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Dissertation
Evaluation of chimeric antigen receptor design to identify affinity and spatial binding requirements for optimal T cell effector function
Doctor of Philosophy (Ph.D.), Drexel University
Sep 2024
DOI:
https://doi.org/10.17918/00010627
Abstract
Chimeric antigen receptors (CARs) are artificial immunoreceptors that drive T cell-mediated tumor destruction. CAR-modified T cells have demonstrated unprecedented clinical success, leading to six FDA-approved CAR T cell therapies targeting hematological malignancies. In many cases, however, CARs elicit suboptimal T cell effector function. CAR T cell therapies have been less impactful in patients with solid tumors, yielding no current FDA-approved treatments for these indications. While CAR T cells have positively disrupted the landscape of medicine, many structural modifications are needed to improve the modality. Antigen binding affinity and spacer length are important biophysical properties of CAR design that directly impact T cell effector function. These parameters play a critical role in determining a CAR's clinical performance, yet optimal affinity and spacer characteristics remain unclear. Furthermore, it’s unknown whether the ideal CAR design parameters are universal or target specific. Here, we describe our strategy to assess the influence of antigen binding affinity and spacer length on T cell effector function. We explore multiple tumor models to learn whether our results may be widely applied or restricted to individual indications. We identified high affinity anti-BCMA and anti-DLL3 antibodies to represent hematological and solid tumor models, respectively. Each parental antibody was CDR sequence-manipulated and then recombinantly expressed as scFv-Fc fusion constructs. SPR measurements were performed to identify affinity variant scFvs for spacer domain pairing and CAR design construction. The anti-BCMA and anti-DLL3 affinity and spacer variant CARs were tested in a matrixed format for T cell effector function, in vitro. The results of our studies indicated, for both tumor models, that high affinity CARs (<100nM Kd) paired with an intermediate length spacer (IgG1 Fc, CH2-CH3 domains) elicited superior tumor killing and T cell expansion. These CARs also displayed the strongest cellular avidity when measured in a conjugation assay, suggesting a relationship between cellular avidity and T cell functional performance. Collectively, our findings suggest a widely applicable threshold for optimal CAR spacer and antigen binding properties. However, further evaluation is required to support the theory. The principles identified in this study may be used to improve CAR design to better fit the needs of patients.
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Details
- Title
- Evaluation of chimeric antigen receptor design to identify affinity and spatial binding requirements for optimal T cell effector function
- Creators
- Nicholas William Mazzanti
- Contributors
- Kara L. Spiller (Advisor)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xv, 120 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- School of Biomedical Engineering, Science, and Health Systems (1997-2026); Drexel University
- Other Identifier
- 991021901914304721