Gunn, Rebecca; (2023) Novel Biopolymers for the Activation and Expansion of T-cells: Towards 3D Scaffolds for Cell Therapy. Doctoral thesis (Eng.D), UCL (University College London).

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Abstract

Chimeric Antigen Receptor (CAR) T-cell therapy is an emerging immunotherapy that exhibits promising results against haematological malignancies. Activation is a crucial step in the manufacturing as it renders T-cells susceptible to transduction, initiates expansion, and the development of effector functions. Ex vivo activation is achieved through stimulation of the T-cell receptor complex and co-stimulation, typically provided by anti-CD3 and anti-CD28 antibodies. Antibody coated magnetic beads are currently the most common activation technology, however, they require an additional process step for removal and release testing for residual beads. Furthermore, the beads are expensive, non-biodegradable, and evidence suggests that they are not optimal for antibody presentation due to aggregation. SpheriTech’s biopolymers are low-cost peptide-based polymers that can be cast into a 3D porous structure to which anti-CD3 and anti-CD28 can be bound. The porous structure allows T-cells to be passed through, eliminating the need for a removal step and increasing the surface area for antibody presentation. This thesis aimed to establish SpheriTech’s biopolymer as an alternative manufacturing option. Fundamental characterisation and initial proof-of-concept studies of two iterations of SpheriTech’s biopolymers are presented; termed polymer A and B. Characterisation included structural characterisation, antibody distribution, and biocompatibility. The first polymer was found to possess a macro-porous structure with a median pore diameter of 7.8 ± 0.2 μm, when produced at densities of 0.04 to 0.07 g/cm3; and polymer B was found to possess a super-macro-porous structure with a median pore diameter of 134 μm. aCD3 and aCD28 were successfully attached to both polymers and confirmed with fluorescence imaging. T-cell cultures stimulated with aCD3/aCD38 polymer A displayed a 19 % population expression of activation marker, CD69, 24 hours post-stimulation. Though polymer A demonstrated the potential for T-cell activation; it was limited by a low cell recovery of T-cells (51 %) from the structure. Polymer B was subsequently investigated and exhibited an improved cell recovery of 97 %. Activation and expansion of primary human T-cells were demonstrated with polymer B in both a non-porous and a porous 3D scaffold format. T-cell cultures stimulated with non-porous aCD3/aCD28 polymer B resulted in a 27 and 44 % expression of activation markers CD25 and CD69, respectively, 3 days post-stimulation. Stimulation resulted in an 8.3-fold expansion with > 90 % viability over 14 days, which was comparable to Dynabeads. Expanded T-cell populations were found to be skewed towards CD8+ T-cells (CD4:CD8 ratio of 0.72), and exhibited a 3.6 % decrease in the expression of exhaustion marker, PD-1, than when activated with Dynabeads. The dominant sub-populations of CD4+ and CD8+ T-cells were found to be TEM (40 %) and TCM (38 %), respectively. T-cell cultures expanded with a porous aCD3/aCD28 polymer B scaffold resulted in an 11-fold expansion with a viability of 88 % after 14 days. Expanded T-cell populations had a CD4:CD8 ratio of 1.2 and consisted predominantly of clinically relevant TCM (50 - 84 %) within both CD8+ and CD4+ populations. Stimulation of T-cells with the polymer scaffold was found to induce IFNγ secretion, indicating the expansion of T-cells with an advantageous cytotoxic function. Hence, this thesis has demonstrated the proof-of-concept of a novel T-cell activation and expansion technology using SpheriTech’s biopolymer matrices.

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