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

Background

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.

Methods

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.

Results

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.

Conclusions

In vitro and in vivo 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.