275 Second-generation CAR-T with different co-stimulatory domains indicate distinct immunological features in B16F10 murine melanoma

Background

T cell therapeutics engineered with a chimeric antigen receptor (CAR-T) incorporate either CD28 (28z) or 4-1BB (BBz) as co-stimulatory domains. Preclinical studies, primarily using immunodeficient xenograft mouse models, have yielded conflicting results when comparing 28z and BBz CAR-T, demonstrating various functional differences or similarities. Given that CAR-T cells interact with multiple components of the immune system, using an immunocompetent animal model offers deeper insights into the behavior of CAR-T cells within a complex immune environment, as opposed to the commonly used immunodeficient models. In this study, we compared the pharmacological and immunological profiles of second-generation 28z and BBz CAR-T cells using an immunocompetent C57BL/6 mouse model.

Methods

Mice bearing human CD19-expressing B16F10 melanoma were preconditioned with cyclophosphamide and subsequently administered either CD19-targeted 28z or BBz CAR-T cells. The pharmacokinetic profile was assessed by quantifying CAR-T cell levels in the blood, while the pharmacodynamic assessment was performed by measuring 48 immune-related markers in plasma using the Luminex platform. The immunological profile was evaluated by comparing the phenotypic and polyfunctional characteristics of 28z and BBz CAR-T cells.

Results

Results showed that 28z CAR-T cells demonstrated superior anti-tumor activity and significantly higher persistence in the blood compared to BBz CAR-T cells. Both CAR-T cell levels peaked at 7 days post-infusion, rapidly declining to one-third of the peak by 15 days post-infusion. Notably, immune-related proteins (including cytokines and chemokines) had distinct dynamics depending on co-stimulatory domain incorporated in the CAR construct. While the memory phenotypes were similar between the two constructs, 28z CAR-T cells exhibited greater polyfunctionality than BBz CAR-T cells.

Conclusions

In conclusion, 28z and BBz CAR-T cell treatments exhibit distinct differences in anti-melanoma efficacy, proliferation rates, pharmacodynamic profile, and polyfunctionality when evaluated in an immunocompetent syngeneic mouse model. Understanding the underlying causes and consequences of these differences in immunocompetent animal models may enhance the development of effective CAR-T therapeutics for patients with solid tumors.