Functional stimulation of CAR T cells with artificial antigen presenting scaffolds

Engineered T-cell therapies for cancer have advanced rapidly over the past decade, mainly due to the remarkable success of chimeric antigen receptor (CAR)-engineered T cells in treating hematological malignancies. However, this therapy still faces significant challenges in achieving durable clinical responses. One of the key limitations is the poor persistence and cytotoxic capacity of the adoptive T-cell products. A successful adoptive T cell therapy requires an infusion product with a favorable phenotype to enhance in vivo persistence and cytotoxicity, which are critical for effective tumor regression. Preclinical and clinical studies in recent years have highlighted the importance of functional stimulation parameters, including T-cell receptor (TCR) stimulation, the type of co-stimulation, and cytokine exposure. These factors strongly influence T-cell differentiation and the resulting phenotypic and functional profiles. Advances in biomaterials aim to address these challenges by developing artificial antigen-presenting platforms designed to deliver optimized stimulatory signals, thereby producing more potent T-cell infusion products. This thesis presents the development and validation of two such artificial antigen-presenting platforms.

In Manuscript I, we developed and characterized a three-dimensional spherical dextran nanoparticle platform called T-Expand. This nanoparticle was conjugated with CD3 and CD28 stimulatory antibodies using click chemistry and evaluated for its ability to expand CAR-engineered T-cell products. Unlike the micro-sized Dynabeads™ , that are widely used in clinical settings for polyclonal T-cell expansion and CAR T-cell production, T-Expand is biocompatible and biodegradable. Using CD19 CAR T cells as a model, we investigated the effects of T-Expand on polyclonal activation and synapse formation on T-cell, in comparison to the conventional Dynabeads™. Our results demonstrated that T-Expand-expanded CAR T cells exhibit a therapy-favorable phenotype, with enhanced proliferation, persistence, and cytotoxicity.

In Manuscript II, we optimized the expansion of CAR/TCR-engineered T-cell products using dextran-based artificial antigen-presenting scaffolds (Ag.scaffold), previously described for the antigen-specific expansion of CD8+ viral and tumor-specific T cells. Leveraging the "plug-and-play" design of this technology, we examined the impact of antigen-mediated expansion on the phenotypic and functional profiles of T cells engineered with CRISPR-Cas9 knock-in or lentiviral vectors. Additionally, we compared these scaffold-expanded T-cell products with counterparts expanded using conventional, nonspecific cytokine cocktail protocols. Our findings demonstrated that the Ag.scaffold technology not only significantly enriched the expansion products for the target CAR/TCR but also markedly enhanced their anti-tumor efficacy.

Altogether, this thesis underscores the critical need for optimized platforms T-cell stimulation and expansion to improve the clinical efficacy of engineered adoptive T cell therapies. Through detailed investigations, we examined the effects of different stimulation strategies on the phenotype, proliferative capacity, and anti-tumor efficacy of CAR/TCR T-cell products. These insights provide a deeper understanding of the functional stimulation, required for manufacturing superior T-cell infusion products for cancer therapy.