T cell receptor based targeting of immunoglobulin constant domains for treatment of Multiple Myeloma
Multiple myeloma (MM) is a malignancy of the plasma cells characterized by the production and secretion of monoclonal immunoglobulins. The majority (54%) of MM cases produce an IgG heavy chain containing immunoglobulin, IgA heavy chains are less frequent (22%) but IgA production is associated with worse clinical outcomes. Therefore, we propose that the constant domains of the IgG and IgA heavy chains are promising targets for treatment of patients suffering from MM. In MM, surface immunoglobulin expression is absent, but peptides derived from immunoglobulins will still be presented in HLA on the cell surface generating possibilities for T cell receptor (TCR) based targeting of immunoglobulins.
Peptides derived from IgG and IgA constant domains presented in HLA were identified from a previously established HLA-peptidome database of B-cell malignancies. Target epitopes were selected and peptide-HLA-tetramers were used to isolate T cell clones from HLA-mismatched healthy donors. T cells were isolated over an HLA-barrier to allow identification of high affinity T cells specific for these non-mutated peptides. Potent immunoglobulin specific T cell clones were selected based on different functional assays. TCRs of promising T cell clones were sequenced, transferred into CD8 T cells and assessed for functionality.
Using peptide elution data, four IgA and four IgG derived peptides were identified that are presented in HLA-A2 and B7 respectively. Each epitope was included in approximately ten T cell isolations from HLA-mismatched healthy donor PBMCs. For IgA in HLA-B7, 14 peptide specific T cell clones were identified. Nine of these clones recognized IgA Td K562 cells, from which the two most potent T cell clones were selected based on recognition of IgA expressing MM cell line UM6. For IgG in HLA-A2, 13 peptide specific T cell clones were identified of which only four recognized IgG transduced K562 cells. One of these T cell clones recognized IgG expressing cell line UM3. The three selected most potent IgA and IgG specific T cell clones were included in an in vitro killing assay of UM3 (IgG) and UM6 (IgA) MM cell lines. Potent and immunoglobulin isotype specific killing was observed highlighting clinical promise of these T cell clones. TCRs were sequenced and functionality was confirmed by specific tetramer staining and peptide titration experiments after transfer to CD8 T cells.
In conclusion, we successfully isolated T cell clones recognizing the constant domain of IgA and IgG. Potent toxicity of MM cell lines was observed demonstrating promise for the treatment of MM patients. Future experiments need to be performed to assess the cytotoxic potential of TCR Td T cells towards patient derived MM samples.