Abstract: CRISPR-Cas9 gene editing provides a powerful tool to enhance the natural ability of human T cells to fight cancer. We report a first-in-human phase 1 clinical trial to test the safety and feasibility of multiplex CRISPR-Cas9 editing to engineer T cells in three patients with refractory cancer. Two genes encoding the endogenous T cell receptor (TCR) chains, TCRα ( TRAC ) and TCRβ ( TRBC ), were deleted in T cells to reduce TCR mispairing and to enhance the expression of a synthetic, cancer-specific TCR transgene (NY-ESO-1). Removal of a third gene encoding programmed cell death protein 1 (PD-1; PDCD1 ), was performed to improve antitumor immunity. Adoptive transfer of engineered T cells into patients resulted in durable engraftment with edits at all three genomic loci. Although chromosomal translocations were detected, the frequency decreased over time. Modified T cells persisted for up to 9 months, suggesting that immunogenicity is minimal under these conditions and demonstrating the feasibility of CRISPR gene editing for cancer immunotherapy.

Abstract: Background: Autologous T cells genetically modified with a lentiviral vector to express affinity-enhanced T cell receptors (TCR) or chimeric antigen receptors have shown great promise for the treatment of cancer. NY-ESO-1 is a cancer testis antigen with little normal tissue expression but with aberrant expression in MM, sarcomas, and melanomas. An HLA-A201 restricted TCR recognizing the NY-ESO-1/LAGE-1 157-165 epitope (SLLMWITQC) kills NY-ESO positive cell lines and has been used to treat 25 patients with MM after ASCT with expansion, persistence, antigen-directed functionality and long-term safety and antitumor activity (Nat Med 2015, Blood Adv 2019). We hypothesized removal of the genes encoding the endogenous TCR, TCRα (TRAC) and TCRβ (TRBC), would enhance NY-ESO TCR expression and reduce TCR mispairing and with removal of PD-1 (PDCD1) would enhance activity and persistence. We previously demonstrated CRISPR/Cas9 and TCRα, TCRβ and PDCD1 targeting gRNAs could be successfully introduced via electroporation in preclinical models to disrupt gene expression (Clin Cancer Res 2017). We therefore began a phase 1 pilot clinical trial for pts with advanced MM and sarcoma of NY-ESO-1 TCR-expressing T cells with CRISPR/Cas9 TCRα, TCRβ and PDCD1 edited genes to assess safety, feasibility and activity (NCT03399448). Methods: Adults with HLA-A*0201 and expressing NY-ESO-1 and/or LAGE-1 antigen with advanced MM, synovial sarcoma, and myxoid/round cell liposarcoma (MRCL) with adequate performance and organ function and, for MM relapsed or refractory to at least 3 prior regimens and, for MRCL, proven metastatic disease or surgically inoperable local recurrence, were enrolled. Autologous T cells were transfected with Cas9 protein complexed with single guide RNAs against TRAC, TRBC and PDCD1 and subsequently transduced to express NY-ESO-1-specific TCR at the University of Pennsylvania. Frequency of NYCE T cells in final product was measured by flow cytometric dextramer analysis. Once cells were successfully manufactured and released, pts received fludarabine 30mg/m2 and cyclophosphamide 300mg/m2 daily on day -4,-3,-2. On Day 0 pts received a single infusion of thawed NYCE T cells as an out-patient. Pts were monitored closely for the first 28 days, monthly till 6 mo and then followed every 3 mo for adverse events, antitumor response and survival, NYCE T cell expansion, persistence, trafficking, phenotype and function, and immunogenicity. An assessment after accrual of the first 3 subjects in this ongoing trial was planned and is reported here. Results: 3 pts, 2 with MM and 1 with MRCL, have received NYCE T cells. Pt 1 is a 67 y/o F with IgG kappa MM with lytic bone lesions, and a +17q after 8 lines of therapy including 3 ASCTs, lenalidomide, pomalidomide, bortezomib, carfilzomib, daratumumab, and panobinostat. Pt 2 is a 65 y/o M with a recurrent MRCL manifested by abdominal and pelvic involvement after neo-adjuvant doxorubicin, multiple resections and radiation treatments with progression at time of enrollment. Pt 3 is a 62 y/o F with kappa light chain MM with lytic bone lesions and plasmacytomas and a +1q after 7 lines of therapy including lenalidomide, pomalidomide, bortezomib, carfilzomib, daratumumab, 2 ASCTs and an immunoconjugate . Manufacturing for these pts resulted in satisfactory products with 89.4 to 96% viability, transduction efficiency by qPCR of 0.04 to 0.2 copies/cell , residual Cas9 concentration 0 to 0.37 ng/ml, dextramer 0.4 to 1.8% NY-ESO-1 expression. TRAC, TRBC, and PDCD1 disruption efficiency was 44.3 to 49.4, 3.61 to 15.7 and 15.6 to 20.2% respectively. Pts tolerated treatment well without neurotoxicity or CRS. By day +60 pt 1 progressed by IMWG. Pt 2 received 1 U PRBC. By day +90 he remained with stable disease by serial CT scans. Pt 3 is too early to evaluate. Serial qPCR for copies of lentiviral transcripts in peripheral blood and tumor biopsies for pts 1+2 showed in vivo expansion, stable persistence and tumor targeting (Figure). Conclusion: Early results of a phase 1 trial of NYCE T cells infused in 3 pts with advanced MM and MRCL show safety and feasibility and viable, expanding, and persisting CRISPR/Cas9 gene edited T cells that trafficked to tumor. The persistence of the NYCE T cells suggests that immunogenicity from multiplexed gene-editing using Cas9 is minimal under these conditions. Further characterization of phenotype and function of these cells and clinical outcomes will be presented. Figure Disclosures Stadtmauer: Celgene: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Novartis: Consultancy, Research Funding; Tmunity: Research Funding; Abbvie: Research Funding. Cohen:Poseida Therapeutics, Inc.: Research Funding. Lacey:Novartis: Patents & Royalties: Patents related to CAR T cell biomarkers; Tmunity: Research Funding; Novartis: Research Funding. Melenhorst:Incyte: Research Funding; Novartis: Research Funding, Speakers Bureau; Parker Institute for Cancer Immunotherapy: Research Funding; Genentech: Speakers Bureau; Stand Up to Cancer: Research Funding; IASO Biotherapeutics, Co: Consultancy; Simcere of America, Inc: Consultancy; Shanghai Unicar Therapy, Co: Consultancy; Colorado Clinical and Translational Sciences Institute: Membership on an entity's Board of Directors or advisory committees; National Institutes of Health: Research Funding. Fraietta:Tmunity: Research Funding; Cabaletta: Research Funding; LEK Consulting: Consultancy. Mangan:amgen: Speakers Bureau; takeda: Speakers Bureau; celgene: Speakers Bureau; janssen: Speakers Bureau. Lancaster:novartis: Research Funding. Suhoski:novartis: Research Funding. Fesnak:Novartis: Research Funding. Young:novartis: Research Funding. Chew:tmunity: Other: Scientific Founder, Research Funding; novartis: Research Funding. Zhao:Tmunity: Membership on an entity's Board of Directors or advisory committees, Research Funding; novartis: Research Funding. Hwang:Novartis: Research Funding; Tmunity: Research Funding. Hexner:novartis: Research Funding. June:Novartis: Research Funding; Tmunity: Other: scientific founder, for which he has founders stock but no income, Patents & Royalties.

Abstract: 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 〈 PR or early progression on imid/PI induction) responding to first- or second-line therapy, 〈 1y from diagnosis, and unexposed to cytotoxic chemo (except low-dose cy). PhB patients are randomized to receive CART-BCMA +/- CTL119. Both products are dosed at 5x108 CAR+ cells in 3 divided doses (10%, 30%, 60%) after cy 300 mg/m2 + fludarabine 30 mg/m2 daily x 3d (cy/flu). Pts receive maintenance (maint) lenalidomide or pomalidomide beginning d30 or upon recovery from toxicity. Here we report initial results of 6 PhA and 4 PhB pts. 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 〈 1000 and plt 〈 50K at day 30, whereas 4/4 PhB pts recovered ANC & plts by day 30. Aside from CRS, serious AEs ≥possibly related to CAR T cells included pneumonia, sepsis-associated AKI, CMV, pancreatitis, and bone pain, all occurring in PhA pts. Among patients with IMWG-measurable disease, ≥PR was observed in 5/5 PhA pts, including 1 pt with VGPR despite only 10% CART-BCMA dose (pt 05), and 3/3 PhB pts. Responses are ongoing in 2/6 PhA and 3/4 PhB patients (table). Initiation of maint was feasible in 4/6 PhA (median start d67) and 4/4 PhB (median start d48) pts; no recurrence of CRS was observed with maint initiation. All pts exhibited in vivo expansion of both CART19 and CART-BCMA, including those with low disease burden; no re-expansion was observed with maint initiation. Among 5 patients with ongoing responses at day 90 who were evaluable for MRD by flow cytometry, 4 had MRD with detectable BCMA expression (dim in 2 pts, bright in 1 pt, variable in 1 pt), and 1 was MRD-negative (sensitivity ~10-5); 4/5 exhibited absence of normal plasma cells, suggesting possible ongoing anti-BCMA immune surveillance but resistance of residual MM PCs. Among 8 patients who received both CART-BCMA and CTL119 in either PhA or PhB, 5 had ongoing absence of circulating B cells at last follow-up, including two pts with ongoing responses at 1y and 4m, providing evidence for long-term CAR T cell activity in MM patients. 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.

Abstract: Background Immunotherapy with anti-CD19 CART cells (CART19) induces complete remission (CR) in the minority of patients with CLL; however, these CRs tend to be durable (Porter Sci Tr Med 2015). Based on preclinical evidence of synergy, we combined anti-CD19 CAR T cells with ibrutinib to test the hypothesis that pre- and concurrent treatment enhances the CR rate. Methods This is a pilot trial of autologous anti-CD19 CAR T cells in adults with CLL/SLL who were not in CR despite at least 6 months of ibrutinib. T cells were lentivirally transduced to express a CAR comprising CD3z, 4-1BB, and humanized anti-CD19 scFv (CTL119). Pts underwent lymphodepleting chemotherapy up to 1 week before infusion, followed by planned infusion of 1-5x108 CTL119 cells dosed as 10%, 30% and 60% of the total planned dose over 3 days, with doses beyond dose#1 given only in the absence of fever or cytokine release syndrome (CRS). Results CTL119 manufacturing was successful for all pts. Twenty pts were enrolled and 19 were infused (one pt was not infused due to intercurrent large cell transformation and newly diagnosed adenocarcinoma). Of the 19, 15 were male, the median age was 62 (range 42-76); and 5 were on 1st line ibrutinib. Of the remaining 14, the median number of prior therapies was 2 (range 1-16), and 3 pts had received prior murine CART19 therapy (CTL019) without ibrutinib. Eleven pts had abnormalities of chromosome 17p or TP53. An additional 3 pts had abnormalities of chromosome 11q22 or ATM. All pts had marrow involvement (median 21% range 7-63%). For 9 pts who had enlarged nodes at baseline, the median cross-sectional area was 1471 mm2 (range 178-2220). All pts received at least 2 CTL119 doses; 14 patients received all 3 and 5 received 2 doses. The median number of CART cells given was 5.3x106/kg (range 2.0-7.5). Median peak CART cell number by qPCR was 90,990 copies/ug genomic DNA (range 965-210,556) and by flow cytometry was median 536 CART cell/ul blood (range, 0-3640). 18/19 (95%) pts experienced CRS, with a median Penn CRS Grade of 2. CRS was grade 1-2 and 3-4 in 15 and 3 pts, respectively. Two pts received tocilizumab. Of 5 pts with encephalopathy, 2 were CTCAE gr 1, 2 gr 2 and 1 gr 4. One pt died on D14 from a cardiac arrhythmia during severe neurotoxicity after resolution of CRS. There were 49 gr 3 and 22 gr 4 toxicities in total. As of 7/16/18, 18/19 pts are alive (95%) and 12 pts have been followed for at least 12 months. The median follow-up for the 18 surviving pts is 18.5 months (range 8-28). Per iwCLL criteria, at 3 months 14 pts were evaluable and their responses were CR (n=6), PR (n=4), SD (n=3), PD (n=1). Marrow responses at month 3 were available in 18 and showed a morphologic CR in 17 pts (94%); of these 15 also had no measurable residual disease (MRD) by 9-color flow cytometry. MRD was also assessed at 3 months by deep sequencing of the immunoglobulin heavy chain locus (limit of detection 1 B cell in 1x106 nucleated cells). 14/18 pts were MRD negative, and the remaining 4 had 3.36, 4.76, 1.79, and 0.48 log10 reduction of the leukemic clonotype relative to the baseline sample. LN biopsies from 2 pts 3 and 10 months after CTL119 confirmed absence of the CLL clonotypes in this compartment as well. At 12 months, 11 pts had evaluable marrows of which 10 (91%) were in morphologic CR and 1 showed morphologic relapse. Of the 10 in morphologic CR, three pts showed low MRD positivity (3.58, 2.34, 3.79 log10 reduction) and the rest remained MRD-ve. Of the 3 pts who had received murine CTL019 previously, 2 were in MRD+ve CR at 12 months and one was refractory to humanized CTL119. Six pts discontinued ibrutinib at a median 8 months (range 3-12) due to toxicity (n=2) or pt choice (n=4). Five pts remain MRD-ve at short followup. In total, 16/18 pts remain in morphologic and/or flow CR at last followup. Conclusion In patients not achieving CR despite at least 6 months of ibrutinib who were treated with humanized CART19, we found an iwCLL CR rate of 43% and a bone marrow remission rate of 94% including a 78% MRD negative response by deep sequencing. This compares favorably to prior CART19 cell studies in patients with progressive CLL (iwCLL CR rates of 21-29%). CRS was frequent but mild-moderate and did not commonly require anti-cytokine therapy. These results suggest that the combination of CTL119 with ibrutinib results in a high rate of sustained responses and high rates of MRD-ve marrow response in patients with CLL. This combination will be further tested in larger studies. Figure. Figure. Disclosures Gill: Carisma Therapeutics: Equity Ownership; Extellia: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding. Frey:Servier Consultancy: Consultancy; Novartis: Consultancy. Mato:Johnson & Johnson: Consultancy; Portola: Research Funding; Acerta: Research Funding; AstraZeneca: Consultancy; AbbVie: Consultancy, Research Funding; Pharmacyclics, an AbbVie Company: Consultancy, Research Funding; Regeneron: Research Funding; TG Therapeutics: Consultancy, Research Funding; Celgene: Consultancy; Medscape: Honoraria; Prime Oncology: Honoraria. Lacey:Novartis Pharmaceuticals Corporation: Patents & Royalties; Tmunity: Research Funding; Parker Foundation: Research Funding; Novartis Pharmaceuticals Corporation: Research Funding. Melenhorst:Novartis: Patents & Royalties, Research Funding; Incyte: Research Funding; Tmunity: Research Funding; Shanghai UNICAR Therapy, Inc: Consultancy; CASI Pharmaceuticals: Consultancy. Davis:Novartis Institutes for Biomedical Research, Inc.: Patents & Royalties. Schuster:Gilead: Membership on an entity's Board of Directors or advisory committees; Physician's Education Source, LLC: Honoraria; Novartis Pharmaceuticals Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Consultancy, Honoraria, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Nordic Nanovector: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Dava Oncology: Consultancy, Honoraria; Genentech: Honoraria, Research Funding; OncLive: Honoraria. Siegel:Novartis: Research Funding. Isaacs:Novartis: Employment. June: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; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding. Porter:Kite Pharma: Other: Advisory board; Genentech: Other: Spouse employment; Novartis: Other: Advisory board, Patents & Royalties, Research Funding.

Abstract: 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