Collagen-binding anti-GITR antibody reshapes immunosuppressive microenvironment and prolongs survival in recurrent glioblastoma

Recurrent glioblastoma (rGBM) remains lethal with median survival under six months, due to immunotherapy resistance driven by immunosuppressive tumor microenvironment (TME) with elevated regulatory T cells (Tregs). Despite the development of Treg-targeting antibodies such as anti-GITR (Glucocorticoid-induced TNFR-related protein) antibody, their poor delivery to brain tumors has restricted therapeutic efficacy, necessitating novel drug delivery strategies. We aimed to identify anchors for selective antibody delivery to tumors and focused on Collagen type I and III (COLI&III), which are minimal in normal brain but highly upregulated in rGBM.We engineered a novel antibody, CBD-alphaGITR, by conjugating the COLI&III-binding domain (CBD) of von Willebrand Factor (vWF) to an anti-GITR antibody. vWF naturally binds to exposed collagen only at sites of vascular injury, inflammation, and tumor while sparing normal tissues, providing a potential mechanism for tumor-specific drug delivery. We investigated the tumor-selective accumulation, immunosuppressive microenvironment remodeling, and antitumor effects of CBD-alphaGITR using clinically relevant syngeneic rGBM mouse models, humanized mouse models, and patient-derived 3D tumor slice cultures.Using a fluorescently labeled biodistribution assay, CBD-alphaGITR selectively accumulated in rGBM tumors but not in normal brain or COLI/III-expressing organs. Single-cell RNA sequencing revealed that CBD-alphaGITR dramatically decreased GITR+ Tregs and exhausted CD8+ T cells (which express moderate levels of GITR), thereby reversing the immunosuppressive TME and promoting the recruitment of anti-tumor macrophages and type 1 conventional dendritic cells. CBD-alphaGITR treatment (300ug, i.v. 1x) achieved approximately 20% complete remission (CR) in two independent rGBM mouse models, while alphaGITR (non-CBD) antibody showed no survival benefit. Furthermore, combining CBD-alphaGITR with PD-1 inhibitor further enhanced antitumor efficacy, achieving 67% CR in rGBM mouse models. We have developed a humanized CBD-alphaGITR for clinical application, which demonstrated extended survival in humanized rGBM mouse models and significant human Treg reduction in patient-derived 3D tumor slice cultures.GITR antibody modified for COLI&III binding achieved selective tumor accumulation, immune reprogramming, and robust tumor control. This delivery strategy can be applied to other antibodies and has the potential to significantly advance treatment approaches for rGBM.