T cell engaging bispecific antibody (TCB) is an effective immunotherapy for cancer treatment. Through co-targeting CD3 and tumor-associated antigen (TAA), TCB can redirect CD3⁺ T cells to eliminate tumor cells regardless of the specificity of T cell receptor. Tissue factor (TF) is a TAA that involved in tumor progression. Here, we designed and characterized a novel TCB targeting TF (TF-TCB) for the treatment of TF-positive tumors. In vitro, robust T cell activation, tumor cell lysis and T cell proliferation were induced by TF-TCB. The tumor cell lysis activity was dependent upon both CD3 and TF binding moieties of the TF-TCB, and was related to TF expression level of tumor cells. In vivo, in both tumor cell/human peripheral blood mononuclear cells (PBMC) co-grafting model and established tumor models with poor T cell infiltration, tumor growth was strongly inhibited by TF-TCB. T cell infiltration into tumors was induced during the treatment. Furthermore, efficacy of TF-TCB was further improved by combination with immune checkpoint inhibitors. For the first time, our results validated the feasibility of using TF as a target for TCB and highlighted the potential for TF-TCB to demonstrate efficacy in solid tumor treatment.

Background: Trophoblast cell surface antigen 2 (TROP2) is widely overexpressed in epithelial cancers and associated with poor prognosis, representing an attractive target for cancer treatment. Recent clinical studies reported favorable outcomes of TROP2-targeting antibody–drug conjugates. However, heterogeneity of human cancers and development of resistance encourage the exploration of alternative cytotoxic payloads. In this regard, bacterial immunotoxin is a viable treatment option owing to the unique and potent mechanism of action. Methods: We first constructed three immunotoxins by fusing deimmunized Pseudomonas Exotoxin A (PE24) to novel TROP2-targeting single-chain variable fragments identified through phage display, which were screened for high binding and cytotoxic activity against tumor cell lines with distinct TROP2 expression. The lead immunotoxin T3PE was engineered by introducing extra disulfide bonds and an albumin-binding domain (ABD) to generate disulfide-stabilized T3PE (dsT3PE and dsT3PE_ABD_CC). Then, both variants were characterized in stability, cell binding, internalization, pharmacokinetic profiles, and in vitro and in vivo antitumor activities. Results: T3PE bound strongly to TROP2 (EC₅₀ = 0.3824 μg/mL) and exerted potent cytotoxicity against NCI-N87 cells (EC₅₀ = 0.09074 μg/mL), but it displayed poor serum stability and was prone to aggregation. Introduction of disulfide bonds stabilized the immunotoxins while preserving the potent cytotoxic activity, and fusion of ABD extended the plasma half-life 6-fold in mice. Additionally, five doses of either dsT3PE or dsT3PE_ABD_CC at 3 mg/kg could significantly reduce tumor progression in the NCI-N87 mouse xenograft model. Conclusions: Together, these results demonstrated the therapeutic potential of these novel anti-TROP2 immunotoxins against TROP2-overexpressing 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.

Given the continuously mutating nature of SARS-CoV-2, the sensitivity of most monoclonal therapeutic antibodies has decreased to the mutants. Bispecific antibodies (BsAbs), with their unique antiviral mechanisms that enable simultaneous binding to two epitopes, offer a distinct advantage in combating the continuous viral mutation by preventing immune escape and enhancing viral neutralization. In this study, we synthesized 67 bsAbs based on the epitope distribution from antibodies isolated using single B-cell cloning from convalescent patients and phage display, 11 of which showed superior neutralization of WA1/2020 compared to their parent antibodies. One bispecific antibody (9A6-6C3), exhibiting 100-fold greater neutralizing activity than its parent antibodies, efficiently neutralized various SARS-CoV-2 variants (IC50 < 100ng/mL). Structural analysis indicates that 9A6 binds to the H-RBD epitope, encountering spatial conflict with the NTD of neighboring S monomer, while 6C3 is capable of binding to a conserved loop on S2. In vitro evidence demonstrates that 9A6-6C3 promotes the disassembly of the S protein, exposing S2, which likely contributes to its broad-spectrum neutralizing activity. In summary, we discovered a potential broad-spectrum mechanism and presented an epitope design strategy for bsAbs, offering valuable insights for the design and development of bsAbs in the fight against COVID-19.