Development of recombinant antibodies targeting T cell co-inhibitory protein TIM-3

Abstract

Immune checkpoint inhibition is a new frontier of cancer therapy. It involves reactivation of exhausted T cells by inhibiting the binding of checkpoint proteins to their respective partners. Since patients treated using monoclonal antibodies against cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1) developed resistance, new immune checkpoint targets such as T cell immunoglobulin mucin 3 (TIM-3) are being researched. TIM-3 is part of the TIM family of immunoregulatory proteins and is associated with regulation of the immune response in autoimmunity and cancer. Clinical trials conducted using anti-TIM-3 monoclonal antibodies showed significant anti-tumour efficacy. Phage display biopanning was used to obtain a polyclonal population of canine single chain variable fragment antibodies using either plate or bead-bound TIM-3. The phage titre was calculated for the original phage library and for the 4 rounds of panning to determine if it remained constant throughout. The specificity of the phages to the target protein TIM-3 was determined by using IgG and VISTA as controls. As expected, the phages bound only to the target protein. To investigate whether using bead-bound TIM-3 in biopanning was more efficient and effective than using plate-bound TIM-3, flow cytometry and enzyme-linked immunosorbent assay were used. The conclusive results revealed that biopanning using bead-bound TIM-3 was significantly more efficient and effective than using plate-bound TIM-3.