Utilising recombinant TAPBPR technology to investigate MHC class I peptide editing and therapeutic applications

Change log
Aflalo, Aure 

Major histocompatibility complex class I (MHC-I) molecules play a crucial role in the immune response to infection and malignancy by presenting peptides to CD8+ T cells. Optimal peptide:MHC-I complexes are generated in the MHC-I antigen processing and presentation pathway. In this pathway, TAPBPR shapes the presented peptidome by catalysing peptide exchange on MHC-I molecules and promoting the recycling of sub-optimally loaded MHC-I molecules to the peptide loading complex. TAPBPR has been shown to preferentially bind to and exchange peptides on HLA-A molecules compared to HLA-B and -C molecules, and the TAPBPR K22-D35 loop impacts both the mechanism of peptide editing and preference of TAPBPR for certain MHC-I molecules. Furthermore, it was previously shown that recombinant soluble TAPBPR (sTAPBPR) can be used to load exogenous peptide onto MHC-I molecules at the cell surface. Preliminary data also indicated that by fusing sTAPBPR to an antibody fragment, peptide loading could be improved and targeted to cells expressing the antibody target. Thus, these constructs provide an interesting translational prospect for immunotherapy.

During my PhD, I performed an in-depth characterisation of the efficiency and targetability of sTAPBPR fused to a nanobody targeting green fluorescent protein (sTAPBPR-αGFPNB) as a proof-of-concept. I confirmed and expanded on previous data, showing that peptide loaded by sTAPBPR-αGFPNB can elicit an antigen-specific CD8+ T cell response leading to targeted cell killing. To validate this technology with a therapeutically relevant target, I generated multiple sTAPBPR-antibody fusion proteins targeting human epidermal growth factor receptor 2 (HER2) and characterised their ability to load peptide on MHC-I molecules on HER2-positive breast cancer cells.

Secondly, I sought to use the increased efficiency of sTAPBPR-αGFPNB to investigate peptide editing on MHC-I molecules with a low affinity for TAPBPR. I showed that sTAPBPR-αGFPNB can edit peptides on a wider range of HLA molecules than sTAPBPR, including HLA-B and -C molecules, indicating that tethering of TAPBPR to the plasma membrane is required for peptide editing on MHC-I molecules with a low affinity for TAPBPR. I also found that while some MHC-I molecules require the TAPBPR K22-D35 loop for efficient peptide editing, others undergo improved peptide editing when the tapasin E11-K20 loop is substituted, suggesting potential complementary functions of the two peptide editors.

Finally, I analysed the effect of TAPBPR depletion on the presented peptidomes of a panel of HLA-A and -B molecules. Loss of TAPBPR led to a reduced affinity of peptides presented by HLA-B*44:02 and B*44:05, and a decrease in the abundance of peptides containing a C-terminal tryptophan. This indicates that TAPBPR promotes the presentation of high-affinity peptides and may facilitate the loading of peptides with particular sequence motifs.

Overall, my work has advanced the pre-clinical development of TAPBPR-based technology for immunotherapy and expanded our understanding of the mechanisms of peptide editing by TAPBPR.

Boyle, Louise
Antigen presentation, HLA, Immunology, Immunotherapy, MHC, TAPBPR
Doctor of Philosophy (PhD)
Awarding Institution
University of Cambridge
Wellcome Trust PhD studentship (220012/Z/19/Z])