Background The clinical success of PD-1- and CTLA-4- immune checkpoint inhibitors highlights the key contribution of immunosuppression to limiting effective anti-tumor responses. However, as many patients do not respond to anti-PD1 or CTLA4 therapy^1-3 novel therapeutics that target additional immune-suppressive mechanisms are needed. Regulatory T cells (Tregs) inhibit immune responses in the tumor microenvironment via multiple suppressive mechanisms.^{4 5} Existing Treg-targeting agents lack specificity for intratumoral Tregs and can also deplete effector cells, a property that has likely contributed to the lack of clinical activity observed to date. CCR8 (C-C chemokine receptor 8) is selectively expressed on highly activated intratumoral Tregs, its high expression correlates with poor prognosis in multiple human tumor types^{6 7} and depletion of CCR8+ Tregs in preclinical models elicited potent anti-tumor activity. These observations provided rationale for the development of a CCR8-specific human depleting antibody.

Methods Human FOXP3 and CCR8 expression was correlated across multiple tumor types using TCGA datasets and expression of CCR8 evaluated in primary tumor explants and PBMCs by flow cytometry. The efficacy of anti-CCR8 antibody treatment was evaluated in the MC38 and CT26 murine tumor models. The depletion of Tregs following anti-CCR8 treatment was assessed by flow cytometry. Flow cytometric-based binding assays were performed using cell lines expressing human or cynomolgus CCR8. Purified human NK cells were co-cultured with CCR8+ target cells and flow cytometry used to evaluate antibody-dependent killing activity.

Results CCR8 expression was highly correlated with FoxP3 across multiple cancer subtypes and was low to absent on effector T cells. Importantly, CCR8 was not detected on any peripheral human leukocyte subset. In murine tumor models, anti-CCR8 antibody treatment reduced tumor growth in a dose- and Fc-gamma-receptor-dependent manner and resulted in complete regressions and the development of memory. Tumor shrinkage was associated with a reduction in intratumoral Tregs and increased representation of intratumoral CD8 T cells. FPA157 is a highly specific human and cynomolgus crossreactive CCR8 antibody that does not bind closely related chemokine receptors. FPA157 was engineered to enhance antibody-dependent cell-mediated cytotoxicity (eADCC) and elicited potent NK-mediated killing of target cells expressing CCR8 at levels observed on human intratumoral

Tregs.

Conclusions FPA157 is a CCR8-specific monoclonal antibody with eADCC activity that is being developed for the treatment of cancer. Depletion of CCR8+ Tregs induced substantial anti-tumor activity in pre-clinical models, thus supporting the clinical evaluation of FPA157 as a novel approach to alleviate immune suppression in the microenvironment of human solid tumors.

References

1. Hellmann MD, Ciuleanu TE, Pluzanski A, Lee JS, Otterson GA, Audigier-Valette C, Minenza E, Linardou H, Burgers S, Salman P, Borghaei H, Ramalingam SS, Brahmer J, Reck M, O’Byrne KJ, Geese WJ, Green G, Chang H, Szustakowski J, Bhagavatheeswaran P, Healey D, Fu Y, Nathan F, Paz-Ares L. Nivolumab plus Ipilimumab in lung cancer with a high tumor mutational burden. N Engl J Med 2018;378(22):2093-2104.

2. Wolchok JD, Chiarion-Sileni V, Gonzalez R, Rutkowski P, Grob JJ, Cowey CL, Lao CD, Wagstaff J, Schadendorf D, Ferrucci PF, Smylie M, Dummer R, Hill A, Hogg D, Haanen J, Carlino MS, Bechter O, Maio M, Marquez-Rodas I, Guidoboni M, McArthur G, Lebbé C, Ascierto PA, Long GV, Cebon J, Sosman J, Postow MA, Callahan MK, Walker D, Rollin L, Bhore R, Hodi FS, Larkin J. Overall survival with combined nivolumab and ipilimumab in advanced melanoma. N Engl J Med 2017;377(14):1345-1356.

3. Motzer RJ, Tannir NM, McDermott DF, Arén Frontera O, Melichar B, Choueiri TK, Plimack ER, Barthélémy P, Porta C, George S, Powles T, Donskov F, Neiman V, Kollmannsberger CK, Salman P, Gurney H, Hawkins R, Ravaud A, Grimm MO, Bracarda S, Barrios CH, Tomita Y, Castellano D, Rini BI, Chen AC, Mekan S, McHenry MB, Wind-Rotolo M, Doan J, Sharma P, Hammers HJ, Escudier B; CheckMate 214 Investigators. Nivolumab plus ipilimumab versus sunitinib in advanced renal-cell carcinoma. N Engl J Med 2018 Apr 5;378(14):1277-1290.

4. Teng MW, Ngiow SF, von Scheidt B, McLaughlin N, Sparwasser T, Smyth MJ. Conditional regulatory T-cell depletion releases adaptive immunity preventing carcinogenesis and suppressing established tumor growth [published correction appears in Cancer Res. 2010; 70(23):10014]. Cancer Res 2010;70(20):7800-7809.

5. Simpson TR, Li F, Montalvo-Ortiz W, Sepulveda MA, Bergerhoff K, Arce F, Roddie C, Henry JY, Yagita H, Wolchok JD, Peggs KS, Ravetch JV, Allison JP, Quezada SA. Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma. J Exp Med 2013;210(9):1695-710.

6. Plitas G, Konopacki C, Wu K, Bos PD, Morrow M, Putintseva EV, Chudakov DM, Rudensky AY. Regulatory T cells exhibit distinct features in human breast cancer. Immunity 2016;45(5):1122-1134.

7. De Simone M, Arrigoni A, Rossetti G, Gruarin P, Ranzani V, Politano C, Bonnal RJP, Provasi E, Sarnicola ML, Panzeri I, Moro M, Crosti M, Mazzara S, Vaira V, Bosari S, Palleschi A, Santambrogio L, Bovo G, Zucchini N, Totis M, Gianotti L, Cesana G, Perego RA, Maroni N, Pisani Ceretti A, Opocher E, De Francesco R, Geginat J, Stunnenberg HG, Abrignani S, Pagani M. Transcriptional landscape of human tissue lymphocytes unveils uniqueness of tumor-infiltrating T regulatory cells. Immunity 2016;45(5):1135-1147.