Search

Remission durability following single-antigen targeted chimeric antigen receptor (CAR) T-cells is limited by antigen modulation, which may be overcome with combinatorial targeting. Building upon our experiences targeting CD19 and CD22 in B-cell acute lymphoblastic leukemia (B-ALL), we report on our phase 1 dose-escalation study of a novel murine stem cell virus (MSCV)-CD19/CD22-4-1BB bivalent CAR T-cell (CD19.22.BBζ) for children and young adults (CAYA) with B-cell malignancies. Primary objectives included toxicity and dose finding. Secondary objectives included response rates and relapse-free survival (RFS). Biologic correlatives included laboratory investigations, CAR T-cell expansion and cytokine profiling. Twenty patients, ages 5.4 to 34.6 years, with B-ALL received CD19.22.BBζ. The complete response (CR) rate was 60% (12 of 20) in the full cohort and 71.4% (10 of 14) in CAR-naïve patients. Ten (50%) developed cytokine release syndrome (CRS), with 3 (15%) having ≥ grade 3 CRS and only 1 experiencing neurotoxicity (grade 3). The 6- and 12-month RFS in those achieving CR was 80.8% (95% confidence interval [CI]: 42.4%-94.9%) and 57.7% (95% CI: 22.1%-81.9%), respectively. Limited CAR T-cell expansion and persistence of MSCV-CD19.22.BBζ compared with EF1α-CD22.BBζ prompted laboratory investigations comparing EF1α vs MSCV promoters, which did not reveal major differences. Limited CD22 targeting with CD19.22.BBζ, as evaluated by ex vivo cytokine secretion and leukemia eradication in humanized mice, led to development of a novel bicistronic CD19.28ζ/CD22.BBζ construct with enhanced cytokine production against CD22. With demonstrated safety and efficacy of CD19.22.BBζ in a heavily pretreated CAYA B-ALL cohort, further optimization of combinatorial antigen targeting serves to overcome identified limitations (www.clinicaltrials.gov #NCT03448393).

Regulatable CAR platforms could circumvent toxicities associated with CAR-T therapy, but existing systems have shortcomings including leakiness and attenuated activity. Here, we present SNIP CARs, a protease-based platform for regulating CAR activity using an FDA-approved small molecule. Design iterations yielded CAR-T cells that manifest full functional capacity with drug and no leaky activity in the absence of drug. In numerous models, SNIP CAR-T cells were more potent than constitutive CAR-T cells and showed diminished T cell exhaustion and greater stemness. In a ROR1-based CAR lethality model, drug cessation following toxicity onset reversed toxicity, thereby credentialing the platform as a safety switch. In the same model, reduced drug dosing opened a therapeutic window that resulted in tumor eradication in the absence of toxicity. SNIP CARs enable remote tuning of CAR activity, which provides solutions to safety and efficacy barriers that are currently limiting progress in using CAR-T cells to treat solid tumors.

CD19-directed CAR T-cells have been remarkably successful in treating patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) and transformed follicular lymphoma (t-FL). In this cohort study, we treated 60 patients with axicabtagene ciloleucel or tisagenlecleucel. Complete and partial metabolic responses (CMR/PMR) were obtained in 40% and 23% of patients, respectively. After 6.9 months of median follow-up, median progression-free survival (mPFS) and overall survival (mOS) were estimated at 3.1 and 12.3 months, respectively. Statistical analyses revealed that CMR, PFS, and OS were all significantly associated with age-adjusted international prognostic index (aaIPI, p &lt, 0.05). T-cell subset phenotypes in the apheresis product tended to correlate with PFS. Within the final product, increased percentages of both CD4 and CD8 CAR+ effector memory cells (p = 0.02 and 0.01) were significantly associated with CMR. Furthermore, higher CMR/PMR rates were observed in patients with a higher maximal in vivo expansion of CAR T-cells (p = 0.05) and lower expression of the LAG3 and Tim3 markers of exhaustion phenotype (p = 0.01 and p = 0.04). Thus, we find that aaIPI at the time of infusion, phenotype of the CAR T product, in vivo CAR T-cell expansion, and low levels of LAG3/Tim3 are associated with the efficacy of CAR T-cell therapy in DLBCL patients.

Chimeric antigen receptor (CAR)-T cell therapy has shown remarkable results in the treatment of hematological malignancies. However, several challenges remain, including achieving predictable efficacy and safety profiles and extending this approach to solid tumors. As such, the development of approaches that generate engineered T cells with superior persistence and efficacy are needed. Currently, the generation of CAR-T requires that they be activated and expanded ex vivo, generally via beads coated with anti-CD3/anti-CD28 mAbs. T cell activation provides three fundamental signals; (i) primary triggering of TCR signals; (ii) secondary co-stimulatory signals and, (iii) synthesis of cytokines to direct T cell expansion and differentiation. We hypothesize that a CAR-T response can be modulated by tuning the: (i) pattern, (ii) magnitude and, (iii) duration of the input T cell activation signals. To this end, we used a novel, soluble, tunable activation platform based on DNA hybridization - utilizing concatenated single stranded DNA (ssDNA) polymers to cluster complementary oligonucleotide-modified antibodies targeted at T-cell surface receptors. This DNA-Based-T-cell-Activation (DBTA) platform was compared with conventional bead-bound CD3/CD28 antibodies (Dynabead) in the generation and function of T cells engineered to express a CD19-targeted CAR harboring a 4-1BB co-stimulatory domain. The two methods did not result in any significant differences in CAR-T expansion nor in memory/activation phenotypes. Notably, though, these cells exhibited higher cytokine secretion in response to CAR stimulation, and in vitro killing of DBTA-generated CART was significantly higher than that of bead-activated CART. Indeed, only the former showed cytotoxicity against Nalm6 leukemic cells expressing low levels of the CD19 target antigen CART. Furthermore, in an in vivo NSG model with Nalm6 leukemia, the adoptive transfer of low numbers of DBTA-generated CART (2e6) resulted in leukemia eradication and delayed leukemia relapse. Mechanistically, the enhanced function of DBTA-generated CAR was associated with increased metabolic activity in the pre-infusion product. These results suggest that calibrating the activation potential of CAR-T cells can result in the generation of a product with enhanced therapeutic potential. Citation Format: Mehdi Benzaoui, Sooraj R. Achar, Vandana Keskar, Anup Sood, Brian M. Davis, Steven L. Highfill, Grégoire Altan-Bonnet, Christopher D. Chien, Naomi Taylor. Novel DNA-based-T-cell-activation for the generation of chimeric antigen receptor T cells with enhanced anti-leukemia cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB103.