Epidermal growth factor receptor variant III (EGFRvIII) is highly and specifically expressed in a subset of lethal glioblastoma (GBM), making the receptor a unique therapeutic target for GBM. Recently, bispecific antibodies (BsAbs) have shown exciting clinical benefits in cancer immunotherapy. Here, we report remarkable results for GBM treatment with a BsAb constructed by the “BAPTS” method. The BsAb was characterized through LC/MS, SEC-HPLC, and SPR. Furthermore, the BsAb was evaluated in vitro for bioactivities through FACS, antigen-dependent T-cell-mediated cytotoxicity, and a cytokine secretion assay, as well as in vivo for antitumor activity and pharmacokinetic (PK) parameters through immunodeficient NOD/SCID and BALB/c mouse models. The results indicated that the EGFRvIII-BsAb eliminated EGFRvIII-positive GBM cells by recruiting and stimulating effector T cells secreting cytotoxic cytokines that killed GBM cells in vitro. The results demonstrated the antitumor potential and long circulation time of EGFRvIII-BsAb in NOD/SCID mice bearing de2–7 subcutaneously heterotopic transplantation tumors and BALB/c mice. In conclusion, our experiments in both in vitro and in vivo have shown the remarkable antitumor activities of EGFRvIII-BsAb, highlighting its potential in clinical applications for the treatment of GBM. Additional merits, including a long circulation time and low immunogenicity, have also made the novel BsAb a promising therapeutic candidate.

Lewis Y antigen, a glycan highly expressed on most epithelial cancers, was targeted for cancer treatment but lacked satisfactory results in some intractable and refractory cancers. Thus, it is highly desirable to develop an effective therapy against these cancers, hopefully based on this target. In this work, we constructed a novel T cell-engaging bispecific antibody targeting Lewis Y and CD3 (m3s193 BsAb) with the IgG-[L]-scfv format. In vitro activity of m3s193 BsAb was evaluated by affinity assay to target cells, cytotoxicity assay, cytokines releasing assay, and T cells proliferation and recruiting assays. Anti-tumor activity against gastric cancer was evaluated in vivo by subcutaneous huPBMCs/tumor cells co-grafting model and huPBMCs intravenous injecting model. In vitro, m3s193 BsAb appeared to have a high binding affinity to Lewis Y positive cells and Jurkat cells. The BsAb showed stronger activity than its parent mAb in T cell recruiting, activation, proliferation, cytokine release, and cytotoxicity. In vivo, m3s193 BsAb not only demonstrated higher therapeutic efficacy in the huPBMCs/tumor co-grafting gastric carcinoma model than the parent mAb but also eliminated tumors in the model of intravenous injection with huPBMCs. Strong anti-tumor activity of m3s193 BsAb revealed that Lewis Y could be targeted in T cell-engaging BsAb for gastric cancer therapy.

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: The efficacy of chemotherapy in triple-negative breast cancer (TNBC) is limited by intrinsic resistance and the tumor microenvironment (TME). Accumulating evidence reveals a mechanistic connection between programmed death-ligand 1 (PD-L1) and c-x-c motif chemokine receptor 4 (CXCR4), which dominate stroma barriers, immune escape, and cancer metastasis. Earlier studies have shown that dual suppression of c-x-c motif ligand 12 (CXCL12)/CXCR4 and programmed cell death-1 (PD-1)/PD-L1 pathways regulates extracellular matrix (ECM) deposition, activation of cancer-associated fibroblasts (CAFs), and epithelial–mesenchymal transition (EMT) of pancreatic cancer cells. Methods: We combined BsNb PX4, a bispecific nanobody targeting PD-L1 and CXCR4, with paclitaxel or gemcitabine in multiple tumor cell lines and human peripheral blood mononuclear cell (hPBMC)-reconstituted xenograft mouse models. Antitumor activity was assessed by CCK-8, flow cytometry, and ELISA, and immune cell infiltration and TME remodeling were examined by immunofluorescence, immunohistochemistry, cytokine assays, and RNA-seq. Results: In MDA-MB-231 cells, BsNb PX4 synergistically enhanced paclitaxel-induced growth inhibition and apoptosis via G₂/M cycle arrest. This combinatorial strategy profoundly remodeled tumor immunity by expanding CD8⁺ T cells and depleting Foxp3⁺ CD4⁺ regulatory T cells (Tregs), while concurrently restoring T-cell cytotoxicity and skewing the cytokine balance toward an antitumor state, with elevated IFN-γ and reduced TGF-β1. Notably, compared with paclitaxel monotherapy, the combination significantly elevated intratumoral CD8⁺ T-cell infiltration, decreased Treg abundance, and exerted robust inhibitory effects on tumor growth and metastasis in humanized TNBC xenografts. Conclusions: These findings reveal that dual blockade of PD-L1 and CXCR4 acts synergistically with chemotherapy by triggering tumor cell apoptotic effects and reversing the immunosuppressive microenvironment, thereby emerging as a promising therapeutic strategy for TNBC.

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.