T Cell Receptors Specific for the Intracellular Transcription Factor Bob1 Allow Efficient Targeting of Human B Cell Leukemia and Multiple Myeloma
Authors: Jahn Lorenz , Hombrink Pleun , Kester Michel G.D. , van der Steen Dirk M. , Hagedoorn Renate S. , Hassan Chopie , Schoonakker Marjolein P. , van der Pijl Erwin , van Veelen Peter A. , Falkenburg J.H. Frederik , Heemskerk Mirjam H.M. 
Published: October 2019

Abstract
Therapeutic reactivity of CD20-specific monoclonal antibodies (mAb) or CD19-specific chimeric antigen receptor (CAR)-transduced T cells is exerted by targeting extracellular antigens. However, loss of CD20 and CD19 expression or absence of these molecules on other malignancies such as multiple myeloma restricts their application.
Here, we identified the intracellular transcription factor Bob1 encoded by gene POU2AF1 as a suitable target for immunotherapy. Bob1 is highly expressed in CD19+ B cells, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL) and multiple myeloma (MM) and is absent in the non-B lineages including CD34+ hematopoietic progenitor cells (HPCs), T cells, fibroblasts, keratinocytes and gastrointestinal tract. Bob1 is localized intracellularly but HLA-presented Bob1-derived peptides are accessible on the cell surface to T cell receptors (TCRs) and can thus be recognized by T cells. From the HLA-presented ligandome (Mol Cell Proteomics, 2013;12:1829) we identified naturally processed Bob1-derived peptides displayed in HLA-A*0201 (HLA-A2) and in HLA-B*0702 (HLA-B7). Since auto-reactivity towards self-antigens such as Bob1 is prevented by depleting high-avidity T cells recognizing self-antigens in self-HLA, we exploited the immunogenicity of these peptides presented in allogeneic HLA. From a total of 3 x 109 peripheral blood mononuclear cells from 6 different HLA-A2/B7-negative healthy donors, we isolated and clonally expanded more than 1000 CD8+ T cells binding to peptide-MHC-tetramers composed of the Bob1-derived peptides bound to HLA-A2 or HLA-B7. The T cell clones were tested for stringent peptide-specificity by stimulation with Bob1-negative K562 cells expressing either HLA-A2 or B7 unloaded or pulsed with Bob1-derived peptides. This resulted in the selection of 15 T cell clones highly specific for Bob1. To identify the T cell clones of highest avidity, T cell clones were compared for peptide-sensitivity by testing the recognition of stimulator cells loaded with titrated amounts of Bob1-derived peptides and of Bob1-expressing HLA-A2/B7-positive EBV-transformed B cells. T cell clone 4G11 was selected because of high sensitivity and specificity for Bob1-derived peptide Bob144 presented in HLA-B7 and T cell clone 3C10 specifically recognized peptide Bob1245 bound to HLA-A2. Bob1-dependent recognition was demonstrated by transduction of Bob1 into cell lines that otherwise lack Bob1 expression. To investigate whether harmful toxicities could be caused by these T cell clones, we tested their reactivity against a wide panel of Bob1-negative stimulator cells demonstrating absence of recognition of HLA-B7-positive CD34+ HPCs, T cells, monocytes, immature and mature dendritic cells, and fibroblasts even under simulated inflamed conditions. Stringent HLA-B7-restricted recognition was observed for clone 4G11 when tested against a stimulator panel expressing a wide range of common and rare HLA class I and II molecules. These data illustrate a safe reactivity profile with little chance of off-target toxicity. To test their clinical applicability, clone 4G11 and 3C10 were tested for recognition of various primary B cell malignancies. Clone 4G11 efficiently recognized HLA-B7-positive primary ALL, CLL and mantle cell lymphoma while clone 3C10 recognized HLA-A2-positive primary B cell malignancies albeit to a lesser degree. Furthermore, reproducible strong recognition of purified primary HLA-B7-positive multiple myeloma could be demonstrated for clone 4G11. Therefore, T cell clone 4G11’s TCR may be used for immunotherapy by administering TCR-transduced T cells to multiple myeloma patients. To test whether introduction of 4G11’s TCR confers Bob1-reactivity onto recipient cells, the TCR was cloned into a retroviral vector. Highly specific reactivity against HLA-B7-positive Bob1-expressing target cells could be installed to TCR-transduced recipient T cells.
In summary, we identified the intracellular transcription factor Bob1 encoded by gene POU2AF1 as a suitable target for TCR-based immunotherapies of B cell malignancies and multiple myeloma. Bob1-specific T cell clone 4G11 efficiently recognized primary B cell leukemia and multiple myeloma. TCR gene transfer approaches using Bob1-specific TCRs can bring novel treatment modalities for patients with B cell malignancies or multiple myeloma.

Disclosures
No relevant conflicts of interest to declare.