Development of monoclonal antibodies targeting T cell coinhibitory proteins TIM-3 and TIM-4
Immune checkpoint inhibition (ICI) therapy uses antibodies to block checkpoint proteins from binding with their partner proteins to help overcome dysfunctional T cells and promote the recognition and elimination of tumour cells. The exploration of novel immune checkpoint targets, like T cell immunoglobulin mucin 3 and 4 (TIM-3 and TIM-4), stems from trying to improve the efficacy of ICI therapies (e.g. against cytotoxic T-lymphocyte antigen 4 (CTLA-4) and/or programmed cell death protein 1 (PD-1)) which are currently being used to treat certain cancers like breast, colon and lung cancer either as sole agents or in combination with other immune checkpoint inhibitors or alternative treatments. Targeting TIM-3 and TIM-4 may reverse immune tolerance and suppress tumour cell growth and migration but no effective TIM-4 inhibitors have been developed yet. In this study, monoclonal single chain variable fragment (scFv) antibodies against TIM-3-Fc and TIM-4 were identified using phage display biopanning which isolated antibodies from large immunoglobulin gene repertoires displayed on the surface of bacteriophages. Thirty monoclones from each of the polyclonal pools of anti-TIM-3-Fc and anti-TIM-4 scFv antibodies displayed on phages were picked for validation. Enzyme-linked immunosorbent assay was used to determine the specificity of the phage clones to the target proteins TIM-3-Fc and TIM-4 respectively versus non-target proteins severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike S1 and SARS-CoV-2 receptor-binding domain proteins respectively. This revealed 21 anti-TIM-3-Fc scFv antibody phage monoclones with affinity and specificity to TIM-3-Fc and 8 anti-TIM-4 scFv antibody phage monoclones binding with affinity and specificity to TIM-4. Finally, sanger sequencing was used to determine the DNA sequence of the antiTIM-3-Fc and anti-TIM-4 scFv antibodies and to compare the resulting amino acid sequences to confirm that different scFv antibodies were identified for TIM-3-Fc versus TIM-4.
Immunotherapy, Monoclonal antibodies, Bacteriophages
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