Background 4–1BB (CD137; TNFRSF9) is 50- to 55-kDa protein expressed in most case on activated T cells and NK cells. B7H3 (CD276), a B7 superfamily member, is rarely expressed in normal tissues but overexpressed in many types of tumors. Here, we developed a bispecific antibody (bsAb) targeting both B7H3 and 4–1BB with an intact human IgG1 Fc.
Methods HK056–001 was constructed in IgG-scFv format. We assessed the binding activities of HK056–001 to tumor cells expressing varying levels of B7H3 in cell binding assay and examined its impact on the activation of 4–1BB signaling through B7H3 crosslinking in reporter gene assay. Subsequently, we quantified the cytotoxicity of NK cells induced by HK056–001 in vitro. T cell activation and target cell lysis activities were evaluated by co-culture of human PBMC with hB7H3-expressing tumor cells. IFN-γ was measured using ELISA kit, and target cell lysis was measured with CCK-8. The anti-tumor activity of HK056–001 was also evaluated in a MC38-hB7H3 tumor model in vivo. Finally, the risk of cytokine release storm (CRS) was assessed in vitro by ELISA.
Results HK056–001, with its optimized affinity, demonstrates an affinity for B7H3 that is an order of magnitude greater than that of 4–1BB, making it highly effective in tumor localization. It can bind to various cancer cells expressing B7H3 and 4–1BB expressing CHO-K1 cells, thereby facilitating the interaction between B7H3+ cells and 4–1BB+ cells. The activation level of 4–1BB signaling by HK056–001 depends on the expression level of B7H3. HK056–001, with its intact Fc function, can exert cytotoxic effect via NK cells. Besides, HK056–001 can also elicit T cell activation, and thus lead to target cell lysis. But these effects are specifically observed in the presence of B7H3+ cells. In MC38-hB7H3 tumor models, HK056–001 exhibits a dose-dependent anti-tumor activity and a more pronounced growth inhibition effect compared to HK056–002, its Fc function-deficient counterpart. Moreover, HK056–001 induces immune cell infiltration but does not trigger systemic immune response. In addition, HK056–001 shows the ability to protect mice against tumor re-challenge. In vitro studies demonstrate that HK056–001 dose not induce cytokine release from PBMCs in the absence of tumor cells.
Conclusions HK056–001, a B7H3×4–1BB bsAb with human IgG1 Fc fragment prevents tumor development by killing tumor cells directly via effector functions mediated by NK and cytotoxic T cells. These results indicate that this bsAb holds the potential to be developed as a novel clinical therapy for cancer types with B7H3 expression.
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