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Background T reg within TME (TIL-Treg) suppresses tumor immunity as demonstrated in preclinical modeling, e.g. depleting-antibodies and gene-disruptions.1–5 Various TIL-Treg-depleting-antibodies against receptors (highly)-expressed in TIL-Treg (CCR8, OX40, CD25, CD4) have been tested for cancer immunotherapy without success in clinics, although highly potent in preclinical models.6 One interpretation is the presence of inhibitory Fc-receptor (FcgRIIB) in humans, absent in mice.7 An alternative to overcome this is to devise an ADC against these targets to enable robust depletion TIL-Treg without impact from FcgRIIB. One attempt by CD25-ADC (ADCT-301) failed for severe toxicities. A new CD25-ADC (PF-08046032) with attenuated affinity to CD25 and a less toxic VC-MMAE demonstrated preferential killings of TIL-Treg over peripheral Treg or CD8+ T cells, expecting widened therapeutic window. OX40-mAbs have widely been tested in the clinics with good safety profiles.6 8 We recently described a novel high affinity OX40-mAb that efficiently internalizes into OX40+ cell lines, prompting us to hypothesize that ADC of this mAb, HX111, could potentially be a safe cancer treatment by efficiently depleting TIL-Treg.Methods hu9C12, a novel humanized OX40-mAb/its Fc-disabled derivative, and a mouse OX40-mAb, OX86/its Fc-disabled derivative, were conjugated to VC-MMAE with an average DAR value of 4 to form ADCs (HX111, Fc-disabled-HX111, OX86-VC-MMAE, Fc-disabled-OX86-VC-MMAE) respectively. These ADCs, along with their corresponding mAbs, were tested in syngeneic MC38 tumor models with or without humanization for anti-tumor activity.Results We first assessed OX86, Fc-disabled-OX86 and Fc-disabled-OX86-VC-MMAE in MC38 model to test our hypothesis. The results demonstrated significant antitumor activity for both OX86, likely due to its ADCC-mediated TIL-T reg depletion, and Fc-disabled OX86-VC-MMAE, likely due to its ADC-mediated TIL-Treg depletion, but none for Fc-disabled OX86. These results are consistent with our hypothesis that OX40-ADC can be a potential anti-tumor agent via MOA of TIL-Treg depletion. Considering the complement MOAs of PD1-mAb, targeting TIL-Teff and reversing T-cell exhaustion, and OX40-ADC, targeting and depleting TIL-Treg, the combo treatments of Fc-disabled OX86-VC-MMAE and mPD1-mAb indeed exerted synergistic antitumor activity, significantly stronger than either agent alone. With the encouraging mouse surrogate data, we next set out to test the corresponding human OX40 targeting agents, HX111 and all the relevant control agents, in human OX40 knock-in mice. We plan to present the preliminary data of these studies during the meeting.Conclusions OX40-ADC can potentially deplete TIL-T reg to enhance tumor immunity/anti-tumor activities in animal models. HX111 could be such a candidate drug for tumor treatment, with enhanced efficacy when combined with PD-1 blockades.References Jin Y, An X, Mao B, Sun R, Kumari R, Chen X, Shan Y, Zang M, Xu L, Muntel J, et al. Different syngeneic tumors show distinctive intrinsic tumor-immunity and mechanisms of actions (MOA) of anti-PD-1 treatment. Sci Rep . 2022;12:3278. 10.1038/s41598-022-07153-z.Marabelle A, Kohrt H, Sagiv-Barfi I, Ajami B, Axtell RC, Zhou G, Rajapaksa R, Green MR, Torchia J, Brody J, et al. Depleting tumor-specific tregs at a single site eradicates disseminated tumors. J Clin Invest. 2013;123:2447–2463. 10.1172/JCI64859.Nagira Y, Nagira M, Nagai R, Nogami W, Hirata M, Ueyama A, Yoshida T, Yoshikawa M, Shinonome S, Yoshida H, et al. S-531011, a novel anti-human CCR8 antibody, induces potent antitumor responses through depletion of tumor-infiltrating CCR8-expressing regulatory T cells. Mol Cancer Ther. 2023;22, 1063–1072. 10.1158/1535-7163.MCT-22-0570.Ueyama A, Nogami W, Nashiki K, Haruna M, Miwa H, Hagiwara M, Nagira M, Wada H, Nagira Y. Immunotherapy targeting CCR8+ regulatory T cells induces antitumor effects via dramatic changes to the intratumor CD8+ T cell profile. J Immunol. 2023;211:673–682. 10.4049/jimmunol.2300067.Zhang Z, Wang G, Shao X, Wu H, Su X, Zhu L, Ji Z. A novel prognostic biomarker CCR8 for gastric cancer and anti-CCR8 blockade attenuate the immunosuppressive capacity of tregs in vitro. Cancer Biother Radiopharm. 2023;38:415–424. 10.1089/cbr.2022.0095.Gutierrez M, Moreno V, Heinhuis KM, Olszanski AJ, Spreafico A, Ong M, Chu Q, Carvajal RD, Trigo J, Ochoa de Olza M, et al. OX40 agonist BMS-986178 alone or in combination with nivolumab and/or ipilimumab in patients with advanced solid tumors. Clin Cancer Res. 2021;27:460–472. 10.1158/1078-0432.CCR-20-1830.Knorr DA, Blanchard L, Leidner RS, Jensen SM, Meng R, Jones A, Ballesteros-Merino C, Bell RB, Baez M, Marino A, et al. FcgammaRIIB is an immune checkpoint limiting the activity of treg-targeting antibodies in the tumor microenvironment. Cancer Immunol Res. 2024;12:322–333. 10.1158/2326-6066.CIR-23-0389.Guttman-Yassky E, Simpson EL, Reich K, Kabashima K, Igawa K, Suzuki T, Mano H, Matsui T, Esfandiari E, Furue M. An anti-OX40 antibody to treat moderate-to-severe atopic dermatitis: a multicentre, double-blind, placebo-controlled phase 2b study. Lancet. 2023;401:204–214. 10.1016/S0140-6736(22)02037-2.