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Chimeric antigen receptor (CAR) T-cell therapy for multiple myeloma targeting B-cell maturation antigen (TNFRSF17; BCMA) induces high overall response rates; however, relapse occurs commonly. A reservoir of multiple myeloma cells lacking sufficient BCMA surface expression (antigen escape) may be implicated in relapse. We demonstrate that simultaneous targeting of an additional antigen—here, G protein-coupled receptor class-C group-5 member-D (GPRC5D)—can prevent BCMA escape–mediated relapse in a model of multiple myeloma. To identify an optimal approach, we compare subtherapeutic doses of different forms of dual-targeted cellular therapy. These include; (i) parallel-produced and pooled mono-targeted CAR T cells, (ii) bicistronic constructs expressing distinct CARs from a single vector, and (iii) a dual-scFv “single-stalk” CAR design. When targeting BCMA-negative disease, bicistronic and pooled approaches had the highest efficacy, whereas for dual-antigen–expressing disease, the bicistronic approach was more efficacious than the pooled approach. Mechanistically, expressing two CARs on a single cell enhanced the strength of CAR T-cell/target cell interactions. Significance: Myeloma frequently relapses post-CAR T-cell therapy; antigen escape–mediated relapse can be mitigated with upfront dual-targeting (BCMA/GPRC5D). A bicistronic vector encoding two CARs avoids the challenge of parallel manufacturing separate CAR T-cell products, while providing superior efficacy; this dual-targeted approach may enhance the durability of responses to cellular therapy for myeloma. See related commentary by Simon and Riddell, p. 130. This article is highlighted in the In This Issue feature, p. 127

Chimeric antigen receptor (CAR) T-cell therapy has shown remarkable responses in B-cell malignancies. However, many patients suffer from limited response and tumor relapse due to lack of persisting CAR T cells and immune escape. These clinical challenges have compromised the long-term efficacy of CAR T-cell therapy and call for the development of novel CAR designs. We demonstrated that CAR T cells secreting a cytokine interleukin-36γ (IL-36γ) showed significantly improved CAR T-cell expansion and persistence, and resulted in superior tumor eradication compared with conventional CAR T cells. The enhanced cellular function by IL-36γ was mediated through an autocrine manner. In addition, activation of endogenous antigen-presenting cells (APCs) and T cells by IL-36γ aided the formation of a secondary antitumor response, which delayed the progression of antigen-negative tumor challenge. Together, our data provide preclinical evidence to support the translation of this design for an improved CAR T-cell-mediated antitumor response.

BackgroundWaldenström macroglobulinemia (WM) is an incurable disease and, while treatable, can develop resistance to available therapies and be fatal. Chimeric antigen receptor (CAR) T cell therapy directed against the CD19 antigen has demonstrated efficacy in relapsed or refractory B lymphoid malignancies, and is now approved for B cell acute lymphoblastic leukemia and certain B cell lymphomas. However, CAR T therapy has not been evaluated for use in WM.Methods and resultsWe performed preclinical studies demonstrating CAR T cell activity against WM cells in vitro, and developed an in vivo murine model of WM which demonstrated prolonged survival with use of CAR T therapy. We then report the first three patients with multiply relapsed and refractory WM treated for their disease with CD19-directed CAR T cells on clinical trials. Treatment was well tolerated, and observed toxicities were consistent with those seen in CAR T treatment for other diseases, and no grade 3 or higher cytokine release syndrome or neurotoxicity events occurred. All three patients attained at least a clinical response to treatment, including one minimal residual disease-negative complete response, though all three eventually developed recurrent disease between 3 and 26 months after initial treatment.ConclusionsThis report summarizes preclinical and clinical activity of CD19-directed CAR T therapy in WM, demonstrating early tolerability and efficacy in patients with WM, and representing a possible treatment option in patients with heavily pretreated and relapsed or refractory WM. Larger studies evaluating CAR T therapy in WM are warranted, along with further evaluation into mechanisms of resistance to CAR T therapy.

In the New England Journal of Medicine, Wang et al. report favorable ZUMA-2 trial results of CAR T cells for patients with relapsed and refractory mantle cell lymphoma following prior failed Bruton's tyrosine kinase inhibitor therapy, with an overall response rate of 93% and an expected safety profile.

Chimeric antigen receptor (CAR) T-cell therapy has dramatically shifted the landscape of treatment for lymphoid malignancies, especially diffuse large B-cell lymphoma (DLBCL) and acute lymphoblastic leukemia (ALL). However, there continue to be significant limitations of this therapy, such as incomplete or nonsustained responses and severe toxicities in a subset of patients. Furthermore, expanding the role of CAR T-cell therapy to new disease types is an important next step. In this review, we will highlight landmark trials for anti-CD19 CAR T cells and first-in-human trials of novel CARs, as well as discuss promising innovative CAR designs that are still undergoing preclinical development. Lastly, we will discuss toxicity and mechanisms of CAR T-cell resistance and failure, as well as potential future treatment approaches to these common issues.

Immunotherapy for ovarian cancer is an area of intense investigation since the majority of women with relapsed disease develop resistance to conventional cytotoxic therapy. The paucity of safe and validated target antigens has limited the development of clinically relevant antibody-based immunotherapeutics for this disease. Although MUC16 expression is almost universal in High Grade Serous Ovarian Cancers, engagement of the shed circulating MUC16 antigen (CA-125) presents a theoretical risk of systemic activation and toxicity. We designed and evaluated a series of bispecific tandem single-chain variable fragments specific to the retained portion of human MUC16 ectodomain (MUC16ecto) and human CD3. These MUC16ecto- BiTEDs retain binding in the presence of soluble MUC16 (CA-125) and show cytotoxicity against a panel of ovarian cancer cells in vitro. MUC16ecto- BiTEDs delay tumor progression in vivo and significantly prolong survival in a xenograft model of ovarian peritoneal carcinomatosis. This effect was significantly enhanced by antiangiogenic (anti-VEGF) therapy and immune checkpoint inhibition (anti-PD1). However, the combination of BiTEDs with anti-VEGF was superior to combination with anti-PD1, based on findings of decreased peritoneal tumor burden and ascites with the former. This study shows the feasibility and efficacy of MUC16ecto- specific BiTEDs and provides a basis for the combination with anti-VEGF therapy for ovarian cancer.

Immunosuppressive tumor microenvironment (TME) and ascites-derived spheroids in ovarian cancer (OC) facilitate tumor growth and progression, and also pose major obstacles for cancer therapy. The molecular pathways involved in the OC-TME interactions, how the crosstalk impinges on OC aggression and chemoresistance are not well-characterized. Here, we demonstrate that tumor-derived UBR5, an E3 ligase overexpressed in human OC associated with poor prognosis, is essential for OC progression principally by promoting tumor-associated macrophage recruitment and activation via key chemokines and cytokines. UBR5 is also required to sustain cell-intrinsic β-catenin-mediated signaling to promote cellular adhesion/colonization and organoid formation by controlling the p53 protein level. OC-specific targeting of UBR5 strongly augments the survival benefit of conventional chemotherapy and immunotherapies. This work provides mechanistic insights into the novel oncogene-like functions of UBR5 in regulating the OC-TME crosstalk and suggests that UBR5 is a potential therapeutic target in OC treatment for modulating the TME and cancer stemness., Ovarian cancer cells often metastasize to the peritoneal cavity, forming spheroid-like structures and promoting a highly immunosuppressive tumor microenvironment. Here, the authors show that the ubiquitin ligase UBR5 is required for ovarian cancer growth and metastasis, sustaining spheroid formation and the infiltration of immunosuppressive tumor associated macrophages.

While effective in specific settings, adoptive chimeric antigen receptor (CAR) T cell therapy for cancer requires further improvement and optimization. Our previous results show that CD40L-overexpressing CAR T cells mobilize endogenous immune effectors, resulting in improved antitumor immunity. However, the cell populations required for this protective effect remain to be identified. Here we show, by analyzing Batf3−/− mice lacking the CD103+ conventional dendritic cell type 1 (cDC1) subpopulation important for antigen cross-presentation, that CD40L-overexpressing CAR T cells elicit an impaired antitumor response in the absence of cDC1s. We further find that CD40L-overexpressing CAR T cells stimulate tumor-resident CD11b−CD103− double-negative (DN) cDCs to proliferate and differentiate into cDC1s in wild-type mice. Finally, re-challenge experiments show that endogenous CD8+ T cells are required for protective antitumor memory in this setting. Our findings thus demonstrate the stimulatory effect of CD40L-overexpressing CAR T cells on innate and adaptive immune cells, and provide a rationale for using CD40L-overexpressing CAR T cells to improve immunotherapy responses., CD40L-expressing chimeric antigen receptor (CAR) T cells show enhanced anti-tumor immunity, but the cellular mechanisms are still unclear. Here we show, by analyzing mice deficient of conventional dendritic cell type 1 (cDC1) that cDC1s are induced by CD40L+ CAR T cells to prime endogenous CD8 T cells for a stronger anti-tumor immune response.

Chimeric Antigen Receptor (CAR) T cell therapies – adoptive T cell therapies that have been genetically engineered for a new antigen-specificity - have displayed significant success in treating patients with hematologic malignancies, leading to three recent US Food and Drug Administration approvals. Based on the promise generated from these successes, the field is rapidly evolving to include new disease indications and CAR designs, while simultaneously reviewing and optimizing toxicity and management protocols. As such, this review provides expert perspective on the significance and clinical considerations of CAR T cell therapies in order to provide timely information to clinicians about this revolutionary new therapeutic class.

Background: There is compelling rationale to use bridging radiotherapy (BRT) between leukapheresis and chimeric antigen receptor T-cell infusion for cytoreduction and palliation of relapsed/refractory B cell malignancies. There is also preclinical evidence suggesting possible immune synergy with the combination of BRT and CAR T. Existing BRT series are small and generally focused on diffuse large B-cell lymphoma (DLBCL). We report a large cohort of RT-bridged patients including early experience with BRT for non-DLBCL histologies. Methods: We analyzed 43 patients with DLBCL (n=35), mantle cell lymphoma (MCL; n=6), and Burkitt's lymphoma (BL; n=2). All received RT for any intent within 1 month prior to leukapheresis up until CAR T infusion. PET response was evaluated by Lugano criteria within the BRT treatment field ("in field"), outside of the BRT field ("out of field") and overall at 5 time points: pre-BRT, pre-CAR T infusion, and day +30 (n=41), +90 (n=30), and +180 (n=19). Survival analysis was per Kaplan-Meier and clincodemographic associations with survival were assessed by Cox univariate proportional hazards. Results: Patients received axicabtagene (n=20, 48%), tisagenlecleucel (n=11, 26%), lisocabtagene (n=8, 19%), brexucabtagene (n=2, 5%), and an experimental CAR T product (n=1, 2%). Overall, patients had a median of 3 prior therapies (range 1-9). Prior to BRT, most (74%) had advanced stage III/IV relapsed/refractory disease and 17 (40%) had bulky (>7.5 cm) lesions. There was heterogeneity in BRT-treatment parameters; most commonly treated sites were head/neck (n=10), pelvis/groin (n=8), and abdomen (n=7). Median BRT dose was 30 Gy (range 4-54), with 20 Gy in 5 fractions as the most common regimen (n=12). 18 (42%) received BRT to all sites of PET avid disease ("comprehensive BRT"). 16 (37%) patients also received systemic bridging therapy, including concurrently (n=10) with BRT. With a short 14d median time to restaging PET, in field overall response rate (ORR) post BRT was 82% (complete response, CR; n=9, 27% or partial response, PR; n=18, 55%) [Figure 1]. Reflective of the population referred for bridging, 21 (64%) had out of field progression post BRT and pre-CAR T. Post-CAR T, 2 patients (5%) had grade 3 cytokine release syndrome, 4 (9%) had grade 3 neurotoxicity, and no severe toxicity within the BRT field was noted. Following CAR T, ORR was 72% (CR: n=25, 60%; PR: n=5, 12%). Median post CAR T follow-up was 9 mos (range 1-40), with 44% (n=19) of patients having no evidence of disease, 44% (n=19) deceased, and 12% (n=5) alive with disease at last contact. 33 (77%) patients never progressed within the BRT field. Of the 27 (63%) patients who relapsed, 10 were CD19- and 9 (33%) had disease in the BRT field at first progression. In DLBCL patients, progression free survival (PFS) was 83%, 60%, and 51% and overall survival (OS) was 100%, 89% and 69%, respectively at Day +30, +90, and +180. Univariate analysis showed that pre-BRT ECOG performance status ≥2 (HR: 12.0, p Conclusion: BRT has broad utility for aggressive B-cell malignancies and is associated with excellent pre-CAR T local control and no serious toxicity within the irradiated sites. Most patients had durable post-CAR T local control. Prospective studies are planned to clarify outcomes and evaluate mechanistic synergies. Future studies are needed to define optimal patient characteristics for systemic vs. radiotherapy bridging and elucidate the best BRT strategy for maximizing local control and potential immune augmentation. Figure 1 Figure 1. Disclosures Palomba: Kite: Consultancy; Novartis: Consultancy; Notch: Honoraria, Other: Stock; Seres: Honoraria, Other: Stock, Patents & Royalties, Research Funding; Lygenesis: Honoraria; PCYC: Consultancy; BeiGene: Consultancy; Pluto: Honoraria; Wolters Kluwer: Patents & Royalties; Juno: Patents & Royalties; Ceramedix: Honoraria; Magenta: Honoraria; Priothera: Honoraria; Nektar: Honoraria; WindMIL: Honoraria; Rheos: Honoraria. Shouval: Medexus: Consultancy. Batlevi: Regeneron: Current holder of individual stocks in a privately-held company; Viatris: Current holder of individual stocks in a privately-held company; TouchIME: Honoraria; BMS: Current holder of individual stocks in a privately-held company; Moderna: Current holder of individual stocks in a privately-held company; Pfizer: Current holder of individual stocks in a privately-held company; Medscape: Honoraria; TG Therapeutics: Consultancy; Seattle Genetics: Consultancy; Life Sciences: Consultancy; Kite Pharma: Consultancy; Dava Oncology: Honoraria; Karyopharm: Consultancy; Juno/Celgene: Consultancy; Bayer: Research Funding; Memorial Sloan Kettering Cancer Center: Current Employment; ADC Therapeutics: Consultancy; GLG Pharma: Consultancy; Xynomic: Research Funding; Roche/Genentech: Research Funding; Novartis: Research Funding; Epizyme: Research Funding; Janssen: Research Funding; Autolus: Research Funding. Brentjens: BMS: Consultancy, Patents & Royalties, Research Funding; Gracell Biotechnologies, Inc: Consultancy, Ended employment in the past 24 months; sanofi: Patents & Royalties; Caribou: Patents & Royalties. Dahi: Kite / Gilead: Membership on an entity's Board of Directors or advisory committees. Giralt: JENSENN: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; PFIZER: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; AMGEN: Membership on an entity's Board of Directors or advisory committees; JAZZ: Membership on an entity's Board of Directors or advisory committees; SANOFI: Membership on an entity's Board of Directors or advisory committees; CELGENE: Membership on an entity's Board of Directors or advisory committees; Actinnum: Membership on an entity's Board of Directors or advisory committees. Park: Kura Oncology: Consultancy; Intellia: Consultancy; Autolus: Consultancy; Artiva: Consultancy; Novartis: Consultancy; Amgen: Consultancy; PrecisionBio: Consultancy; Curocel: Consultancy; Affyimmune: Consultancy; BMS: Consultancy; Minerva: Consultancy; Servier: Consultancy; Kite Pharma: Consultancy; Innate Pharma: Consultancy. Scordo: i3 Health: Other: Speaker; Kite - A Gilead Company: Membership on an entity's Board of Directors or advisory committees; Omeros Corporation: Consultancy; Angiocrine Bioscience: Consultancy, Research Funding; McKinsey & Company: Consultancy. Sauter: Gamida Cell: Consultancy; Celgene: Consultancy, Research Funding; Novartis: Consultancy; Precision Biosciences: Consultancy; Kite/Gilead: Consultancy; Genmab: Consultancy; GSK: Consultancy; Bristol-Myers Squibb: Research Funding; Spectrum Pharmaceuticals: Consultancy; Juno Therapeutics: Consultancy, Research Funding; Sanofi-Genzyme: Consultancy, Research Funding. Shah: Janssen: Research Funding; Amgen: Research Funding. Perales: NexImmune: Honoraria; Kite/Gilead: Honoraria, Other; Karyopharm: Honoraria; Bristol-Myers Squibb: Honoraria; Cidara: Honoraria; Incyte: Honoraria, Other; Equilium: Honoraria; Celgene: Honoraria; MorphoSys: Honoraria; Medigene: Honoraria; Omeros: Honoraria; Novartis: Honoraria, Other; Nektar Therapeutics: Honoraria, Other; Miltenyi Biotec: Honoraria, Other; Merck: Honoraria; Sellas Life Sciences: Honoraria; Servier: Honoraria; Takeda: Honoraria.

Background: Patients with diffuse large B cell lymphoma (DLBCL) who do not achieve a complete response (CR) following CD19-targeted Chimeric Antigen Receptor T cell (CAR T) therapy are less likely to obtain durable benefit. Greater tumor burden pre-CAR T has been shown to predict lower CR rate and shorter survival. Recently, patterns of failure studies have identified baseline lesion characteristics including size, SUV, and extranodal location as associated with increased risk of post-CAR T failure. It is not yet known how bridging radiotherapy (BRT) prior to CAR T can alter subsequent patterns of failure. Methods: We reviewed DLBCL patients treated from 2017 to 2021 with BRT for any intent in the period from 30d pre-leukapheresis to CAR T infusion. Comprehensive BRT fields were defined as no avidity above liver mean outside of the BRT field. Bulky disease was defined as a max diameter of ≥7.5cm in any dimension. PET response was evaluated by Lugano criteria. Pattern of failure analysis was performed to identify failure sites as pre-existing (present pre-CAR T) vs. new and as in-field, marginal, or distant with respect to BRT. Marginal was defined as failure outside of the area receiving prescription dose but within 1cm of BRT field edge. One patient with a cutaneous target was excluded from size-based analyses. Significance was assessed by Fisher's exact test or T-test. Results: Thirty-five patients were identified with a median age of 66. Most were advanced stage (74%) at BRT with a median of 3 prior lines of systemic therapy. Twelve (35%) had bulky disease at any site pre-BRT (median max diameter 5.9 cm; range 1.1 - 24) with highest overall SUV median 19.8 (range 3.4 - 47); 83% (n=29) had ≥1 extranodal site (bone: n=13; CNS: n=4). Most common BRT sites were head/neck (n=10), pelvis (n=6), and extremity (n=5). BRT targeted the largest lesion in 85% (median 5.8 cm; 32% bulky) and the site of highest SUV in 81% (median 18.7); 49% (n=17) of targets were extranodal. BRT fields were comprehensive in 39%; median radiation treatment volume was 751cc (range 18 - 5856). Twenty-four patients (63%) received ≥30 Gy (median 30 Gy; range 20 - 54). Systemic therapy was given during the bridging period to 31% (n=11). Patients received axicabtagene (n=20), tisagenlecleucel (n=11), lisocabtagene (n=3), or experimental CAR T (n=1). On PET after BRT and pre-infusion (median 12d from BRT), 85% (n=23) had achieved partial response (PR) or better in-field (CR: n=7) though 63% had kinetically active disease with out-of-field progression (PD) and 19% (n=5) had marginal PD. Despite short interval restaging, there was a significant reduction in patients with bulky disease after BRT both within the irradiated field (p=0.007) and at any site (p=0.01). (Fig 1a) A median 81% SUV reduction in the irradiated site was noted with a significant decrease in max SUV in both the irradiated (p Conclusion: BRT significantly reduced the number of patients with bulky disease at any site at time of CAR T and significantly reduced the overall highest lesional SUV. Marginal failure rates of 19% and 23% at interim scan post-BRT and after CAR T, respectively in conjunction with the pattern of post CAR T failure predominantly in pre-existing sites, may suggest a need for more generous fields when a BRT approach is used in this population with rapidly proliferating disease. Further work and larger sample sizes are needed to evaluate the impact of dose on local control in bulky lesions as well as to investigate whether comprehensive BRT affects outcomes or patterns of failure. Figure 1 Figure 1. Disclosures Palomba: Juno: Patents & Royalties; Notch: Honoraria, Other: Stock; Novartis: Consultancy; Kite: Consultancy; PCYC: Consultancy; Wolters Kluwer: Patents & Royalties; Seres: Honoraria, Other: Stock, Patents & Royalties, Research Funding; Magenta: Honoraria; Ceramedix: Honoraria; WindMIL: Honoraria; Nektar: Honoraria; Rheos: Honoraria; Priothera: Honoraria; BeiGene: Consultancy; Lygenesis: Honoraria; Pluto: Honoraria. Shouval: Medexus: Consultancy. Batlevi: Kite Pharma: Consultancy; Viatris: Current holder of individual stocks in a privately-held company; Dava Oncology: Honoraria; TG Therapeutics: Consultancy; ADC Therapeutics: Consultancy; Regeneron: Current holder of individual stocks in a privately-held company; TouchIME: Honoraria; Juno/Celgene: Consultancy; Medscape: Honoraria; BMS: Current holder of individual stocks in a privately-held company; Seattle Genetics: Consultancy; Life Sciences: Consultancy; Pfizer: Current holder of individual stocks in a privately-held company; Karyopharm: Consultancy; Moderna: Current holder of individual stocks in a privately-held company; Memorial Sloan Kettering Cancer Center: Current Employment; Bayer: Research Funding; GLG Pharma: Consultancy; Xynomic: Research Funding; Roche/Genentech: Research Funding; Novartis: Research Funding; Epizyme: Research Funding; Janssen: Research Funding; Autolus: Research Funding. Brentjens: Gracell Biotechnologies, Inc: Consultancy, Ended employment in the past 24 months; BMS: Consultancy, Patents & Royalties, Research Funding; sanofi: Patents & Royalties; Caribou: Patents & Royalties. Dahi: Kite / Gilead: Membership on an entity's Board of Directors or advisory committees. Giralt: JAZZ: Membership on an entity's Board of Directors or advisory committees; AMGEN: Membership on an entity's Board of Directors or advisory committees; JENSENN: Membership on an entity's Board of Directors or advisory committees; PFIZER: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; SANOFI: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; CELGENE: Membership on an entity's Board of Directors or advisory committees; Actinnum: Membership on an entity's Board of Directors or advisory committees. Park: BMS: Consultancy; Kite Pharma: Consultancy; Innate Pharma: Consultancy; Novartis: Consultancy; Minerva: Consultancy; Servier: Consultancy; Kura Oncology: Consultancy; PrecisionBio: Consultancy; Amgen: Consultancy; Intellia: Consultancy; Artiva: Consultancy; Curocel: Consultancy; Autolus: Consultancy; Affyimmune: Consultancy. Scordo: Angiocrine Bioscience: Consultancy, Research Funding; Kite - A Gilead Company: Membership on an entity's Board of Directors or advisory committees; i3 Health: Other: Speaker; Omeros Corporation: Consultancy; McKinsey & Company: Consultancy. Sauter: Bristol-Myers Squibb: Research Funding; GSK: Consultancy; Gamida Cell: Consultancy; Precision Biosciences: Consultancy; Kite/Gilead: Consultancy; Celgene: Consultancy, Research Funding; Genmab: Consultancy; Novartis: Consultancy; Spectrum Pharmaceuticals: Consultancy; Juno Therapeutics: Consultancy, Research Funding; Sanofi-Genzyme: Consultancy, Research Funding. Shah: Amgen: Research Funding; Janssen Pharmaceutica: Research Funding. Perales: Merck: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Cidara: Honoraria; Miltenyi Biotec: Honoraria, Other; Kite/Gilead: Honoraria, Other; Equilium: Honoraria; Incyte: Honoraria, Other; Medigene: Honoraria; Karyopharm: Honoraria; Takeda: Honoraria; Servier: Honoraria; NexImmune: Honoraria; Novartis: Honoraria, Other; Nektar Therapeutics: Honoraria, Other; Sellas Life Sciences: Honoraria; Omeros: Honoraria; MorphoSys: Honoraria.

Introduction: Cellular immunotherapy with CD19-targeted chimeric antigen receptor (CAR) T cells has provided new therapeutic options for patients with high-risk hematologic malignancies. Following this therapy, patients may experience disease relapse or CAR-mediated toxicity due to cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS). Recent studies have confirmed that the intestinal microbiome can modulate the anti-tumor immune response to chemotherapy, immune checkpoint blockade, graft-versus-host disease after allogeneic hematopoietic cell transplantation, and adoptive cellular therapy. The contribution of the intestinal microbiome on the function of CAR T cells in vivo both with respect to their anti-tumor function and their propensity to induce toxicities is not known. Hence, in a multi-center study we analyzed the association between clinical outcomes and (1) antibiotic exposure prior to CAR T cell infusion and (2) the composition and diversity of the fecal microbiome. Methods and Results: We retrospectively collected clinical data and antibiotic exposures from patients with acute lymphoblastic leukemia (ALL, n=91) and non-Hodgkin lymphoma (NHL, n=137) treated with investigational or commercial CD19 CAR T cells at Memorial Sloan Kettering Cancer Center (MSK) and the University of Pennsylvania (Penn). We considered any antibiotic exposure between day -30 and the day of CAR T cell infusion. We focused our analysis on anaerobe-targeting antibiotics used in the setting of neutropenic fever: piperacillin-tazobactam, imipenem-cilastatin, and meropenem (here referred to as "P-I-M"). We found that forty-seven (20.6%) of 228 patients were exposed to P-I-M in the four weeks before CAR T cell infusion. Patient characteristics at the time of CAR T cell infusion were similar between the P-I-M-exposed and not-exposed groups, although a worse performance status was observed in patients with NHL treated with P-I-M. We found that overall survival (OS) was significantly decreased following CAR T cell infusion in patients exposed to P-I-M (Fig 1A; OS HR, 2.58; 95% CI, 1.68 - 3.98; p= We also prospectively collected baseline fecal samples prior to cell infusion from CD19 CAR T cells recipients (n=48) at MSK and Penn. Samples were submitted for 16S RNA sequencing of the V4-V5 region on the Illumina MiSeq platform and the amplicon sequence variants (ASVs) were annotated according to the NCBI 16S database using BLAST. In comparison to healthy controls (n=30), we found that alpha-diversity was significantly lower in fecal samples from CAR T cell patients (p= 0.0023, Fig 1B) and the composition of fecal samples was significantly different (p= Conclusion: Our results suggest that exposure to antibiotics, in particular P-I-M, in the four weeks before therapy was associated with worse survival. Profiling of the baseline fecal microbiome samples by 16S revealed that CD19 CAR T cell patients presented with evidence of an altered fecal microbiome as measured by lower alpha-diversity and a composition that is distinct from that of healthy controls. Finally, we identified bacterial taxa that were associated with Day 100 CR and CAR-mediated toxicity. Our findings indicate that the intestinal microbiome can affect the efficacy of CD19 CAR T cell therapy and provides a rationale to target the intestinal microbiome to improve clinical outcomes of patients treated with cellular therapies. Figure 1 Figure 1. Disclosures Smith: Janssen: Consultancy, Honoraria. Gomes: Xbiome: Current Employment. Schluter: Postbiotics Plus LLC: Other: cofounder. Park: Kura Oncology: Consultancy; BMS: Consultancy; Servier: Consultancy; Autolus: Consultancy; Curocel: Consultancy; Artiva: Consultancy; Kite Pharma: Consultancy; Amgen: Consultancy; Novartis: Consultancy; Affyimmune: Consultancy; Intellia: Consultancy; Innate Pharma: Consultancy; Minerva: Consultancy; PrecisionBio: Consultancy. Palomba: Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Kite Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Jain: Targeted Healthcare Communications: Consultancy; Bristol Myers Squibb: Other: for advisory board participation; CareDx: Other: for advisory board participation; CTI Biopharma: Research Funding; Syneos Health: Research Funding. Pennisi: Gilead Sciences: Consultancy. Perales: Miltenyi Biotec: Honoraria, Other; Novartis: Honoraria, Other; Omeros: Honoraria; NexImmune: Honoraria; Bristol-Myers Squibb: Honoraria; Merck: Honoraria; Celgene: Honoraria; Takeda: Honoraria; Kite/Gilead: Honoraria, Other; Medigene: Honoraria; Nektar Therapeutics: Honoraria, Other; Cidara: Honoraria; Servier: Honoraria; Sellas Life Sciences: Honoraria; Karyopharm: Honoraria; MorphoSys: Honoraria; Equilium: Honoraria; Incyte: Honoraria, Other. Garfall: Amgen: Honoraria; CRISPR Therapeutics: Research Funding; GlaxoSmithKline: Honoraria; Janssen: Honoraria, Research Funding; Novartis: Research Funding; Tmunity: Research Funding. Landsburg: Triphase: Research Funding; Morphosys: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Other: DSMB member; Incyte: Membership on an entity's Board of Directors or advisory committees; ADCT: Membership on an entity's Board of Directors or advisory committees; Curis: Research Funding; Takeda: Research Funding. Gerson: Kite: Consultancy; Pharmacyclics: Consultancy; Abbvie: Consultancy; TG Therapeutics: Consultancy. Svoboda: Imbrium: Consultancy; Genmab: Consultancy; Astra Zeneca: Consultancy, Research Funding; Atara: Consultancy; BMS: Consultancy, Research Funding; Adaptive: Consultancy, Research Funding; Incyte: Research Funding; Merck: Research Funding; Pharmacyclics: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; TG: Research Funding. Giralt: AMGEN: Membership on an entity's Board of Directors or advisory committees; PFIZER: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; SANOFI: Membership on an entity's Board of Directors or advisory committees; CELGENE: Membership on an entity's Board of Directors or advisory committees; JAZZ: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; JENSENN: Membership on an entity's Board of Directors or advisory committees; Actinnum: Membership on an entity's Board of Directors or advisory committees. Gill: Interius Biotherapeutics: Current holder of stock options in a privately-held company, Research Funding; Novartis: Other: licensed intellectual property, Research Funding; Carisma Therapeutics: Current holder of stock options in a privately-held company, Research Funding. Rivière: FloDesign Sonics: Other: Provision of Services; Centre for Commercialization of Cancer Immunotherapy: Other: Provision of Services; Fate Therapeutics: Other: Provision of Services, Patents & Royalties; The Georgia Tech Research Corporation (GTRC): Other: Provision of Services (uncompensated); Juno Therapeutics: Patents & Royalties. Porter: Kite/Gilead: Membership on an entity's Board of Directors or advisory committees; Wiley and Sons Publishing: Honoraria; Tmunity: Patents & Royalties; Novartis: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; ASH: Membership on an entity's Board of Directors or advisory committees; DeCart: Membership on an entity's Board of Directors or advisory committees; Genentech: Current equity holder in publicly-traded company, Ended employment in the past 24 months; American Society for Transplantation and Cellular Therapy: Honoraria; National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees. Schuster: Abbvie: Consultancy, Research Funding; Acerta Pharma: Consultancy; AstraZeneca: Consultancy; Adaptive Biotechnologies: Research Funding; BeiGene: Consultancy; Celgene: Consultancy, Honoraria, Research Funding; DTRM: Research Funding; Genetech: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Incyte: Research Funding; Juno Theraputics: Consultancy, Research Funding; Loxo Oncology: Consultancy; Merck: Research Funding; Nordic Nanovector: Consultancy; Novartis: Consultancy, Honoraria, Patents & Royalties, Research Funding; Pharmaclcyclics: Research Funding; Tessa Theraputics: Consultancy; TG Theraputics: Research Funding. Sadelain: NHLBI Gene Therapy Resource Program: Other: Provision of Services (uncompensated); Fate Therapeutics: Other: Provision of Services (uncompensated), Patents & Royalties; Atara Biotherapeutics: Patents & Royalties; Ceramedix: Patents & Royalties; Mnemo Therapeutics: Patents & Royalties; Takeda Pharmaceuticals: Other: Provision of Services, Patents & Royalties; St. Jude Children's Research Hospital: Other: Provision of Services; Juno Therapeutics: Patents & Royalties; Minerva Biotechnologies: Patents & Royalties. Frey: Novartis: Research Funding; Kite Pharma: Consultancy; Sana Biotechnology: Consultancy; Syndax Pharmaceuticals: Consultancy. Brentjens: Gracell Biotechnologies, Inc: Consultancy, Ended employment in the past 24 months; BMS: Consultancy, Patents & Royalties, Research Funding; sanofi: Patents & Royalties; Caribou: Patents & Royalties. June: AC Immune, DeCART, BluesphereBio, Carisma, Cellares, Celldex, Cabaletta, Poseida, Verismo, Ziopharm: Consultancy; Novartis: Patents & Royalties; Tmunity, DeCART, BluesphereBio, Carisma, Cellares, Celldex, Cabaletta, Poseida, Verismo, Ziopharm: Current equity holder in publicly-traded company. Pamer: Diversigen: Other: Advisory board; Bristol Myers Squibb, Celgene, Seres Therapeutics, MedImmune, Novartis and Ferring Pharmaceuticals: Honoraria. Peled: DaVolterra: Consultancy; MaaT Pharma: Consultancy; CSL Behring: Consultancy; Seres Therapeutics: Research Funding. Ruella: BMS, BAYER, GSK: Consultancy; Novartis: Patents & Royalties; AbClon: Consultancy, Research Funding; Tmunity: Patents & Royalties; viTToria biotherapeutics: Research Funding. van den Brink: WindMILTherapeutics: Honoraria; Pluto Therapeutics: Current holder of stock options in a privately-held company, Other: has consulted, received honorarium from or participated in advisory boards ; Priothera: Research Funding; Forty-Seven, Inc.: Honoraria; MagentaTherapeutics: Honoraria; GlaskoSmithKline: Other: has consulted, received honorarium from or participated in advisory boards; Ceramedix: Other: has consulted, received honorarium from or participated in advisory boards ; Merck & Co, Inc: Honoraria; Synthekine (Spouse): Other: has consulted, received honorarium from or participated in advisory boards; Kite Pharmaceuticals: Other; Amgen: Honoraria; Frazier Healthcare Partners: Honoraria; Seres: Other: Honorarium, Intellectual Property Rights, Research Fundingand Stock Options; Rheos: Honoraria; Therakos: Honoraria; Jazz Pharmaceuticals: Honoraria; Notch Therapeutics: Honoraria; Nektar Therapeutics: Honoraria; Wolters Kluwer: Patents & Royalties; Juno Therapeutics: Other; DKMS (nonprofit): Other; Pharmacyclics: Other; Da Volterra: Other: has consulted, received honorarium from or participated in advisory boards; Novartis (Spouse): Other: has consulted, received honorarium from or participated in advisory boards; Lygenesis: Other: has consulted, received honorarium from or participated in advisory boards .

Background: BCMA targeted CAR T cell therapy has shown promising results in patients with relapsed/refractory multiple myeloma (RRMM), but relapses are common. Additional treatment options with novel therapeutic targets or mechanisms of action are needed. Here we report on the safety and efficacy of MCARH109, the first-in-class G Protein Coupled Receptor Class C Group 5 Member D (GPRC5D) targeted CAR T cell therapy (Smith EL et al. Sci. Trans Med 2019) in RRMM including patients who relapsed post BCMA targeted CAR T cell therapy. Methods: This is a phase I first-in-human, dose escalation trial of MCARH109; patients received lymphodepleting chemotherapy with fludarabine 30 mg/m 2 daily and cyclophosphamide 300 mg/m 2 daily for 3 days followed by a single infusion of MCARH109. The trial followed a standard 3+3 design with the following dose cohorts to date: 25X10 6, 50X10 6, 150X10 6, 450X10 6 viable CAR + T cells. The primary objective is to assess safety of MCARH109; secondary objectives include anti-myeloma efficacy, expansion and persistence of MCARH109 using quantitative polymerase chain reaction (qPCR) on peripheral blood and bone marrow samples. Results: 18 patients with RRMM were enrolled and underwent apheresis between September 15, 2020 and July 14, 2021. 12 patients have completed MCARH109 infusion to date, with 6 patients currently undergoing manufacturing and pending treatment. Of the 12 patients treated, median age was 59 (37-76) years and patients received a median of 8 (4-14) lines of therapy. 11 (92%) were penta-exposed, all patients were triple refractory, and 7 (58%) had prior treatment with BCMA targeted therapy including 6 (50%) who received prior BCMA CAR T therapy. 3 (25%) patients had non-secretory myeloma and 6(50%) patients had extramedullary plasmacytoma at baseline. 11 (92%) were refractory to last line of therapy and 11 (92%) patients received bridging therapy after apheresis prior to MCARH109 infusion; all patients were refractory to bridging therapy. There were no dose limiting toxicities. Cytokine release syndrome (CRS) grade 1-3 occurred in 11 (92%) patients with only one patient with grade 3 event; 4 (25%) patients received tocilizumab and 1 (8%) received dexamethasone for the treatment of CRS (Table). There were no neurologic toxicities reported to date; 3 (25%) patients had grade 1 nail changes possibly related to MCARH109 (Table). As of July 28, 2021, all treated patients have been followed for at least 2 weeks (median: 13.0 weeks; range: 2.0-39.1 weeks) and 10 (83%) had at least a minimal response or better (2 responses unconfirmed): 2 minimal response, 3 partial response, 3 very good partial response, 2 stringent complete response (sCR). 5 (56%) of the first 9 patients were minimal residual disease (MRD) negative in the bone marrow by multicolor flow cytometry (sensitivity: 10 -5). 6 (100%) patients with prior BCMA CAR T therapy had a response with 2 patients achieving sCR. We also noted robust MCARH109 expansion in the peripheral blood using qPCR across the first 3 dose levels with available data (peak expansion vector copy number/mL, median: 404,467; range: 44,670- 3,560,000; Table). With a median follow-up of 13 weeks, 9 (75%) patients are progression free and followed without additional therapy. Conclusions: MCARH109 is the first-in-class GPRC5D targeted CAR T cell therapy for MM and has a very manageable safety profile with no serious or unexpected toxicities; this dose escalation study is ongoing with additional patients planned for treatment at higher doses. Efficacy is promising in heavily pre-treated RRMM, reflected in high rates of clinical response as well as MRD-negativity, including at doses as low as 25x10 6 CAR T cells. Clinically important, all 6 patients who relapsed after BCMA CAR T therapy responded to GPRC5D targeted CAR T therapy, including 2 patients who achieved sCR. Figure 1 Figure 1. Disclosures Mailankody: Allogene Therapeutics: Research Funding; Physician Education Resource: Honoraria; Bristol Myers Squibb/Juno: Research Funding; Takeda Oncology: Research Funding; Fate Therapeutics: Research Funding; Jansen Oncology: Research Funding; Evicore: Consultancy; Legend Biotech: Consultancy; Plexus Communications: Honoraria. Shah: Janssen: Research Funding; Celgene/BMS: Research Funding. Lesokhin: pfizer: Consultancy, Research Funding; Iteos: Consultancy; Trillium Therapeutics: Consultancy; Genetech: Research Funding; Serametrix, Inc: Patents & Royalties; bristol myers squibb: Research Funding; Janssen: Honoraria, Research Funding; Behringer Ingelheim: Honoraria. Korde: Medimmune: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding. Hassoun: Celgene, Takeda, Janssen: Research Funding. Hultcrantz: GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Research Funding; Daiichi Sankyo: Research Funding; Intellisphere LLC: Consultancy; Curio Science LLC: Consultancy. Shah: Amgen: Research Funding; Janssen: Research Funding. Landau: Takeda, Janssen, Caelum Biosciences, Celgene, Pfizer, Genzyme: Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding; Genzyme: Honoraria. Scordo: Angiocrine Bioscience: Consultancy, Research Funding; Omeros Corporation: Consultancy; Kite - A Gilead Company: Membership on an entity's Board of Directors or advisory committees; i3 Health: Other: Speaker; McKinsey & Company: Consultancy. Roshal: Auron Therapeutics: Other: Ownership / Equity interests; Provision of services; Celgene: Other: Provision of services; Physicians' Education Resource: Other: Provision of services. Landgren: Janssen: Other: IDMC; Janssen: Honoraria; Janssen: Research Funding; Amgen: Honoraria; Celgene: Research Funding; Amgen: Research Funding; Takeda: Other: IDMC; GSK: Honoraria. Dogan: Physicians' Education Resource: Honoraria; Seattle Genetics: Consultancy; Peer View: Honoraria; Takeda: Consultancy, Research Funding; Roche: Consultancy, Research Funding; EUSA Pharma: Consultancy. Giralt: Actinnum: Membership on an entity's Board of Directors or advisory committees; JENSENN: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; AMGEN: Membership on an entity's Board of Directors or advisory committees; CELGENE: Membership on an entity's Board of Directors or advisory committees; PFIZER: Membership on an entity's Board of Directors or advisory committees; JAZZ: Membership on an entity's Board of Directors or advisory committees; SANOFI: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Park: Autolus: Consultancy; Kite Pharma: Consultancy; PrecisionBio: Consultancy; Minerva: Consultancy; Curocel: Consultancy; Intellia: Consultancy; Amgen: Consultancy; Affyimmune: Consultancy; Innate Pharma: Consultancy; Novartis: Consultancy; Servier: Consultancy; Kura Oncology: Consultancy; Artiva: Consultancy; BMS: Consultancy. Rivière: FloDesign Sonics: Other: Provision of Services; Juno Therapeutics: Patents & Royalties; The Georgia Tech Research Corporation (GTRC): Other: Provision of Services (uncompensated); Centre for Commercialization of Cancer Immunotherapy: Other: Provision of Services; Fate Therapeutics: Other: Provision of Services, Patents & Royalties. Brentjens: BMS: Consultancy, Patents & Royalties, Research Funding; Gracell Biotechnologies, Inc: Consultancy, Ended employment in the past 24 months; sanofi: Patents & Royalties; Caribou: Patents & Royalties. Smith: Eureka Therapeutics: Consultancy; Fate Therapeutics: Research Funding; Chimeric Therapeutics: Consultancy; Novarits: Consultancy; Sanofi: Patents & Royalties: GPRC5D antibody based therapies; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: CAR T cells for MM. OffLabel Disclosure: MCARH109 is an experimental GPRC5D targeted CART therapy

While recent progress has been made in the management of multiple myeloma, it remains a highly fatal malignancy especially among patients with relapsed-refractory disease. Immunotherapy with adoptive T cells targeting myeloma-associated antigens are at various stages of development and have brought about a new hope for cure. This is a review on the emerging field of adoptively transferred engineered T cell based approaches, with an in-depth focus on chimeric antigen receptors (CAR) targeting multiple myeloma. The recent results from CAR T cells targeting B cell maturation antigen are encouraging but eventual resistance to the CAR T cell therapies remain problematic. With newer approaches in therapies for multiple myeloma, the role of transplantation is evolved to form a platform for T cell therapies.

Background: Autologous CAR T cell therapy targeting the B-cell specific surface antigen CD19 has demonstrated favorable clinical responses in relapsed or refractory (R/B) B-cell lymphomas (BCL). However, despite 40-60% initial complete response (CR) rates, only a subset of patients experience durable remissions, and there is a need to further improve the efficacy of CAR therapies by preventing relapse and attaining a deeper CR. We hypothesized that the redundancy of CD28 and CD3V signaling in a CAR design incorporating all 3 CD3Vimmunoreceptor tyrosine-based activation motifs (ITAMs) might foster counterproductive T cell differentiation and exhaustion, and therefore created a new CD19 CAR construct with calibrated CAR activation potential by mutating 2 of the 3 ITAMs, termed 1XX. In systemic ALL mouse models, 19-28z1XX CAR induced effective tumor eradication at low CAR T cell doses with improved survival compared to conventional 19-28z CAR. Further preclinical studies demonstrated that the enhanced therapeutic benefit resulted from the reduced strength of activation mediated by the 19-28z1XX CAR, achieving a favorable balance of effector and memory functions, thereby enhancing persistence of functional CAR T cells and promoting effective elimination of CD19+ leukemia at lower T cell doses than needed with 19-28z CAR T cells (Feucht J et al. Nat Med 2019). To further improve the persistence of functional CAR T cells, we screened different humanized CD19-directed scFv in the context of a 19-28z1XX CAR design and proved high specificity and functionality of 19-28z1XX CARs containing a novel humanized scFv T2 - termed 19(T2)28z1XX. Study Design and Methods: This study is a single center Phase I clinical trial of 19(T2)28z1XX in patients with R/R B-cell malignancies at Memorial Sloan Kettering Cancer Center (NCT04464200). Key disease eligibility criteria include R/R diffuse large B cell lymphoma (DLBCL), high grade BCL, primary mediastinal BCL, indolent BCL and chronic lymphocytic leukemia (CLL). Patients with prior CD19 CAR therapies are eligible as long as expression of CD19 is confirmed. Key exclusion criteria include ongoing immunosuppression such as systemic GvHD therapy and active CNS disease. The study uses a 3+3 dose-escalation design to identify the maximum tolerated dose for BCL. There are 5 planned flat-dose levels. Patients will receive conditioning chemotherapy consisting of 3 days of fludarabine and cyclophosphamide followed by a single infusion of 19(T2)28z1XX CAR T cells. In the dose-escalation phase, patients with DLBCL, high grade BCL, and primary mediastinal BCL are eligible to participate. Once the recommended phase 2 dose (RP2D) is determined, the study will open to dose expansion phase with two cohorts. Cohort 1 includes DLBCL, high grade BCL and primary mediastinal BCL (i.e. same eligibility criteria as the dose-escalation phase). Cohort 2 will include patients with indolent BCL, CLL, and Richter's transformation. The dose-expansion part of the trial is designed to further characterize the safety, efficacy, and pharmacokinetics of 19(T2)28z1XX CAR in multiple indications. The primary objective of the trial is to evaluate safety and tolerability and determine the recommended Phase 2 dose of 19(T2)28z1XX. Key secondary objectives include evaluation of 19(T2)28z1XX's efficacy and cellular kinetics. Exploratory objectives include assessment of B cell aplasia, and analysis of serum cytokines. The trial has begun enrollment in August 2020. The investigators are hopeful this study will lead to development of improved CD19 CAR T cell therapy with enhanced efficacy and favorable toxicity profiles with lower infused T cell dose. Disclosures Park: AstraZeneca: Consultancy; Servier: Consultancy, Research Funding; Autolus: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Novartis: Consultancy; Minverva: Consultancy; Artiva: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Research Funding; Kite: Consultancy, Research Funding; Incyte: Consultancy, Research Funding; Genentech/Roche: Research Funding; Juno Therapeutics: Research Funding; GSK: Consultancy; Intellia: Consultancy; Allogene: Consultancy. Riviere:Fate Therapeutics Inc.: Consultancy, Other: Ownership interest , Research Funding; FloDesign Sonics: Consultancy, Other: Ownership interest; Juno Therapeutics: Other: Ownership interest, Research Funding; Takeda: Research Funding; Atara: Research Funding. Palomba:Genentech: Research Funding; Juno Therapeutics, a Bristol-Meyers Squibb Company: Honoraria, Research Funding; Regeneron: Research Funding; Novartis: Honoraria; Merck: Honoraria; Celgene: Honoraria; Pharmacyclics: Honoraria. Brentjens:BMS: Research Funding; Gracell Therapeutics: Consultancy; Juno Therapeutics (a Bristol Myers Squibb company): Patents & Royalties. Sadelain:Atara: Patents & Royalties, Research Funding; Fate Therapeutics: Patents & Royalties, Research Funding; Minerva: Other: Biotechnologies , Patents & Royalties; Mnemo: Patents & Royalties; Takeda: Patents & Royalties, Research Funding. OffLabel Disclosure: Cyclophosphamide and fludarabine will be used as conditioning therapy prior to 19(T2)28z1XX CAR T cell administration.

T-cell receptor (TCR)–modified T-cell gene therapy can target a variety of extracellular and intracellular tumor-associated antigens, yet has had little clinical success. A potential explanation for limited antitumor efficacy is a lack of T-cell activation in vivo. We postulated that expression of proinflammatory cytokines in TCR-modified T cells would activate T cells and enhance antitumor efficacy. We demonstrate that expression of interleukin 18 (IL18) in tumor-directed TCR-modified T cells provides a superior proinflammatory signal than expression of interleukin 12 (IL12). Tumor-targeted T cells secreting IL18 promote persistent and functional effector T cells and a proinflammatory tumor microenvironment. Together, these effects augmented overall survival of mice in the pmel-1 syngeneic tumor model. When combined with sublethal irradiation, IL18-secreting pmel-1 T cells were able to eradicate tumors, whereas IL12-secreting pmel-1 T cells caused toxicity in mice through excessive cytokine secretion. In another xenograft tumor model, IL18 secretion enhanced the persistence and antitumor efficacy of NY-ESO-1–reactive TCR-modified human T cells as well as overall survival of tumor-bearing mice. These results demonstrate a rationale for optimizing the efficacy of TCR-modified T-cell cancer therapy through expression of IL18. See related commentary by Wijewarnasuriya et al., p. 732

Although clinical responses with CD19-targeting chimeric antigen receptor (CAR) T-cell treatment have been observed in patients with certain hematologic malignancies, high rates of disease relapse highlight the necessity to understand and improve mechanisms of CAR T-cell failure. Because T-cell dysfunction is thought to contribute to CAR T-cell treatment failure, understanding what mechanisms drive T cells into this dysfunctional state may aid optimal design of efficacious CAR T cells. Dysfunctional CAR T cells have been characterized as having upregulated inhibitory receptors and decreased cytolytic capabilities. Previous studies have identified a role for sustained CAR CD3ζ signaling in CAR T-cell dysfunction. Here, we demonstrate a mechanism that drives dysfunction in CAR T cells through excessive costimulation. Fully activated CD19-targeted CAR T cells were rendered dysfunctional upon stimulation with both endogenous CD28 stimulation and CAR-mediated CD28 costimulation. Costimulation-driven dysfunction of CAR T cells was demonstrated in a syngeneic immunocompetent mouse model, in which CAR T cells were activated with signals 1 (CD3ζ), 2 (CD28), and 3 (IL12). Thus, we show that CAR T-cell dysfunction can be driven through excessive CD28 and 4-1BB costimulation. See related article by Drakes et al., p. 743

Multiple myeloma (MM) remains generally incurable, calling for the development of novel treatment strategies such as chimeric antigen receptor (CAR) T cell therapy. Most clinically tested CAR T cell therapies for MM target B cell maturation antigen (BCMA), but despite high response rates, many patients relapse (Raje N. NEJM 2019). BCMA negative-low MM cells are implicated as a reservoir preceding relapse (Brudno J. JCO 2018; Cohen A. JCI 2019). Our aims are to (1) evaluate whether upfront simultaneous targeting of an additional antigen such as G protein-coupled receptor class C group 5 member D (GPRC5D; Smith EL. Sci Trans Med 2019) can mitigate BCMA escape-mediated relapse in MM, and (2) compare dual targeting strategies to identify an optimal approach. Dual targeting for CD19/CD22 malignancies has been investigated, and multiple approaches are feasible; however, approaches have yet to be comprehensively compared head to head. Here, we compare 2 parallel production and 3 single-vector dual targeting strategies (Fig. 1A). To enhance clinical translatability, all strategies are built on the BCMA(125)/4-1BBζ CAR (BCMA scFv 125; Smith EL. Mol Ther 2018), which is currently under multi-center clinical investigation (NCT03430011; Mailankody S. ASH 2018). We confirmed that all dual targeted approaches lyse, proliferate, and secrete polyfunctional cytokines specifically in response to BCMA and GPRC5D mono- and dual-positive cell lines and/or primary patient MM aspirate samples. Activity in vivo was confirmed using the bone marrow-tropic OPM2 MM model (endogenously BCMA+GPRC5D+). In all experiments MM cells (2 x 106) were injected IV into NSG mice and engrafted/expanded for 14 days before treatment. A high dose of all dual targeted CAR T cell approaches (3 x 106 CAR+) induced long-term disease control (median overall survival (mOS) BCMA(125) non-signaling del control 32d vs other groups mOS not reached; p < 0.05). Prevention of latent BCMA escape-mediated relapse was evaluated by re-challenge of previously treated long-surviving mice with 2 x 106 OPM2 BCMA CRISPR KO (OPM2BCMA KO) cells at day 100 without re-treatment. While mice previously treated with BCMA(125)/41BBζ CAR T cells succumbed to OPM2BCMA KO disease, dual targeted approaches prevented OPM2BCMA KO growth (mOS BCMA mono-targeted arm 37d post re-challenge vs other groups mOS not reached; p < 0.05). To better recapitulate human MM and distinguish among dual targeting approaches, we modeled established BCMA heterogeneous disease by spiking 5-10% OPM2BCMA KO into bulk OPM2WT cells for injection. Each OPM2 population was modified to express distinct luciferases for simultaneous in vivo monitoring by bioluminescent imaging (BLI). Treatment with a moderate (5 x 105) dose of CAR T cells eradicated OPM2WT cells in all groups, but anti-GPRC5D CARs with CD28 co-stimulation, whether included within a mixed T cell population or in a bicistronic construct (Fig. 1A ii, iv), failed to control OPM2BCMA KO cells (Fig. 1B). Correspondingly, 4-1BB-only containing CAR T cells had increased in vivo expansion (2.1-4.1-fold increase CAR T cell BLI at day 7 over CD28 containing groups; p < 0.05). As this result is likely from greater activation-induced cell death in the CD28-containing approaches that was not rescued by 4-1BB, we later compared 4-1BB-only containing approaches (Fig. 1A i, iii, v). These 3 dual targeting approaches effectively controlled OPM2WT disease at moderate (1 x 106 CAR+) and low (2.5 x 105 CAR+) doses. However, when using a sub-therapeutic dose (2.5 x 105 CAR+) in the OPM2BCMA KO-spiked model, the tandem scFv-single stalk design was least effective in controlling OPM2BCMA KO disease (Fig 1C). At a dose that is sub-therapeutic to control OPM2WT disease (1 x 105 CAR+), the bicistronic dual 4-1BB design (Fig. 1A iii) was more effective in eradicating tumor compared with the parallel production approach (6-fold difference tumor BLI at day 28; p < 0.05). These results indicate that upfront dual targeting of BCMA/GPRC5D with CAR T cells can mitigate BCMA escape-mediated relapse in a model of MM. While parallel infusion of separate BCMA- and GPRC5D-targeted CAR T cells is effective, a single bicistronic vector encoding two 4-1BB-containing CARs avoids the practical challenges of parallel manufacturing, and uniquely may provide superior anti-MM efficacy. Figure Disclosures Fernandez de Larrea: Takeda: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding. Brentjens:JUNO Therapeutics: Consultancy, Patents & Royalties, Research Funding; Celgene: Consultancy. Smith:Celgene: Consultancy, Patents & Royalties, Research Funding; Fate Therapeutics and Precision Biosciences: Consultancy.

We present a case of a patient with multiply relapsed, refractory myeloma whose clinical course showed evidence of a synergistic abscopal-like response to chimeric antigen receptor (CAR) T-cell therapy and localized radiotherapy (XRT). Shortly after receiving B-cell maturation antigen (BCMA)–targeted CAR T-cell therapy, the patient required urgent high-dose steroids and XRT for spinal cord compression. Despite the steroids, the patient had a durable systemic response that could not be attributed to XRT alone. Post-XRT findings included a second wave of fever and increased CRP and IL6, beginning 21 days after CAR T cells, which is late for cytokine-release syndrome from CAR T-cell therapy alone on this trial. Given this response, which resembled cytokine-release syndrome, immediately following XRT, we investigated changes in the patient's T-cell receptor (TCR) repertoire over 10 serial time points. Comparing T-cell diversity via Morisita's overlap indices (CD), we discovered that, although the diversity was initially stable after CAR T-cell therapy compared with baseline (CD = 0.89–0.97, baseline vs. 4 time points after CAR T cells), T-cell diversity changed after the conclusion of XRT, with >30% newly expanded TCRs (CD = 0.56–0.69, baseline vs. 4 time points after XRT). These findings suggest potential synergy between radiation and CAR T-cell therapies resulting in an abscopal-like response.

CD19-directed chimeric antigen receptor (CAR) T cells are clinically effective in a limited set of leukemia patients. However, CAR T-cell therapy thus far has been largely restricted to targeting extracellular tumor-associated antigens (TAA). Herein, we report a T-cell receptor-mimic (TCRm) CAR, termed WT1-28z, that is reactive to a peptide portion of the intracellular onco-protein Wilms Tumor 1(WT1), as it is expressed on the surface of the tumor cell in the context of HLA-A*02:01. T cells modified to express WT1-28z specifically targeted and lysed HLA-A*02:01+ WT1+ tumors and enhanced survival of mice engrafted with HLA-A*02:01+, WT1+ leukemia or ovarian tumors. This in vivo functional validation of TCRm CAR T cells provides the proof-of-concept necessary to expand the range of TAA that can be effectively targeted for immunotherapy to include attractive intracellular targets, and may hold great potential to expand on the success of CAR T-cell therapy.

The past several years have been marked by extraordinary advances in clinical applications of immunotherapy. In particular, adoptive cellular therapy utilizing chimeric antigen receptor (CAR) modified T cells targeted to CD19 has demonstrated substantial clinical efficacy in children and adults with relapsed or refractory B cell acute lymphoblastic leukemia (B-ALL), and durable clinical benefit in a smaller subset of patients with relapsed or refractory chronic lymphocytic leukemia (CLL) or B cell non-Hodgkin lymphoma (B-NHL). Early phase clinical trials are presently assessing CAR T cell safety and efficacy in additional malignancies. Herein, we discuss clinical results from the largest series to date investigating CD19-targeted CAR T cells in B-ALL, CLL, and B-NHL, including discussion of differences in CAR T cell design and production and treatment approach, as well as clinical efficacy, nature of severe cytokine release syndrome and neurologic toxicities, and CAR T cell expansion and persistence. We additionally review the current and forthcoming use of CAR T cells in multiple myeloma and several solid tumors, and highlight challenges and opportunities afforded by the current state of CAR T cell therapies, including strategies to overcome inhibitory aspects of the tumor microenvironment and enhance antitumor efficacy.

The chimeric antigen receptor (CAR) represents the epitome of cellular engineering and is one of the best examples of rational biologic design of a synthetic molecule. The CAR is a single polypeptide with modular domains, consisting of an antibody-derived targeting moiety, fused in line with T cell-derived signaling domains, allowing for T cell activation upon ligand binding. T cells expressing a CAR are able to eradicate selectively antigen-expressing tumor cells in a MHC-independent fashion. CD19, a tumor-associated antigen (TAA) present on normal B cells, as well as most B cell-derived malignancies, was an early target of this technology. Through years of experimental refinement and preclinical optimization, autologously derived CD19-targeting CAR T cells have been successfully, clinically deployed, resulting in dramatic and durable antitumor responses but not without therapy-associated toxicity. As CD19-targeted CAR T cells continue to show clinical success, work at the bench continues to be undertaken to increase further the efficacy of this therapy, while simultaneously minimizing the risk for treatment-related morbidities. In this review, we cover the history and evolution of CAR technology and its adaptation to targeting CD19. Furthermore, we discuss the future of CAR T cell therapy and the need to ask, as well as answer, critical questions as this treatment modality is being translated to the clinic.

Significant progress has been made in the field of cancer cell therapy over the last 2 decades. Specifically, a patient's own T cells may be genetically engineered to express an artificial T-cell receptor, termed a chimeric antigen receptor (CAR), which, when expressed in a T cell, can alter the specificity of the T cell. A CAR consists of a binding domain, typically derived from a monoclonal antibody, fused to T-cell signaling domains including a co-stimulatory domain, CD28 or 4-1BB, fused to a distal CD3 zeta chain cytoplasmic signaling domain. CAR T cells targeted to the CD19 antigen, expressed on most B-cell malignancies, have demonstrated the greatest clinical efficacy, and commercial CAR T-cell products have recently been FDA approved in the treatment of relapsed and refractory B-cell lymphomas as well as B-cell acute lymphoblastic leukemias. Similarly, CAR T cells targeting the BCMA antigen, expressed on most multiple myeloma tumor cells, have demonstrated impressive antitumor responses in relapsed and refractory disease in both early-phase clinical trials as well as ongoing registration trials. While response rates in the context of CAR T cells targeted to CD19 and BCMA have been impressive, unfortunately the durability of response in some patients is limited and others fail to respond to treatment entirely. The complete list of etiologies responsible for treatment failures remains to be fully defined. Likely causes for treatment failures include the loss of target antigen expression by the tumor, limited persistence of CAR T cells in vivo, and suppression of CAR T cells in the context of an immune-suppressive tumor microenvironment. While this list is likely incomplete, investigators in the CAR T cell field are working on designing next-generation CAR T cells that could address these current limitations. Armored CAR T cells are CAR T cells further modified to enhance persistence and cytotoxicity. Specifically, CAR T cells may be modified to express or secrete biologically active cytokines or ligands designed to modulate the tumor microenvironment and recruit endogenous antitumor immune effectors to enhance the antitumor immune response. Examples of armored CAR T cells include cytokine-secreting CAR T cells (IL-12, IL-18), co-stimulatory ligand-expressing CAR T cells (4-1BBL, CD40L), CAR T cells that secrete single-fragment-length antibodies (scFvs) targeted to immune checkpoint receptors (PD-1), and CAR T cells that express dominant negative chimeric receptors (Fas, TGF-B). Preclinical studies in the context of these armored CAR T cells have demonstrated markedly enhanced antitumor activity in mice when compared to T cells modified to express the CAR alone. Outcomes of planned and ongoing early-stage clinical trials are pending but may markedly change the field in the foreseeable future. Citation Format: Renier J. Brentjens. CARs and armored CARs: Improving CAR T-cell therapy for cancer [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr IA15.

Despite recent setbacks in the clinical development of immunotherapy for gynecologic malignancies, the promise of durable responses with this form of treatment has led to a redoubling of our efforts to understand and develop new immunotherapeutic modalities. Some of the reasons behind the relative lack of success of immunotherapy in ovarian cancer include low tumor antigenic burden; a hostile tumor microenvironment (TME) consisting of physical barriers to immune infiltration; suppressive ligands, cytokines, chemokines, and other immunosuppressive cellular elements. There are ongoing efforts to overcome many of these limitations simultaneously. For instance, several clinical trials are exploring the combination of immune checkpoint blockade with chemotherapy, poly (ADP-ribose) polymerase (PARP) inhibitors, or both in an attempt to increase tumor antigenic burden. Other approaches are centered around targeting known tumor-associated antigens such as MUC16. MUC16 is amplified in 5-10% of ovarian cancers. The predominant extracellular portion of MUC16 is cleaved and secreted (referred to as CA-125), although a significant portion of the antigen is retained on the surface of the tumor (referred to as MUC16ecto). We have developed a variety of MUC16ecto- directed therapies, including chimeric antigen receptor (CAR) T-cells and bispecific T-cell engager cells (BiTEs). Targeting MUC16ecto solves the problem of low tumor mutational burden in ovarian cancer, but further optimizations are required to overcome the immunosuppressive TME. For our CAR T-cell therapy, we devised and evaluated three separate approaches to overcome the TME in ovarian cancer. MUC16ecto-directed CAR T-cells modified to secrete interleukin-12 (IL-12) showed superior efficacy in advanced syngeneic and xenograft murine models of metastatic ovarian cancer. We found that autocrine and paracrine activity of IL-12 potentiated the activity of these CAR T-cells and mediated resistance to PD-L1 suppression. Furthermore, we found that secretion of IL-12 in the TME promoted differentiation of proinflammatory tumor-associated macrophages and led to the deletion of other suppressive macrophages in the TME. Similarly, MUC16ecto-directed CAR T-cells modified to secrete interleukin-18 (IL-18) significantly improved survival in low and high tumor burden syngeneic models of metastatic ovarian cancer. We also evaluated CAR T-cells engineered to secrete anti-PD-1 blocking single-chain variable fragments (scFv) and showed that this approach significantly improved efficacy in relevant preclinical models of metastatic ovarian cancer. Furthermore, this approach fostered CAR T-cell persistence leading to resistance to tumor rechallenge. Unsurprisingly, using CAR T-cells as a delivery vehicle for delivery of immune checkpoint blockade promoted bystander effects on endogenous immune cells and mitigated systemic anti-PD-1 leak. We next evaluated MUC16ecto-specific BITEs and found a significant reduction in tumor burden in xenogeneic models of metastatic ovarian cancer. Adjunctive therapies to augment our MUC16ecto-specific BiTEs are currently in testing, along with other MUC16-directed immunotherapeutic strategies. Citation Format: Oladapo O. Yeku, Sarwish Rafiq, Mythili Koneru, Terence J. Purdon, Mei Song, Pei Wang, Ronald Hendrickson, Renier J. Brentjens, David Spriggs. MUC16-directed immunotherapy for ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr IA21.

Summary Chimeric antigen receptor (CAR) T cells provide great efficacy in B cell malignancies. However, improved CAR T cell therapies are still needed. Here, we engineered tumor-targeted CAR T cells to constitutively express the immune-stimulatory molecule CD40 ligand (CD40L) and explored efficacy in different mouse leukemia/lymphoma models. We observed that CD40L+ CAR T cells circumvent tumor immune escape via antigen loss through CD40/CD40L-mediated cytotoxicity and induction of a sustained, endogenous immune response. After adoptive cell transfer, the CD40L+ CAR T cells displayed superior antitumor efficacy, licensed antigen-presenting cells, enhanced recruitment of immune effectors, and mobilized endogenous tumor-recognizing T cells. These effects were absent in Cd40−/− mice and provide a rationale for the use of CD40L+ CAR T cells in cancer treatment.

8062 Background: Immunotherapy with anti CD20 is often associated with mild easily manageable infusion reactions. In rare cases, patients experience severe drug hypersensitivity reactions (DHR) serum sickness or anaphylaxis. These in turn may lead to discontinuation of the drug. In our experience, switching to a different anti-CD20 agent is a feasible alternative to discontinuation or desensitization protocols. Methods: From our pharmacology database we identified all the patients that received rituximab and/or obinutuzumab, and/or ofatumumab, and/or all the patients who received a flat dose of less than 50 mL of the same drugs and were followed at our institution. From the medical record, we identified all the cases where the anti-CD20 antibody was changed due to allergy, serum sickness or other types of DHR, and all those who received minimal doses of anti-CD20 in the context of a desensitization protocol. DHRs were evaluated either by an allergist, or by retrospective review following the World Allergy Organization guidelines. Our primary comparison, was to assess the proportion of pts able to completed planned infusion of abs using either approach (Fisher’s exact Test). Results: Among 343 patients receiving at least two different anti-CD20 antibodies or a flat dose of < 50 mL, we identified 44 patients experiencing severe DHRs needing intervention. At the time of the reaction, 16 (36%) received the anti-CD20 as single agent, 24 (54%) in combination with chemotherapy, 4 (9%) in combination with ibrutinib or lenalidomide. In 9 (20%) patients the reaction was defined as anaphylactoid (8 rituximab; 1 obinutuzumab) and in 8 (18%) patients, all receiving rituximab, as serum sickness. Episodes of DHR were addressed with either desensitization (n = 29) or change of anti-CD20 agent (n = 25), 9 patients received both of these approaches, one patient switched anti-CD20 antibodies twice. Overall, 21 desensitizations were successful (72.4%), 8 failed; 23 changes of anti-CD20 were successful (92%) and 2 failed (p = 0.09). Conclusions: In patients with DHR use of an alternative anti-CD20 antibody is safe and is an alternative or complementary approach to anti-CD20 desensitization.

CAR T cell therapy of cancers promises to revolutionize oncology by harnessing the powers of synthetic biology and immunotherapy in a single agent. CARs are synthetic receptors composed of an extracellular antigen binding domain and one or more intracellular signaling domains which act in concert to activate the T cell upon antigen recognition. CARs targeting B cell associated CD19 demonstrated robust in vivo cytolytic activity, expansion, and persistence upon antigen exposure paving the way for clinical application of this technology and ultimately FDA approval for pediatric and young adult acute lymphoblastic leukemia as well as patients with relapsed or refractory diffuse large B cell lymphoma. However, these successes have not yet been replicated in the arena of solid tumors. Unlike hematologic malignancies, solid tumors present numerous challenges in the form of an immunosuppressive tumor microenvironment. In this chapter, we will highlight clinical application of CAR T cells in solid tumors, discuss hurdles that have impeded CAR T cell function in these malignancies, and propose methods to overcome these limitations.

Clinical trials utilizing T cells genetically engineered to express a tumor-targeted T-cell receptor (TCR) illustrate the promise of this therapy in treating cancer patients. However, there are obstacles that must be overcome to improve the efficacy of TCR gene therapy. Two separate signals are needed to completely activate T cells and drive T-cell proliferation and differentiation. Co-stimulatory molecules present on the surface of cancer cells can become downregulated as an escape mechanism, thus depriving the T cells of complete activation and leading to T-cell apoptosis or anergy. This project aims to armor tumor-associated antigen-specific T cells with proinflammatory cytokines, specifically IL-12 and IL-18. We hypothesize that this genetic modification will provide an alternate form of co-stimulation that will augment antitumor efficacy. Tumor-associated antigen specific pmel-1 T cells transduced with retroviral constructs were shown to be more functional through increased secretion of IFNγ when co-cultured with antigen-positive tumor cells. Additionally, the armored pmel T cells were able to significantly enhance lysis of the B16F10 melanoma cell line in vitro. Subsequently, the in vivo efficacy of the armored T cells was assessed in a syngeneic, immune-competent B16F10 melanoma mouse model. The armored pmel T cells were confirmed to significantly delay the B16F10 tumor progression and enhance the survival of these mice in comparison to control pmel T cells. These results provide future insight into applying the concept of armored TCR T cells to a therapeutically relevant human tumor target to enhance TCR-modified T-cell antitumor responses. Citation Format: Dylan J. Drakes, Sarwish Rafiq, Terence Purdon, Renier Brentjens. Engineering armored TCR-modified T cells to enhance anti-tumor efficacy [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 A37.

MUC16, a subunit of CA125 that is overexpressed on epithelial ovarian cancers, can be targeted by T cells via the incorporation of the 4h11m28m chimeric antigen receptor (CAR) into the T-cell surface. However, the microenvironment of ovarian cancers consists of regulatory T cells, myeloid-derived suppressor cells, and tumor-associated macrophages that act to immunosuppress CAR T-cell function, thus abrogating CAR T-cell efficacy. To overcome this immunosuppression, our lab has developed IL-12 and IL-18 secreting CAR T cells, which have shown enhanced efficacy in a syngeneic ovarian cancer model when compared to second-generation CAR T cells. While IL-18 secreting CAR T cells have proven to be superior to IL-12 secreting CAR T cells in various leukemia and lymphoma mouse models, this observation does not translate to the ovarian cancer model. Mechanistic studies into the effects of IL-18 and IL-12 in a syngeneic ovarian cancer model are currently under way in order to better understand what each cytokine is doing to the tumor microenvironment as well as to the CAR T cell. With better understanding of the effects of IL-18 and IL-12, we can contribute to the immunobiology of these cytokines as well as harness their efficacy to apply to other tumor models. Citation Format: Christina E. Bebernitz, Renier J. Brentjens. Comparing IL-12 and IL-18 secreting CAR T-cell efficacy [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 A31.

Within the past several years, renewed interest in immunotherapy has been observed in multiple fields of oncology, including antibody-based therapeutics (e.g., checkpoint blockade) and adoptive cellular therapies. In the field of adult leukemias, such interest has been driven by the limitations of presently available therapies to induce durable remissions reliably in patients with relapsed and refractory leukemia. The adoptive transfer of genetically modified autologous T-cells aims to rapidly establish specific antitumor activity. This strategy requires targeting of autologous T-cells by means of a transgene-encoded antigen receptor, consisting of a chimeric antigen receptor (CAR), as will be discussed herein, or T-cell receptor (TCR) chains. CD19 is nearly universally expressed by B-ALL, CLL, and hairy cell leukemia, while not expressed on normal tissues other than B-cells, including multipotent hematopoietic progenitor cells. Multiple generations of CARs have been developed and investigated in clinical studies and will be discussed in this review. In this chapter, we review clinical outcomes of adults with leukemia treated with CAR T-cells, toxicities associated with CAR T-cell administration, present challenges limiting therapeutic efficacy, and future directions, including novel targets and enhancements to improve antileukemic activity.

T cells can be genetically modified to target tumors through the expression of a chimeric antigen receptor (CAR). The clinical benefit of CAR T cells has now been reported by several groups targeting the CD19 antigen in patients with hematologic malignancies. However, in solid tumors, CAR T cells have been less effective at inducing complete tumor responses and have mediated a high rate of on-target/ off-tumor toxicity. To understand this discrepancy, we must understand the design and implementation of CAR T cells for cancer immunotherapy. The basic design of CAR T cells consists of two fundamental domains: the antigen-binding portion (commonly composed of a single-chain variable fragment [scFv] derived from a monoclonal antibody [mAb]) joined to one or more intracellular T-cell signaling domains. To be effective after infusion, CAR T cells must expand, persist, exhibit enduring antitumor cytotoxicity, withstand and/or counteract an immunosuppressive tumor microenvironment, and overcome targeted tumor antigen escape. In designing CAR T cells for cancer immunotherapy, all of these factors must be harmonized to generate the optimal CAR T cell.

Adoptive cell therapy with genetically modified T cells expressing a chimeric antigen receptor (CAR) is a promising therapy for patients with B-cell acute lymphoblastic leukemia. However, CAR-modified T cells (CAR T cells) have mostly failed in patients with solid tumors or low-grade B-cell malignancies including chronic lymphocytic leukemia with bulky lymph node involvement. Herein, we enhance the antitumor efficacy of CAR T cells through the constitutive expression of CD40 ligand (CD40L, CD154). T cells genetically modified to constitutively express CD40L (CD40L-modified T cells) demonstrated increased proliferation and secretion of proinflammatory TH1 cytokines. Further, CD40L-modified T cells augmented the immunogenicity of CD40(+) tumor cells by the upregulated surface expression of costimulatory molecules (CD80 and CD86), adhesion molecules (CD54, CD58, and CD70), human leukocyte antigen (HLA) molecules (Class I and HLA-DR), and the Fas-death receptor (CD95). Additionally, CD40L-modified T cells induced maturation and secretion of the proinflammatory cytokine interleukin-12 by monocyte-derived dendritic cells. Finally, tumor-targeted CD19-specific CAR/CD40L T cells exhibited increased cytotoxicity against CD40(+) tumors and extended the survival of tumor-bearing mice in a xenotransplant model of CD19(+) systemic lymphoma. This preclinical data supports the clinical application of CAR T cells additionally modified to constitutively express CD40L with anticipated enhanced antitumor efficacy.

B cell maturation antigen (BCMA) has recently been identified as an important multiple myeloma (MM)-specific target for chimeric antigen receptor (CAR) T cell therapy. In CAR T cell therapy targeting CD19 for lymphoma, host immune anti-murine CAR responses limited the efficacy of repeat dosing and possibly long-term persistence. This clinically relevant concern can be addressed by generating a CAR incorporating a human single-chain variable fragment (scFv). We screened a human B cell-derived scFv phage display library and identified a panel of BCMA-specific clones from which human CARs were engineered. Despite a narrow range of affinity for BCMA, dramatic differences in CAR T cell expansion were observed between unique scFvs in a repeat antigen stimulation assay. These results were confirmed by screening in a MM xenograft model, where only the top preforming CARs from the repeat antigen stimulation assay eradicated disease and prolonged survival. The results of this screening identified a highly effective CAR T cell therapy with properties, including rapid in vivo expansion (>10,000-fold, day 6), eradication of large tumor burden, and durable protection to tumor re-challenge. We generated a bicistronic construct including a second-generation CAR and a truncated-epithelial growth factor receptor marker. CAR T cell vectors stemming from this work are under clinical investigation.

Chimeric antigen receptors (CARs) are recombinant receptors composed of an antibody-derived extracellular single-chain variable fragment linked to a costimulatory signaling domain combined with the...

SUMMARY Chimeric antigen receptor (CAR) T cell therapy has proven clinically beneficial against B cell acute lymphoblastic leukemia and non-Hodgkin’s lymphoma. However, suboptimal clinical outcomes have been associated with decreased expansion and persistence of adoptively transferred CAR T cells, antigen-negative relapses, and impairment by an immunosuppressive tumor microenvironment. Improvements in CAR T cell design are required to enhance clinical efficacy, as well as broaden the applicability of this technology. Here, we demonstrate that interleukin-18 (IL-18)-secreting CAR T cells exhibit enhanced in vivo expansion and persistence and significantly increase long-term survival in syngeneic mouse models of both hematological and solid malignancies. In addition, we demonstrate that IL-18-secreting CAR T cells are capable of modulating the tumor microenvironment, as well as enhancing an effective endogenous anti-tumor immune response. IL-18-secreting CART cells represent a promising strategy to enhance the clinical outcomes of adoptive T cell therapy., Graphical abstract In Brief Avanzi et al. generate CAR T cells that secrete IL-18 and show improved activity in syngeneic hematologic and solid tumor models without prior preconditioning. They further show enhanced recruitment and anti-tumor activity of endogenous T cells.

Chimeric antigen receptor (CAR) T cell therapy has shown limited efficacy for the management of solid tumor malignancies. In ovarian cancer, this is in part due to an immunosuppressive cytokine and cellular tumor microenvironment which suppresses adoptively transferred T cells. We engineered an armored CAR T cell capable of constitutive secretion of IL-12, and delineate the mechanisms via which these CAR T cells overcome a hostile tumor microenvironment. In this report, we demonstrate enhanced proliferation, decreased apoptosis and increased cytotoxicity in the presence of immunosuppressive ascites. In vivo, we show enhanced expansion and CAR T cell antitumor efficacy, culminating in improvement in survival in a syngeneic model of ovarian peritoneal carcinomatosis. Armored CAR T cells mediated depletion of tumor associated macrophages and resisted endogenous PD-L1-induced inhibition. These findings highlight the role of the inhibitory microenvironment and how CAR T cells can be further engineered to maintain efficacy.

Adoptive cellular therapy (ACT) is now considered a bona fide treatment modality within the evolving field of anticancer immunotherapy. Great advances have enabled the adoptive transfer of tumour-selective lymphocytes for the treatment of a variety of malignancies. Unfortunately, this selectivity has led to the emergence of antigen-loss variants. New strategies need to be employed to minimize the incidence of this phenomenon, enabling the full potential of ACT to be realized.

Acquired resistance to CD19-targeted CAR T cell therapy can occur through mutations in the CD19 gene or after insertion of a chimeric antigen receptor (CAR) into leukemia cells, resulting in the adjacent receptor being masked by the CAR.

2520 Background: Autologous chimeric antigen receptor (CAR) T cell therapy has shown to be effective in CD19+ relapsed or refractory (R/R) B-ALL but requires a 2-4 week period of cell processing and manufacture. During this “bridging period,” patients are vulnerable to disease progression and complications. We sought to characterize bridging strategies in our published study of CD19 CAR T cell therapy in adults with R/R ALL (Park et al., NEJM, 2018). Methods: We performed a retrospective review of adult patients with R/R ALL treated with 19-28z CAR T therapy at MSKCC. Bridging therapy was defined as any therapy given from trial enrollment to cell infusion and classified as either high intensity (remission-inducing or myelosuppressive regimens, eg hyper-CVAD or high-dose cytarabine based regimens) or low intensity (maintenance and/or less myelosuppressive regimens, eg POMP, Blinatumomab, TKI). Results: Of 53 patients who received CAR T cell infusion, 19 were bridged with a high intensity regimen and 34 with a low intensity regimen. There was no difference in number of prior therapies, pre-bridging chemotherapy disease burden, and prior transplant status between groups. High intensity therapy was associated with a higher rate of Gr3-4 infectious complications during the bridging period (78% vs 32%, p < 0.002 by Fisher’s Exact) but not with response to bridging or CAR T cell therapy, relapse free survival, post-CAR Gr3-4 cytokine release syndrome (CRS) or neurotoxicity (NT). Patients in both groups who converted from morphologic to molecular disease during bridging (n=9) had a decreased rate of eventual severe CRS (0% vs 41%, p=0.01) or NT (0% vs 55%, p 0.05) or a combined endpoint of CAR T cell infusion or alternative therapy including transplant (80% vs 86%, p> 0.05). Conclusions: High intensity bridging therapy is associated with a high risk of infectious complications without a clear benefit in outcome in R/R ALL receiving CD19 CAR T cells. Selection of bridging regimen therefore requires consideration of previous treatments and patient status to maximize the efficacy and safety. Clinical trial information: NCT01044069.

2511 Background: We conducted a phase I dose escalation trial of first-in-human autologous chimeric antigen receptor (CAR) T-cell immunotherapy targeting mesothelin (MSLN), a cell-surface antigen that is highly expressed in pleural cancers- malignant pleural mesothelioma (MPM) and metastatic lung and breast cancers. Methods: A single dose of CD28-costimulated MSLN CAR T cells with the I-caspase-9 safety gene was administered intrapleurally in patients with MSLN-expressing pleural tumors. Following a 3+3 design, patients were treated in dose escalating cohorts (dose range 3E5 to 1E7 CAR T cells/kg) following IV cyclophosphamide lymphodepletion (first 3 patients did not receive cyclophosphamide). A subset of MPM patients received subsequent anti-PD-1 therapy, off-protocol, which we have shown to prolong CAR T-cell functional persistence in preclinical models. Results: Twenty patients (18 MPM, 1 lung cancer, 1 breast cancer) were treated (prior lines of therapy 1–8, 35% received ≥3 lines of therapy). No CAR T-cell–related toxicities higher than grade 1 were observed. Intense monitoring for on-target, off-tumor toxicity by clinical (chest or abdominal pain), radiological (CT/PET or echocardiogram for pericardial effusion, ascites), laboratory (troponin elevation), and EKG evaluation found no evidence of toxicity. Fourteen MPM patients received subsequent anti-PD1 therapy (1–21 cycles, pretreatment tumor PD-L1 < 10% in all patients except one), with 1 patient developing grade 3 pneumonitis that responded to steroid treatment. CAR T cells were detected in the peripheral blood of 13 of 14 patients (1-39 weeks). At data cut-off date (Jan 31, 2019), among 14 MPM patients that received combination therapy (follow-up 13-77 weeks, median 31 weeks), best responses included 2 patients with complete metabolic response on PET (62 and 39 weeks ongoing); 5 partial responses and 4 stable disease by investigator assessment. Conclusions: Intrapleurally administered MSLN-targeted CAR T cells were safe. Encouraging antitumor activity of MSLN-targeted CAR T-cell therapy was observed when combined with anti-PD1 therapy and shows promise for future development of this approach. Clinical trial information: NCT02414269.

The genetic modification of autologous T cells with chimeric antigen receptors (CARs) represents a breakthrough for gene engineering as a cancer therapy for hematologic malignancies. By targeting the CD19 antigen, we have demonstrated robust and rapid anti-leukemia activity in patients with heavily pre-treated and chemotherapy-refractory B cell acute lymphoblastic leukemia (B-ALL). We demonstrated rapid induction of deep molecular remissions in adults, which has been recently confirmed in a case report involving a child with B-ALL. In contrast to the results when treating B-ALL, outcomes have been more modest in patients with chronic lymphocytic leukemia (CLL) or other non-hodgkin's lymphoma (NHL). We review the clinical trial experience targeting B-ALL and CLL and speculate on the possible reasons for the different outcomes and propose potential optimization to CAR T cell therapy when targeting CLL or other indolent NHL. Lastly, we discuss the pre-clinical development and potential for clinical translation for using CAR T cells against multiple myeloma and acute myeloid leukemia. We highlight the potential risks and benefits by targeting these poor outcome hematologic malignancies.