Initial Results of a First-in-Human, Phase I Study Point-of-Care Manufacturing of Trispecific CAR-T Cells Targeting CD19/20/22 in B-Cell Malignancies
Authors: Vasu Sumithira , Bezerra Evandro , Denlinger Nathan , Szuminski Nicole , Schneider Dina , Dash Pradyot , Wirthlin Louisa , Epperla Narendranath , Sawalha Yazeed , Woyach Jennifer A. , Rogers Kerry A. , Bhat Seema A , Behbehani Gregory K , Larkin Karilyn T. , Kittai Adam S , Chan Wing , Krull Ashley Angell , Nishat Shamama , O'Donnell Lynn , Alinari Lapo , de Lima Marcos 
Published: December 2024

Introduction: Targeting CD19, 20, 22 which are ubiquitously expressed on B-cell malignancies may decrease the risk of antigen-negative and antigen positive relapses. Our preliminary data showed feasibility of in-house manufacturing of Trispecific CAR-Ts and confirmed their specificity and killing activity in in vitro and in vivo B-cell lymphoma models. Here we report the results of a first-in-human phase I study evaluating Trispecific CAR-T cells targeting CD19, 20 or 22 (NCT05418088).

Methods: The duoCAR20.19.22D94 (Lentigen Technology, Inc) designed for Trispecific targeting of CD19, CD20, and CD22 is comprised of two separate CAR molecules co-expressed in each transduced T cell: a tandem second-generation CAR comprised of scFv targeting antigens CD20 and CD19, respectively, hinge and transmembrane domain derived from CD8, OX40 co-stimulatory domain, and CD3ζ activation domain, and a first generation CD22-targeting CAR with anti CD22 scFv binder domain, CD8- derived hinge and transmembrane domains and CD3ζ activation domain. Manufacturing of Trispecific CAR-T cells utilized the Miltenyi DuoCAR lentiviral vector, and CliniMACS Prodigy device using TransAct T cell reagent. The day after apheresis, both manufacturing and lymphodepleting chemotherapy (Cyclophosphamide 60mg/kg IV on day -6, Fludarabine 25mg/m2 IV on days -5 to -3) was initiated. Protocol initially allowed CAR-naïve patients and was later amended to include Commercial CD19 CAR-T failures. Cohort A included B-cell NHL with lesions &lt; 5 cm, CLL, B-PLL and utilized a fresh product. Cohort B included B-ALL, Richter's transformation (RT) and B-cell NHL with lesions &gt; 5cm. 40% of the CAR-T product was infused as a fresh infusion on day 0 and the remainder was infused as a cryopreserved product on day +7. Prophylactic tocilizumab was administered 30 minutes prior to receiving CAR-T cells for both cohorts. There were three dose levels (DL): 0.5 x 10e6/kg, 1.0x 10e6/kg and 2.0 x 10e6/kg.

Results: 17 patients were eligible and 16 started lymphodepleting chemotherapy. Successful product manufacturing was achieved in 15/16 patients. One patient had a manufacturing failure following apheresis and subsequent recollection also resulted in unsuccessful manufacturing. Median transduction efficiency was 26% (range: 11.25%-43.1%), and median fold expansion was 5 (range: 1.81- 11.46). Median time to manufacturing was 7 days (range: 6-10 days). Cohort A included 3 patients with CLL, 1 with B-PLL, 1 with DLBCL and 1 with FL. Cohort B included 2 patients with DLBCL, 3 with RT, 2 had ALL, 2 had MCL and 1 had lymphoid blast crisis from CML.

Out of 15 patients, CR was achieved at day 30 in 2 patients, by day 90 in 4 patients, with a total of 6 patients who achieved CR. CR was observed in MCL, FL, DLBCL and B-ALL. Among 9 NHL (3 with RT) patients, 5 patients achieved CR and 3/5 remain in CR beyond 1 year post CAR-T. CR was seen at dose level (DL) 1 (n=2), DL 2 (n=2) and DL3 (n=2). Response was noted in a B-ALL patient who had progressed after commercial CD19 CART. CRS was seen in 3 patients, max grade 2 (2 patients G1, 1 patient G2) and 3 patients required tocilizumab, ICANS Grade 1 was seen in 1 patient. No HLH was observed. None of thepatients required steroids.One death was observed from intracranial hemorrhage in a B-ALL patient with persistent disease at day 32. No responses were observed in Richter's transformation. Stable disease was observed at day 30 in CLL and B-PLL. Two patients in CR developed other malignancies- one pleomorphic sarcoma and prostate cancer, both deemed unrelated to Trispecific CAR-T.

Of those who relapsed, 3 patients had a biopsy to evaluate CD19, 20 or 22 expression pre-and post CAR-T. In one patient, CD20 positivity changed to CD20 dim expression upon relapse. In another patient, CD20 negative status changed to CD20 + after CAR-T. We studied persistence data of circulating Trispecific CAR-T cells using PCR. We measured a maximum of 600 copies per ng DNA by day 20, with the highest levels of expansion detected in the higher dose levels.

Conclusions: Initial results from this first-in-human Trispecific CAR-T targeting CD19/20/22 show feasibility of manufacturing and that the product is safe with very low incidence of CRS/ICANS and no HLH. Despite lack of persistence of cells beyond day 30, durable remissions have been observed. Remissions have been observed in MCL, FL, DLBCL and B-ALL. Further investigations into mechanisms of response vs. resistance are ongoing.