Triple-negative breast cancer (TNBC) is a highly aggressive subset of breast cancer with limited therapeutic options. However, its immune evasion mechanisms, characterized by the over-expression of the immune checkpoint molecules PD-L1 and CD47, can be targeted in order to facilitate cancer elimination by cells of innate and adaptive immunity. In this paper, we describe the design, preparation, and evaluation of three novel dual-targeting fusion proteins that were based on the structure frame of prototype IAB (innate and adaptive dependent bispecific fusion protein) and the “Orcutt-type IgG-scFv” molecular model. Three molecules with different spatial conformations were designed to improve antigen–antibody affinity by the addition of Ag–Ab binding sites from the variable region sequences of the anti-PD-L1 monoclonal antibody (mAb) atezolizumab and CV1, a high-affinity receptor of CD47. The results showed that the best-performing among the three proteins designed in this study was protein Pro3; its CV1 N-terminus and Fc domain C-terminus were not sterically hindered. Pro3 was better at boosting T cell proliferation and the engulfment of macrophages than the IAB prototype and, at the same time, retained a level of ADCC activity similar to that of IAB. Through improved design, the novel constructed dual-targeting immunomodulatory protein Pro3 was superior at activating the anti-tumor immune response and has thus shown potential for use in clinical applications.

Background: B7-H3, a type I transmembrane glycoprotein belonging to the B7 superfamily, is an attractive target for antitumor therapies. B7-H3 demonstrates aberrant overexpression in various types of solid tumors while limited and low expression in normal human organs. Various types of treatment targeting B7-H3 have been reported. Among these treatments, antibody-drug conjugate (ADC) has shown potent activity and several clinical trials including DS7300a and MGC018 are currently ongoing. Methods: Here, we constructed CD276-8 ADC composed of anti-B7-H3 antibody CD276-8 with moderate affinity, enzymatically cleavable tetra-peptide–based linker and DXd. Characteristics including in vitro binding affinity and internalization activity was assessed by Bio-Layer Interferometry (BLI), flow cytometry and high content analysis (HCA). The cytotoxicity of CD276-8 ADC was evaluated by the cell-lines with B7-H3 expressed. Pharmacokinetics profiles and antitumor activity were evaluated in mice models in vivo. Finally, developability of CD276-8 ADC was assessed with plasma stability, accelerate stability and freeze-thawing studies using LC-MS and HPLC. Results: Characterization in vitro demonstrated the moderate affinity and acceptable internalization activity of CD276-8 ADC. In addition, CD276-8 ADC appeared potent antitumor activities in B7-H3-positive cell line-derived xenograft (CDX) models with acceptable pharmacokinetics profiles, although it showed less potent cytotoxicity in various cell lines in vitro, indicating acceptable developability. Conclusions: We developed CD276-8 ADC, a B7-H3-targeting ADC with moderate affinity, which delivers the TOP1 inhibitor DXd. This design enabled superior tumor penetration and favorable pharmacokinetics, resulting in potent antitumor efficacy in vivo. Our study highlights affinity optimization as a crucial strategy for enhancing ADC efficacy, positioning CD276-8 ADC as a promising therapeutic for B7-H3-expressing solid tumors.

Background: B7-H3, a type I transmembrane glycoprotein belonging to the B7 superfamily, is an attractive target for antitumor therapies. B7-H3 demonstrates aberrant overexpression in various types of solid tumors while showing limited and low expression in normal human organs. Various types of treatment targeting B7-H3 have been reported. Among these treatments, antibody–drug conjugates (ADCs) have shown potent activity, and several clinical trials, including DS7300a and MGC018, are currently ongoing. Methods: Here, we constructed CD276-8 ADC, composed of the anti-B7-H3 antibody CD276-8 with moderate affinity, an enzymatically cleavable tetra-peptide-based linker and DXd. Characteristics, including in vitro binding affinity and internalization activity, were assessed by bio-layer interferometry (BLI), flow cytometry and high content analysis (HCA). The cytotoxicity of CD276-8 ADC was evaluated in cell lines expressing B7-H3. Pharmacokinetic profiles and antitumor activity were evaluated in mouse models in vivo. Finally, the developability of CD276-8 ADC was assessed with plasma stability, accelerated stability and freeze–thaw studies using LC-MS and HPLC. Results: Characterization in vitro demonstrated the moderate affinity and acceptable internalization activity of CD276-8 ADC. In addition, CD276-8 ADC exhibited potent antitumor activities in B7-H3-positive cell line-derived xenograft (CDX) models with acceptable pharmacokinetic profiles, although it showed less potent cytotoxicity in various cell lines in vitro, indicating acceptable developability. Conclusions: We developed CD276-8 ADC, a B7-H3-targeting ADC with moderate affinity, which delivers the TOP1 inhibitor DXd. This design combined moderate affinity and acceptable pharmacokinetics, resulting in potent antitumor efficacy in vivo. Our study suggests that affinity optimization could be a useful consideration for enhancing ADC efficacy, positioning CD276-8 ADC as a promising therapeutic for B7-H3-expressing solid tumors.