A modular “Catch-and-Play” platform for rapid T-cell engager target assembly for personalised cancer treatment

Bispecific T-cell engagers (also referred to as BiTEs) are bi/multi-specific antibodies that are a clinically used cancer immunotherapy, which enforce formation of an immunological synapse between T-cells and tumour cells by antigenic engagement on the respective cells, resulting in cancer cell apoptosis. 1 , 2 Tumour heterogeneity, however, is a major challenge for identifying the most appropriate cancer target antigen for each patient. Discovery of new tumour targets outweighs new BiTE approval, calling for rapid screening tools to evaluate novel targets and BiTE combinational formats. Although several multifunctional BiTEs platforms have been explored, a simple, rapid, and safe platform needs to be developed. Here we introduce an albumin-based “Catch-and-Play” modular SpyCatcher/SpyTag conjugation platform for potential rapid assembly of interchangeable targeting modules personalised to the patient tumour antigenic profile, and an FcRn-driven half-life extension to reduce the required dose (Fig. 1a ). Fig. 1 FcRn-driven half-life extension and T-cell engager anti-tumour effects of the modular "Catch and Play" platform. a Schematic representation of the “Catch-and-Play” modular platform design and the therapeutic mechanism of action. b FcRn-driven cellular recycling of recombinant human albumin, SpyCatcher-OKT3-Albumin and Albu-Catch-T in a FcRn-expressing dermal human microvascular endothelium cell line (HMEC-1-FcRn) and the pharmacokinetics of Nb 11-SpyTag, SpyCatcher-OKT3-Albumin, and Albu-Catch-T in BALB/c mice ( N = 6). The data is shown as mean ± SD. A one-way ANOVA with multiple comparisons and Turkey’s post hoc correction was used to investigate statistical differences between the constructs **** p < 0.0001, ns-no significance. c In vitro cancer cell lysis induction on addition of either Nb 11-SpyTag, SpyCatcher-OKT3-Albumin or Albu-Catch-T with human PBMCs mixed with MDA-MB-231 cells at a E:T ratio of 10:1. A one-way ANOVA with multiple comparisons and Turkey’s post hoc correction was used to investigate statistical differences between the constructs **** p < 0.0001, ns-no significance. Microscopic time-lapse imaging of Albu-Catch-T activated T-cell-mediated killing of target MDA-MB-231 cells (DiI (Red) labelled) by human PBMCs (CFSE (Green) labelled). d In vivo efficacy in a humanised breast cancer orthotopic BALB/c nude mouse model. At day 10, either PBS, Cetuximab, Nb 11-SpyTag, SpyCatcher-OKT3-Albumin, or Albu-Catch-T mixed with PBMCs were injected (200 µL volume containing 666.67 mole protein) into the tail veins every 3 days for a total of 6 doses (Albu-Catch-T, Cetuximab, Nb 11-SpyTag, and SpyCatcher-OKT3-Albumin groups, N = 6; PBS group, N = 4). Tumour volumes were recorded every 3 days from day 10 and normalised to day 10 to show tumour volume fold-increase. At day 20 isolated tumour tissue was weighed. Generalised linear mixed models were used to investigate statistical differences between the constructs, **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.1, ns-no significance Full size image