Abstract:
CAR-T Cells have completely changed the therapeutic landscape in B-phenotypic malignancies and demonstrated their clinical efficacy in lymphatic leukemia, lymphomas, and myelomas. However, conventional CAR-T cells have their limitations due to a lack of controllability and limited versatility. To achieve temporal and qualitative control of CAR-T function, as well as the ability for multiplex targeting, we have developed the Adapter CAR (AdCAR) system. AdCAR-T cells are redirected to surface antigens via biotinylated adapter molecules (AM) in the context of a specific linker structure, referred to as the Linker-Label Epitope (LLE). AdCAR-T cells are non-functional without the adapter molecules and mediate their anti-cancer function only in the presence of adapter molecules.
In the first project of my thesis the AdCAR System was evaluated in terms of CAR specific activation, cytokine secretion, cytolysis, combinatorial targeting and exhaustion. Alternative target antigens were analyzed by flow cytometry on three NHL cell lines, and AMs were generated by biotinylation of Immunoglobulin G (IgG) 1 against CD19, CD20, CD22, ROR-1, CD276, CD79B, and CD10. AdCAR T cells demonstrated specific activation and cytokine secretion comparable with conventional CD19CAR-T cells. We found that quantitative low molar biotinylation is important to prevent cross-activation. In a CD19 knockout model, we demonstrate the great advantage of combinatorial targeting with the AdCAR in contrast to the mono-targeted CD19CAR to prevent antigen escape.
The second project we focused on combinatorial targeting of Acute myeloid leukemia (AML). AML-associated antigen expression on 30 pediatric AML samples was analyzed by multicolor flow cytometry. CD33, CD38, CD371, IL1RAP and CD123 were identified as the most frequently expressed antigens. High variability among patient samples and also within leukemic cells from the same patient suggests that single antigen targeting is destined to lead to antigen escape and therapy failure. We generated AMs against CD33, CD38, CD123, CD135 and CD371 and validated them in vitro and in vivo on AML cell lines. In two PDX model preserving the intratumoral heterogeneity of the primary disease, single antigen targeting against either CD33, CD38, or CD371 led to antigen negative relapse of the disease. In contrast, a combinatorial targeting of CD33, CD38, and CD371 resulted in disease clearance and lasting remission. In conclusion, we were able to shown that the AdCAR technology enables controllable, flexible, combinatorial, and selective targeting.
