HEV-Targeted Antibody-Drug Conjugate Promotes Long-Term Cardiac Allograft Acceptance

Abstract

BACKGROUND: The entrance of naive T cells into lymph nodes (LNs) is a crucial step for induction of heart transplant acceptance under costimulatory blockade. Specialized blood vessels within the LN known as high endothelial venules (HEVs) mediate this process. HEVs express a key glycoprotein containing 6-sulfo sialyl Lewis X, the binding site for L-selectin on the membrane of naive T cells. The proper formation of this carbohydrate requires sulfation by N-acetylglucosamine 6-O-sulfotransferases, particularly CHST4. Given the critical role of CHST4 in HEVs and transplant immunity, we aimed to develop a first-in-class antibody-drug conjugate (ADC) targeting the CHST4/HEV axis to deliver immunoregulatory therapeutics to LNs and promote long-term cardiac transplant survival. METHODS: We used CHST4 knockout mice and inducible diphtheria toxin receptor mouse models to investigate the role of CHST4 in HEV function during heart allograft transplantation. To assess the impact of CHST4 deficiency on heart transplant outcomes, we used fully major histocompatibility complex–mismatched and single major histocompatibility complex class II–mismatched models to study acute and chronic heart transplant rejection, respectively. We developed a first-in-class, dual-payload antibody-drug conjugate aimed at promoting regulatory T cells by conjugating rapamycin and tubastatin A to the HEV-targeting MHA112 antibody. The efficacy of this ADC was tested in murine heart allograft models. RESULTS: Loss of CHST4 expression by HEVs impaired the entry of naive T cells into LNs and consequently reduced regulatory T cell formation, thereby disrupting cardiac transplant acceptance under costimulatory blockade. Treatment with the HEV-targeted ADC carrying rapamycin and tubastatin A prolonged murine heart allograft survival and suppressed chronic rejection, a major barrier to long-term graft success. Mechanistically, this ADC enhanced regulatory T cell induction, reduced proinflammatory T cells within lymph nodes, and decreased immune cell infiltration in cardiac allografts, achieving these effects with substantially lower doses of rapamycin and tubastatin A compared with administration of the free drugs. CONCLUSIONS: Our findings uncover a key mechanistic role for CHST4 in HEVs in facilitating the regulatory T cell generation within the LN and promoting heart transplant acceptance. Moreover, we introduce a promising CHST4-based, HEV-targeted ADC therapy as a potential strategy to suppress heart transplant rejection.