DOI: 10.25549/usctheses-c40-362597
title: Engineering chimeric antigen receptor T cells towards enhanced safety and efficacy in cancer immunotherapy

description: Adoptive transfer of chimeric antigen receptor (CAR) engineered T cells is an emerging cancer immunotherapy and has achieved many encouraging results in clinical trials for cancer treatment, especially in treating hematological malignancies. CAR-T cells can be redirected to specifically recognize tumor-associated antigens (TAAs) and induce high levels of antitumor activity. Despite its great potential, CAR-T cell therapy has some major limitations to overcome. CAR-T cells often have less satisfactory efficacy in solid tumor treatment and they may cause dangerous side effects sometime, such as on-target off-tumor toxicities resulting from low-level expression of TAAs in healthy tissues. These adverse effects have raised considerable safety concerns and limited the clinical application of this otherwise very promising therapeutic modality. Further optimization of CAR to enhance its safety and efficacy could help to expand this therapeutic success to more patients and more cancer types. ❧ The first two studies included in this dissertation use epithelial growth factor receptor (EGFR) specific CARs as model. EGFR is overexpressed in many types of tumor and is an attractive target antigen. However, since it is expressed in low levels in healthy tissues, most of targeted therapeutics for EGFR have considerable side effects. ❧ To minimize the on-target off-tumor toxicities of anti-EGFR CAR T cells, we have designed a masked CAR (mCAR) in the first study. It consists of a masking peptide that blocks the antigen-binding site and a protease-sensitive linker that can be cleaved in the tumor microenvironment. mCAR T cells can only recognize target antigens at the tumor site. They showed dramatically reduced antigen binding and antigen-specific activation in the absence of proteases, but normal levels of binding and activity upon treatment with certain proteases in vitro. Masked CAR-T cells also showed antitumor efficacy in vivo comparable to that of unmasked CAR. Our study demonstrates the feasibility of improving the safety profile of conventional CARs and may also inspire future design of CAR molecules targeting broadly expressed TAAs. ❧ In the second study, we designed novel adnectin-based CARs. Most CAR constructs used in clinical trials incorporate single-chain variable fragment (scFv) as the extracellular antigen recognition domain. However, the immunogenicity of nonhuman scFv could cause host rejection against CAR T cells and compromise their persistence and efficacy. The limited availability of scFvs and slow discovery of new monoclonal antibodies also limit the development of novel CAR constructs. Adnectin, a class of affinity molecules derived from the 10th type III domain of human fibronectin, can be efficiently adapted to target proteins of interest with high specificity and affinity. Adnectin could be an alternative to scFv as an antigen binding moiety in the design of CAR molecules. We constructed adnectin-based CARs targeting EGFR and compared the results to scFv-based CAR. Adnectin-based CAR T cells exhibited equivalent cell-killing activity against target H292 lung cancer cells in vitro and had comparable antitumor efficacy in xenograft tumor-bearing mice in vivo. With optimal affinity-tuning, adnectin-based CAR showed higher selectivity to target cells with high EGFR expression compared to those with low expression. This new design of adnectin CARs can potentially facilitate the development of T cell immunotherapy for cancer and other diseases. ❧ Hematopoietic stem cells (HSCs) engineered with CARs could have long-term engraftment and hence continuously replenish anti-cancer effector cells, such as myeloid, NK and T cells with CAR expression. In the third study, we mainly focused on enhancing ex vivo expansion of cord blood HSCs. Umbilical cord blood (UCB) has been established as an important source of hematopoietic stem cells (HSC) for transplantation during the past two decades. However, cord blood transplantation in adult recipients is severely limited due to the insufficient number of HSC in each UCB unit. Many efforts have been made to expand HSCs ex vivo to achieve sufficient cell dose. Yet the most common expansion conditions using serum free medium with cytokine cocktail often caused compromised re-constitutive potential of the cells after expansion. In this study, we use a culture system based on fibrin gel, a biologically compatible matrix, to culture cord blood HSCs. Compared to the conventional culture condition, fibrin gel based culture system can promote ex vivo expansion of UCB HSC with less differentiation and loss of repopulating ability. Our results suggest that fibrin gel matrix is suitable for ex vivo expansion of HSCs and it may extend the clinical application of cord blood HSCs to more patients.