Broadly Applicable TCR Based Therapy for Multiple Myeloma Targeting the Jchain

We identified the immunoglobulin joining chain (Jchain) as a suitable target antigen for immunotherapeutic strategies for multiple myeloma (MM) because it was highly expressed in 4/5 MM cases and expression in healthy tissues was restricted to the B cell lineage. In healthy B cells, Jchain functions as a linker protein between the monomers of dimeric IgA and pentameric IgM when secreted by plasma cells. Similar to healthy plasma cells, Jchain production in MM occurs independent of the immunoglobulin isotype produced by the MM cells. Analysis of a publicly available microarray dataset, containing 133 primary MM samples, confirmed Jchain expression in 81% of the cases (GEO accession: GSE13591). Jchain derived peptides presented in HLA molecules may therefore be suitable antigens for TCR mediated T cell therapy of MM. In this study the HLA-peptidome of MM cell lines U266 and UM9 was established and analyzed for Jchain derived peptides presented in commonly expressed HLA molecules. In total this resulted in identification of 9 Jchain peptides presented by HLA-A1, -A2, -A3, -A11 or -A24.

To avoid immunogenic tolerance of the HLA-matched setting and allow identification of potent T cell clones targeting Jchain peptides, immunogenicity of peptides presented in foreign HLA alleles was exploited. PBMCs from healthy donors negative for HLA alleles of interest were co-incubated with PE-labeled pHLA tetramers and an anti-PE MACS enrichment step was performed. From the pHLA-tetramer enriched fraction pHLA-Tetramer^pos, CD8^pos T cells were single cell sorted and expanded. Screening for peptide specificity resulted in identification of multiple T cell clones recognizing Jchain peptides presented in HLA-A1, -A24, -A3 and -A11. Next, 8 promising clones were identified based on recognition of Jchain transduced target cells and peptide titration experiments. Safety screenings revealed clear cross-reactivity for 2 out of 8 clones which were discarded to prevent toxicity. Per peptide-HLA restriction, the most potent clones that did not demonstrate off-target recognition were selected. These clones all recognized and lysed Jchain expressing MM cell lines. TCRs of selected T cell clones were sequenced, cloned into retroviral vectors, and successfully transferred to CD8 T cells. Jchain TCR T cells recognized and effectively lysed MM cell lines U266 and UM9 when the appropriate HLA allele was expressed. Additionally, Jchain or target HLA negative cells, including healthy subsets of hematopoietic and non-hematopoietic origins were not recognized. Peripheral blood B cells are positive for Jchain and co-culture with Jchain TCR T cells resulted in cytokine production as well as lysis of B cells, these data suggest that treatment with Jchain TCR T cells will result in depletion of the healthy B cell compartment of patients.

Ultimately, 4 out of 6 patient derived Jchain positive MM samples were strongly lysed by Jchain TCR T cells (Figure 1). Remarkably, 2 of 3 HLA-A24^pos MM samples were not lysed, suggesting a disruption of Jchain-A24 epitope presentation in a proportion of samples. To investigate in vivo targeting of MM cells, mice bearing U266 tumors were treated with Jchain TCR T cells. Compared to control treated mice, tumors in Jchain TCR treated mice rapidly shrunk upon treatment resulting in a 70- to 209-fold tumor burden reduction (Figure 2).

To conclude, Jchain specific T cells were successfully isolated from the HLA-mismatched repertoire of healthy donors. Jchain TCR T cells effectively targeted MM samples in vitro as well as in vivo in a pre-clinical mouse model. Except for recognition of Jchain expressing B cells, off-tumor reactivity of Jchain TCR T cells was not observed. Taken together these data show promise for Jchain targeting TCR T-cells as therapy for MM.

Griffioen:Miltenyi Biotec B.V & Co. KG: Research Funding.