499 Development and efficacy of a bispecific antibody targeting BTN1A1 and PD-L1 in cancer immunotherapy

<h3>Background</h3> BTN1A1, also known as Butyrophilin 1A1, is an immune checkpoint protein that can be targeted to create new and effective immunotherapy options. We observed that BTN1A1 and PD-L1 expression are mutually exclusive in a variety of cancer cells. BTN1A1 expression in cancer cells is associated with low or absent levels of PD-L1, and vice versa. Extensive research in patient tumor samples and cell lines has consistently shown a negative correlation between BTN1A1 and PD-L1. It has been suggested that patients may benefit from simultaneously targeting of tumors expressing both BTN1A1 and PD-L1. Recently, the phase 1 clinical trial of Nelmastobart, a BTN1A1 antibody, was successfully completed, and as a next step, we successfully developed a bispecific antibody targeting BTN1A1 and PD-L1. Additionally, 3D cultures and mouse models were used as multiple evaluation platforms to understand the mechanism of interaction with immune cells for targeting BTN1A1 and PD-L1. <h3>Methods</h3> We have developed a bi-specific antibody that targets BTN1A1 and PD-L1 simultaneously. Colon cancer organoids were processed with hPBMCs and bi-specific antibodies targeting BTN1A1 and PD-L1. Patient-derived colon cancer organoids were grown in Matrigel. After evaluating the size and structure of the organoids, we observed morphological changes in the organoids following treatment with hPBMCs, the bi-specific antibody, and 5-FU. Additionally, we performed allogeneic transplants of MC38 and CT26 cells into the right flank of mice. Around 10 days later, the mice were administered STC109 (an antibody targeting mouse BTN1A1), anti-PD-L1, and 5-FU. In all experiments, we analyzed the synergistic effects of anti-BTN1A1, anti-PD-L1, and other treatments alone or in combination in relation to the tumor immune context. <h3>Results</h3> In all experiments, inhibition of BTN1A1 and PD-L1 by bi-specific antibodies (hSTC810 and anti-PD-L1) exhibited more dramatic anticancer efficacy than immune checkpoint treatment alone. In particular, significant upregulation of genes involved in the innate immune response leads to T cell activation and tumor elimination. In organoid and mouse models, bi-specific anti-BTN1A1 and anti-PD-L1 therapy enhanced the antitumor activity of hPBMCs and synergized with chemotherapy treatments. Opal multiple immunofluorescence analysis revealed that hSTC810 treatment effectively removes general tumors and YAP-positive tumors. <h3>Conclusions</h3> <i>In vitro</i> and<i> in vivo</i> data show that treatment with bispecific antibodies (hSTC810 and anti-PD-L1) significantly enhances antitumor activity compared to treatment alone. Our findings suggest that this represents a new immune checkpoint capable of overcoming antitumor resistance by targeting a broad spectrum of colon cancers using bispecific antibodies.