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.

7004 Background: A substantial proportion of patients (pts) with relapsed/refractory (R/R) non-Hodgkin lymphomas (NHL) will not derive a long-term benefit from the existing anti-CD19 chimeric antigen receptor (CAR) T cells. To enhance therapeutic efficacy, we have engineered huCART19-IL18, a 4th generation 4-1BB anti-CD19 construct, armored with the ability to secrete the pro-inflammatory cytokine, IL-18. Methods: This is a first-in-human trial using huCART19-IL18 for CD19+ B-cell malignancies (NCT04684563). Expedited 3-day manufacturing is utilized to limit T-cell exhaustion. To be eligible for the NHL cohort, pts must be R/R to prior anti-CD19 CAR T cells if indicated by FDA label. Dose levels (DL) between 3x106 and 3x108 of huCART19-IL18+ cells are administered as a single IV infusion following lymphodepleting chemotherapy. Bridging therapy is optional. Responses are first assessed at 3 months (mo) using Lugano criteria. Results: As of January 20, 2024, 21 pts with CD19+ NHL were infused with huCART19-IL18. Characteristics include median age 64 yrs (47-74), 76% male, 9 (43%) DLBCL, 6 (29%) FL, 3 (14%) MCL, 2 (10%) tFL, 1 (5%) HGBCL. Median number of prior Rx was 7 (4-14) with 20 (95%) pts R/R to prior anti-CD19 CAR T cells. Manufacturing of DL5 (3x108) was not feasible due to inability to achieve the target dose in 4/6 (67%) pts assigned to DL5. 18 (86%) pts received bridging. 3 pts received DL1 (3x106), 4 pts DL2 (7x106), 1 pt non-defined dose (2.8x107), 6 pts DL3 (3x107), 5 pts DL4 (7x107), 2 pts DL5 (3x108). No study-related deaths occurred in 21 safety-evaluable pts. CRS occurred in 15 (71%) pts: G1 in 8 (38%), G2 in 4 (19%), G3 in 3 (14%). ICANS occurred in 3 (14%) pts: G1 in 2 (10%), G2 in 1 (5%). The most common G3 adverse events at least possibly related to huCART19-IL18 included fatigue (38%), hypotension (29%), and low fibrinogen (23%). 20 pts are efficacy evaluable with median (m) follow-up of 15 mo (3-31). The 3 mo ORR was 80% (90% CI: 60-93%), with CR 50% (90% CI: 30-70%) and PR 30% (90% CI: 14-51%). mDOR was 10 mo (5.5-NR). mPFS was 8.7 mo (90% CI 5-NR), and mOS was NR (90% CI 25 mo-NR). We detected continued persistence of huCART19-IL18 in pts with 24 mo follow-up. No correlation between cell dose and outcome was identified, but response rates and mean expansion (copies/µg gDNA) were higher in pts previously exposed to CD28 CAR than those who had prior 4-1BB CAR (Table). Conclusions: Treatment with huCART19-IL18 has an acceptable safety profile and produced durable remissions in heavily pre-treated pts with R/R NHL despite prior CAR T-cell therapy. The subtype of the preceding CAR product may influence the expansion and effectiveness of huCART19-IL18. Clinical trial information: NCT04684563 . [Table: see text]

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

Introduction: Lymphoma (NHL) patients (pts) relapsing/refractory (R/R) to anti-CD19 chimeric antigen receptor T-cells (CART) represent a challenging group in need of effective therapies. HuCART19-IL18 is a 4th generation 4-1BB CART product designed to express humanized anti-CD19 CAR and secrete interleukin 18, a pro-inflammatory cytokine shown to enhance CART efficacy in pre-clinical models. Its humanized scFv may allow for better persistence, and the additional use of a novel expedited 3-day manufacturing protocol may improve the product’s potency. Methods: We are conducting a first-in-human trial of huCART19-IL18 in pts ≥18 years old with CD19+ R/R B-cell NHL or CLL, who have had at least 2 prior lines of therapy including failure of prior CART. Using a modified Bayesian optimal interval dose titration design, we are exploring doses between 3 and 300 million huCART19-IL18+ cells. The product is administered as a single IV infusion following lymphodepleting (LD) chemotherapy. Bridging therapy is optional and huCART19-IL18 re-treatment is permitted for pts not achieving complete response (CR). Responses are assessed at 3, 6, 9, and 12 months (mo) using Lugano criteria for NHL and revised iwCLL criteria for CLL. Results: As of 3 March 2023, 16 pts have enrolled. 15 pts had huCART19-IL18 manufactured and all achieved a minimum protocol-defined dose. The 13 pts infused to date include 5 DLBCL, 4 FL, 2 MCL, 1 HGBCL, 1 THRBCL pts. The median age is 65 years (53–74), 77% male, 92% had prior anti-CD19 CART (axi-cel 6, tisa-cel 4, brex-cel 1, tisa-cel+liso-cel 1) with 67% relapsed and 33% refractory to prior CART. The median number of prior therapies was 8 (4–14); 11 (85%) pts received bridging therapy. LD chemotherapy was used in 12 (92%) pts. Three were treated at DL1 (3 × 106 CART+ cells), 2 at DL2 (7 × 106), 1 at dose between DL2-3 (2.8 × 107), 6 at DL3 (3 × 107) and 1 at DL4 (7 × 107). Two pts underwent re-treatment (at DL1 and DL3). Twelve pts are evaluable for safety. There were no study related deaths. Most common possibly related non-hematologic G3/4 toxicities included infection (17%), CRS-related hypoxia (17%) and hypotension (17%). CRS was seen in 7 (58%) pts (G1 in 4, G2 in 2, G3 in 1) and ICANS in 2 (17%) pts (G1 in 1, G2 in 2), which were transient/reversible with 3 (25%) pts requiring tocilizumab. 11 pts are evaluable for efficacy with 3-month overall response rate 82% (90% CI: 53–97) and CR rate 55% (90% CI: 27–80). 1 pt with PR at 3 mo achieved sustained CR after huCART-IL18 re-treatment. At median follow-up 12 mo (3–20) for evaluable pts, responses are durable (median not reached) and all pts are alive as shown in Figure 1. Correlative analyses of CART expansion/persistence and cytokine levels are ongoing. The research was funded by: Institutional funds from the University of Pennsylvania Keywords: aggressive B-cell non-Hodgkin lymphoma, cellular therapies Conflicts of interests pertinent to the abstract J. Svoboda Consultant or advisory role: Seagen, Pharmacyclics, Incyte, Genmab, BMS, Atara, Astra Zeneca, Adaptive, ADCT Research funding: TG, Seagen, Pharmacyclics, Merck, Incyte, BMS, Astra Zeneca, Adaptive D. L. Landsburg Consultant or advisory role: Karyopharm, Epizyme, Morphosys, Calithera, ADCT Research funding: Curis, Triphase E. A. Chong Consultant or advisory role: Beigene, Tessa, BMS, KITE, Novartis S. K. Barta Consultant or advisory role: Janssen, Kyowa Kirin, Affimed, Daiichi Sankyo Honoraria: Kyowa Kirin, Acrotech, Seagen S. D. Nasta Research funding: Roche, Gilead, Rafael, Pharmacyclics M. Ruella Employment or leadership position: viTToria Consultant or advisory role: BMS, GSK, Bayer, NanoString, AbClon Research funding: Beckman Coulter, NanoString, AbClon E. O. Hexner Consultant or advisory role: Blueprint Medicines, PharmaEssentia Research funding: Blueprint Medicines, Samus Therapeutics, Novartis, Tmunity M. M. Davis Consultant or advisory role: Novartis, Cellares Research funding: Tmunity N. Frey Consultant or advisory role: Kite Pharmaceuticals, Sana Biotechnology, Mneumo therapeutics, Pfizer B. Levine Employment or leadership position: Tmunity Therapeutics, Capstan Therapeutics Consultant or advisory role: Akron, Avectas, Brammer Bio, Immuneel, Immusoft, In8bio, Ori Biotech, Oxford Biomedica, UTC Therapeutics, Vycellix D. L. Porter Consultant or advisory role: DeCART, BMS, Bluebird Bio, Kadmon, Angiocrine, Mirror Biologics, Genentech, Jazz, Janssen, Gerson Lerham, Incyte, Gilead, Novartis Stock ownership: Roche, Tmunity Honoraria: Elsevier, Adecept Bio Research funding: Novartis S. J. Schuster Consultant or advisory role: BMS, Genentech, Nordic Nanovector, Loxo, Roche, AstraZeneca, Fate, Genmab, Mustang Biotech, BeiGene, Regeneron, MorphoSys, Novartis, Incyte, Legend Biotech Research funding: Merck, AbbVie, BMS, Genentech, TG, DTRM, Adaptive, Roche, Pharmacyclics, Novartis, Incyte C. H. June Employment or leadership position: Tmunity, Capstan Therapeutics, Poseida Consultant or advisory role: AC Immune, BluesphereBio, Cabaletta, Carisma, Cellares, Alaunos, Verismo Research funding: Capstan Therapeutics

Background: Efforts to augment the efficacy of cell therapies include development of 4th generation chimeric antigen receptor (CAR) T-cells delivering a transgenic activator protein to the tumor target. Interleukin-18 (IL-18) is a pro-inflammatory cytokine shown to enhance CAR T-cell proliferative potency and antitumor activity in pre-clinical models (Hu et al, Cell Reports 2017). Methods: We are conducting a first-in-human trial using huCART19-IL18, a 4th generation autologous CAR T-cell product transduced by lentiviral vector to co-express humanized anti-CD19 CAR and IL-18 in patients (pts) with relapsed/refractory B-cell non-Hodgkin lymphomas (NHL) or chronic lymphocytic leukemia (CLL) (NCT04684563). We are using a Bayesian optimal interval dose titration design exploring doses between 3 and 300 million huCART19-IL18 cells per pt. The ex vivo culture time for manufacturing is reduced to 3 days to further improve T-cell activity/persistence and to shorten the time from apheresis to treatment. For this ongoing phase 1 trial, pts must be ≥ 18 years old, have CD19+ relapsed/refractory B-cell NHL or CLL, and have had at least 2 lines of therapy including failure of prior CAR T-cell therapy (if indicated by FDA label). The primary objective is to define the recommended phase 2 dose and evaluate the safety of huCART19-IL18; secondary objectives are feasibility, efficacy, and characterization of pharmacokinetics. Following apheresis, optional bridging therapy is permitted. HuCART19-IL18 cells are administered as a single IV infusion 2-5 days after lymphodepleting chemotherapy (LD). Pts with clinical benefit are eligible to receive retreatment. Dose-limiting toxicity (DLT) observation period is 28 days after infusion. Responses are assessed using Lugano criteria for NHL and revised iwCLL criteria for CLL at months (M) 3, 6, 9, and 12. Results: As of July 14, 2022, 9 pts have enrolled, and 8 pts have been infused and are evaluable for safety (DLBCL 3, MCL 2, THRBCL 1, HGBL 1, FL 1). At enrollment, median age was 65 years (56-75), 75% were males, median ECOG PS was 1 (0-1), the median number of prior therapies was 6.5 (range 4-13), 7/8 (88%) pts relapsed after prior CAR-T (3 post axi-cel, 3 post tisa-cel, 1 post brex-cel). The best responses to prior CAR T-cell therapy were CR in 3, PD in 3, PR in 1. Seven pts (88%) received systemic bridging therapy including 5 (63%) who also had radiation. Pts receiving LD were treated with bendamustine (90 mg/m2 x 2 days). With staggered enrollment to allow for DLT evaluation, the median time from apheresis to infusion was 47 days (26-82). The first pt was infused with DL1A (3x106 cells) without LD. Subsequently, 2 were infused with DL1B (3x106 cells), 1 with DL2 (7x106), and 2 with DL3 (3x107), all after LD. Manufacturing for 2 products did not meet the target dose but exceeded minimum infusible dose and pts were treated: 1 with DL2 (7x106) and 1 with dose between DL2 and DL3 (2.8x107). Cytokine release syndrome (CRS) occurred in 4 (50%) pts: Grade (G)1 in 2, G2 in 1, G3 in 1 with median onset at 7.5 days (2-8) and median duration of 5.5 days (5-11); 2 pts required anti-cytokine therapy. Neurotoxicity occurred in 2 (25%) pts: 1 on day 20 lasting for 2 days (G1) and 1 on day 8 for 6 days (G2). Other non-hematologic G3 or higher adverse events at least possibly related to huCART19-IL18 included infections in 2 (25%), hypotension in 2 (25%), and AST elevation in 1 (12.5%) in the setting of CRS. There have been no study-related deaths. Of 7 pts who are evaluable for response (DLBCL 3, THRBCL 1, MCL 2, FL 1), the ORR at M3 is 100% (CR 57%, PR 43%). Of the 3 pts with PR at M3, 1 pt was re-treated with huCART19-IL18 at M4 (achieved CR at M3 after re-treatment and remains in CR), 1 pt was taken off study in PR to pursue alternative therapy, and 1 pt progressed at M5 with CD19-negative disease and is receiving alternative therapy. None of the 4 pts who achieved CR at M3 have progressed to date including FL pt refractory to axi-cel who is in sustained CR over 12 M after huCART19-IL18 at DL1B (Figure 1A). All pts are alive at median follow-up of 8 M (1.9-14.1). Figure 1B shows huCART19-IL18 cell expansion and persistence for each pt. Conclusions: In this first-in-human study, huCART19-IL18 shows a manageable toxicity profile and encouraging early efficacy across all dose levels in heavily pretreated pts with CD19+ NHL including those who did not respond to prior 2nd generation CAR T-cell products. Enrollment continues at DL3. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal