Poster Session II: Gene therapy, cellular immunotherapy and vaccination - Biology & translational research
Antigen-loss tumor escape variants are a major concern when targeting cancer, while tumor-specific targeting is crucial to prevent unwanted toxicity. Thus, targeting essential cancer- or lineage-specific genes might represent attractive targets for antineoplastic therapies since antigen-loss is less likely to occur, and toxicity is limited. Here, we describe B cell lineage-specific transcriptional coactivator BOB1 to meet these important criteria. Interestingly, in our lab we have successfully identified several distinct T cell receptors (TCRs) that are BOB1-specific with demonstrable preclinical efficacy.
With this study we aimed to demonstrate that the transcriptional coactivator BOB1 is essential for survival of B cell malignancies with a germinal center origin, and that these neoplasms are recognized by our recently and newly identified BOB1-specific TCRs in the context of HLA-B*07:02 and HLA-B*35:01, respectively.
Using a CRISPR/Cas9 puromycin plasmid containing a sgRNA or by applying a doxycycline-inducible system, we set out to target BOB1 in different B cell malignancies. Upon targeting, we selected puromycin resistant single-cell clones or tracked cell growth upon induction, and used sanger-sequencing to identify possible indel mutations. Besides single-cell analysis, Tracking of Indels by DEcomposition (TIDE) was used to accurately quantify the editing efficacy and most prominent types of indel mutation in different pools of B cell tumors. Finally, we performed in vitro co-cultures with BOB1-specific TCR transduced CD8 T cells and a plethora of B cell malignancies, including myeloma and lymphoma, and assessed preclinical efficacy targeting myeloma in vivo.
Remarkably, for each type of B cell tumor used, we were unable to generate BOB1-deficient clones, unveiling the importance for cell survival. In line, using the TIDE algorithm for easy detection of predominant types of indel mutations in targeted pools of distinct types of B cell malignancies, we found unmutated BOB1 sequences only (>90%), while generally a mere 5% is expected to be unaltered for any irrelevant gene. Finally, we further established the essentiality of BOB1 in a broad panel of B cell malignancies containing inducible CRISPR/Cas9, which uniformly resulted in rapid cell death upon induction. Interestingly and importantly, using our recently and newly identified TCRs recognizing peptides derived from BOB1 in the context of HLA-B*07:02 and HLA-B*35:01, we observed highly specific and efficient targeting of a vast majority of B cell malignancies in vitro and in vivo.
Altogether, we postulate BOB1 as an essential B cell lineage-specific target, fortifying gene transfer of our unique TCRs into CD8+ cytotoxic T cells as an efficient therapeutic treatment option for a large variety of B cell neoplasms.