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We conducted a phase I clinical trial of anti-BCMA chimeric antigen receptor T cells (CART-BCMA) with or without anti-CD19 CAR T cells (huCART19) in multiple myeloma (MM) patients responding to third- or later-line therapy (phase A, N = 10) or high-risk patients responding to first-line therapy (phase B, N = 20), followed by early lenalidomide or pomalidomide maintenance. We observed no high-grade cytokine release syndrome (CRS) and only one instance of low-grade neurologic toxicity. Among 15 subjects with measurable disease, 10 exhibited partial response (PR) or better; among 26 subjects responding to prior therapy, 9 improved their response category and 4 converted to minimal residual disease (MRD)–negative complete response/stringent complete response. Early maintenance therapy was safe, feasible, and coincided in some patients with CAR T-cell reexpansion and late-onset, durable clinical response. Outcomes with CART-BCMA + huCART19 were similar to CART-BCMA alone. Collectively, our results demonstrate favorable safety, pharmacokinetics, and antimyeloma activity of dual-target CAR T-cell therapy in early lines of MM treatment.Significance:CAR T cells in early lines of MM therapy could be safer and more effective than in the advanced setting, where prior studies have focused. We evaluated the safety, pharmacokinetics, and efficacy of CAR T cells in patients with low disease burden, responding to current therapy, combined with standard maintenance therapy.This article is highlighted in the In This Issue feature, p. 101

Supplemental tables: (1) Subject characteristics. (2) Cytogenetic profiles and high-risk features. (3) Prior treatment exposures and refractoriness. (4) CAR T cell product characteristics. (5) Products that did not meet target dose. (6) Adverse events of grade 3-4. (7) Cytokine release syndrome and ICANS. (8). Maintenance therapy. Supplemental Figures: (1) Study schematic and subject disposition, (2) Correlates of manufacturing success, (3) Hematopoietic recovery, (4) Post-infusion T cell phenotypes, (5) Correlates of in vivo expansion and manufacturing success, (6) Late post-infusion CAR T cell re-expansion, (7) Soluble BCMA, (8) Late-onset clinical responses, (9) MM cell BCMA expression, (10) Pre- and post-treatment Sox2-specific T cell responses in CART-BCMA monotherapy patients, (11) Pre- and post-treatment Sox2-specific T cell responses in CART-BCMA + huCART19 combination therapy patients, (12) Sustained post-treatment SOX2-specific T-cell responses.

We conducted a phase I clinical trial of anti-BCMA chimeric antigen receptor T cells (CART-BCMA) with or without anti-CD19 CAR T cells (huCART19) in multiple myeloma (MM) patients responding to third- or later-line therapy (phase A, N = 10) or high-risk patients responding to first-line therapy (phase B, N = 20), followed by early lenalidomide or pomalidomide maintenance. We observed no high-grade cytokine release syndrome (CRS) and only one instance of low-grade neurologic toxicity. Among 15 subjects with measurable disease, 10 exhibited partial response (PR) or better; among 26 subjects responding to prior therapy, 9 improved their response category and 4 converted to minimal residual disease (MRD)–negative complete response/stringent complete response. Early maintenance therapy was safe, feasible, and coincided in some patients with CAR T-cell reexpansion and late-onset, durable clinical response. Outcomes with CART-BCMA + huCART19 were similar to CART-BCMA alone. Collectively, our results demonstrate favorable safety, pharmacokinetics, and antimyeloma activity of dual-target CAR T-cell therapy in early lines of MM treatment.Significance:CAR T cells in early lines of MM therapy could be safer and more effective than in the advanced setting, where prior studies have focused. We evaluated the safety, pharmacokinetics, and efficacy of CAR T cells in patients with low disease burden, responding to current therapy, combined with standard maintenance therapy.This article is highlighted in the In This Issue feature, p. 101

This phase I study investigated the safety and activity of lentiviral-transduced chimeric antigen receptor (CAR)-modified autologous T cells redirected against mesothelin (CART-meso) in patients with malignant pleural mesothelioma, ovarian carcinoma, and pancreatic ductal adenocarcinoma. Fifteen patients with chemotherapy-refractory cancer (n = 5 per indication) were treated with a single CART-meso cell infusion. CART-meso cells were engineered by lentiviral transduction with a construct composed of the anti-mesothelin single-chain variable fragment derived from the mouse monoclonal antibody SS1 fused to intracellular signaling domains of 4-1BB and CD3zeta. Patients received 1–3 × 10(7) or 1–3 × 10(8) CART-meso cells/m(2) with or without 1.5 g/m(2) cyclophosphamide. Lentiviral-transduced CART-meso cells were well tolerated; one dose-limiting toxicity (grade 4, sepsis) occurred at 1–3 × 10(7)/m(2) CART-meso without cyclophosphamide. The best overall response was stable disease (11/15 patients). CART-meso cells expanded in the blood and reached peak levels by days 6–14 but persisted transiently. Cyclophosphamide pre-treatment enhanced CART-meso expansion but did not improve persistence beyond 28 days. CART-meso DNA was detected in 7/10 tumor biopsies. Human anti-chimeric antibodies (HACA) were detected in the blood of 8/14 patients. CART-meso cells were well tolerated and expanded in the blood of all patients but showed limited clinical activity. Studies evaluating a fully human anti-mesothelin CAR are ongoing.

Pancreatic ductal adenocarcinoma (PDAC) is resistant to T-cell mediated immunotherapy. We engineered T cells to transiently express an mRNA encoding a chimeric antigen receptor (CAR) specific for mesothelin—a protein that is over-expressed by PDAC cells. We performed a phase 1 study to evaluate the safety and efficacy of adoptive cell therapy with autologous mesothelin-specific CAR T cells (CARTmeso cells) in 6 patients with chemotherapy-refractory metastatic PDAC. Patients were given intravenous CARTmeso cells 3 times weekly for 3 weeks. None of the patients developed cytokine release syndrome or neurologic symptoms and there were no dose limiting toxicities. Disease stabilized in 2 patients, with progression-free survival times of 3.8 and 5.4 months. We used FDG-positron emission tomography/computed tomography imaging to monitor the metabolic active volume (MAV) of individual tumor lesions. The total MAV remained stable in 3 patients and decreased by 69.2% in 1 patient with biopsy-proven mesothelin expression; in this patient, all liver lesions had a complete reduction in FDG uptake at 1 month compared to baseline, although there was no effect on the primary PDAC. Transient CAR expression was detected in patients’ blood after infusion and led to expansion of new immunoglobulin G proteins. Our results provide evidence for the potential anti-tumor activity of mRNA CARTmeso cells, as well as PDAC resistance to the immune response. Keywords: immune response heterogeneity, immune therapy, anti-tumor immunity, pancreatic cancer treatment

BACKGROUND: Anti-BCMA CAR T cells are effective in relapsed/refractory multiple myeloma (RRMM), but most patients eventually progress. Extensive prior therapy and high disease burden in RRMM pts may compromise CAR T cell safety, feasibility, and efficacy, and rare BCMA-neg/CD19+ MM cells with enhanced clonogenic potential may mediate relapse after anti-BCMA CARs. We therefore initiated a trial of anti-BCMA CAR T cells (CART-BCMA) as consolidation of response to prior MM therapy, including high-risk (HR) pts responding to first-line therapy, and combined CART-BCMA with anti-CD19 CAR T cells (CTL119). Both CART-BCMA and CTL119 are 4-1BB/CD3z-based CARs transduced via lentiviral vector. CART-BCMA was previously reported (Cohen et al JCI 2019) and exhibited 64% response rate at 5x108 cell dose following cyclophosphamide (cy) alone as lymphodepleting chemo in RRMM. CTL119 is a humanized version of tisagenlecleucel. METHODS: To assess safety/feasibility of CART-BCMA + CTL119, Phase A (PhA) is evaluating the combination in pts responding (≥MR) to ≥3rd line therapy (or ≥2nd line if exposed to all major agents). After early PhA results showed no excessive toxicity, Phase B (PhB) began enrolling HR pts (R-ISS 3, complex karyotype, PC leukemia, or RESULTS: 6/7 enrolled PhA pts were infused; 1 pt died due to CNS progression before infusion. 4/4 enrolled PhB pts were infused (2 CART-BCMA alone, 2 CART-BCMA + CTL119). See table for age & prior # therapies; 9/10 had HR cytogenetics or were R-ISS 3. Regarding mfg feasibility, 2 PhA pts (05, 07) failed to meet full target doses; all 4 PhB pts achieved full dose. Two subjects (01, 03) had the 3rd (60%) dose held due to early fevers. CRS was gr 0-2 by ASTCT criteria and was observed in pts with both high and low disease burden (table). No neurologic toxicity was observed. Gr 3-4 toxicities were mainly hematologic; 3/6 PhA pts had either ANC CONCLUSIONS: Preliminary results support safety and high initial response-rate of CART-BCMA + CTL119 after cy/flu in MM. In pts with low disease burden responding to prior therapy, including first-line therapy, CAR T cells expanded in vivo and generated clinical responses with low-grade CRS and no neurotoxicity. Preliminarily, hematologic toxicity and feasibility of maint seem more favorable in first-line setting. Addition of CTL119 did not clearly prevent progression after CART-BCMA in the PhA population (3-9 prior lines); data from PhB with randomization +/- CTL119 are immature. Accrual is ongoing, and updated results will be presented at the meeting. Table. Disclosures Garfall: Surface Oncology: Consultancy; Novartis: Patents & Royalties: inventor on patents related to tisagenlecleucel (CTL019) and CART-BCMA, Research Funding; Janssen: Research Funding; Amgen: Research Funding; Tmunity: Honoraria, Research Funding. Cohen:Poseida Therapeutics, Inc.: Research Funding. Lacey:Novartis: Research Funding; Tmunity: Research Funding; Novartis: Patents & Royalties: Patents related to CAR T cell biomarkers. Tian:Novartis: Research Funding. Hwang:Novartis: Research Funding; Tmunity: Research Funding. Vogl:Active Biotech: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Karyopharm Therapeutics: Consultancy; Takeda: Consultancy; Celgene: Consultancy. Lancaster:novartis: Research Funding. Nelson:Novartis: Research Funding. Ferthio:Novartis: Research Funding. Fesnak:Novartis: Research Funding. Melenhorst:National Institutes of Health: Research Funding; IASO Biotherapeutics, Co: Consultancy; Simcere of America, Inc: Consultancy; Incyte: Research Funding; Shanghai Unicar Therapy, Co: Consultancy; Colorado Clinical and Translational Sciences Institute: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding, Speakers Bureau; Stand Up to Cancer: Research Funding; Parker Institute for Cancer Immunotherapy: Research Funding; Genentech: Speakers Bureau. Young:novartis: Research Funding. Levine:Brammer Bio: Membership on an entity's Board of Directors or advisory committees; Vycellix: Membership on an entity's Board of Directors or advisory committees; Incysus: Membership on an entity's Board of Directors or advisory committees; Cure Genetics: Consultancy; Novartis: Consultancy; Novartis: Consultancy, Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership; CRC Oncology: Consultancy; Avectas: Membership on an entity's Board of Directors or advisory committees. Brogdon:Novartis: Employment. Isaacs:Novartis: Employment. June:Tmunity: Other: scientific founder, for which he has founders stock but no income, Patents & Royalties; Novartis: Research Funding. Milone:Novartis: Patents & Royalties, Research Funding. Stadtmauer:Amgen: Consultancy; Novartis: Consultancy, Research Funding; Tmunity: Research Funding; Abbvie: Research Funding; Celgene: Consultancy; Takeda: Consultancy; Janssen: Consultancy.

Background: Relapsed/refractory (rel/ref) MM is associated with progressive immune dysfunction, including reversal of CD4:CD8 T cell ratio and acquisition of terminally-differentiated T cell phenotypes. BCMA-directed CAR T cells have promising activity in MM, but the factors that predict for robust in vivo expansion and responses are not known. In a phase 1 study of CART-BCMA (autologous T cells expressing a human BCMA-specific CAR with CD3ζ/4-1BB signaling domains) in refractory MM patients (median 7 priors, 96% high-risk cytogenetics), we observed partial response (PR) or better in 12/25 (47%) (Cohen et al, ASH 2017, #505). Recently, we demonstrated in CLL pts receiving CD19-directed CAR T cells that certain T cell phenotypes prior to generation of the CAR T product were associated with improved in vivo expansion and clinical outcomes (Fraietta et al, Nat Med 2018). We thus sought to identify pre-treatment clinical or immunological features associated with CART-BCMA expansion and/or response. Methods: Three cohorts were enrolled: 1) 1-5 x 108 CART cells alone; 2) cyclophosphamide (Cy) 1.5 g/m2 + 1-5 x 107 CART cells; and 3) Cy 1.5 g/m2 + 1-5 x 108 CART cells. Phenotypic analysis of peripheral blood (PB) and bone marrow (BM) mononuclear cells, frozen leukapheresis aliquots, and phenotype and in vitro kinetics of CART-BCMA growth during manufacturing were performed by flow cytometry. CART-BCMA in vivo expansion was assessed by flow cytometry and qPCR. Responses were assessed by IMWG criteria. Results: Responses (≥PR) were seen in 4/9 pts (44%, 1 sCR, 2 VPGR, 1 PR) in cohort 1; 1/5 (20%, 1 PR) in cohort 2; and 7/11 (64%, 1 CR, 3 VGPR, 3 PR) in cohort 3. As of 7/9/18, 3/25 (12%) remain progression-free at 11, 14, and 32 months post-infusions. As previously described, responses were associated with both peak in vivo CART-BCMA expansion (p=0.002) as well as expansion over first month post-infusion (AUC-28, p=0.002). No baseline clinical or MM-related characteristic was significantly associated with expansion or response, including age, isotype, time from diagnosis, # prior therapies, being quad- or penta-refractory, presence of del 17p or TP53 mutation, serum hemoglobin, BM MM cell percentage, MM cell BCMA intensity, or soluble BCMA concentration. Treatment regimen given before leukapheresis or CART-BCMA infusions also had no predictive value. We did find, however, that higher CD4:CD8 T cell ratios within the leukapheresis product were associated with greater in vivo CART-BCMA expansion (Spearman's r=0.56, p=0.005) and clinical response (PR or better; p=0.014, Mann-Whitney). In addition, and similar to our CLL data, we found that a higher frequency of CD8 T cells within the leukapheresis product with an "early-memory" phenotype of CD45RO-CD27+ was also associated with improved expansion (Spearman's r=0.48, p=0.018) and response (p=0.047); Analysis of manufacturing data confirmed that higher CD4:CD8 ratio at culture start was associated with greater expansion (r=0.41, p=0.044) and, to a lesser degree, responses (p=0.074), whereas absolute T cell numbers or CD4:CD8 ratio in final CART-BCMA product was not (p=NS). In vitro expansion during manufacturing did associate with in vivo expansion (r=0.48, p=0.017), but was not directly predictive of response. At the time of CART-BCMA infusion, the frequency of total T cells, CD8+ T cells, NK cells, B cells, and CD3+CD56+ cells within the PB or BM was not associated with subsequent CART-BCMA expansion or clinical response; higher PB and BM CD4:CD8 ratio pre-infusion correlated with expansion (r=0.58, p=0.004 and r=0.64, p=0.003, respectively), but not with response. Conclusions: In this study, we found that CART-BCMA expansion and responses in heavily-pretreated MM patients were not associated with tumor burden or other clinical characteristics, but did correlate with certain immunological features prior to T cell collection and manufacturing, namely preservation of normal CD4:CD8 ratio and increased frequency of CD8 T cells with a CD45RO-CD27+ phenotype. This suggests that patients with less dysregulated immune systems may generate more effective CAR T cell products in MM, and has implications for optimizing patient selection, timing of T cell collection, and manufacturing techniques to try to overcome these limitations in MM patients. Disclosures Cohen: Celgene: Consultancy; Novartis: Research Funding; Oncopeptides: Consultancy; Janssen: Consultancy; Poseida Therapeutics, Inc.: Research Funding; Bristol Meyers Squibb: Consultancy, Research Funding; Kite Pharma: Consultancy; GlaxoSmithKline: Consultancy, Research Funding; Seattle Genetics: Consultancy. Melenhorst:Parker Institute for Cancer Immunotherapy: Research Funding; novartis: Patents & Royalties, Research Funding; Casi Pharmaceuticals: Consultancy; Incyte: Research Funding; Shanghai UNICAR Therapy, Inc: Consultancy. Garfall:Amgen: Research Funding; Kite Pharma: Consultancy; Bioinvent: Research Funding; Novartis: Research Funding. Lacey:Novartis Pharmaceuticals Corporation: Patents & Royalties; Parker Foundation: Research Funding; Tmunity: Research Funding; Novartis Pharmaceuticals Corporation: Research Funding. Davis:Novartis Institutes for Biomedical Research, Inc.: Patents & Royalties. Vogl:Karyopharm Therapeutics: Consultancy. Pruteanu:Novartis: Employment. Plesa:Novartis: Research Funding. Young:Novartis: Patents & Royalties, Research Funding. Levine:Novartis: Consultancy, Patents & Royalties, Research Funding; CRC Oncology: Consultancy; Incysus: Consultancy; Tmunity Therapeutics: Equity Ownership, Research Funding; Brammer Bio: Consultancy; Cure Genetics: Consultancy. June:Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Stadtmauer:Takeda: Consultancy; Celgene: Consultancy; Amgen: Consultancy; AbbVie, Inc: Research Funding; Janssen: Consultancy. Milone:Novartis: Patents & Royalties.

A phase I study to evaluate the safety and feasibility of an intravenous infusion of autologous T cells transduced to express a chimeric antigen receptor directed against mesothelin (CART-meso) in patients with mesothelin expressing tumors is being conducted at the University of Pennsylvania. Here we report on the first five patients treated with CART-meso T cell infusion. Five patients with recurrent advanced stage cancers (two serous ovarian, two epithelial mesothelioma, and one pancreatic) who had received 4-12 prior regimens, were treated with a single infusion of CART-meso cells. Apheresed autologous T cells were transduced with a lentiviral vector expressing the CAR construct composed of an extracellular anti-mesothelin single chain variable fragment derived from mouse monoclonal antibody (SS1) fused to the intracellular signaling domains of 4-1BB (CD137) and TCRzeta. Each patient received a single dose of 1-3 × 10^7 CART-meso cells/m2. No lymphodepletion with chemotherapy was given prior to infusion. All subjects enrolled were successfully infused and there were no acute adverse events (AE) with infusion. To date, Grade 4 AEs have included sepsis(1), anemia(1), pleural effusions(1), tachypnea(1), dyspnea(1), and aspiration(1) and Grade 3 AEs have included sepsis(1), leukocytosis(2), DIC(1), tachycardia(1), dyspnea(1), hypotension(1), dusky extremities(1), fatigue(1), ascites(1), peritonitis(1), elevated LFTs(1), portal vein hypertension(1), and acute kidney injury(1). After infusion, CART-meso T cells were transiently detectable by PCR in the peripheral blood in all patients up to Day 21-28 after infusion. CART-meso cells were found to traffic to tumor sites within the peritoneum and liver, as well as to off-tumor on-target sites such as pericardial fluid, but without clinical toxicity of pericarditis. Expansion of CART-meso cells was also observed in the pleural fluid of one patient who had a known malignant pleural effusion. Cytokine profiling of the peripheral blood revealed elevations of IL-6 and VEGF with significant elevations seen in the one patient with malignant pleural effusion. No elevations in TNFα or IFNγ were found. Anti-tumor efficacy is suggested by the clearing of malignant cells in the pleural fluid of one patient on Day 21 and Day 26 after infusion, as well as radiological and clinical evidence of stable to decreased burden of disease in one patient. Patient follow-up for 1-3 months post infusion has revealed no evidence of long-term toxicities to date. These interim results suggest that intravenous infusion of CART-meso T cells without lymphodepletion is feasible and safe. In these five patients with metastatic mesothelin expressing cancers, the infusion was well tolerated with no off-tumor on-target toxicities. Laboratory and clinical findings suggest infused T cells are effective and functional. Citation Format: Janos L. Tanyi, Andrew R. Haas, Gregory L. Beatty, Mark A. Morgan, Caitlin J. Stashwick, Mark H. O'Hara, David L. Porter, Marcela V. Maus, Bruce L. Levine, Simon F. Lacey, Anne Marie Nelson, Maureen McGarvey, Naseem DS Kerr, Gabriela Plesa, Carl H. June. Safety and feasibility of chimeric antigen receptor modified T cells directed against mesothelin (CART-meso) in patients with mesothelin expressing cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT105. doi:10.1158/1538-7445.AM2015-CT105