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The production of autologous T cells expressing a chimaeric antigen receptor (CAR) is time-consuming, costly and occasionally unsuccessful. T-cell-derived induced pluripotent stem cells (TiPS) are a promising source for the generation of ‘off-the-shelf’ CAR T cells, but the in vitro differentiation of TiPS often yields T cells with suboptimal features. Here we show that the premature expression of the T-cell receptor (TCR) or a constitutively expressed CAR in TiPS promotes the acquisition of an innate phenotype, which can be averted by disabling the TCR and relying on the CAR to drive differentiation. Delaying CAR expression and calibrating its signalling strength in TiPS enabled the generation of human TCR– CD8αβ+ CAR T cells that perform similarly to CD8αβ+ CAR T cells from peripheral blood, achieving effective tumour control on systemic administration in a mouse model of leukaemia and without causing graft-versus-host disease. Driving T-cell maturation in TiPS in the absence of a TCR by taking advantage of a CAR may facilitate the large-scale development of potent allogeneic CD8αβ+ T cells for a broad range of immunotherapies.

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.

Supplementary Data Combined

Table S1: Detailed descriptions of all grade neurotoxicity; Table S2: Correlation between CAR T cell phenotype and neurotoxicity; Table S3: Correlation between CSF cytokines and CSF protein concentrations; Table S4: Definitions of CRS

Figure S1: Detailed time course of neurotoxicity for each patient; Figure S2: Serum cytokines in relation to tocilizumab and corticosteroid administration; Figure S3: Hematopoietic toxicity and coagulopathy in severe neurotoxicity; Figure S4: Angiopoietin 1 and 2 alterations in severe neurotoxicity

With the US Food and Drug Administration (FDA) approval of four CD19- and one BCMA-targeted chimeric antigen receptor (CAR) therapy for B cell malignancies, CAR T cell therapy has finally reached the status of a medicinal product. The successful manufacturing of autologous CAR T cell products is a key requirement for this promising treatment modality. By analyzing the composition of 214 apheresis products from 210 subjects across eight disease indications, we found that high CD14+ cell content poses a challenge for manufacturing CAR T cells, especially in patients with non-Hodgkin's lymphoma and multiple myeloma caused by the non-specific phagocytosis of the magnetic beads used to activate CD3+ T cells. We demonstrated that monocyte depletion via rapid plastic surface adhesion significantly reduces the CD14+ monocyte content in the apheresis products and simultaneously boosts the CD3+ content. We established a 40% CD14+ threshold for the stratification of apheresis products across nine clinical trials and demonstrated the effectiveness of this procedure by comparing manufacturing runs in two phase 1 clinical trials. Our study suggests that CD14+ content should be monitored in apheresis products, and that the manufacturing of CAR T cells should incorporate a step that lessens the CD14+ cell content in apheresis products containing more than 40% to maximize the production success.

Malignant pleural diseases, comprising metastatic lung and breast cancers and malignant pleural mesothelioma (MPM), are aggressive solid tumors with poor therapeutic response. We developed and conducted a first-in-human, phase I study of regionally delivered, autologous, mesothelin-targeted chimeric antigen receptor (CAR) T-cell therapy. Intrapleural administration of 0.3M to 60M CAR T cells/kg in 27 patients (25 with MPM) was safe and well tolerated. CAR T cells were detected in peripheral blood for >100 days in 39% of patients. Following our demonstration that PD-1 blockade enhances CAR T-cell function in mice, 18 patients with MPM also received pembrolizumab safely. Among those patients, median overall survival from CAR T-cell infusion was 23.9 months (1-year overall survival, 83%). Stable disease was sustained for ≥6 months in 8 patients; 2 exhibited complete metabolic response on PET scan. Combination immunotherapy with CAR T cells and PD-1 blockade agents should be further evaluated in patients with solid tumors. Significance: Regional delivery of mesothelin-targeted CAR T-cell therapy followed by pembrolizumab administration is feasible, safe, and demonstrates evidence of antitumor efficacy in patients with malignant pleural diseases. Our data support the investigation of combination immunotherapy with CAR T cells and PD-1 blockade agents in solid tumors. See related commentary by Aldea et al., p. 2674. This article is highlighted in the In This Issue feature, p. 2659

CD19-targeted chimeric antigen receptor (CAR) T-cell therapy has become a breakthrough treatment of patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). However, despite the high initial response rate, the majority of adult patients with B-ALL progress after CD19 CAR T-cell therapy. Data on the natural history, management, and outcome of adult B-ALL progressing after CD19 CAR T cells have not been described in detail. Herein, we report comprehensive data of 38 adult patients with B-ALL who progressed after CD19 CAR T therapy at our institution. The median time to progression after CAR T-cell therapy was 5.5 months. Median survival after post–CAR T progression was 7.5 months. A high disease burden at the time of CAR T-cell infusion was significantly associated with risk of post–CAR T progression. Thirty patients (79%) received salvage treatment of post–CAR T disease progression, and 13 patients (43%) achieved complete remission (CR), but remission duration was short. Notably, 7 (58.3%) of 12 patients achieved CR after blinatumomab and/or inotuzumab administered following post–CAR T failure. Multivariate analysis revealed that a longer remission duration from CAR T cells was associated with superior survival after progression following CAR T-cell therapy. In summary, overall prognosis of adult B-ALL patients progressing after CD19 CAR T cells was poor, although a subset of patients achieved sustained remissions to salvage treatments, including blinatumomab, inotuzumab, and reinfusion of CAR T cells. Novel therapeutic strategies are needed to reduce risk of progression after CAR T-cell therapy and improve outcomes of these patients.

Chimeric antigen receptor (CAR) T cells have demonstrated clinical benefit in patients with relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). We undertook a multicenter clinical trial to determine toxicity, feasibility, and response for this therapy. A total of 25 pediatric/young adult patients (age, 1-22.5 years) with R/R B-ALL were treated with 19-28z CAR T cells. Conditioning chemotherapy included high-dose (3 g/m2) cyclophosphamide (HD-Cy) for 17 patients and low-dose (≤1.5 g/m2) cyclophosphamide (LD-Cy) for 8 patients. Fifteen patients had pretreatment minimal residual disease (MRD

Anti-CD19 chimeric antigen receptor (CAR) T cell therapy has led to unprecedented responses in patients with high-risk hematologic malignancies. However, up to 60% of patients still experience disease relapse and up to 80% of patients experience CAR-mediated toxicities, such as cytokine release syndrome or immune effector cell-associated neurotoxicity syndrome. We investigated the role of the intestinal microbiome on these outcomes in a multicenter study of patients with B cell lymphoma and leukemia. We found in a retrospective cohort (n = 228) that exposure to antibiotics, in particular piperacillin/tazobactam, meropenem and imipenem/cilastatin (P-I-M), in the 4 weeks before therapy was associated with worse survival and increased neurotoxicity. In stool samples from a prospective cohort of CAR T cell recipients (n = 48), the fecal microbiome was altered at baseline compared to healthy controls. Stool sample profiling by 16S ribosomal RNA and metagenomic shotgun sequencing revealed that clinical outcomes were associated with differences in specific bacterial taxa and metabolic pathways. Through both untargeted and hypothesis-driven analysis of 16S sequencing data, we identified species within the class Clostridia that were associated with day 100 complete response. We concluded that changes in the intestinal microbiome are associated with clinical outcomes after anti-CD19 CAR T cell therapy in patients with B cell malignancies.

Spearheaded by the therapeutic use of chimeric antigen receptors (CARs) targeting CD19, synthetic immunology has entered the clinical arena. CARs are recombinant receptors for antigen that engage cell surface molecules through the variable region of an antibody and signal through arrayed T cell activating and costimulatory domains. CARs allow redirection of T cell cytotoxicity against any antigen of choice, independent of MHC expression. Patient T cells engineered to express CARs specific for CD19 have yielded remarkable outcomes in subjects with relapsed/refractory B cell malignancies, setting off unprecedented interest in T cell engineering and cell-based cancer immunotherapy. In this review, we present the challenges to extend the use of CAR T cells to solid tumors and other pathologies. We further highlight progress in CAR design, cell manufacturing and genome editing, which in aggregate hold the promise of generating safer and more effective genetically instructed immunity. Novel engineered cell types, including innate T cell types, natural killer (NK) cells, macrophages and induced pluripotent stem (iPS) cell-derived immune cells, are on the horizon, as are applications of CAR T cells to treat autoimmunity, severe infections and senescence-associated pathologies. This article is protected by copyright. All rights reserved.

Replication-competent retrovirus (RCR) is a safety concern for individuals treated with retroviral gene therapy. RCR detection assays are used to detect RCR in manufactured vector, transduced cell products infused into research subjects, and in the research subjects after treatment. In this study, we reviewed 286 control (n = 4) and transduced cell products (n = 282) screened for RCR in the National Gene Vector Biorepository. The transduced cell samples were submitted from 14 clinical trials. All vector products were previously shown to be negative for RCR prior to use in cell transduction. After transduction, all 282 transduced cell products were negative for RCR. In addition, 241 of the clinical trial participants were also screened for RCR by analyzing peripheral blood at least 1 month after infusion, all of which were also negative for evidence of RCR infection. The majority of vector products used in the clinical trials were generated in the PG13 packaging cell line. The findings suggest that screening of the retroviral vector product generated in PG13 cell line may be sufficient and that further screening of transduced cells does not provide added value. Keywords: retrovirus, safety testing, replicating virus, lentivirus

CD19-specific chimeric antigen receptor (CAR) T-cell therapy is highly effective against relapsed or refractory acute lymphoblastic leukemia (ALL), but is hindered by neurotoxicity. In 53 adult patients with ALL, we found a significant association of severe neurotoxicity with high pretreatment disease burden, higher peak CAR T-cell expansion, and early and higher elevations of proinflammatory cytokines in blood. Patients with severe neurotoxicity had evidence of blood–cerebrospinal fluid (CSF) barrier disruption correlating with neurotoxicity grade without association with CSF white blood cell count or CAR T-cell quantity in CSF. Proinflammatory cytokines were enriched in CSF during severe neurotoxicity with disproportionately high levels of IL6, IL8, MCP1, and IP10, suggesting central nervous system–specific production. Seizures, seizure-like activity, myoclonus, and neuroimaging characteristics suggested excitatory neurotoxicity, and we found elevated levels of endogenous excitatory agonists in CSF during neurotoxicity.Significance: We detail the neurologic symptoms and blood, CSF, and neuroimaging correlates of neurotoxicity associated with CD19 CAR T cells and identify neurotoxicity risk factors. Our findings implicate cellular components other than T cells and suggest novel links between systemic inflammation and characteristic neurotoxicity symptoms. Cancer Discov; 8(8); 958–71. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 899

FT819 is a first-of-kind, allogeneic, off-the-shelf CAR T-cell therapy derived from a clonal master induced pluripotent stem cell (iPSC) line precisely engineered to insert a novel 1XX anti-CD19 chimeric antigen receptor (CAR) under the regulation of the T-cell receptor alpha constant (TRAC) locus for optimized control of anti-tumor activity and to completely delete T-cell receptor (TCR) expression to eliminate the potential of graft-versus-host disease (GvHD). Unlike conventional allogeneic CAR T-cell therapies which require repeatedly sourcing of T cells from various donors as the starting material, the use of a clonal master engineered iPSC line serves as a renewable starting cell source and ensures routine mass production of a uniformly engineered, homogenous CAR T-cell product for broad patient access. T cell-derived iPSCs were generated using a proprietary non-integrating cellular reprogramming system and genetically modified to integrate a novel anti-CD19 1XX CAR into both alleles of the TRAC gene. After single cell subcloning, each engineered iPSC clone was screened for multiple critical quality attributes including pluripotency, identity, genomic stability, cassette integration, on/off-target integration, T-cell differentiation propensity, and CAR T-cell function. Accordingly, the ideal single cell-derived engineered iPSC clone was selected as the clonal master iPSC line for FT819 and was converted into a master cell bank (MCB). The iPSC MCB serves as a renewable source for the routine GMP manufacture of FT819 drug product. The FT819 production process consists of three stages: 1) generation of CD34-expressing hematopoietic progenitor cells from iPSCs (>90% CD34+ cells post enrichment); 2) lineage-specification to T cells followed by T-cell expansion (>5e5 fold expansion); and 3) fill/finish and cryopreservation of the drug product. As an example, in an initial small-scale manufacturing campaign, a total of 2.5 × 10 10 FT819 CAR T-cells were generated and filled and finished starting from one vial of the MCB. The FT819 drug product was tested on safety, identity, purity, and potency. The final product was comprised of CD45+CD7+ lymphocytes (>99%), with homogeneous CAR expression (>99% CAR+) and lacking expression of TCRαβ (not detected) on the cell surface. Importantly, there were no residual iPSCs detected in the FT819 drug product. The FT819 drug product exhibited potent and consistent effector function against NALM6 leukemia cells. The FT819 drug product is currently being used in a landmark Phase I study (NCT04629729), the first-ever iPSC-derived T-cell therapy to undergo clinical investigation, for the treatment of patients with relapsed/refractory B-cell lymphoma, chronic lymphocytic leukemia and precursor B-cell acute lymphoblastic leukemia. In summary, FT819 is a first-of-kind, off-the-shelf, CAR T-cell therapy uniquely derived from a clonal multiplexed-engineered master iPSC line. The novel manufacturing paradigm enables mass production of a uniformly engineered, homogenous cell therapy product that is available on-demand for broad patient access. A multi-center Phase 1 study of FT819 is currently ongoing for the treatment of B-cell malignancies. Key Words: cancer immunotherapy, cell therapy, CAR-T, CD19, allogeneic, induced pluripotent stem cell, iPSC, clonal master iPSC line, engineered, off-the-shelf, cGMP, production, manufacturing, FT819 Disclosures Yuan: Fate Therapeutics, Inc.: Current Employment. Clarke: Fate Therapeutics, Inc.: Current Employment. Lai: Fate Therapeutics, Inc.: Current Employment. Chang: Fate Therapeutics, Inc.: Current Employment. Yang: Fate Therapeutics, Inc.: Current Employment. Hsia: Fate Therapeutics, Inc.: Current Employment. Abujarour: Fate Therapeutics, Inc.: Current Employment. Lee: Fate Therapeutics, Inc.: Current Employment. van der Stegen: Fate Therapeutics, Inc.: Current Employment. Shaked: Fate Therapeutics, Inc.: Current Employment. Jalloh: Fate Therapeutics, Inc.: Current Employment. Moreno: Fate Therapeutics, Inc.: Current Employment. ORourke: Fate Therapeutics, Inc.: Current Employment. Sung: Fate Therapeutics, Inc.: Current Employment. Gutierrez: Fate Therapeutics, Inc.: Current Employment. Rezner: Fate Therapeutics, Inc.: Current Employment. Eberhart: Fate Therapeutics, Inc.: Current Employment. Magdaleno: Fate Therapeutics, Inc.: Current Employment. Farnan: Fate Therapeutics, Inc.: Current Employment. Plavsic: Fate Therapeutics, Inc.: Current Employment. Bressi: Fate Therapeutics, Inc.: Current Employment. Rivière: 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); FloDesign Sonics: Other: Provision of Services; Juno Therapeutics: Patents & Royalties. Valamehr: Fate Therapeutics, Inc.: Current Employment.

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

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.

The marketing approval of genetically engineered hematopoietic stem cells (HSCs) as the first-line therapy for the treatment of severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID) is a tribute to the substantial progress that has been made regarding HSC engineering in the past decade. Reproducible manufacturing of high-quality, clinical-grade, genetically engineered HSCs is the foundation for broadening the application of this technology. Herein, the current state-of-the-art manufacturing platforms to genetically engineer HSCs as well as the challenges pertaining to production standardization and product characterization are addressed in the context of primary immunodeficiency diseases (PIDs) and other monogenic disorders.

Genetically engineered T cells are powerful new medicines, offering hope for curative responses in patients with cancer. Chimaeric antigen receptors (CARs) are a class of synthetic receptors that reprogram lymphocyte specificity and function. CARs targeting CD19 have demonstrated remarkable potency in B cell malignancies. Engineered T cells are applicable in principle to many cancers, pending further progress to identify suitable target antigens, overcome immunosuppressive tumour microenvironments, reduce toxicities, and prevent antigen escape. Advances in the selection of optimal T cells, genetic engineering, and cell manufacturing are poised to broaden T-cell-based therapies and foster new applications in infectious diseases and autoimmunity.

Background & Aim Chimeric antigen receptors (CARs) are synthetic receptors that redirect and reprogram T cells to mediate tumor rejection. CD19 targeted CAR T cells have showed remarkable effacy for chemorefractory/relapsed B cell malignancies. Recombinant retroviral vectors mediated- CAR gene transfer is currently the standard method to generate cGMP grade CAR T cells. However, CAR gene expression is highly variable, owing to position effects and vector incorporation variations. CAR T cells engineered in this manner are capable of tumor eradication, but are prone to tonic signaling and accelerated exhaustion. We have developed a novel genetic engineering strategy to insert CAR genes into a precise genomic location in human peripheral blood T cells. Methods, Results & Conclusion T cells are electroporated with Cas9 mRNA or Cas9 protein and a guide RNA, followed by transduction with a recombinant Adeno-Associated Virus encoding the CAR sequence to facilitate the insertion of the CAR gene upstream of the constant region of TCR alpha chain. This results in the endogenous TCR promoter (TRAC) controlled CAR expression and abrogation of TCR surface expression. This strategy not only allows uniform CAR expression, but also delays T-cell differentiation and exhaustion, leading to enhanced T cell function and anti-tumor efficacy. The edited cells vastly outperformes retrovirally modified CAR T cells in a pre-B ALL NALM6 mouse model. The targeting of CARs to the TCR locus also provides a safer therapeutic T cell by minimizing the risks of insertional oncogenesis, and TCR-induced autoimmunity and alloreactivity (thus spanning both autologous and allogeneic T cell applications). In addition, we have incorporated in the CAR a mutant CD3z chain encoding a single immunoreceptor tyrosine-based activation motif that improves the balance between effector and memory T cells composition. We are in the process of translating this novel approach into the clinical setting by establishing cGMP conditions and protocols for the manufacturing of TRAC-CAR T cells. Using the 4D-LV electroporator, we have demonstrated that we can knockout the TCR at large scale (100 E06 CD3+ T cells) with high efficiency (70-80%) as that obtained at small scale. We have evaluated AAV6 to deliver the CAR transgene in the TCR locus. Data will be presented on optimizing the manufacturing of the TRAC-CAR T cells and on evaluating their anti-tumor efficacy in vivo.

High-dose chemotherapy followed by autologous stem cell transplantation (HDT-ASCT) is the standard of care for relapsed or chemorefractory diffuse large B-cell lymphoma (rel/ref DLBCL). Only 50% of patients are cured with this approach. We investigated whether CD19-specific chimeric antigen receptor (CAR) T cells administered following HDT-ASCT may enhance progression-free survival (PFS). Methods: Eligibility for this study includes poor-risk rel/ref aggressive B-NHL chemosensitive to salvage therapy with: 1) FDG-PET (+) or 2) bone marrow involvement. Patients underwent BEAM conditioned HDT-ASCT and followed by 19-28z CAR-T cells on days +2 and +3. Results: Of 15 subjects treated on study, dose-limiting toxicity was observed at both dose levels (5 x106 and 1 x107 19-28z CAR-T/kg). Ten of 15 subjects experienced CAR T cell-induced neurotoxicity and/or cytokine-release syndrome (CRS), which were associated with greater CAR T cell persistence (p=0.05) but not peak CAR T cell expansion. Serum IFN-g elevation (pl0.001) and possibly IL-10 (p=0.07) were associated with toxicity. The 2-year PFS is 30% (95% CI: 20-70%). Two subjects with progression of disease (POD) were CD19 (-) on re-biopsy. Subjects given decreased naive-like (CD45RA+CCR7+) CD4+ and CD8+ CAR T cells experienced superior PFS (p=0.02 and 0.04, respectively). There was no association between CAR T cell peak expansion, persistence or cytokine changes and PFS. Conclusions: 19-28z CAR T cells following HDT-ASCT was associated with a high-incidence of reversible neurotoxicity and CRS. Following HDT-ASCT, effector CD4+ and CD8+ immunophenotypes may improve disease control. Phenotype selection and/or multiple infusions may be the focus of the next clinical trial. This study is registered at www.clinicaltrials.gov as #NCT01840566.

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.

BACKGROUND: Subgroups of patients with relapsed or refractory (R/R) chronic lymphocytic leukemia (CLL) exhibit suboptimal outcomes after standard therapies, including oral kinase inhibitors. We and others have previously reported on the safety and efficacy of autologous CD19-targeted CAR T cells for these patients. Here, we report safety and long-term follow-up of CAR T cell therapy with or without conditioning chemotherapy for patients with R/R CLL and indolent B cell non-Hodgkin lymphoma (B-NHL). METHODS: We conducted a phase I clinical trial investigating CD19-targeted CAR T cells incorporating a CD28 costimulatory domain (19–28z). Seventeen of twenty patients received conditioning chemotherapy prior to CAR T cell infusion. Five patients with CLL received ibrutinib at the time of autologous T cell collection and/or CAR T cell administration. RESULTS: This analysis included 16 patients with R/R CLL and 4 patients with R/R indolent B-NHL. Cytokine release syndrome (CRS) was observed in all 20 patients, but grade 3 and 4 CRS and neurological events were uncommon (10% for each). Ex vivo expansion of T cells and proportions of CAR T cells with the CD62L(+)CD127(+) immunophenotype were significantly greater (P = 0.047; CD8 subset, P = 0.0061, CD4 subset) in patients on ibrutinib at leukapheresis. Three of twelve evaluable CLL patients receiving conditioning chemotherapy achieved complete response (CR) (2 had minimal residual disease–negative CR). All patients achieving CR remained progression free at median follow-up of 53 months. CONCLUSION: Conditioning chemotherapy and 19–28z CAR T cells were acceptably tolerated across investigated dose levels in heavily pretreated patients with R/R CLL and indolent B-NHL, and a subgroup of patients achieved durable CR. Ibrutinib therapy may modulate autologous T cell phenotype. TRIAL REGISTRATION: ClinicalTrials.gov NCT00466531. FUNDING: Juno Therapeutics and NIH/National Cancer Institute Cancer Center Support Grant (P30-CA08748).

Early clinical results of chimeric antigen receptor (CAR) T cell therapy targeting B cell maturation antigen (BCMA) for multiple myeloma (MM) appear promising, but relapses associated with residual low-to-negative BCMA-expressing MM cells have been reported, necessitating identification of additional targets. The orphan G protein–coupled receptor, class C group 5 member D (GPRC5D), normally expressed only in the hair follicle, was previously identified as expressed by mRNA in marrow aspirates from patients with MM, but confirmation of protein expression remained elusive. Using quantitative immunofluorescence, we determined that GPRC5D protein is expressed on CD138(+) MM cells from primary marrow samples with a distribution that was similar to, but independent of, BCMA. Panning a human B cell–derived phage display library identified seven GPRC5D-specific single-chain variable fragments (scFvs). Incorporation of these into multiple CAR formats yielded 42 different constructs, which were screened for antigen-specific and antigen-independent (tonic) signaling using a Nur77-based reporter system. Nur77 reporter screen results were confirmed in vivo using a marrow-tropic MM xenograft in mice. CAR T cells incorporating GPRC5D-targeted scFv clone 109 eradicated MM and enabled long-term survival, including in a BCMA antigen escape model. GPRC5D(109) is specific for GPRC5D and resulted in MM cell line and primary MM cytotoxicity, cytokine release, and in vivo activity comparable to anti-BCMA CAR T cells. Murine and cynomolgus cross-reactive CAR T cells did not cause alopecia or other signs of GPRC5D-mediated toxicity in these species. Thus, GPRC5D(109) CAR T cell therapy shows potential for the treatment of advanced MM irrespective of previous BCMA-targeted therapy.

Hematopoietic stem cell (HSC) gene therapy has effectively become a therapeutic option largely due to the resounding clinical successes in patients with primary immunodeficiencies (PIDs) such as X-linked severe immunodeficiency (SCID-X1), adenosine deaminase deficiency (ADA-SCID), Wiskott-Aldrich syndrome (WAS), and chronic granulomatous disease (CGD). HSC gene therapy is also being investigated in patients with metabolic diseases such as X-linked adrenoleukodystrophy (ALD) and metachromatic leukodystrophy (MLD) and inherited blood disorders such as β-thalassemia and sickle cell disease (SCD) where some therapeutic benefits have been reported more recently. Safer and more efficient self-inactivating (SIN) γ-retroviral and lentiviral vectors have been developed to overcome the genotoxicity imparted by γ-retroviral vectors with intact long terminal repeat (LTR). The discovery and maturation of gene-editing platforms, including zinc-finger nuclease (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, offer exciting prospective strategies for further improving gene therapy by targeting the repair of diseased genes.

Parkinson's disease is characterized by the loss of dopaminergic neurons in the substantia nigra leading to disabling deficits. Dopamine neuron grafts may provide a significant therapeutic advance over current therapies. We have generated midbrain dopamine neurons from human embryonic stem cells and manufactured large-scale cryopreserved dopamine progenitors for clinical use. After optimizing cell survival and phenotypes in short-term studies, the cell product, MSK-DA01, was subjected to an extensive set of biodistribution, toxicity, and tumorigenicity assessments in mice under GLP conditions. A large-scale efficacy study was also performed in rats with the same lot of cells intended for potential human use and demonstrated survival of the grafted cells and behavioral amelioration in 6-hydroxydopamine lesioned rats. There were no adverse effects attributable to the grafted cells, no obvious distribution outside the brain, and no cell overgrowth or tumor formation, thus paving the way for a future clinical trial.

Chimeric Antigen Receptor (CAR) T cells are a new treatment paradigm for relapsed/refractory hematopoietic malignancies. However, their autologous nature imposes manufacturing constraints that can delay CAR T cell availability and increase their cost. We previously established proof of principle that αβ T cell-derived induced pluripotent stem cells (TiPSCs) can provide a self-renewing source for in vitro CAR T cell production (Themeli, Nat Biotechnol, 2013). The use of cloned TiPSC further enhances the feasibility of verifying genome integrity of the genetically engineered stem cells and should in principle yield highly homogenous cell products. Using αβ T cell-derived TiPSCs transduced with a well-defined CD19-specific CAR (1928z; Park, NEJM, 2018), we previously demonstrated that TiPSCs can be differentiated into CAR T cells. These T cells retained their endogenous T cell receptor (TCR) and also displayed characteristics of innate lymphoid cells. We have now examined how the timing of CAR expression as well as the CAR signaling strength influence T cell lineage commitment, enabling better control towards αβ T cell lineage commitment. αβ T cell lineage development depends in part on a precisely orchestrated interactions between NOTCH and (pre)TCR signaling, the timing and strength of which are crucial for αβ lineage commitment. Because TiPSCs harbor rearranged TCRα and TCRβ genes, mature TCR expression occurs earlier than if it required VDJ recombination, skewing differentiation towards acquiring innate features including CD4-CD8- double-negative or CD8αα single-positive phenotypes. We show that providing strong NOTCH stimulation counteracts the effects of early antigen receptor expression, facilitating CD4+CD8αβ+ double positive (DP) formation. We hypothesized that CAR signaling in the absence of ligand binding (tonic signaling) may mimic a TCR signal, the strength and timing of which could re-direct lineage commitment. We therefore investigated CARs providing different levels of signaling strength and the impact of delaying the onset of CAR expression. Tonic CAR signaling was measured in peripheral blood T cells expressing 1928z or 1928z-1XX, a construct in which the second and third ITAM in the CD3ζ domain have been mutated to be non-functional (Feucht, Nat Med, 2019), following either retroviral transduction (SFG vector) orTRAC-targeted cDNA integration, placing CAR expression under the transcriptional control of the TCRα promoter (Eyquem, Nature, 2017). CAR signaling in the absence of antigen exposure, measured by phosphorylation of ITAM3, ERK1/2 and ZAP70, was reduced by bothTRAC-targeting and reduction of functional ITAMs, with additive effects when combined inTRAC-1928z-1XX. Three of these engineering strategies (virally expressed 1928z,TRAC-1928z andTRAC-1928z-1XX) were evaluated in the context of TiPSC-derived T cell differentiation. Virally expressed 1928z (resulting in constitutive CAR expression throughout differentiation) resulted in the predominant generation of innate-like CD8αα T cells, associated with the absence of early T cell lineage markers such as CD5, CD2 and CD1a. Delayed expression of 1928z throughTRACtargeting resulted in increased CD5, CD2 and CD1a, but did not yield any more CD4+CD8αβ+ DP cells. In TiPSC expressingTRAClocus-encoded 1928z-1XX, a greater DP population emerged, from which CD8αβ single-positive T cells could be induced. Phenotypic analyses of clonal TRAC-1928z-1XX TiPSC lines further establish the interplay between CAR and NOTCH1 in determining αβ lineage commitment. Together these data show that early TCR and CAR expression skew T cell lineage commitment towards an innate-like T cell fate, which can be overcome by controlling the strength and timing of NOTCH, TCR and CAR signaling. These studies pave the way for the predetermined generation of a variety of CAR T cell types endowed with different functional attributes. Disclosures Whitlock: Fate Therapeutics Inc.:Current Employment, Current equity holder in publicly-traded company.Clarke:Fate Therapeutics Inc.:Current Employment, Current equity holder in publicly-traded company.Valamehr:Fate Therapeutics, Inc:Current Employment, Current equity holder in publicly-traded company.Riviere:Juno Therapeutics:Other: Ownership interest, Research Funding;Takeda:Research Funding;Fate Therapeutics Inc.:Consultancy, Other: Ownership interest , Research Funding;FloDesign Sonics:Consultancy, Other: Ownership interest;Atara:Research Funding.Sadelain:Atara:Patents & Royalties, Research Funding;Fate Therapeutics:Patents & Royalties, Research Funding;Mnemo:Patents & Royalties;Takeda:Patents & Royalties, Research Funding;Minerva:Other: Biotechnologies , Patents & Royalties.

Summary The lack of persistence of infused T cells is a principal limitation of adoptive immunotherapy in man. Interleukin (IL)-15 can sustain memory T cell expansion when presented in complex with IL-15Rα (15Rα/15). We developed a novel in-vitro system for generation of stable 15Rα/15 complexes. Immunologically quantifiable amounts of IL-15 were obtained when both IL-15Rα and IL-15 genes were co-transduced in NIH 3T3 fibroblast-based artificial antigen-presenting cells expressing human leucocyte antigen (HLA) A:0201, β2 microglobulin, CD80, CD58 and CD54 [A2-artificial antigen presenting cell (AAPC)] and a murine pro-B cell line (Baf-3) (A2-AAPC15Rα/15and Baf-315Rα/15). Transduction of cells with IL-15 alone resulted in only transient expression of IL-15, with minimal amounts of immunologically detectable IL-15. In comparison, cells transduced with IL-15Rα alone (A2-AAPCRα) demonstrated stable expression of IL-15Rα; however, when loaded with soluble IL-15 (sIL-15), these cells sequestered 15Rα/15 intracellularly and also demonstrated minimal amounts of IL-15. Human T cells stimulated in vitro against a viral antigen (CMVpp65) in the presence of 15Rα/15 generated superior yields of high-avidity CMVpp65 epitope-specific T cells [cytomegalovirus-cytotoxic T lymphocytes (CMV-CTLs)] responding to ≤ 10− 13 M peptide concentrations, and lysing targets cells at lower effector : target ratios (1 : 10 and 1 : 100), where sIL-15, sIL-2 or sIL-7 CMV-CTLs demonstrated minimal or no activity. Both soluble and surface presented 15Rα/15, but not sIL-15, sustained in-vitro expansion of CD62L+ and CCR7+ central memory phenotype CMV-CTLs (TCM). 15Rα/15 complexes represent a potent adjuvant for augmenting the efficacy of adoptive immunotherapy. Such cell-bound or soluble 15Rα/15 complexes could be developed for use in combination immunotherapy approaches.

The beta-thalassemias are inherited anemias caused by mutations that severely reduce or abolish expression of the beta-globin gene. Like sickle cell disease, a related beta-globin gene disorder, they are ideal candidates for performing a genetic correction in patient hematopoietic stem cells (HSCs). The most advanced approach utilizes complex lentiviral vectors encoding the human β-globin gene, as first reported by May et al. in 2000. Considerable progress toward the clinical implementation of this approach has been made in the past five years, based on effective CD34+ cell mobilization and improved lentiviral vector manufacturing. Four trials have been initiated in the United States and Europe. Of 16 evaluable subjects, 6 have achieved transfusion independence. One of them developed a durable clonal expansion, which regressed after several years without transformation. Although globin lentiviral vectors have so far proven to be safe, this occurrence suggests that powerful insulators with robust enhancer-blocking activity will further enhance this approach. The combined discovery of Bcl11a-mediated γ-globin gene silencing and advances in gene editing are the foundations for another gene therapy approach, which aims to reactivate fetal hemoglobin (HbF) production. Its clinical translation will hinge on the safety and efficiency of gene targeting in true HSCs and the induction of sufficient levels of HbF to achieve transfusion independence. Altogether, the progress achieved over the past 15 years bodes well for finding a genetic cure for severe globin disorders in the next decade.

Genetic engineering of T cells using a chimeric antigen receptor targeting CD19 antigen (CAR19) is now a well-established treatment of B cell malignancies. While cellular immunotherapies are entering front line treatment, substantial limitations currently hamper the broad application of adoptive T cell therapies in diverse patient population including dysfunctional starting material, lack of product consistency and purity post genetic engineering and inefficient quantity produced for true on-demand availability. FT819 is a first-of-kind off-the-shelf CAR19-T cell product generated from a renewable pluripotent stem cells for large-scale clinical manufacturing. We previously reported the engineering and characterization of the FT819 clonal master cell bank (MCB) derived from a single cell comprising targeted integration of a novel CD19 1XX CAR into the T-cell receptor (TCR) α constant locus to provide optimally regulated CAR expression and elimination of graft versus host (GvH) response. Here we preview the nonclinical study for the original investigational new drug application of FT819. Derived in a manufacturing process analogous to pharmaceutical drug product development, pilot runs from the MCB demonstrated FT819 can be consistently and uniformly manufactured in cGMP compliance, cryopreserved at clinical scale to support off-the-shelf clinical application with greater than 1e5 fold increase in cellular yield from the starting MCB and can be thawed and directly used for facilitated treatment. Repeatedly, FT819 displayed a uniform product profile of ≥95% CAR+, TCR-, CD45+, CD7+ and CD3+ [intracellular] with majority of CD8 T cells expressing CD8β. FT819 global gene expression profile displayed high similarity to primary CAR19-T cells confirming its identity as a T lymphocyte. Functional assessment demonstrated that FT819 possesses potent antigen specific cytolytic activity against leukemia and lymphoma cell lines (p=0.0004). Additional specificity studies demonstrated on-target, off-tumor cytolysis of CD19+ B cells in mixed lymphocyte reaction assay (85% lysis of CD19+ B cells versus < 2% lysis of T cells). Inability of FT819 to produce a GvH response was confirmed in a co-culture assay with anti-TCR crosslinking antibodies. Disseminated leukemia xenograft mouse studies demonstrated the ability of directly thawed and infused FT819 to control tumor growth (p=0.0003 at day 21). In a systemic administered leukemia model FT819 also showed sustained localization in the bone marrow up to 45 days post injection. Ongoing in vivo studies will assess long-term survival and avoidance of GvH disease. Collectively, these studies demonstrate that FT819 is a potent, consistent and uniform CAR19 T cell product and can be effectively and safely used off-the-shelf in the treatment of B cell malignancies with an original Phase 1 clinical trial planned in 2020. Citation Format: Mili Mandal, Raedun Clarke, Sjoukje van der Stegen, Chia-Wei Chang, Yi-Shin Lai, Alec Witty, Mushtaq Husain, Cheng-Jang Wu, Bi-Huei Yang, Chad Dufaud, Gloria Hsia, Helena Shaked, Laurel Stokely, Helen Chu, Mochtar Pribadi, Gilberto Hernandez, Jason ORourke, Alma Gutierrez, Ramzey Abujarour, Tom Lee, Jolanta Stefanski, Juan Zhen, Meilan Wu, Isabelle Riviere, Michel Sadelain, Bahram Valamehr. FT819 path to IND: First-of-kind off-the-shelf CAR19 T-cell for B cell malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3245.

Introduction CAR T-cells provide benefit in patients (pts) with R/R hematologic malignancies. Limitations including: cost, production complexity, and life-threatening toxicity impede this therapy. An "off-the-shelf" CAR product could overcome these limitations. Objectives/Methods We developed an "off-the-shelf" CD19-specific CAR T-cell by transducing the 19-28z CAR into donor EBV-specific cytotoxic lymphocytes (19-28z CAR EBV-CTL). Pts with R/R B-cell malignancies were stratified into two treatment cohorts: cohort 1 (relapse after allo/auto HSCT) or cohort 2 (CAR-EBV-CTLs as consolidative therapy after auto-HSCT; NCT 01430390). Objectives: determine dose limiting toxicity (primary endpoint), optimal dose for multiple infusions, and disease response (dz evaluation: 1 month (B-ALL/CLL) or 3 months (NHL)). Results Ten pts were treated with 19-28z CAR-EBV-CTLs with seven in cohort 1 (B-ALL n=5; Burkitt lymphoma n=1; CLL n=1), and three in cohort 2 (PMBCL n=2; DLBCL n=1). Median age at treatment was 14.7 yrs (range 1.3-70.5 yrs). Median dose infused: 2.4 × 106 EBV-CTLs/kg (range = 0.6 – 7.5 × 106 EBV-CTLs/kg) with variable transduction (median 20.5%; range 7.4-41%). Six pts received multiple doses (median 2, range 1-3) with 3 × 106 T cells/kg determined to be optimal (allowing for multiple doses per cell line). EBV-CTL source: primary HSCT donors (n=4) and 3rd Party donors (n=6). HLA-matching between (pts to donor): 10/10-match (n=3); 6/10 (n=2); 5/10 (n=1), 4/10 (n=2), and 2/10 (n=2). Eight of the pts were EBV-seropositive. Response was seen in 70% (7/10) of treated pts including pts with B-ALL (n=2), CLL (n=1) and NHL (n=4). The clinical response was striking in pts treated for NHL (100% CR; 4/4) and recipients of 3rd Party cells (83% CR; 5/6) who exhibited durable response (NHL median OS 30.8; range 8-72.9 months and 3rd Party recipient median OS 23.6, range 8-72.9 - Figure 1). CRS or neurotoxicity did not occur post infusion and no DLT was noted in the trial. Two pts developed diffuse skin rash which was biopsy positive (n=1; HLA-matched HSCT donor) and biopsy negative (n=1; haploidentical 3rd Party donor) for GVHD and one pt (cohort 2) developed reversible pneumonitis following external beam radiation/auto-HSCT/CAR T-cells. Symptoms resolved after a course of topical corticosteroids (skin rash) and systemic steroids (pneumonitis). Conclusions We have successfully treated ten pts with encouraging preliminary results: CR in 70% (7/10) of treated pts including 100% (4/4) in NHL and 83% in recipients of 3rd party products. The absence of CRS and ICANS (neurotoxicity) is a salient feature of this therapy and this trial demonstrates that a readily accessible source of "off-the-shelf" CAR T cell product can be made available for pts without alternative therapy.

Long-term follow-up of adoptive transfer of autologous T cells expressing a chimeric antigen receptor (CAR) directed to CD19 antigen has demonstrated encouraging, durable clinical outcome in various B cell malignancies. However, to make such CAR-T cells available to a broader base and to reach a more diverse patient population, challenges associated with product consistency, cost of manufacture, precision genetic engineering and on-demand availability still need to be addressed. FT819 is a first-of-kind off-the-shelf CAR-T cell product candidate derived from a renewable master pluripotent cell line. FT819 comprises precise genetic engineering of multiple targeting events at the single cell level and is produced using a clonally-derived master cell bank (MCB) that serves as the starting material to support consistent and reproducible clinical manufacturing. The engineered features of FT819 include the targeted integration of a novel CD19 1XX-CAR into the T-cell receptor α constant (TRAC) locus to provide antigen specificity, enhanced efficacy and temporally-regulated CAR expression driven by an endogenous (TCR) promoter. Such features are designed to also eliminate the possibility of graft versus host disease (GvHD) by nullifying the TCR. To develop the MCB for FT819, αβ T cells were reprogrammed into induced pluripotent stem cells (iPSCs) and subsequently engineered to direct CD19 1XX-CAR into the TRAC locus with knockout of the TCR. To generate clonal lines, engineered iPSCs were sorted by flow cytometry for various markers and single cells were seeded into individual wells of feeder-free 96-well plates. Engineered iPSC clones were screened for integration of CAR into the TRAC locus by amplifying the genomic DNA flanking the homologous recombination site and confirmed by a SNP phasing assay. Clones were further screened for random integration of donor template by quantitative PCR and the CAR copy number was confirmed by droplet digital PCR. Genome stability of each clone was also confirmed by karyotype analysis. Overall, the described screening initiative surveyed 774 clones to select the ideal MCB for FT819. Utilizing our stage-specific T cell differentiation and expansion protocol, we demonstrated that T cells derived from the FT819 (FT819-iTs) expanded greater than 100,000-fold during the clinical manufacturing process and the cells expressed greater than 95% T lymphocyte markers such as CD45, CD7, intracellular CD3, and TRAC-regulated CAR. Further modifications to the T cell differentiation protocol resulted in enhanced expression of CD8 αβ from less than 25% to greater than 70% of the total population. In addition, expression of CD2, CD5, and CD27 was increased by approximately 5- to 20-fold. In vitro functional studies showed that FT819-iTs possess antigen specificity as confirmed by cytokine release and cytotoxic T lymphocytes (CTL) assays. Upon stimulation with a wild type acute lymphoblastic leukemia line, Nalm-6, FT819-iTs expressed 30% CD107a/b compared to 2% when stimulated by Nalm-6 CD19KO. In an in vitro CTL assay, greater than 80% of Nalm-6 WT cells were lysed with effector to target (E:T) ratio at 10:1 as compared to Nalm-6-CD19KO, which showed less than 10% lysis at the same E:T ratio. Finally, in an in vivo tumor model, FT819-iTs generated from our original and modified T cell differentiation protocols showed similar tumor burden control and prolonged survival rate when compared to primary CAR-T cells (days of survival >80days, p>0.1). In a more stringent in vivo model, FT819-iTs generated from the modified differentiation protocol demonstrated higher anti-tumor response and better animal survival rate compared to iTs from the original T cell differentiation protocol (Day 30 p Disclosures Chang: Fate Therapeutics: Employment. Van Der Stegen:Memorial Sloan Kettering Cancer Center: Employment. Mili:Fate Therapeutics: Employment. Clarke:Fate Therapeutics: Employment. Lai:Fate Therapeutics: Employment. Witty:Fate Therapeutics: Employment. Lindenbergh:Memorial Sloan Kettering Cancer Center: Employment. Yang:Fate Therapeutics: Employment. Husain:Fate Therapeutics: Employment. Shaked:Fate Therapeutics: Employment. Groff:FATE THERAPEUTICS: Employment. Stokely:Fate Therapeutics: Employment. Abujarour:Fate Therapeutics, Inc.: Employment. Lee:Fate Therapeutics, Inc.: Employment. Chu:Fate Therapeutics: Employment. Pribadi:Fate Therapeutics, Inc.: Employment. ORourke:Fate Therapeutics: Employment. Gutierrez:Fate Therapeutics: Employment. Riviere:Juno Therapeutics: Consultancy, Equity Ownership, Research Funding; Fate Therapeutics: Consultancy; Memorial Sloan Kettering Cancer Center: Employment. Sadelain:Memorial Sloan Kettering Cancer Center: Employment; Fate Therapeutics: Consultancy, Patents & Royalties; Juno Therapeutics: Consultancy, Patents & Royalties, Research Funding. Valamehr:Fate Therapeutics, Inc: Employment.

Introduction: Autologous chimeric antigen receptor (CAR) T cell therapy has shown to be effective in CD19+ relapsed or refractory (R/R) B-ALL patients (pts). However, relapses are common and the role of post-CAR allogeneic hematopoietic stem cell transplant (alloHSCT) remains unclear, particularly in adults with relapsed B-ALL. We previously reported that there was no survival benefit with post-CAR alloHSCT in overall adult ALL population (Park J et al. NEJM 2018), but it is unclear whether there is a subset of pts who may benefit the most from post-CAR alloHSCT. Therefore, we sought to identify clinical features that are associated with a better long-term survival in adult B-ALL pts treated with CD19 CAR T cells followed by alloHSCT. Methods: We performed a retrospective chart review of adult pts with R/R ALL treated with 19-28z CAR T therapy at MSKCC (NCT01044069), with a particular focus on pts who achieved minimal residual disease (MRD) negative CR to the CAR therapy and subsequently proceeded to alloHSCT. Clinical data was updated with the data cutoff date of July 15, 2019. We examined the association between overall survival and the following clinical factors: impact of age, prior HSCT status, presence of Philadelphia chromosome (Ph), disease status at the time of CAR therapy (MRD vs. morphologic), severity of cytokine release syndrome (CRS) and neurotoxicity (NTX) experienced during CAR therapy. Survival was calculated from the time of CAR T cell infusion. Continuous variables were analyzed univariately using cox PH models. Categorical variables were analyzed using Kaplan Meier methodology and log rank tests. Results: Of the 53 pts who received 1928z CAR T cells, 16 pts proceeded to alloHSCT after achieving MRD negative CR to the CAR therapy. The median age of these pts was 38 (range, 23-66), and baseline disease and pt characteristics as well as response and toxicity to CAR T therapy are listed in Table 1. Of the 16 pts, 6 pts remain alive in remission; 4 pts died of relapse (median time to relapse: 104.5 days, 57-194), and 6 pts died of transplant-related mortality (TRM) in remission (n=4, infection; n=1, pulmonary VOD; n=1, respiratory failure of unclear etiology). Detailed disease and treatment characteristics by the outcome categories are listed in Table 2. For those pts who are alive in remission, the median survival is 47.5 months (range, 39.7-92.4). Among the examined clinical variables, we found younger age and no severe NTX (i.e. grade 0-2) after CAR therapy to be significantly associated with improved overall survival (p=0.014 and 0.05, respectively) but prior lines of therapy, time to HSCT, prior HSCT, Ph chromosome, disease status at the time of T cell therapy and severity of CRS did not impact the survival after alloHSCT. Conclusions: In this retrospective analysis, we found that younger age and no severe NTX to CD19 CAR therapy were associated with improved overall survival following alloHSCT. Number of relapses following alloHSCT (4 of 16 pts) appears to be lower compared to the previously reported number of relapse in the adult ALL cohort without alloHSCT (17 of 26 pts). However, the occurrence of TRM due to infection is high, likely a reflection of heavily pretreated pt population and possibly further immunosuppression from CAR + alloHSCT. The link between severe NTX and worse survival is unclear but could be related to prolonged corticosteroid use and high disease burden. While no definitive conclusion can be drawn due to a small sample size, our data suggests a certain subset of adult pts with R/R B-ALL may benefit more from alloHSCT as a consolidation therapy following CD19 CAR T cells, and acute and late infectious complications following alloHSCT should be carefully monitored. These findings should be validated prospectively and compared between different CAR constructs. Disclosures Park: Allogene: Consultancy; Amgen: Consultancy; AstraZeneca: Consultancy; Autolus: Consultancy; GSK: Consultancy; Incyte: Consultancy; Kite Pharma: Consultancy; Novartis: Consultancy; Takeda: Consultancy. Riviere:Fate Therapeutics: Consultancy; Juno Therapeutics: Consultancy, Equity Ownership, Research Funding; Memorial Sloan Kettering Cancer Center: Employment. Sadelain:Memorial Sloan Kettering Cancer Center: Employment; Fate Therapeutics: Consultancy, Patents & Royalties; Juno Therapeutics: Consultancy, Patents & Royalties, Research Funding. Brentjens:JUNO Therapeutics: Consultancy, Patents & Royalties, Research Funding; Celgene: Consultancy.

Patients with residual chronic lymphocytic leukemia (CLL) following initial purine analog-based chemoimmunotherapy exhibit a shorter duration of response and may benefit from novel therapeutic strategies. We and others have previously described the safety and efficacy of autologous T cells modified to express anti-CD19 chimeric antigen receptors (CARs) in patients with relapsed or refractory B cell acute lymphoblastic leukemia and CLL. Here we report the use of CD19-targeted CAR T cells incorporating the intracellular signaling domain of CD28 (19-28z) as a consolidative therapy in 8 patients with residual CLL following first-line chemoimmunotherapy with pentostatin, cyclophosphamide, and rituximab. Outpatients received low-dose conditioning therapy with cyclophosphamide (600 mg/m(2)), followed by escalating doses of 3 × 10(6), 1 × 10(7), or 3 × 10(7) 19-28z CAR T cells/kg. An objective response was observed in 3 of 8 patients (38%), with a clinically complete response lasting more than 28 months observed in two patients. Self-limited fevers were observed post-CAR T cell infusion in 4 patients, contemporaneous with elevations in interleukin-6 (IL-6), IL-10, IL-2, and TGF-α. None developed severe cytokine release syndrome or neurotoxicity. CAR T cells were detectable post-infusion in 4 patients, with a longest observed persistence of 48 days by qPCR. Further strategies to enhance CAR T cell efficacy in CLL are under investigation.

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.

The β-thalassemias are inherited blood disorders that result from insufficient production of the β-chain of hemoglobin. More than 200 different mutations have been identified. β-Thalassemia major requires life-long transfusions. The only cure for severe β-thalassemia is to provide patients with hematopoietic stem cells. Globin gene therapy promises a curative autologous stem cell transplantation without the immunologic complications of allogeneic transplantation. The future directions of gene therapy include enhancement of lentiviral vector-based approaches, fine tuning of the conditioning regimen, and the design of safer vectors. Progress in genetic engineering bodes well for finding a cure for severe globin disorders.

CD19-specific chimeric antigen receptor (CAR) T cells induce high rates of initial response among patients with relapsed B-cell acute lymphoblastic leukemia (ALL) and long-term remissions in a subgroup of patients.We conducted a phase 1 trial involving adults with relapsed B-cell ALL who received an infusion of autologous T cells expressing the 19-28z CAR at the Memorial Sloan Kettering Cancer Center (MSKCC). Safety and long-term outcomes were assessed, as were their associations with demographic, clinical, and disease characteristics.A total of 53 adults received 19-28z CAR T cells that were manufactured at MSKCC. After infusion, severe cytokine release syndrome occurred in 14 of 53 patients (26%; 95% confidence interval [CI], 15 to 40); 1 patient died. Complete remission was observed in 83% of the patients. At a median follow-up of 29 months (range, 1 to 65), the median event-free survival was 6.1 months (95% CI, 5.0 to 11.5), and the median overall survival was 12.9 months (95% CI, 8.7 to 23.4). Patients with a low disease burden (5% bone marrow blasts) before treatment had markedly enhanced remission duration and survival, with a median event-free survival of 10.6 months (95% CI, 5.9 to not reached) and a median overall survival of 20.1 months (95% CI, 8.7 to not reached). Patients with a higher burden of disease (≥5% bone marrow blasts or extramedullary disease) had a greater incidence of the cytokine release syndrome and neurotoxic events and shorter long-term survival than did patients with a low disease burden.In the entire cohort, the median overall survival was 12.9 months. Among patients with a low disease burden, the median overall survival was 20.1 months and was accompanied by a markedly lower incidence of the cytokine release syndrome and neurotoxic events after 19-28z CAR T-cell infusion than was observed among patients with a higher disease burden. (Funded by the Commonwealth Foundation for Cancer Research and others; ClinicalTrials.gov number, NCT01044069 .).

Supplemental Digital Content is available in the text., The successful genetic engineering of patient T cells with γ-retroviral vectors expressing chimeric antigen receptors or T-cell receptors for phase II clinical trials and beyond requires the large-scale manufacture of high-titer vector stocks. The production of retroviral vectors from stable packaging cell lines using roller bottles or 10- to 40-layer cell factories is limited by a narrow harvest window, labor intensity, open-system operations, and the requirement for significant incubator space. To circumvent these shortcomings, we optimized the production of vector stocks in a disposable fixed-bed bioreactor using good manufacturing practice–grade packaging cell lines. High-titer vector stocks were harvested over 10 days, representing a much broader harvest window than the 3-day harvest afforded by cell factories. For PG13 and 293Vec packaging cells, the average vector titer and the vector stocks’ yield in the bioreactor were higher by 3.2- to 7.3-fold, and 5.6- to 13.1-fold, respectively, than those obtained in cell factories. The vector production was 10.4 and 18.6 times more efficient than in cell factories for PG13 and 293Vec cells, respectively. Furthermore, the vectors produced from the fixed-bed bioreactors passed the release test assays for clinical applications. Therefore, a single vector lot derived from 293Vec is suitable to transduce up to 500 patients cell doses in the context of large clinical trials using chimeric antigen receptors or T-cell receptors. These findings demonstrate for the first time that a robust fixed-bed bioreactor process can be used to produce γ-retroviral vector stocks scalable up to the commercialization phase.

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...