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Prostate-specific membrane antigen (PSMA)-targeted radionuclide therapy (RNT) may increase tumor immunogenicity. We aimed at exploiting this effect by combining RNT with immunotherapy in a mouse model of prostate cancer (PC). Methods: C57BL/6-mice bearing syngeneic RM1-PGLS tumors were treated with 225 Ac-PSMA617, an anti-PD-1 antibody, or both. Therapeutic efficacy was assessed by tumor volume measurements (CT), time to progression (TTP), and survival. Results: PSMA RNT or anti-PD-1 alone tended to prolong TTP (isotype control, 25 d; anti-PD-1, 33.5 d [P 5 0.0153]; RNT, 30 d [P 5 0.1038]) and survival (control, 28 d; anti-PD-1, 37 d [P 5 0.0098]; RNT, 32 d [P 5 0.1018]). Combining PSMA RNT and anti-PD-1 significantly improved disease control compared with either monotherapy. TTP was extended to 47.5 d (P # 0.0199 vs. monotherapies), and survival to 51.5 d (P # 0.0251 vs. monotherapies). Conclusion: PSMA RNT and PD-1 blockade synergistically improve therapeutic outcomes in our PC model, supporting the evaluation of RNT and immunotherapy combinations for PC patients.

3066 Background: Fibroblast activation protein (FAP)-expressing cancer-associated fibroblasts (CAF), a major component of tumor stroma, confer treatment resistance, promote local progression, metastasis and immunosuppression. Because FAP is selectively expressed in the tumor stroma of many cancers, radiolabeled small molecule ligands targeting FAP are being explored for their use as pan-cancer theranostic agents. The objective was to establish the spectrum of FAP expression across various cancers by immunohistochemistry (IHC) and to explore whether 68Ga-FAPi-46 PET image biodistribution faithfully reflects tumor FAP expression from resected tumor and non-tumor specimens. Methods: This study was a prospective, exploratory, imaging trial in cancer patients. Referred volunteer patients scheduled to undergo surgical resection of the primary tumor and/or metastases were eligible. Patients underwent one whole body 68Ga-FAPi-46 PET/CT scan. Subsequently, patients underwent surgical resection of the primary tumor and/or metastasis. The outcome measure was the correlation of 68Ga-FAPi-46 PET maximum standardized uptake value (SUVmax) with FAP IHC score in patient-matched cancer and non-cancer tissue. Results: The frequency of FAP expression across 14 cancers on tissue microarrays ranged from 25 to 100% (mean 76.6±25.3%). For imaging and IHC correlation, fifteen patients with the following cancer types were prospectively included: colorectal (n = 4), head and neck (n = 3), pancreas (n = 2), breast (n = 2),stomach (n = 1), esophagus (n = 2) and uterus (n = 1). All 15 patients underwent surgery following their 68Ga-FAPi-46 PET scan within a mean time interval of 16.1±14.4 days (range 1 – 50 days). For two patients the tumor was deemed unresectable. 68Ga-FAPi-46 SUVmax and IHC scores were higher in cancer tissue than in normal tissue: mean 68Ga-FAPi-46 SUVmax 7.4±4.6 (range 1.5-15.9) vs 1.6±1.2 (range 0.4-5.1), (p < 0.001) and mean FAP IHC score 2.38±0.65 vs 0.54±0.66 (p < 0.001), respectively. The FAP IHC scores strongly correlated with 68Ga-FAPi-46 SUVmax (p = 0.001, repeated measures correlation r = 0.85 (95% CI 0.53-0.95), p < 0.001). Conclusions: 68Ga-FAPi-46 PET biodistribution across multiple cancers strongly correlates with FAP tissue expression as measured by IHC. This translational validation paves the way for large scale prospective trials on the use of 68Ga-FAPi-46 PET/CT as a biomarker and stratification tool for FAP-targeted therapies. Clinical trial information: NCT04147494.

FlhDC is a heterohexameric complex that acts as a master regulator of flagellar biosynthesis genes in numerous bacteria. Previous studies have identified a single flhDC operon encoding this complex. However, we found that two flhDC loci are present throughout Paraburkholderia and two additional flhC copies also present in P. unamae. Systematic deletion analysis in P. unamae of the different flhDC copies showed that one of the operons, flhDC1 , plays the predominant role, with deletion of its genes resulting in a severe inhibition of motility and biofilm formation. Expression analysis using promoter- lacZ fusions and real-time quantitative PCR support the primary role of flhDC1 in flagellar gene regulation, with flhDC2 a secondary contributor. Phylogenetic analysis shows the presence of the flhDC1 and flhDC2 operons throughout Paraburkholderia . In contrast, Burkholderia and other bacteria only carry the copy syntenous with flhDC2 . The varying impact each copy of flhDC has on downstream processes indicates that regulation of FlhDC in P. unamae, and likely other Paraburkholderia, is regulated at least in part by the presence of multiple copies of these genes. IMPORTANCE Motility is important in the colonization of plant roots by beneficial and pathogenic bacteria, with flagella playing essential roles in host cell adhesion, entrance, and biofilm formation. Flagellar biosynthesis is energetically expensive. Its complex regulation by the FlhDC master regulator is well-studied in peritrichous flagella expressing enterics. We report the unique presence throughout Paraburkholderia of multiple copies of flhDC . In P. unamae , the flhDC1 copy showed higher expression and greater effect on swim motility, flagellar development, and regulation of downstream genes, than the flhDC2 copy that is syntenous to flhDC in E. coli and pathogenic Burkholderia spp. The flhDC genes have evolved differently in these plant-growth promoting bacteria, giving an additional layer of complexity in gene regulation by FlhDC.