (UTD) macrophages. We demonstrate that primary human macrophages, which are resistant to most viral vectors, are efficiently transduced by the chimeric fiber adenoviral vector Ad5f35 (~70% in 10 normal donors). Using Ad5f35, we engineered primary human macrophages with a CD3-zeta-based CAR against HER2. Anti-HER2 primary human CAR macrophages demonstrated targeted phagocytosis against HER2+ but not HER2- cell lines, with phagocytic activity dependent on both the CAR and antigen densities. Furthermore, CAR, but not UTD, macrophages led to potent dose-dependent killing of three distinct HER2-high cell lines in vitro. We sought to test the efficacy of anti-HER2 primary human macrophages in xenograft models of human HER2+ ovarian cancer. A single dose of CAR, but not UTD macrophages, led to tumor regression and improved overall survival in both intraperitoneal and disseminated models of disease. We show that macrophage transduction with Ad5f35, a double-stranded DNA virus, leads to a broad gene expression change, an interferon signaling signature, and phenotypic clustering toward classically activated M1 macrophages. CAR macrophages upregulated co-stimulatory ligand and antigen processing/presentation genes and led to enhanced T-cell stimulation in vitro and in vivo. Lastly, CAR, but not UTD, macrophages showed a broad resistance for M2 conversion in response to immunosuppressive cytokines. In conclusion, we show that primary human CAR macrophages are capable of targeted tumor phagocytosis, lead to improved overall survival in xenograft models, and demonstrate enhanced T-cell stimulation. CAR macrophages are a novel cell therapy platform for the treatment of human cancer. This abstract is also being presented as Poster B29. Citation Format: Michael Klichinsky, Marco Ruella, Olga Shestova, Andrew Best, Kristin Blouch, Xueqing M. Lu, Saad S. Kenderian, Miriam Y. Kim, Roddy O'Connor, Stephen Wallace, Miroslaw Kozlowski, Dylan M. Marchione, Maksim Shestov, Benjamin A. Garcia, Carl June, Saar Gill. Human chimeric antigen receptor (CAR) macrophages for cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR07.

Background: With the incorporation of positron emission tomography (PET) imaging as part of the standard staging evaluation of follicular lymphoma (FL), it is generally recommended to obtain the diagnostic biopsy from a lesion with the highest standardized uptake value (SUV) to rule out de novo histologic transformation (HT). In some cases such an approach might be impractical, while in other cases a biopsy from a diseased area with a high SUV may still demonstrate an indolent histology. To date, there is no data to guide treatment choices in patients (pts) with FL with high SUVmax but with no documented evidence of HT. Specifically, it remains unclear whether anthracycline-containing regimens, such as R-CHOP, provide a better outcome than R-Bendamustine (BR). Furthermore, it is unknown whether rituximab (R) maintenance is beneficial in this setting. Therefore, we aimed to compare the efficacy of R-CHOP vs BR in newly diagnosed FL pts with high SUVmax at baseline PET and to clarify the role of maintenance. Patients and Methods: We retrospectively identified 261 consecutive pts with newly diagnosed biopsy-proven FL1-3A and a SUVmax≥13 on baseline PET, who were treated with frontline R-CHOP (n=183) or BR (n=78) at 7 US cancer centers based on the physician's choice. Progression-free survival (PFS) was calculated from starting treatment until progression, relapse or death. Cut-offs of SUVmax ≥13≤18 and SUVmax>18 were used for sub-analysis (Haematologica;2020;105:1907-1913). Results: Median age was 59 years. The baseline characteristics of the two groups differed significantly for a younger age (58 vs 62 years, p=0.009), a higher rate of B-symptoms (26% vs 10%, p=0.005) and baseline SUVmax>18 (49% vs 36%, p=0.04) in the R-CHOP group. A diagnostic biopsy was obtained from the site at the highest SUV in 129 (71%) and 43 (55%) pts, respectively (p=0.01). These suggest a potential selection of R-CHOP in pts with adverse features. At the end of therapy (EoT), R-CHOP treated pts achieved higher PET-CT complete response rates (CR 82% vs 69%, p=0.02). This superiority held in pts with diagnostic biopsy at the highest SUV site (CR 83% vs 67%, p=0.03), but not in the others (CR 80% vs 71%, p=0.37). Progression occurred at EoT in 11 and 14 pts receiving R-CHOP and BR (6% vs 18%, p=0.02), with a higher rate of HT in the BR group (13% vs 4%, p=0.006). After a median follow-up of 76 months (range, 4-208 months), there was no significant difference in PFS between R-CHOP and BR treated pts (hazard ratio [HR] 1.22, p=0.34). PFS was strongly associated with FLIPI 2 (HR 2.32, p=0.01) and 3-5 (HR 2.69, p=0.003) in the univariate as well as in the multivariate analysis (FLIPI 2 HR 2.19, p=0.02; FLIPI 3-5 HR 2.51, p=0.01). There was a trend toward overall survival (OS) benefit in the R-CHOP group (Fig.1A). In the univariate analysis for OS, BR regimen (HR 2.12, p=0.03), age >60 (HR 2.54, p=0.006), FLIPI 3-5 (HR 5.13, p=0.03) and biopsy at the highest SUV site (HR 0.44, p=0.01) were prognostic, however none of those remained significant in the multivariate analysis (BR regimen HR 1.84, p=0.09; age >60 HR 1.92, p=0.08; FLIPI 3-5 HR 3.11, p=0.14; biopsy at the highest SUV site HR 0.60, p=0.13; SUVmax>18 HR 1.87, p=0.055). Among pts with EoT response after R-CHOP or BR (n=170 and n=64, respectively), one third in each group (36% vs 41%, respectively) received R-maintenance for a median of 8 administrations (range, 2-12). A significant PFS advantage (landmark analysis) was observed with R-maintenance (Fig.1B; HR 0.53, p=0.02); however, this did not translate in survival benefit (HR 0.67, p=0.49). In a multivariable model, R-maintenance (HR 0.53, p=0.02), FLIPI 2 (HR 2.47, p=0.03) and 3-5 (HR 2.79, p=0.01) remained independent prognosticators of PFS, while no parameters significantly affected OS. The 2-year HT rate (10% vs 17%) and cumulative incidence did not statistically differ between R-CHOP and BR groups (Fig.1C, p=0.159). Early progression (POD24) occurred in 43 and 22 pts who received R-CHOP and BR, respectively (24% vs 28%) and resulted in a significantly higher risk of death (HR 4.12, p=0.006). Conclusion: In newly diagnosed FL pts with SUVmax≥13 at baseline PET, R-CHOP was more effective in achieving CR and reducing the risk of early HT compared with BR, and it was associated with a trend toward survival advantage. Rituximab maintenance significantly prolonged PFS. A prospective evaluation with a larger population will be needed to confirm our findings. Disclosures Joffe: Epizyme: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees. Ruella:Abclon, BMS, NanoString: Consultancy; Abclon: Consultancy, Research Funding; UPenn/Novartis: Patents & Royalties. Svoboda:Adaptive: Consultancy; Astra-Zeneca: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Imbrium: Consultancy; Incyte: Research Funding; Pharmacyclics: Consultancy, Research Funding; Merck: Research Funding; Seattle Genetics: Consultancy, Research Funding; TG: Research Funding; Genmab: Consultancy; Atara: Consultancy. Witzig:Immune Design: Research Funding; Spectrum: Consultancy; Karyopharm Therapeutics: Research Funding; Acerta: Research Funding; Celgene: Consultancy, Research Funding; AbbVie: Consultancy; MorphSys: Consultancy; Incyte: Consultancy. Smith:TG Therapeutics: Consultancy, Research Funding; Janssen: Consultancy; BMS: Consultancy; Celgene: Consultancy, Research Funding; Pharmacyclics: Research Funding; Karyopharm: Consultancy, Research Funding; FortySeven: Research Funding; Acerta: Research Funding; Genentech/Roche: Consultancy, Other: Support of parent study and funding of editorial support, Research Funding. Armand:Affimed: Consultancy, Research Funding; Otsuka: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; IGM: Research Funding; Infinity: Consultancy; Genentech: Research Funding; Sigma Tau: Research Funding; Celgene: Consultancy; Adaptive: Consultancy, Research Funding; Tensha: Research Funding; Roche: Research Funding; Merck & Co., Inc.: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy; ADC Therapeutics: Consultancy. Nastoupil:Janssen: Honoraria, Research Funding; LAM Therapeutics: Research Funding; Gilead/KITE: Honoraria; Gamida Cell: Honoraria; TG Therapeutics: Honoraria, Research Funding; Merck: Research Funding; Novartis: Honoraria, Research Funding; Genentech, Inc.: Honoraria, Research Funding; Karus Therapeutics: Research Funding; Bayer: Honoraria; Celgene: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding. Ansell:Takeda: Research Funding; Regeneron: Research Funding; Bristol Myers Squibb: Research Funding; Seattle Genetics: Research Funding; Trillium: Research Funding; Affimed: Research Funding; AI Therapeutics: Research Funding; ADC Therapeutics: Research Funding. Zelenetz:Novartis: Consultancy; Adaptive Biotechnology: Consultancy; Genentech/Roche: Consultancy; Gilead: Consultancy; Gilead: Research Funding; Celgene: Consultancy; Roche: Research Funding; Amgen: Consultancy; BeiGene: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Sandoz: Research Funding; Celgene: Research Funding; MorphoSys: Research Funding; MEI Pharma: Research Funding. Younes:BioPath, Xynomic, Epizyme, and F. Hoffmann-La Roche: Consultancy; Janssen, Curis, Merck, Bristol-Myers Squibb, Syndax Pharmaceuticals, F. Hoffmann-La Roche, Curis (Inst), Johnson & Johnson (Inst), Novartis (Inst): Research Funding; Janssen, AbbVie, Merck, Curis, Epizyme, F. Hoffmann-La Roche, Takeda, Bristol-Myers Squibb, Bayer HealthCare Pharmaceuticals, Celgene, Incyte, Janssen Pharmaceuticals, Merck, Sanofi, Seattle Genetics, Takeda Millennium: Honoraria; AstraZeneca: Current Employment; MSKCC: Ended employment in the past 24 months.

Abstract Acute myeloid leukemia (AML) is challenging to treat with antigen-specific immunotherapy due to the lack of leukemia-specific surface antigens that are absent from normal hematopoiesis. We propose a novel solution to this problem by removing CD33, an AML-associated antigen, from normal hematopoietic stem and progenitor cells (HSPCs) by gene editing, thus rendering them resistant to CD33-targeted therapy. This enables powerful CD33-directed immunotherapy such as chimeric antigen receptor T cells (CAR T cells) to be employed against AML without disrupting normal myeloid function. We developed an optimized protocol to generate CD33 KO HSPCs with CRISPR/Cas9 and obtained up to 90% CD33 KO in primary human CD34+ cells as confirmed by flow cytometry and DNA sequencing. To test whether CD33 KO HSPCs retained multilineage potential, we engrafted NSG mice with CD34+ cells treated with Cas9 and either a CD33-targeting gRNA or a control gRNA. CD33 KO HSPCs were able to engraft and differentiate into both lymphoid and myeloid lineages in the mice to the same degree as controls. Human myeloid cells in the peripheral blood (PB) of the CD33 KO-engrafted mice were deficient for CD33, consistent with the KO efficiency measured in vitro, while expression of other myeloid markers such as CD11b and CD14 were similar to controls (Figure 1A, B). To ensure that long-term repopulating stem cells were present in the CD33 KO HSPCs, we performed secondary transplants of bone marrow from mice after four months of primary engraftment. Persistent engraftment for up to three additional months was observed in both CD33 KO HSPCs and controls, proving that gene editing with CRISPR/Cas9 and the resulting CD33 KO does not affect the self-renewal capability of human hematopoietic stem cells. To determine whether CD33 loss would lead to any functional defects in the myeloid progeny, we investigated the properties of myeloid cells derived from CD33 KO HSPCs differentiated with SCF, GM-CSF, G-CSF, IL-3, IL-6 and EPO. Cell morphology and immunophenotype of the myeloid cells after in vitro differentiation were consistent with normal neutrophils and macrophages. Phagocytosis ability, as measured by uptake of E. coli particles, was identical in CD33 KO myeloid cells as compared to controls. CD33 KO myeloid cells were also able to secrete inflammatory cytokines (IL-1b, IL-8, IL-8, IL-10, IL-12, TNF) in response to LPS stimulation to the same degree as controls. RNA-seq showed a highly concordant gene expression profile of CD33 KO HSPCs and controls (R2=0.99), thus excluding any major impact of CD33 loss on downstream gene expression. Prior studies have shown that CAR T cells targeting CD33 (CART33) can eliminate AML in mouse models but also cause toxicity to normal HSPCs (Kenderian et al, Leukemia 2015). To test our hypothesis that CD33 KO HSPCs can evade CART33-mediated toxicity, we treated mice engrafted with either control or CD33 KO HSPCs with CART33. CD33-expressing cells were eliminated in both control and CD33 KO-engrafted mice, which led to the expected myeloablation in control mice; in contrast CD33 KO-engrafted mice continued to sustain human myeloid cells (Figure 2A). Bone marrow (BM) analysis showed significantly more human stem cells (CD34+38-) and myeloid progenitors (CD34+38+) in CD33 KO-engrafted mice compared to controls after CART33 treatment (Figure 2B). We then proceeded to engraft Molm14, an AML cell line that was engineered to express luciferase, into mice bearing control or CD33 KO hematopoiesis, and studied the effect of CART33 treatment in this model. As expected, AML was eliminated in both groups as measured by bioluminescent imaging (BLI) (Figure 2C), as were peripheral blood monocytes in the control group, while monocytes were still present in the CD33 KO-engrafted mice (Figure 2D). In conclusion, we show that CD33 is dispensable in hematopoietic differentiation, and that absence of CD33 from myeloid progeny does not cause any discernible functional changes. Gene editing to remove CD33 from normal HSPCs allows persistent myelopoiesis during CART33-mediated eradication of AML, thus paving the way to a novel system that maximizes efficacy while minimizing toxicity. We envision future clinical translation of this approach by administering CD33 KO HSPCs as an allogeneic stem cell transplant in combination with CART33 in patients with AML. Disclosures Kenderian: Novartis: Patents & Royalties, Research Funding. Ruella:Novartis: Patents & Royalties. Gill:Novartis: Patents & Royalties, Research Funding.

Abstract Chimeric antigen receptor T cell (CART) therapy has developed as a powerful and potentially curative therapy in hematological malignancies over the last few years. CD19 directed CART cells have resulted in impressive complete response rates of ~90% in acute lymphoblastic leukemia that are durable for the majority of patients. However, the overall response rates in other malignancies such as chronic lymphocytic leukemia are around 50%. This could be partially related to CART exhaustion and dysfunction induced by leukemia cells. In this study, we aim to evaluate the role of inhibitory receptors/pathways in inducing CART cell dysfunction and exhaustion in hematological malignancies. As a tumor model, we used an acute myeloid leukemia (AML) cell line (MOLM14) and primary AML samples and treated them with CD33 or CD123 directed CART cells (second generation CARs using 4-1BB and CD3z signaling domains and a lentiviral vector). Incubation of primary AML samples or MOLM14 cell line with CD33 or CD123 directed CARTs resulted in a significant up-regulation of PD-L1 on tumor cells after 24 hours of incubation (0% on day 0 vs 80% on day 1, P <0.001), and up-regulation of PD-1 and TIM-3 on T cells 3-7 days post co-culture (8% of T cells expressed PD-1 on day 0 vs 43% on day 3, P=0.03 and 13% of T cells expressed TIM-3 on day 0 vs 71% on day 3, p=0.001, Figure 1). For in vivo experiments, we used NSG (NOD-SCID-g-/-) mice and engrafted them with the MOLM14 cell line. Treatment of these AML xenografts with suboptimal doses of CD33 or CD123 CARTs resulted in initial anti-tumor responses, followed by disease relapses in 40-60% of the mice (Figure 2A). T cells were isolated from the bone marrow of these mice and analyzed for differential expression of inhibitory receptors. There was a significantly increased up-regulation of TIM-3 receptors on T cells isolated from mice with relapsed disease compared with T cells isolated from mice in remission after CART cell therapy (Figure 2B). Next, we investigated the role of adding checkpoint blockade to improve T cell function ex vivo after CART cell therapy. Marrows of mice that relapsed after CART cell therapy contained both residual CART cells and leukemia and were used to model the administration of checkpoint blockade in the setting of CART cell exhaustion. Cells were cultured with PD-1 or TIM-3 blocking antibodies or the combination of both (10 ug/ml for 72 hours). CART cell effector functions such as cytokine production and Ki-67 proliferation marker improved in the presence of checkpoint antagonists especially when both PD-1 and TIM-3 blocking antibodies were combined (figure 2C). Finally, we tested the combination of checkpoint blockade with CARTs in AML xenografts. In this approach, we treated MOLM14 xenografts with suboptimal doses of CD33 or CD123 directed CARTs or with control untransduced T cells (UTD), with or without checkpoint blocking antibodies. NSG mice bearing MOLM14 AML xenografts were established. Engraftment was confirmed by bioluminescent imaging. The tumor bearing mice were then treated with suboptimal doses (0.25-0.5 x106 total T cells I.V) of CD33 or CD123 directed CARTs or with control untransduced T cells (UTD). Mice also received PD-1 blockade, TIM-3 blockade or the combination of both on days 3, 6, 9 and 12 post T cell therapy, with the rationale for early checkpoint blockade being based on our in vitro observations of early upregulation of inhibitory ligands on AML after exposure to CART cells. Mice were then followed with serial imaging to assess disease burden. The addition of checkpoint antagonists to untransduced T cells, in particular anti-TIM3, did not lead to an anti-leukemic effect. However, the addition of PD-1 or TIM-3 blockade to CART cell therapy resulted in a synergistic anti-tumor activity as shown in Figure 3. The durable complete response rate was: 45% for treatment with CART123 alone, 80% for treatment with CART123+PD-1 blockade, 100% for treatment with CART123+TIM-3 blockade, and 80% for treatment with CART123+ both PD-1 and TIM-3 blockade). Our preclinical results indicate that PD-1 and TIM-3 pathways are involved in CART exhaustion and dysfunction in AML. Combination of checkpoint inhibitors with CART cells may lead to enhanced efficacy in AML and other hematological malignancies. Current studies are investigating mechanisms of synergy and the role of these combinations in other hematological malignancies. Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures Kenderian: Novartis: Patents & Royalties, Research Funding. Ruella:Novartis: Patents & Royalties, Research Funding. Porter:Novartis: Patents & Royalties, Research Funding. June:University of Pennsylvania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight; Novartis: Research Funding. Gill:Novartis: Patents & Royalties, Research Funding.

Abstract Patients with Hodgkin lymphoma (HL) who relapse or are refractory to first-line therapy have a poor prognosis and some of these patients require innovative therapeutic approaches. Our group and others have demonstrated the clinical efficacy of anti-CD19 chimeric antigen receptor T cells (CART19) for B cell malignancies. However, B cell antigens such as CD19 are typically absent from Hodgkin Reed-Sternberg (HRS) cells. Targeting the CD30 antigen using CART cells has met with limited clinical success to date. In HL the neoplastic HRS cells often represent ~1% of the tumor mass and the remainder is comprised of a highly immunosuppressive tumor microenvironment (TME). Within the TME, tumor-associated macrophages (TAM) have a key role in promoting tumor growth and inhibiting the anti-tumor immune response, and the presence of TAM in HL is associated with a poor prognosis. HL therefore represents an excellent model of tumor-induced immunosuppression that can potentially represent a barrier to successful CART therapy. The objective of this study was therefore to develop an anti-HL CAR T cell immunotherapy that would also overcome the immunosuppression of the HL microenvironment. We sought to define a tumor antigen that was co-expressed on HRS and on TAM. Using immunohistochemistry and flow cytometry we confirmed previous reports that CD123 (the IL-3Rα chain) is expressed on HRS in 50-60% of HL patients and in 100% HL cell lines (HDLM-2, KMH2, SUPHD1, and L428). Importantly, CD123 was also seen on several HL-associated immune cells of the TME, including TAM. Exposure of macrophages to HDLM-2 cells polarized them to an M2-like phenotype (Figure 1 A) with expression of CD123. Luminex analysis of HDLM-2/TAM co-culture supernatants revealed high levels of IL-13, and blockade of IL-13 partially reversed the immunosuppressive phenotype. We then developed a model of TAM-mediated CAR T cell inhibition, using immunosuppressive M2-like macrophages derived by exposure to IL-4 or to HDLM-2 cells. These macrophages could suppress “third party” CART19 proliferation in response to B-acute lymphoblastic leukemia. We then used anti-CD123 CART cells (CART123) to target TAM. Importantly, CART123 actively recognized and killed TAM, thereby resisting TAM-mediated inhibition (as opposed to control CART19 cells) (Figure 1 B). We therefore sought to investigate the utility of anti-CD123 CART for the treatment of HL (initial results for this part presented at ASH 2014). In vitro, CART123 specifically degranulate, proliferate, produce cytokines and kill HL cells. A clinically-relevant xenograft model of HL was generated using luciferase+ HDLM-2 cells in NSG mice. In this model mice showed progressive increase in tumor burden over approximately 6 weeks, reproducing the indolent nature of the human disease. At day ~43 mice were randomized to receive 1.5 million CART123 cells or control T cells. CART123 induced complete and durable eradication of disseminated tumor within 14 days, leading to 100% relapse-free and 100% overall survival at 8 months while mice treated with control T cells had a median survival of 128 days (Figure 2 A). Tumor elimination was associated with extensive CAR T cell expansion as detected by flow cytometry in serial peripheral blood bleedings. In order to prove the formation of an immunological memory, at day 250 CART123 treated mice were re-challenged with the HDLM-2 cell line. The tumor was promptly rejected, associated with a re-expansion of CART123 cells in the peripheral blood (~10 months after T cell injection) leading to an overall survival advantage as compared to controls (Figure 2 B). In summary, we showed that human CD123-redirected T cells display potent therapeutic activity against disseminated HL. In Hodgkin Lymphoma (HL) CD123 is expressed on malignant Reed-Sternberg cells as well as in M2-polarized tumor associated macrophages (TAMs): importantly CART123 can target simultaneously both the HRS and the TAM without being resistant to immunosuppression. We have previously demonstrated that CART123 can lead to myelosuppression in preclinical models, suggesting that our findings could be translated to treat patients with refractory HL in the context of stem cell transplantation using “short-acting” RNA-CAR123 T cells or depletable lentivirally-transduced CART123. Disclosures Ruella: Novartis: Patents & Royalties. Kenderian:Novartis: Patents & Royalties, Research Funding. Wasik:Gilead Sciences: Equity Ownership; Seattle Genetics: Honoraria; Novartis: Research Funding; University of Pennsylvania: Patents & Royalties: NPM-ALK as an omncogene; University of Pennsylvania: Patents & Royalties: CAR T-cells; Gilead Sciences: Research Funding; Pharmacyclics: Research Funding. June:Novartis: Honoraria, Patents & Royalties, Research Funding; Johnson & Johnson: Honoraria; Pfizer: Honoraria; Celldex: Consultancy, Equity Ownership; Immune Design: Consultancy, Equity Ownership; Tmunity Therapeutics: Equity Ownership; Novartis: Honoraria, Patents & Royalties, Research Funding. Gill:Novartis: Patents & Royalties, Research Funding.

Abstract Introduction: Anti-CD19 chimeric antigen receptor T cells (CART19) and bi-specific anti-CD19/CD3 antibodies (blinatumomab) generate unprecedented complete response rates of 45-90% in relapsing/refractory acute lymphoblastic leukemia (r/r B-ALL). However, a subset of patients treated with these targeted approaches will relapse and a significant portion of these relapses are characterized by the loss of detectable CD19 (about 30% of relapses after blinatumomab and up to 50% after CART19), a clear manifestation of immunoediting. Hence, novel effective strategies are needed in order to be able to treat those patients and ideally prevent antigen-loss. CD123, the interleukin-3 receptor alpha, is involved in hematopoiesis and has been shown to be expressed in several hematologic neoplasms, including acute myeloid leukemia (AML) and more recently also B-ALL. Targeting CD123 with chimeric antigen receptor T cells (CART123) was shown to lead to deep and long-term responses in human primary AML xenografts. The goal of this study was to pre-clinically evaluate the impact of targeting CD123 and CD19 with chimeric antigen receptor T cells for the treatment and prevention of CD19-negative leukemia relapses occurring after CD19-directed therapies. Results: We analyzed the expression of CD123 in 42 r/r B-ALL samples and found that CD123 is highly and homogeneously expressed on the surface of most B-ALL blasts (81.75%, range: 5.1-99.6), making it a promising candidate for targeted therapy in B-ALL. Moreover, CD123 was also found to be expressed in the putative leukemia stem cells (LSC), identified as CD34-pos CD38-neg. Notably, in some Ph+ B-ALL samples we found CD19-ve CD123+ve cells with a BCR-ABL translocation by FISH, suggesting that these cells too are malignant. The expression of CD123 was detected in all (n=6) CD19-negative B-ALL blasts analyzed after relapse from CART19 treatment (Figure 1). These findings indicate that CD123 represents an ideal marker to target CD19-neg ALL blasts occurring after CART19 or blinatumomab. Therefore, we generated anti-CD123 chimeric antigen receptor T cells costimulated with 4-1-BB using a lentiviral vector (CART123). We then evaluated the CART123 anti-B-ALL efficacy both in vitro and in vivo against primary B-ALL and cell line (NALM-6). CART123 showed intense anti-B-ALL ex vivo activity, as defined by specific CD107a degranulation, cytokine production, cytotoxicity and proliferation, not statistically different from that of CART19. In order to test the role of CART123 to target CD19-negative relapses we developed a novel in vivo model, engrafting immunodeficient NSG mice with blasts obtained from a patient relapsing with CD19-ve disease after CART19 treatment. At day 14 mice were randomized to receive CART19, CART123 or control T cells (untransduced, UTD). CART19 and control T cell treated mice had no anti-tumor activity, while CART123 led to complete eradication of the disease and long-term survival. We next developed a murine model to test the hypothesis that a combined approach simultaneously targeting CD123 and CD19 could treat and prevent CD19-negative relapses. NSG mice were injected with a mixture of primary CD19-neg and CD19-pos blasts from the same patient that were labeled with different click beetle luciferases (red or green) in order to be able to track the respective clones in vivo. Mice were then randomized to receive UTD, CART19 or the combination of CART19 and CART123 (same total dose of T cells). As shown in Figure 2, mice treated with UTD had progression of both leukemic clones (CD19-pos and CD19-neg) while CART19 showed rapid progression mostly of the CD19-neg disease (red luciferase); on the contrary only mice treated with the combination of CART123 and CART19 showed rapid clearance of the disease, with improved overall survival (64 days for CART19, not reached for CART19+CART123). As a final strategy, we expressed both CAR19 and CAR123 in the same T cells and showed potent anti-leukemia activity (CD107a degranulation 81.7%). Conclusions: Here we demonstrate that CD123 is expressed in CD19-negative B-ALL relapses occurring after CD19-directed therapies, and that combining CART123 cells with CART19 cells is an effective therapy for the treatment and prevention of antigen-loss relapses in B-ALL murine xenografts. Disclosures Ruella: Novartis: Patents & Royalties, Research Funding. Kenderian:Novartis: Patents & Royalties, Research Funding. Scholler:Novartis: Patents & Royalties. Lacey:Novartis: Patents & Royalties, Research Funding. Melenhorst:Novartis: Patents & Royalties, Research Funding. Hunter:Surface Oncology - SAB: Membership on an entity's Board of Directors or advisory committees, Research Funding. Porter:Novartis: Patents & Royalties, Research Funding. June:University of Pennsylvania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight; Novartis: Research Funding. Grupp:Novartis: Consultancy, Research Funding. Gill:Novartis: Patents & Royalties, Research Funding.

Abstract Background. Many children and adults with acute myeloid leukemia (AML) relapse or are incurable with current treatment modalities, highlighting a need for alternative therapies. Chimeric antigen receptor (CAR) T cells targeting CD123 have demonstrated potent anti-leukemia activity in murine xenograft models of human AML. However, CD123-redirected T cell treatment of mice engrafted with normal human hematopoietic cells resulted in profound myeloablation, raising concerns for hematologic toxicity in patients with AML who may be treated with such therapies. We hypothesized that T cell deletion after CD123-redirected T cell-induced eradication of AML could minimize this bystander toxicity without impairing leukemia control, thereby increasing the therapeutic window of anti-AML CAR T cell immunotherapy. Methods. To test this hypothesis, we compared three termination strategies in human AML xenograft models: (1) T cell ablation with the anti-CD52 antibody alemtuzumab after treatment with T cells lentivirally-transduced with anti-CD123-41BB-CD3ζ (CART123), (2) T cell ablation with the anti-CD20 antibody rituximab after treatment with CART123 engineered to co-express CD20 (CART123/CD20), and (3) treatment with "biodegradable" anti-CD123 mRNA-electroporated CAR T cells (RNA-CART123). Mice engrafted with luciferase-expressing human AML cell lines (MOLM14, MOLM13, U937) or primary AML specimens (n=3) were treated with CD123-redirected CAR T cells as above. For T cell depletion studies, alemtuzumab 1 or 5 mg/kg was injected intraperitoneally (IP) at 1-4 weeks after 1x105-106 CART123 to determine optimal dosing and timing of T cell ablation. In subsequent studies, rituximab 10 mg/kg was injected IP 4 weeks after 1x105 -106 CART123/CD20, or 1x107 RNA-CART123 were injected intravenously at 5, 9, and 16 days after AML engraftment. Mice were followed by weekly bioluminescent imaging and/or quantitative flow cytometry analyses of blood, spleen, and/or bone marrow. Results. CART123 treatment of CD123+ AML xenografts induced marked T cell expansion and leukemia eradication in vivo, resulting in long-term animal survival (p<0.0001 vs untransduced T cell-treated controls). Minimal xenogeneic graft-versus-host effects were observed. One dose of alemtuzumab rapidly eliminated T cells in all tested models with best efficacy of 5 mg/kg dosing at 4 weeks post-CART123. CART123/CD20 inhibited AML proliferation with similar kinetics to those of CART123, and 1 dose of rituximab at 4 weeks post-CART123/CD20 infusion rapidly eliminated T cells while preserving leukemia remission. Alemtuzumab or rituximab alone did not inhibit AML proliferation in non-CART123-treated xenograft models vs AML-only controls (p=1.00). Mice with CART123- or CART123/CD20-induced AML remission at time of T cell ablation remained leukemia-free for ≥12 weeks, and animal survival did not differ from that of CD123-redirected CAR T cell-treated mice that did not undergo T cell depletion (p=1.00). In contrast, early depletion of T cells with alemtuzumab at 1, 2, or 3 weeks post-CART123 resulted in failure to eliminate AML with subsequent leukemia progression and animal death. Furthermore, AML rechallenge of animals with alemtuzumab- or rituximab-ablated T cells resulted in rapid leukemia proliferation without T cell re-expansion, confirming completeness of T cell depletion. Non-ablated mice demonstrated CAR T cell re-expansion with rejection of CD123+ leukemia rechallenge (p<0.0001). RNA-CART123 rapidly eliminated AML and facilitated long-term animal survival, although RNA-CART123 had expectedly shorter persistence in vivo than did CART123 or CART123/CD20. Conclusions. Alemtuzumab and rituximab completely eliminated CD123-redirected CAR T cells in human AML xenograft models. Sustained leukemia remission required CART123 or CART123/CD20 persistence for 4 weeks prior to T cell termination via alemtuzumab or rituximab post-CART123 or CART123/CD20, respectively. Ongoing studies are investigating efficacy of T cell elimination in additional xenograft models and against other anti-AML CAR T cell immunotherapies. Results from these studies may help to identify promising T cell termination strategies that will augment efficacy of CAR T cell therapy in patients with AML, particularly prior to stem cell transplantation. RNA-CART123 trials in patients with relapsed/refractory AML will open soon. Disclosures Off Label Use: depletion of CAR T cells with alemtuzumab or rituximab. Kenderian:Novartis: Patents & Royalties, Research Funding. Ruella:Novartis: Patents & Royalties, Research Funding. Aplenc:Sigma Tau: Consultancy. June:Novartis: Patents & Royalties, Research Funding. Grupp:Novartis: Consultancy, Research Funding. Gill:Novartis: Patents & Royalties, Research Funding.