PD-1-cis IL-2R agonism yields better effectors from stem-like CD8+ T cells.

Expansion and differentiation of antigen-experienced PD-1+TCF-1+ stem-like CD8+ T cells into effector cells is critical for the success of immunotherapies based on PD-1 blockade1-4. Hashimoto et al. have shown that, in chronic infections, administration of the cytokine interleukin (IL)-2 triggers an alternative differentiation path of stem-like T cells towards a distinct population of 'better effector' CD8+ T cells similar to those generated in an acute infection5. IL-2 binding to the IL-2 receptor α-chain (CD25) was essential in triggering this alternative differentiation path and expanding better effectors with distinct transcriptional and epigenetic profiles. However, constitutive expression of CD25 on regulatory T cells and some endothelial cells also contributes to unwanted systemic effects from IL-2 therapy. Therefore, engineered IL-2 receptor ß- and γ-chain (IL-2Rßγ)-biased agonists are currently being developed6-10. Here we show that IL-2Rßγ-biased agonists are unable to preferentially expand better effector T cells in cancer models and describe PD1-IL2v, a new immunocytokine that overcomes the need for CD25 binding by docking in cis to PD-1. Cis binding of PD1-IL2v to PD-1 and IL-2Rßγ on the same cell recovers the ability to differentiate stem-like CD8+ T cells into better effectors in the absence of CD25 binding in both chronic infection and cancer models and provides superior efficacy. By contrast, PD-1- or PD-L1-blocking antibodies alone, or their combination with clinically relevant doses of non-PD-1-targeted IL2v, cannot expand this unique subset of better effector T cells and instead lead to the accumulation of terminally differentiated, exhausted T cells. These findings provide the basis for the development of a new generation of PD-1 cis-targeted IL-2R agonists with enhanced therapeutic potential for the treatment of cancer and chronic infections.

A human receptor occupancy assay to measure anti-PD-1 binding in patients with prior anti-PD-1.

Receptor occupancy (RO) assessment by flow cytometry is an important pharmacodynamic (PD) biomarker in the clinical development of large molecules such as monoclonal therapeutic antibodies (mAbs). The total-drug-bound RO assay format directly assesses mAb binding to cell surface targets using anti-drug detection antibodies. Here, we generated a flow cytometry detection antibody specifically binding to mAbs of the IgG1 P329GLALA backbone. Using this reagent, we developed a total-drug-bound RO assay format for RG7769, a bi-specific P329GLALA containing mAb targeting PD-1 and TIM3 on T cells. In its fit-for-purpose validated version, this RO assay has been used in the Phase-I dose escalation study of RG7769, informing on peripheral T cell RO and RG7769 antibody binding capacity (ABC). We assessed RG7769 RO in checkpoint-inhibitor (CPI) naïve patients and anti-PD-1 CPI experienced patients using our novel assay. Here, we show that in both groups, complete T cell RO can be achieved (~100%). However, we found that the maximum number of T cell binding sites for RG7769 pre-dosing was roughly twofold lower in patients recently having undergone anti-PD-1 treatment. We show that this is due to steric hindrance exerted by competing mAbs masking the available drug binding sites. Our findings highlight the importance of quantitative mAb assessment in addition to relative RO especially in the context of patients who have previously received anti-PD-1 treatment.

Beyond checkpoint inhibition: PD-1 cis-targeting of an IL-2Rßγ-biased interleukin-2 variant as a novel approach to build on checkpoint inhibition.

The immunocytokine PD1-IL2v was designed to overcome liabilities and improve efficacy of IL-2 therapies. PD1-IL2v preferentially targets PD-1+ T-cells and acts on antigen-specific stem-like PD-1+ TCF-1+ CD8+ T-cells expanding and differentiating them towards better effectors resulting in superior efficacy in LCMV chronic infection and tumor models compared to checkpoint inhibition.

Simultaneous targeting of PD-1 and IL-2Rßγ with radiation therapy inhibits pancreatic cancer growth and metastasis.

In pancreatic ductal adenocarcinoma (PDAC) patients, we show that response to radiation therapy (RT) is characterized by increased IL-2Rß and IL-2Rγ along with decreased IL-2Rα expression. The bispecific PD1-IL2v is a PD-1-targeted IL-2 variant (IL-2v) immunocytokine with engineered IL-2 cis targeted to PD-1 and abolished IL-2Rα binding, which enhances tumor-antigen-specific T cell activation while reducing regulatory T cell (Treg) suppression. Using PD1-IL2v in orthotopic PDAC KPC-driven tumor models, we show marked improvement in local and metastatic survival, along with a profound increase in tumor-infiltrating CD8+ T cell subsets with a transcriptionally and metabolically active phenotype and preferential activation of antigen-specific CD8+ T cells. In combination with single-dose RT, PD1-IL2v treatment results in a robust, durable expansion of polyfunctional CD8+ T cells, T cell stemness, tumor-specific memory immune response, natural killer (NK) cell activation, and decreased Tregs. These data show that PD1-IL2v leads to profound local and distant response in PDAC.

Deciphering molecular and cellular ex vivo responses to bispecific antibodies PD1-TIM3 and PD1-LAG3 in human tumors.

BACKGROUND: Next-generation cancer immunotherapies are designed to broaden the therapeutic repertoire by targeting new immune checkpoints including lymphocyte-activation gene 3 (LAG-3) and T cell immunoglobulin and mucin-domain containing-3 (TIM-3). Yet, the molecular and cellular mechanisms by which either receptor functions to mediate its inhibitory effects are still poorly understood. Similarly, little is known on the differential effects of dual, compared with single, checkpoint inhibition. METHODS: We here performed in-depth characterization, including multicolor flow cytometry, single cell RNA sequencing and multiplex supernatant analysis, using tumor single cell suspensions from patients with cancer treated ex vivo with novel bispecific antibodies targeting programmed cell death protein 1 (PD-1) and TIM-3 (PD1-TIM3), PD-1 and LAG-3 (PD1-LAG3), or with anti-PD-1. RESULTS: We identified patient samples which were responsive to PD1-TIM3, PD1-LAG3 or anti-PD-1 using an in vitro approach, validated by the analysis of 659 soluble proteins and enrichment for an anti-PD-1 responder signature. We found increased abundance of an activated (HLA-DR+CD25+GranzymeB+) CD8+ T cell subset and of proliferating CD8+ T cells, in response to bispecific antibody or anti-PD-1 treatment. Bispecific antibodies, but not anti-PD-1, significantly increased the abundance of a proliferating natural killer cell subset, which exhibited enrichment for a tissue-residency signature. Key phenotypic and transcriptional changes occurred in a PD-1+CXCL13+CD4+ T cell subset, in response to all treatments, including increased interleukin-17 secretion and signaling toward plasma cells. Interestingly, LAG-3 protein upregulation was detected as a unique pharmacodynamic effect mediated by PD1-LAG3, but not by PD1-TIM3 or anti-PD-1. CONCLUSIONS: Our in vitro system reliably assessed responses to bispecific antibodies co-targeting PD-1 together with LAG-3 or TIM-3 using patients' tumor infiltrating immune cells and revealed transcriptional and phenotypic imprinting by bispecific antibody formats currently tested in early clinical trials.