ATP-binding cassette (ABC) adenosine triphosphatases actively transport a wide variety of compounds across biological membranes. Here, the ABC protein Mdl1 was identified as an intracellular peptide transporter localized in the inner membrane of yeast mitochondria. Mdl1 was required for mitochondrial export of peptides with molecular masses of approximately 2100 to 600 daltons generated by proteolysis of inner-membrane proteins by the m-AAA protease in the mitochondrial matrix. Proteolysis by the i-AAA protease in the intermembrane space led to the release of similar-sized peptides independent of Mdl1. Thus, two pathways of peptide efflux from mitochondria exist that may allow communication between mitochondria and their cellular environment.

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Despite advances with therapies targeting the programmed cell death protein 1 (PD-1) or its ligand (PD-L1), many cancer patients are refractory to or relapse following treatment. Resistance to anti-PD-1 treatment is associated with upregulation of other checkpoint inhibitor receptors such as LAG-3 (Lymphocyte Activation Gene 3). FS118, currently being evaluated in a Phase 1 clinical trial in patients with advanced malignancies (NCT03440437), is a tetravalent bispecific antibody targeting LAG-3 and PD-L1, two immune checkpoint molecules that promote tumour escape from immune surveillance. We have characterised both in vitro and in vivo the functional activity of FS118 and find that this bispecific antibody can overcome PD-L1 and LAG-3 immune suppressive signals. We report a potential novel mechanism of action not observed with the combination of single PD-L1 and LAG-3 antibodies. Our results indicate that FS118 represents a possible novel approach to overcome some of the mechanism of resistance to PD-(L)1 blockade.

Functional polymorphism in the rat peptide transporter associated with antigen processing (TAP) changes the peptide pool available for binding and presentation by a class I MHC allele, RT1.Aa. The peptide binding motif for RT1.Aa, determined by stabilization with synthetic peptides, included a strong preference for arginine at the peptide C terminus. Analysis of natural peptides bound to RT1.Aa by both pool sequencing and anhydrotrypsin chromatography revealed that TAP polymorphism determined the presence or absence of arginine as the peptide C-terminal residue. This result highlights the in vivo impact of TAP-peptide selectivity, and provides evidence against a high rate of generation of new C termini by protease activity in the endoplasmic reticulum.

To generate and characterize a murine GITR ligand fusion protein (mGITRL-FP) designed to maximize valency and the potential to agonize the GITR receptor for cancer immunotherapy. The EC value of the mGITRL-FP was compared with an anti-GITR antibody in an agonistic cell-based reporter assay. We assessed the impact of dose, schedule, and Fc isotype on antitumor activity and T-cell modulation in the CT26 tumor model. The activity of the mGITRL-FP was compared with an agonistic murine OX40L-FP targeting OX40, in CT26 and B16F10-Luc2 tumor models. Combination of the mGITRL-FP with antibodies targeting PD-L1, PD-1, or CTLA-4 was analyzed in mice bearing CT26 tumors. The mGITRL-FP had an almost 50-fold higher EC value compared with an anti-murine GITR antibody. Treatment of CT26 tumor-bearing mice with mGITRL-FP-mediated significant antitumor activity that was dependent on isotype, dose, and duration of exposure. The antitumor activity could be correlated with the increased proliferation of peripheral CD8 and CD4 T cells and a significant decrease in the frequency of intratumoral Tregs. The combination of mGITRL-FP with mOX40L-FP or checkpoint inhibitor antagonists enhanced antitumor immunity above that of monotherapy treatment. These results suggest that therapeutically targeting GITR represents a unique approach to cancer immunotherapy and suggests that a multimeric fusion protein may provide increased agonistic potential versus an antibody. In addition, these data provide, for the first time, early proof of concept for the potential combination of GITR targeting agents with OX40 agonists and PD-L1 antagonists. .