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

Abstract

Anti-CTLA-4 antibodies have successfully elicited durable tumor regression in the clinic; however, long-term benefit is limited to a subset of patients for select cancer indications. The incomplete understanding of their mechanism of action has hindered efforts at improvement, with conflicting hypotheses proposing either antagonism of the CTLA-4:B7 axis or Fc effector-mediated regulatory T cell (Treg) depletion governing efficacy. Here, we report the engineering of a nonantagonistic CTLA-4 binding domain (b1s1e2) that depletes intratumoral Tregs as an Fc fusion. Comparison of b1s1e2-Fc to 9d9, an antagonistic anti-CTLA-4 antibody, allowed for interrogation of the separate contributions of CTLA-4 antagonism and Treg depletion to efficacy. Despite equivalent levels of intratumoral Treg depletion, 9d9 achieved more long-term cures than b1s1e2-Fc in MC38 tumors, demonstrating that CTLA-4 antagonism provided additional survival benefit. Consistent with prior reports that CTLA-4 antagonism enhances priming, treatment with 9d9, but not b1s1e2-Fc, increased the percentage of activated T cells in the tumor-draining lymph node (tdLN). Treg depletion with either construct was restricted to the tumor due to insufficient surface CTLA-4 expression on Tregs in other compartments. Through intratumoral administration of diphtheria toxin in Foxp3-DTR mice, we show that depletion of both intratumoral and nodal Tregs provided even greater survival benefit than 9d9, consistent with Treg-driven restraint of priming in the tdLN. Our data demonstrate that anti-CTLA-4 therapies require both CTLA-4 antagonism and intratumoral Treg depletion for maximum efficacy—but that potential future therapies also capable of depleting nodal Tregs could show efficacy in the absence of CTLA-4 antagonism.

Single cell suppression profiling of human regulatory T cells

“…3i), potentially through its dependence on proliferation. Anti-CTLA4 engagement by antibodies on Tregs has previously been described to either mediate Fc-dependent Treg depletion 59 , antagonistic blocking of CTLA4 function 31 , or Treg destabilization in a glucose and CD28 dependent manner 32 . In our system, we did not see any evidence of Treg depletion, which itself is highly dependent on the microenvironment 59 .…”

“…This prevents the co-stimulatory interaction between CD28 on conventional T cells and CD80/CD86 on APCs, and also frees PD-L1 from CD80 cis-interaction, thus inhibiting T cell activation and proliferation via PD1 [26][27][28] . Blocking CTLA4 with antibodies such as Ipilimumab can enhance T cell activation and proliferation, potentially due to reduced suppressive activity of Tregs 29 through multiple mechanisms such as Fc-dependent Treg depletion 30 , antagonistic blocking of CTLA4 function 31 , or Treg destabilisation 32 .…”

CD40 agonism improves anti-tumor T cell priming induced by the combination of radiation therapy plus CTLA4 inhibition and enhances tumor response

“…Recent clinical trial results testing the combination of anti-PD1 with CD40 agonism and chemotherapy in pancreatic cancer patients have been disappointing in that, whereas each double combination (anti-PD1 + chemotherapy and anti-CD40 + chemotherapy) showed activity, the triple combination of anti-PD1 plus CD40 agonism and chemotherapy did not show any additional benefits. 9 Given the critical role of CTLA-4i in enhancing T cell priming, 10 we provide the preclinical rationale 5 for testing CD40 agonism with CTLA4i and an inducer of immunogenic cell death such as radiation in the clinic.…”

MicroRNAs as regulators of immune checkpoints in cancer immunotherapy: targeting PD-1/PD-L1 and CTLA-4 pathways

“…The effects of CTLA-4 blockade are mainly mediated by the effector T cell compartment, where effector T cells are critical, but Tregs are not. In order to achieve maximum anti-tumor effects, simultaneous blockade of both effector T cells and Tregs is necessary [ 57 – 59 ]. Enhanced specific binding of CTLA-4 to CD80 or CD86 leads to competition with CD28 for binding to important co-stimulatory molecules, inhibiting the formation of co-stimulatory signals.…”