Potentiating Local and Abscopal Antitumor Efficacy through Radiation with FAP-CD40 DARPin and Anti-PD1 Therapy

Abstract

Although the advent of Immune Checkpoint Inhibitors (ICI) has changed the facet of oncology, about 70% of patients develop resistance. CD40 is a potent costimulatory molecule that drives dendritic cells (DCs) and tumor-associated macrophages (TAMs) to prime T-cells. Clinically, agonistic CD40 antibodies have demonstrated antitumor activity, but dose-limiting toxicities have impaired efficacy. Radiation (RT), especially at higher doses, activates DCs and TAMs, but also upregulates Fibroblast Activation Protein (FAP) in the tumor microenvironment (TME). The upregulated FAP expression can be harnessed for the design of targeted drug delivery to the TME. The FAP-CD40 DARPin is a molecule that utilizes FAP and CD40 binding domains to selectively cross-link and activate CD40 in the tumor, thus avoiding systemic CD40 activation. This study aimed to evaluate if RT combined with a murine FAP-CD40 (mFAP-CD40) and ICI could improve survival and local and abscopal responses in a pre-clinical lung adenocarcinoma model. Human FAPxCD40 (MP0317) is being evaluated in Phase I trials. The 344SQ-P tumor cells were bilaterally injected into the right and left hind legs of 129Sv/Ev mice to establish primary and secondary tumors respectively. When primary tumors reached ∼7mm in diameter, they were irradiated with 12 Gy x 3 fractions, while secondary tumors were monitored. mFAP-CD40 (5mg/kg) was injected intraperitoneally at 2, 7, and 11 days after the last fraction of RT. A backbone of α-PD1 ICI was given twice per week starting with RT for 5 shots total. Mice were euthanized when tumors reached 14mm in diameter. Lungs were collected at experimental endpoints, fixed, and enumerated for metastases. For FAP detection in the TME, we first conducted an IHC analysis on 344SQ-P tumors harvested 11 days post RT. Although 5 Gy x 3 was superior to 12 Gy x 3 in upregulating FAP expression compared to unirradiated controls (P = 0.0088), we chose the 12 Gy x 3 dose for subsequent experiments due to its superiority in releasing tumor antigens, priming T-cells, and promoting abscopal responses. In our bilaterally established murine groups, we have recorded the following median survival days: Control = 23; RT = 26; RT+α-PD1 = 33; mFAP-CD40+α-PD1 = 23; and RT+mFAP-CD40+α-PD1 (Triple therapy) = 44 days. These results were further buttressed by the difference in average tumor growth observed between groups; whereby there was a significant delay in tumor growth of both primary (P<0.0001) and secondary tumors (P<0.0001) of the Triple therapy group compared to either RT+α-PD1 or mFAP-CD40+α-PD1 dual therapies. In addition, the Triple therapy cohort had a significantly lower count of lung metastases vs. control (P = 0.0007) and vs. mFAP-CD40+α-PD1 (P = 0.0246) cohorts. mFAP-CD40 DARPin with RT and α-PD1 proved efficacious to control primary and secondary tumors in a murine lung carcinoma model with no detected toxicities.