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Background PDS01ADC (previously designated NHS-IL12) is a tumor-targeting immunocytokine. Promising preclinical results have been shown with murine PDS01ADC, alone and in combination with other therapies, including in poorly immunogenic models. 1–7 A first-in-human clinical trial (NCT01417546) showed PDS01ADC monotherapy to be safe and well tolerated in patients with advanced solid malignancies, with preliminary signs of clinical activity (stable disease in 50% of evaluable patients) observed.8 9 Here, we investigate peripheral blood natural killer (NK) cell phenotypes that are associated with tumor cell lysis in ex-vivo assays, and evaluate patients treated with PDS01ADC for changes in these NK phenotypes and for correlations with clinical response.Methods NK cells from healthy donors (n=12) were evaluated in lysis assays with human SW620 colorectal and H69 small cell lung cancer tumor cell lines and concurrently assessed by multicolor flow cytometry for specific phenotypes that correlated with NK cytotoxicity. Longitudinal peripheral blood collected from patients with advanced malignancies before and after 2 and 4 weeks of PDS01ADC treatment (n=28) was assessed by multicolor flow cytometry for changes in NK cell phenotypes and for correlations with response.Results While total NK cells were not changed in cancer patients receiving PDS01ADC, several specific NK cell phenotypes that positively associate with NK cytotoxicity were increased. For example, Granzyme B + NK cells positively correlated with NK cell lysis of SW620 tumor cells (p=0.0299, r=0.6364) and were increased with PDS01ADC treatment (p<0.0001). A more refined phenotype of NK cells, co-expressing both cytotoxic molecules and activating receptors (Granzyme+Perforin+NKG2D+NKp46+), showed a stronger correlation with NK cell lysis (p=0.0002, r=0.9021), and was both increased in patients upon PDS01ADC treatment and associated with clinical response (median change of +124% vs −21% in patients developing stable disease and progressive disease, respectively, p=0.0688). Other refined NK subsets expressing the inhibitory receptors NKG2A and/or TIGIT, such as NKG2A+TIGIT+Perforin+ NK cells, negatively correlated with lysis (p=0.0078, r=-0.7413), and were decreased with PDS01ADC, with the degree of decrease associated with improved clinical response (p=0.0224).Conclusions PDS01ADC treatment increases peripheral NK cells with specific phenotypes that are positively associated with tumor cell lysis, with increases in some of these phenotypes associating with clinical response. These findings support the potential importance of deep interrogation of the peripheral immunome and demonstrate the role of NK cells in the biologic activity of PDS01ADC. Further studies combining PDS01ADC with other treatments to synergize with these NK cell changes are warranted.References Fallon J, Tighe R, Kradjian G, Guzman W, Bernhardt A, Neuteboom B, Lan Y, Sabzevari H, Schlom J, Greiner JW. The immunocytokine NHS-IL12 as a potential cancer therapeutic. Oncotarget. 2014;5:1869-84.Morillon YM 2nd, Su Z, Schlom J, Greiner JW. Temporal changes within the (bladder) tumor microenvironment that accompany the therapeutic effects of the immunocytokine NHS-IL12. J Immunother Cancer. 2019;7:150.Minnar CM, Chariou PL, Horn LA, Hicks KC, Palena C, Schlom J, Gameiro, SR. Tumor-targeted interleukin-12 synergizes with entinostat to overcome PD-1/PD-L1 blockade-resistant tumors harboring MHC-I and APM deficiencies. 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Nat Commun. 2021;12:5151.Strauss J, Heery CR, Kim JW, Jochems C, Donahue RN, Montgomery AS, McMahon S, Lamping E, Marte JL, Madan RA, Bilusic M, Silver MR, Bertotti E, Schlom J, Gulley JL. First-in-human phase I trial of a tumor-targeted cytokine (NHS-IL12) in subjects with metastatic solid tumors. Clin Cancer Res. 2019;25:99-109.Gatti-Mays ME, Tschernia NP, Strauss J, Madan RA, Karzai FH, Bilusic M, Redman J, Abdul Sater H, Floudas CS, Toney NJ, Donahue RN, Jochems C, Marte JL, Francis D, McMahon S, Lamping E, Cordes L, Schlom J, Gulley JL. A phase I single-arm study of biweekly NHS-IL12 in patients with metastatic solid tumors. Oncologist. 2023;28:364-e217.Ethics Approval All subjects gave written informed consent. Study protocol ( NCT01417546) was approved by the NIH’s IRB, and conducted in accordance with institutional and federal guidelines.