Abstract C090: Development of a multi-functional CAIX-TGFβ targeted radiopharmaceutical for triple-negative breast cancer

Abstract Tumor-targeted radiopharmaceutical therapy (RPT) can induce antitumoral immunogenic responses, but immunosuppressive and radioresistance factors such as transforming growth factor β (TGFβ) can counteract the antitumor responses. Carbonic anhydrase IX (CAIX) has been proposed as a target for RPTs in TNBC, but may be more effective when combined with TGFβ-blockade to counteract resistance. TLX250-TRAP is a novel fusion protein consisting of the ectodomain of TGFβ receptor II (TGFβRIIect) and an anti-CAIX antibody, and with the capacity for radioisotope labeling. We tested CAIX targetability, TGFβ binding, and TGFβR signaling antagonism of TLX250-TRAP using human and mouse TNBC cell lines and evaluated preclinical antitumor response using syngeneic orthotopic murine TNBC models. TLX250-TRAP demonstrated dose-dependent binding to human CAIX-expressing murine EMT6 (EMT6hCAIX+) and 4T1 (4T1hCAIX+) cells, as well as to the endogenous CAIX-expressing human breast cancer cell line MDA-MB-231 under hypoxic and acidic conditions in vitro. TLX250-TRAP captured both human and mouse TGFβs and inhibited TGFβ-induced SMAD2 phosphorylation in both human and mouse TNBCs, and significantly inhibited TGFβ-receptor signaling genes. Using serial µSPECT/CT imaging, isotope lutetium-177 (Lu-177)-labeled TLX250-TRAP (Lu-TLX250-TRAP) efficiently targeted EMT6hCAIX+ and 4T1hCAIX+ tumors in the mammary fat pad from 3 to 168 hours post-IV injection. We found that 240 µCi of Lu-177-labeled Lu-TLX250-TRAP exhibited the most effective antitumor responses with a 25% complete response rate (CRR) compared to the semi-therapeutic dose (120 µCi, CRR: 0%), the lethal dose (400 µCi, CRR: 0%), the non-isotope-labeled TLX250-TRAP (CRR: 0%), or Lu-177-labeled anti-CAIX antibody (null of TGFβRIIect, 240 µCi, CRR: 0%). Lu-TLX250-TRAP reduced the number of white blood cells, red blood cells, neutrophils, and monocytes in blood in weeks 1 and 2 post-IV administration and recovered to the level of the non-isotope-labeled IgG control by week 3. The optimized Lu-TLX250-TRAP (240 µCi of Lu-177-labeled to 400µg of TLX250-TRAP) showed increased intratumoral CD4+ and CD8+ T cells with decreased Regulatory T cell (Treg) infiltration compared to non-isotope-labeled IgG or Lu-177-labeled anti-CAIX antibody (null of TGFβRIIect). CD4+ or CD8+ T cell depletion in mice led to a loss of antitumor responses in the orthotopic TNBC models. These findings demonstrate that TLX250-TRAP binds to CAIX and concurrently inhibits TGFβ receptor signaling. Combined TGFβ-blocking and tumor radiation synergistically enhanced T cell infiltrations and reduced the Treg:CD8 T cell ratio, suggesting TGFβ-mediated immune tolerance can be blocked via the Lu-TLX250-TRAP regimen. Radiation-induced blood cell cytotoxicity at the early stage may require treatment optimization or additional cell survival factors to alleviate the early cytotoxicity. Overall, Lu-177 labeled TLX250-TRAP enables theranostic use to track tumors and promotes therapeutic antitumor immune responses in TNBC. Citation Format: Wonjong Jin, Paul A. Clark, Ria Kumari, Michael P. Wheatcroft, Kwame Twumasi-Boateng, Zachary S. Morris. Development of a multi-functional CAIX-TGFβ targeted radiopharmaceutical for triple-negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr C090.