Author ORCID Identifier

56769892300

Date of Graduation

8-2021

Document Type

Thesis (MS)

Program Affiliation

Biomedical Sciences

Degree Name

Masters of Science (MS)

Advisor/Committee Chair

Katy Rezvani, M.D., Ph.D.

Committee Member

Jianjun Gao, M.D., Ph.D.

Committee Member

Vidya Gopalakrishnan, Ph.D.

Committee Member

Gheath Al-Atrash, D.O., Ph.D.

Committee Member

Marina Konopleva, M.D., Ph.D.

Abstract

Despite the approval of several therapies for metastatic clear cell renal cell carcinoma (ccRCC), disease resistance and relapse are common, and therapies with novel mechanisms of action are urgently needed. Chimeric antigen receptor (CAR) T-cell therapy has shown remarkable responses in hematologic malignancies, but many obstacles hinder success in solid tumors including the paucity of highly specific targets and the hostility of the tumor microenvironment (TME). Moreover, the limitations of generating an autologous cell product, such as cost of manufacture, and the challenges of toxicity with CAR-T cells highlight the need to develop new cell therapy products that are at the same time effective and safe.

We propose an innovative strategy using cord blood (CB)-derived natural killer (NK) cells engineered to target ccRCC as a safe and effective approach. NK cells are attractive contenders for CAR-based cell therapy as they exert potent antitumor cytotoxicity. Unlike T cells, allogeneic NK cells are not associated with graft-versus-host disease (GVHD). Our group has developed a platform to use CBNK cells as vehicles for CAR engineering and has proven the safety and efficacy of this approach in the clinic. We now propose to build on this platform to develop next-generation CAR-NK cells targeting CD70, a pan-cancer antigen which is overexpressed in multiple tumors, including ccRCC, but with limited normal tissue expression. Our group has designed a novel CAR construct that targets CD70, ectopically produces interleukin (IL)15 to support NK cell proliferation and persistence in vivo, and expresses a suicide switch based on inducible caspase 9 (iC9), to address any potential safety concerns (iC9/CAR.CD70/IL15).

However, the translation of CAR-based therapies to solid tumors is still associated with many challenges. Some of these obstacles include trafficking and penetration of NK cells to tumor sites, the persistence of infused NK cells, the lack or the heterogenous nature of antigens in some tumors and finally many obstacles posed by the TME such as upregulation of checkpoints, immunosuppressive cells and metabolic immune suppression. Metabolic immune suppression includes a variety of factors, such as local acidity of the TME. ccRCC is characterized by active glycolysis and subsequently increased acidity that suppresses NK cell cytotoxicity and tumor surveillance. Therefore, in this work, we examine the effects of acidity on NK and CAR-NK cell function as well as the biologic mechanisms through which acidity mediates suppression of NK cell function. We, furthermore, used genetic engineering techniques to target these pathways in NK and CAR-NK cells in order to potentiate their activities in the acidic TME of ccRCC.

We have optimized the protocol for successful CD70-specific CAR transduction and CRISPR-Cas9 gene editing techniques. Our data show significant efficacy of anti-CD70 CAR-NK cells compared to non-transduced NK cells against ccRCC models. We have also utilized CRISPR-Cas9 gene editing to inhibit some of the mechanisms by which acidity negatively affects NK cell function and showed significant potentiation of NK cell therapy with these strategies.

Keywords

Chimeric antigen receptor, natural killer cells, renal cell carcinoma, immunometabolism, genetic engineering, acidity, tumor microenvironment

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