Preview

PDF, English Download (3MB) | Terms of use

Abstract

Ex vivo engineering of patient T cells for the specific redirection toward cancer cells is a promising immunotherapeutic strategy to treat hematological malignancies. In this doctoral thesis, a novel CD22 specific chimeric antigen receptor (CAR) was generated for the adoptive T cell therapy of CD22 positive leukemia and lymphoma. The humanized anti-CD22 (hCD22) single chain variable fragment (scFv) was used as antigen binding domain of a third generation CAR, comprising the signal transduction domains CD3ζ, CD28 and 4-1BB. Due to its high affinity and biophysical stability, the hCD22 scFv was compared to the murine anti-CD22 antibody fragment (mCD22) in terms of scFv and CAR stability. Furthermore, to enhance clinical CAR efficacy and CAR safety, the hCD22 CAR was optimized by mutagenesis. Stability experiments revealed that the mCD22 scFv has a high stability in human serum, comparable to its derived hCD22 scFv. CD22 specific activation of T cells expressing the corresponding hCD22 or mCD22 CAR proved biophysical stability of both scFv derived CARs. By mutating the hCD22 CAR human Fc spacer domain (ΔFc), binding to human Fc receptor expressing cells was blocked, thus reducing on-target, off-tumor CAR related toxicity. The blocking of interleukin-2 (IL-2) secretion caused by the LCK mutation introduced in the hCD22 CAR CD28 signaling domain (ΔCD28) needs to be further investigated as absence of IL-2 release was observed for both the parental and the mutated hCD22 CAR variants. Specific CAR T cell activation was observed for the parental, the ΔFc, the ΔCD28 and the double mutated ΔFc-ΔCD28 hCD22 CAR confirming that both introduced mutations did not affect CAR efficacy in vitro. However, the ΔFc-ΔCD28 hCD22 CAR exhibited a slightly lower anti-tumor efficacy in comparison to the ΔFc, the ΔCD28 and the parental hCD22 CAR. By additionally engineering the hCD22 scFv to further improve the stability of the derived ΔFc-ΔCD28 CAR, CAR T cell activation was not enhanced. This doctoral thesis provides the basis for the clinical development of a novel CD22 CAR T cell therapy for the treatment of CD22 positive leukemia and lymphoma.