Author ORCID Identifier

0000-0001-8510-8608

Date of Graduation

8-2020

Document Type

Thesis (MS)

Program Affiliation

Biomedical Sciences

Degree Name

Masters of Science (MS)

Advisor/Committee Chair

Jeffrey Molldrem, MD

Committee Member

Gheath Al-Atrash, DO, PhD

Committee Member

Sijie Lu, PhD

Committee Member

WIlliam Plunkett, PhD

Committee Member

Stephanie Watowich, PhD

Abstract

Despite recent advances in the field of cancer immunotherapy and chimeric antigen receptor (CAR) therapies for the treatment of acute myeloid leukemia (AML), there is a need for more unique and innovative modalities to improve these therapies. Our group has extensively studied and demonstrated the potential of targeting the leukemia-associated antigen PR1 in the setting of AML. PR1 is an HLA-A2-restricted nonameric peptide derived from serine proteases neutrophil elastase and proteinase 3, is overexpressed on myeloid leukemia cells, and expressed at normal low levels in healthy hematopoietic cells. We have demonstrated efficacy in targeting PR1/HLA-A2 via the novel development of a TCR-like monoclonal antibody named 8F4 and its adaptation into a chimeric antigen receptor (CAR) named h8F4-CAR. As PR1/HLA-A2 is a self-antigen, its targeting renders increased susceptibility to on-target, off-tumor effects which is a common adverse effect seen in other CAR therapies targeting self-antigens. In an attempt to find an innovative manner to avoid this effect and optimize h8F4-CAR against the PR1/HLA-A2 complex, we investigated and conducted foundational studies using the genetic code and its inherent codon bias. This was done by altering serine and arginine residues within the non-variable regions of h8F4-CAR from their endogenous codons into the rare codon isoforms TCG and CGT for serine and arginine, respectively. Synonymous codon modification of h8F4-CAR represents a unique approach and strategy for modulating the affinity/avidity of CAR therapies by introducing rarer, or less frequently used, codons that encode the same residue. Data from on-going studies in which rare synonymous codons were introduced show that expression of h8F4-CAR can be altered. While MFI values across different codon-modified construct iterations vary, immunoblotting results reveal presence of novel bands unique to codon-modified constructs compared to unmodified wildtype h8F4-CAR and control cells. The introduction of synonymous codon modifications, which takes advantage of inherent codon bias and frequency, represents a unique strategy to mitigate on-target, off-tumor effects. This strategy can be used to alter protein or biologic expression, providing a new approach to fine-tune and modulate CAR avidity and potency leading to greater efficacy by increasing therapeutic indices and reducing likelihood of adverse effects. These implications hold promise in not only in CARs but also in many adoptive cellular therapy platforms in which the genetic code can be utilized to improve and enhance therapies leading to favorable outcomes.

Keywords

chimeric antigen receptor, codon modification, CAR-T, CAR-T optimization, acute myeloid leukemia

Available for download on Tuesday, July 27, 2021

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