Alpha-particle radiotherapy combined with anti-CTLA-4 synergistically overcomes radioresistance and induces local and systemic antitumor immunity in PDAC

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in the U.S., with over 90% of patients presenting mutant KRAS (mtKRAS). Increased radioresistance and elevated CTLA-4 expression are hallmarks driven by mtKRAS. Radiation has only shown meaningful efficacy in PDAC at very high doses that are not achievable with conventional radiotherapy due to toxicity to adjacent organs. Additionally, although CTLA-4 upregulation suggests a therapeutic target, PDAC shows limited response to immune checkpoint blockade because of its highly immunosuppressive tumor microenvironment. We propose to sensitize a cold mtKRAS murine PDAC model (KPC) to anti-CTLA-4 (aCTLA-4) by overcoming radioresistance using Diffusing Alpha-emitters Radiation Therapy (Alpha DaRT), an intratumoral source of alpha particles with >1,000-fold greater ionization density than photons. This enables delivery of very high doses directly to the tumor while sparing healthy tissue. To further enhance this effect, an ATR inhibitor will impair DNA repair, increasing susceptibility to treatment. To assess local tumor response, mice received 5×10⁵ cells in the left leg, and after 7 days (day 0) one DaRT source (inert or active) was implanted. ATRi (75 mg/kg) or DMSO was administered orally on days 0, 1, 2, 7, 8, and 9, and aCTLA-4 or IgG was injected intraperitoneally every 3 days starting on day 3 (4 doses). Animals with complete regression (>100 days tumor-free) were rechallenged on day 130. To assess systemic immunity, mice received 5×10⁵ KPC cells in the left leg and 1×10⁵ in the right leg, and after 10 days (day 0) one DaRT source was implanted in the left leg. The drug schedule was identical, except mice received six doses of aCTLA-4. The triple therapy (Alpha DaRT + ATRi + aCTLA-4) induced significantly (p<0.05) greater tumor growth delay compared to control, monotherapies, and duotherapies, excluding Alpha DaRT + aCTLA-4, from day 25 onward. By day 41, all control and monotherapy mice reached euthanasia criteria, whereas 60% and 70% of the Alpha DaRT + aCTLA-4 and triple therapy groups remained alive. Complete tumor regression >100 days was sustained in 40% and 50% of mice, respectively. Upon rechallenge, no tumors regrew in the Alpha DaRT + aCTLA-4 group, and only 1 of 5 in the triple therapy group showed regrowth. A significant (p<0.0001) synergistic effect was observed between Alpha DaRT and aCTLA-4. For systemic immunity, Alpha DaRT + aCTLA-4 and the triple therapy induced greater tumor growth delay in both the irradiated and unirradiated tumors compared to all other treatments. In conclusion, Alpha DaRT combined with aCTLA-4 generates durable local and systemic antitumor responses in mtKRAS PDAC, offering a strategy to overcome KRAS-mediated radioresistance and immune evasion.