Anti-human CTLA-4×TIGIT bispecific antibody: A novel immunotherapy specifically targeting tumor-infiltrating FOXP3+ regulatory T cells

Background: Regulatory T cells (Tregs) are key drivers of immune suppression within the tumor microenvironment (TME). In multiple cancer types, increased numbers of Tregs are associated with poor prognosis in terms of both disease progression and patient survival. This includes lung, breast, pancreatic, hepatocellular, renal, gastric, and cervical cancers. Specifically, the subset of Forkhead box P3 (FOXP3)+ effector Tregs, distinguished by increased expression of immune checkpoint molecules Cytotoxic T-Lymphocyte-Associated Protein 4 (CTLA-4) and T cell Immunoreceptor with Ig and ITIM domains (TIGIT), plays a dominant role in mediating potent immunosuppression within the TME. Current anti-Treg therapies, such as the anti-CTLA-4 monoclonal antibody ipilimumab, show significant clinical efficacy but often induce severe systemic toxicity due to broad depletion of peripheral Tregs. This underscores the unmet medical need for novel agents that selectively target tumor-infiltrating effector Tregs while minimizing systemic adverse effects. Methods: Multiplex Immunohistochemistry (mIHC) assays were performed to identify Tregs within non-small cell lung cancer (NSCLC) tumor samples from 10 donors, using markers including CD4, CD8, FOXP3, TIGIT, CTLA-4, and Pan-Cytokeratin (Pan-CK). TIGIT and CTLA-4 co-expression on tumor-infiltrating Tregs were analyzed by single cell RNA sequencing (scRNA seq) data from NSCLC tumor and normal lung in 12 datasets of 10X genomics and healthy PBMCs from Human Immune Health Atlas. We engineered various formats of anti-human CTLA-4×TIGIT BsAbs. In vitro efficacy was evaluated in hPBMCs and in hCTLA-4 and hTIGIT-expressing stable cell lines, while in vivo effects were assessed in hCTLA-4/hTIGIT double knock-in mice and additional mouse models. Ex vivo analyses were also conducted to characterize immune cell population changes. Results: scRNA-seq analysis revealed that co-expression of CTLA-4 and TIGIT is significantly enriched in tumor-associated Tregs compared to peripheral Tregs. Notably, the frequency of CTLA-4 and TIGIT double-positive cells exceeded that of cells expressing either marker alone. Among inhibitory receptor genes, CTLA-4 and TIGIT exhibited the highest expression levels across T cell subpopulations within NSCLC tumors. The anti-human CTLA-4×TIGIT BsAbs demonstrated enhanced binding affinity under dual expression conditions and exhibited robust antibody-dependent cellular cytotoxicity (ADCC). Furthermore, these BsAbs maintained binding activity in acidic pH conditions and displayed potent antigen blocking efficacy. Conclusions: These findings indicate that the anti-human CTLA-4×TIGIT BsAbs specifically targets tumor-infiltrating Tregs and represents a promising strategy for maximizing specificity while minimizing systemic effects.