Tregs constrain CD8⁺T cell priming required for curative intratumorally anchored anti-4-1BB immunotherapy

Abstract

Although co-stimulation of T cells with agonist antibodies targeting 4-1BB (CD137) improves antitumor immune responses in preclinical studies, clinical development has been hampered by on-target, off-tumor toxicity. Here, we report the development of a tumor-anchored ɑ4-1BB agonist (ɑ4-1BB-LAIR), which consists of an ɑ4-1BB antibody fused to the collagen binding protein LAIR. While combination treatment with an antitumor antibody (TA99) displayed only modest efficacy, simultaneous depletion of CD4+ T cells boosted cure rates to over 90% of mice. We elucidated two mechanisms of action for this synergy: ɑCD4 eliminated tumor draining lymph node Tregs, enhancing priming and activation of CD8+ T cells, and TA99 + ɑ4-1BB-LAIR supported the cytotoxic program of these newly primed CD8+ T cells within the tumor microenvironment. Replacement of ɑCD4 with ɑCTLA-4, a clinically approved antibody that enhances T cell priming, produced equivalent cure rates while additionally generating robust immunological memory against secondary tumor rechallenge.

Both intratumoral regulatory T cell depletion and CTLA-4 antagonism are required for maximum efficacy of anti-CTLA-4 antibodies

“…S5 B ). Similar trends have been reported in the less immunogenic B16/F10 model, although with lower cure rates ( 35 ). The high cure rates resulting from this local DT dosing schedule demonstrate that combined intratumoral and nodal Treg depletion can offer advantages over the use of agents that nonspecifically prime all nodal T cells, including Tregs, and is a promising therapeutic approach.…”

“…As proof of concept, our i.t., low-dose DT administration in Foxp3-DTR mice demonstrates the powerful antitumor efficacy that combined intratumoral and nodal Treg depletion can achieve. This system provides clear mechanistic evidence that supports the idea that nodal Tregs are preventing anti-CTLA-4 antibodies from obtaining their full therapeutic potential ( 35 ). In the absence of CTLA-4 antagonism, DT-mediated nodal Treg depletion indirectly provides priming benefits, as shown by increased effector T cells in the tumor following treatment.…”

“…S4 E and F ). Since Tregs are known to restrain priming in the tdLN, this nodal Treg expansion following 9d9 treatment is partially counteracting the priming of effector T cells ( 31 – 35 ). Therefore, we hypothesized that a therapy which depletes nodal Tregs, in addition to intratumoral Tregs, would result in superior effector T cell priming and have even greater antitumor efficacy than 9d9.…”

“…To avoid the lethal autoimmunity these mice experience with systemic DT administration, we developed a low-dose, i.t. dosing schedule to achieve local (tumor and tdLN) Treg depletion while minimizing systemic depletion ( 35 – 37 , 41 ). To validate our approach, we administered three doses of DT: 75 ng (i.t.…”

“…Tregs also restrain the induction of antitumor immune responses in tdLNs, and since effector T cells and Tregs are primed via the same canonical mechanisms, the tdLN acts as a source of both effector T cells and Tregs ( 26 – 30 ). While some Tregs traffic to the tumor, others accumulate in the tdLN throughout tumor progression, dampening T cell priming, producing anti-inflammatory cytokines, generating immune tolerance against cancer antigens, and even directly killing dendritic cells ( 31 – 35 ). The development of Foxp3-DTR mouse models, where diphtheria toxin receptor (DTR) expression is linked to Foxp3 expression, has allowed for selective Treg depletion via diphtheria toxin (DT) administration ( 36 – 38 ).…”