Therapeutic depletion of CCR8⁺tumor-infiltrating regulatory T cells elicits antitumor immunity and synergizes with anti-PD-1 therapy

Abstract

BackgroundModulation and depletion strategies of regulatory T cells (Tregs) constitute valid approaches in antitumor immunotherapy but suffer from severe adverse effects due to their lack of selectivity for the tumor-infiltrating (ti-)Treg population, indicating the need for a ti-Treg specific biomarker.MethodsWe employed single-cell RNA-sequencing in a mouse model of non-small cell lung carcinoma (NSCLC) to obtain a comprehensive overview of the tumor-infiltrating T-cell compartment, with a focus on ti-Treg subpopulations. These findings were validated by flow cytometric analysis of both mouse (LLC-OVA, MC38 and B16-OVA) and human (NSCLC and melanoma) tumor samples. We generated two CCR8-specific nanobodies (Nbs) that recognize distinct epitopes on the CCR8 extracellular domain. These Nbs were formulated as tetravalent Nb-Fc fusion proteins for optimal CCR8 binding and blocking, containing either an antibody-dependent cell-mediated cytotoxicity (ADCC)-deficient or an ADCC-prone Fc region. The therapeutic use of these Nb-Fc fusion proteins was evaluated, either as monotherapy or as combination therapy with anti-programmed cell death protein-1 (anti-PD-1), in both the LLC-OVA and MC38 mouse models.ResultsWe were able to discern two ti-Treg populations, one of which is characterized by the unique expression of Ccr8 in conjunction with Treg activation markers. Ccr8 is also expressed by dysfunctional CD4+ and CD8+ T cells, but the CCR8 protein was only prominent on the highly activated and strongly T-cell suppressive ti-Treg subpopulation of mouse and human tumors, with no major CCR8-positivity found on peripheral Tregs. CCR8 expression resulted from TCR-mediated Treg triggering in an NF-κB-dependent fashion, but was not essential for the recruitment, activation nor suppressive capacity of these cells. While treatment of tumor-bearing mice with a blocking ADCC-deficient Nb-Fc did not influence tumor growth, ADCC-prone Nb-Fc elicited antitumor immunity and reduced tumor growth in synergy with anti-PD-1 therapy. Importantly, ADCC-prone Nb-Fc specifically depleted ti-Tregs in a natural killer (NK) cell-dependent fashion without affecting peripheral Tregs.ConclusionsCollectively, our findings highlight the efficacy and safety of targeting CCR8 for the depletion of tumor-promoting ti-Tregs in combination with anti-PD-1 therapy.

CCR8 marks highly suppressive Treg cells within tumours but is dispensable for their accumulation and suppressive function

“…Whether the anti-CCR8 antibodies used functioned in part through induction of Treg cell depletion has yet to be formally tested. The hypothesis that therapeutic depletion of CCR8 + cells rather than blockade of CCR8 function leads to induction of anti-tumour immunity is indeed consistent with recently reported findings that administration of anti-CCR8 nanobodies with blocking function does not augment tumour immunity, but does so when provided the capability for ADCC [40].…”

Mechanisms of regulatory T cell infiltration in tumors: implications for innovative immune precision therapies

“… 75 Two recent preclinical studies demonstrated that anti-CCR8 antibodies with Fc-dependent ADCC activity selectively deplete tumor-infiltrating Tregs due to prominent CCR8 expression by the activated Tregs in the TME, leading to long-lasting antitumor immune responses and synergistic antitumor effects with PD-1 blockade. 130 131 …”

Distinct cDC subsets co-operate in CD40 agonist response while suppressive microenvironments and lack of antigens subvert efficacy

“…The elevated abundance of activated CD8 + T cells, was accompanied by both a decreased infiltration of FoxP3 + regulatory T cells (Tregs), as well as a reduced expression of CCR8 on the Tregs (Fig. 1t, u), indicative for reduced suppressive phenotype of these cells [33].…”

“…6g). Both Tregs and neutrophils were shown to suppress CD8 + T cells in LLC tumours [33,42], which could be responsible for the lower initial abundance of CD8 + T cells in LLC tumours and their inability to expand upon αCD40 + αCSF1R treatment (Fig. 6h).…”