LAG-3xPD-L1 bispecific antibody potentiates antitumor responses of T cells through dendritic cell activation.

Several preclinical studies demonstrate that antitumor efficacy of programmed cell death-1 (PD-1)/programmed death-ligand 1 (PD-L1) blockade can be improved by combination with other checkpoint inhibitors. Lymphocyte-activation gene 3 (LAG-3) is an inhibitory checkpoint receptor involved in T cell exhaustion and tumor immune escape. Here, we describe ABL501, a bispecific antibody targeting LAG-3 and PD-L1 in modulating immune cell responses against tumors. ABL501 that efficiently inhibits both LAG-3 and PD-L1 pathways enhances the activation of effector CD4+ and CD8+ T cells with a higher degree than a combination of single anti-LAG-3 and anti-PD-L1. The augmented effector T cell responses by ABL501 resulted in mitigating regulatory-T-cell-mediated immunosuppression. Mechanistically, the simultaneous binding of ABL501 to LAG-3 and PD-L1 promotes dendritic cell (DC) activation and tumor cell conjugation with T cells that subsequently mounts effective CD8+ T cell responses. ABL501 demonstrates its potent in vivo antitumor efficacy in a humanized xenograft model and with knockin mice expressing human orthologs. The immune profiling analysis of peripheral blood reveals an increased abundance of LAG-3hiPD-1hi memory CD4+ T cell subset in relapsed cholangiocarcinoma patients after gemcitabine plus cisplatin therapy, which are more responsive to ABL501. This study supports the clinical evaluation of ABL501 as a novel cancer immunotherapeutic, and a first-in-human trial has started (NCT05101109).

Novel anti-4-1BB×PD-L1 bispecific antibody augments anti-tumor immunity through tumor-directed T-cell activation and checkpoint blockade.

BACKGROUND: Stimulation of 4-1BB with agonistic antibodies is a promising strategy for improving the therapeutic efficacy of immune checkpoint inhibitors (ICIs) or for overcoming resistance to ICIs. However, dose-dependent hepatotoxicity was observed in clinical trials with monoclonal anti-4-1BB agonistic antibodies due to the activation of 4-1BB signaling in liver resident Kupffer cells. METHODS: To avoid this on-target liver toxicity, we developed a novel bispecific antibody (4-1BB×PD-L1 bispecific antibody, termed "ABL503") uniquely designed to activate 4-1BB signaling only in the context of PD-L1, while also blocking PD-1/PD-L1 signaling. RESULTS: Functional evaluation using effector cells expressing both 4-1BB and PD-1 revealed superior biological activity of ABL503 compared with the combination of each monoclonal antibody. ABL503 also augmented T-cell activation in in vitro assays and further enhanced the anti-PD-L1-mediated reinvigoration of tumor-infiltrating CD8+ T cells from patients with cancer. Furthermore, in humanized PD-L1/4-1BB transgenic mice challenged with huPD-L1-expressing tumor cells, ABL503 induced superior anti-tumor activity and maintained an anti-tumor response against tumor rechallenge. ABL503 was well tolerated, with normal liver function in monkeys. CONCLUSION: The novel anti-4-1BB×PD-L1 bispecific antibody may exert a strong anti-tumor therapeutic efficacy with a low risk of liver toxicity through the restriction of 4-1BB stimulation in tumors.

B7-H3×4-1BB bispecific antibody augments antitumor immunity by enhancing terminally differentiated CD8+ tumor-infiltrating lymphocytes.

Cancer immunotherapy with 4-1BB agonists has limited further clinical development because of dose-limiting toxicity. Here, we developed a bispecific antibody (bsAb; B7-H3×4-1BB), targeting human B7-H3 (hB7-H3) and mouse or human 4-1BB, to restrict the 4-1BB stimulation in tumors. B7-H3×m4-1BB elicited a 4-1BB-dependent antitumor response in hB7-H3-overexpressing tumor models without systemic toxicity. BsAb primarily targets CD8 T cells in the tumor and increases their proliferation and cytokine production. Among the CD8 T cell population in the tumor, 4-1BB is solely expressed on PD-1+Tim-3+ "terminally differentiated" subset, and bsAb potentiates these cells for eliminating the tumor. Furthermore, the combination of bsAb and PD-1 blockade synergistically inhibits tumor growth accompanied by further increasing terminally differentiated CD8 T cells. B7-H3×h4-1BB also shows antitumor activity in h4-1BB-expressing mice. Our data suggest that B7-H3×4-1BB is an effective and safe therapeutic agent against B7-H3-positive cancers as monotherapy and combination therapy with PD-1 blockade.

Analysis of spacer regions derived from intramolecular recombination between heterologous loxP sites.

In Cre-loxP recombination system, Cre recombinase binds cooperatively to two 13bp inverted repeats in a 34bp loxP and catalyzes strand exchange in the 8bp spacer region. Up to date, spacer sequences within the recombined loxP sites derived from two loxP sties that have different 8bp spacer regions have never been analyzed. In the present study, we analyzed the spacer sequences within the recombined products, resulted from intramolecular recombination between heterologous loxP sites including M2, M3, M7, M11, and 2272 in vivo and in vitro. From the analyses, it was found that loxP sites with aberrant 8bp spacers can be generated from Cre-mediated recombination between heterologous loxP sites at significantly high frequency, proposing the possibility that recombination between heterologous loxP sites would have not undergone typical formula of Cre-loxP recombination.