Justine D. Mintern

Ubiquitination is a post-translational modification with well described roles in the regulation of immune cell function, such as dendritic cells (DCs). DCs are crucial initiators and regulators of T cell-mediated immune responses. In this study, we applied activity-based probes and proteomics to systematically profile DUBs in an immortalised DC line. Using the lysate-based biotin(bio)-Ubiquitin(Ub)-propargylamide(Prg) probe and proteomics, the activity and abundance of deubiquitinases were measured in unstimulated and CpG-stimulated MutuDCs to simulate responses to bacteria. This analysis was complemented with profiling using the cell-permeable DUB activity-based probe IMP-2373. The combined analysis with bio-Ub-Prg and IMP-2373 provided insights into the dynamics of DUB activity and abundance in DCs following stimulation, identifying several DUBs for further study.

Immune cell phenotyping of TDLN from CT26/HAS tumour-bearing mice at early time points and MC38/OVA-bearing mice at 14d post-dose.

Gating strategies for tumour and lymph node immunophenotyping experiments in MC38/OVA tumour model.

OVA secretion into culture media by MC38/OVA cells confirmed using Western Blot.

Combination therapy with αTIM3 rescues efficacy of αCTLA4 in CT26/HAS tumour mice regardless of dose route.

Immune cell phenotyping of CT26/HAS tumours at early time points and MC38/OVA tumours at 14d post-dose.

Subcutaneous administration is an increasingly patient-preferred, alternative route of administration for monoclonal antibodies (mAb). To overcome the dose-volume restriction with subcutaneous administration and enable the large mAb doses typically required for immunotherapy, recombinant human hyaluronidase PH20 is co-dosed to transiently depolymerize hyaluronan at the injection site. Despite increasing clinical approvals and clinical trials of combination products with PH20, the potential impact of PH20 in facilitating intravenous-to-subcutaneous dose switching is largely unknown. In this study, we investigated whether increased lymphatic drainage via subcutaneous administration with PH20 co-dosing could improve the efficacy of anti-CTLA4 (αCTLA4) by increasing mAb access to the site of antitumor immunomodulation at the tumor-draining lymph node (TDLN). We showed that subcutaneous administration with PH20 significantly enhanced TDLN exposure of αCTLA4. In murine tumor models (CT26/HAS or MC38/OVA), this translated to improved tumor control compared with intravenous, and was either more efficacious or noninferior to subcutaneous alone. Greater efficacy occurred concomitantly with increased cytotoxic effector CD8+ T cells in the tumor and CD62L+ stem-like CD8+ T cells in the TDLN. This was due to increased mAb access to CTLA4+ T-cell populations at the TDLN and was only preserved at a lower mAb dose after subcutaneous administration with PH20. The efficacy advantage of subcutaneous administration with PH20 was primarily apparent for TDLN-targeted mAb (αCTLA4), as combination therapy with a non–TDLN-targeted mAb (αTIM3) was similarly efficacious regardless of dose routes. Overall, our study highlights the potential utility of PH20 to improve TDLN-targeted immunotherapy.

Plasma concentrations of αCTLA4 over the 14d treatment period in CT26/HAS tumour mice are similar across dose routes.

Gating strategies for tumour and lymph node immunophenotyping experiments in CT26/HAS tumour model.

Gating strategies for αCTLA mAb uptake (via detection of IgG2b Fc portion) by CTLA4+ T cells at the TDLN of CT26/HAS tumour model.