Validation of single chain antibodies targeting immune checkpoint proteins CD28 and CTLA-4

Abstract

Cell Differentiation 28 (CD28) and Cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) are both transmembrane receptors predominantly expressed on CD4+ and CD8+ T-cells. CD28 is found on resting T-cells and is a crucial co-stimulatory receptor in the activation of T-cells. Conversely, CTLA-4 plays a major role in preventing autoimmunity, since it is associated with the regulation of the immune response towards self-antigens. Being a homologue of CD28, CTLA-4 receptor outcompetes CD28 with CD80 or CD86 on antigen-presenting cells (APCs), inhibiting T-cell proliferation and the release of cytokines needed to mount an immune response. CTLA-4 receptor is overexpressed in the tumour microenvironment (TME), causing direct suppression of anti-tumour T-cell response. Immune checkpoint inhibitors (ICIs), a type of immunotherapy, act by blocking checkpoint proteins within the TME from interacting with their partner proteins, which reactivates the anti-tumour immune response. Currently, monoclonal antibodies (mAbs) against CTLA-4, namely ipilimumab, have been FDA and EMA approved to be used in metastatic melanoma, however many patients are experiencing resistance. In this study, recombinant antibodies known as single-chain fragment variable (scFv) antibodies were used, which could then be grafted to generate a full-length mAb. These consist of a fusion protein of the variable heavy and light chain portion of the antigen-binding site of an antibody. The biopanning process generated statistically significant polyclonal scFvs with highest affinity for CD28, which were specific for the target protein and did not bind to CTLA-4 protein. This process did not yield statistically significant antibodies targeting CTLA-4-His protein. Biopanning also produced statistically significant polyclonal scFvs targeting CTLA-4-Fc proteins. Therefore, this study will instigate further work aimed at achieving this objective.