Preliminary Safety and Tolerability of CD19x22 CAR-T Cell Therapy in Adolescent and Adult Patients with Relapsed or Refractory B-Cell Non-Hodgkin Lymphoma

Introduction: Although one-third of R/R large B-cell lymphoma (LBCL) patients receiving CD19-directed CAR-T therapy will have durable remission, nearly 60% will be refractory or relapse . Given that CD19 escape is an established mechanism of CAR-T resistance, we developed a bicistronic construct encoding a CD19 CAR incorporating a CD28 costimulatory domain and a CD22 CAR incorporating a 41BB costimulatory domain allowing coexpression of both CARs. Both CAR constructs have been individually validated in the clinic with a well-established safety and efficacy.

Methods: Bicistronic CD19x22 CAR constructs were designed using a 2A ribosomal skip sequence, optimized to generate high and concordant expression of both CARs. The lead construct was tested in a Phase I, 3+3 design, open-label, investigator-initiated trial (IIT) at the University of Colorado Cancer Center. The primary objective was safety, and the secondary objective was efficacy in patients with R/R NHL. Pts with R/R LBCL and mantle cell lymphoma (MCL) who had received at least two lines of prior therapy were eligible and received a single infusion after a 3-days of fludarabine and cyclophosphamide lymphodepleting chemotherapy. CRS and ICANS were graded according to ASTCT 2019 criteria. Response was assessed per Lugano 2014 criteria, with the first evaluation at Day 30.

Results: In preclinical studies, CD19x22 CAR T cells were active against both CD19+ and CD22+ leukemia with expected expansion kinetics in vivo when stimulated through the CD19-CD28 or CD22-41BB CAR alone. Simultaneous engagement of both CARs resulted in enhanced signaling, high in vivo expansion, low exhaustion, and enhanced efficacy. Between June 2022 – July 2024, nine eligible patients received CD19x22 manufactured using Miltenyi Prodigy. All patients had R/R LBCL (DLBCL, n=8; PMBCL, n=1). The median age was 56 (range, 24-84) years. Median prior lines of therapy were 3 (range, 2-8); All pts had ECOG ≤2; 74% Caucasian; 100% had stage III/IV; 22% double hit ; 67% bulky disease, two had prior commercial CD19 CAR; 22% prior Bispecific antibodies; 78% were refractory to last line of therapy. Bridging therapy was utilized in six patients prior to lymphodepletion chemotherapy. One patient was treated off-protocol under emergency expanded access. As of July 2024, five pts (55%) experienced CRS: one at DL1 (3x10⁵ cells/kg) and four at DL2 (1x10⁶ cells/kg); no pts had grade 3 or higher. The median time to CRS onset was Day 9 (range Day 3-15), and the median resolution was on Day 12 (range Day 3-17). CRS was managed with Tocilizumab and steroids. Two patients (22%) experienced ICANS at a DL2 (1x10⁶ cells/kg); none were Grade 3 or higher. The median time to onset of ICANS was Day 18 (range 13-22), with median resolution on Day 21 (range 13-29). The most common treatment-emergent adverse events (TEAE) Grade ≥3 were neutropenia (100%) and thrombocytopenia (12.5%). No infections occurred within the first 30 days post CD19x22 infusion. Immune Effector Cell-Associated Hemophagocytic Lymphohistiocytosis-Like Syndrome (IEC-HS) Grade 5 occurred at DL2 (1x10⁶ cells/kg) in one patient. IEC-HS in this patient followed delayed CRS (onset day 15). This patient was noted to have CD19 dim and CD22 bright R/R LBCL after two lines of therapy. IEC-HS was treated with steroids, anakinra, and ruxolitinib. Since that occurrence of IEC-HS, two additional patients received the same dose level without reoccurrence of IEC-HS. A secondary malignancy (lung cancer) was observed in one patient, seven months post-CAR infusion, and not deemed to be CAR-related. Eight subjects were evaluable for efficacy. Two out of eight (25%) achieved a complete response and one (12.5%) patient achieved a partial response. The median time to response was 3 months. In vivo expansion kinetics and cytokine data were assessed in all nine patients weekly from day 7-28. CAR expansion was observed in all patients with comprehensive kinetics and cell phenotype assessment ongoing.

Conclusions: Thus far, CD19x22 bicistronic CAR-T cell therapy has demonstrated a safety profile similar to other CAR-T cell therapies in patients with R/R NHL and an early promising efficacy profile including post-CAR T-progressing patients. The trial is currently ongoing, and updated data will be presented at the meeting. This CAR is also being tested in Children and Young Adults with Recurrent or Refractory B-Cell Malignancies and is reported separately (NCT05442515)