C. A. Tran1, A. Mandal2, M. Mandal3, W. C. Olson1, J. Shetty2, K. A. Knapp4, E. S. Pires5, S. J. Adair1, T. W. Bauer1, T. N. Bullock4, J. C. Herr2, C. L. Slingluff1 1University of Virginia School of Medicine, Department Of Surgery, Charlottesville, VA, USA 2University of Virginia School of Medicine, Department Of Cell Biology, Charlottesville, VA, USA 3Albemarle High School, Charlottesville, VA, USA 4University of Virginia School of Medicine, Department Of Pathology, Charlottesville, VA, USA 5University of Virginia School of Medicine, Department Of Obstetrics And Gynecology, Charlottesville, VA, USA
Introduction: SAS1B is a protein found in oocytes in adult tissues and also detected in uterine tumor cells, classifying it as a cancer-oocyte neoantigen. Treatment with anti-SAS1B polyclonal antibodies and complement arrested growth of uterine tumor cells, and saporin-immunotoxin directed at SAS1B induced cell death. Study goals were to generate monoclonal antibodies (mAbs) against human SAS1B, develop a cytotoxic reagent targeting SAS1B using mAbs, determine tumor cell types expressing SAS1B, and assess normal tissue SAS1B expression.
Methods: Monoclonal antibodies were generated to recombinant (r) human (h) SAS1B. Mass spectrometry confirmed SAS1B recognition. SAS1B deletion constructs mapped epitopes recognized by SB2/SB5 mAbs to the N-terminal domain. Human cancer cell lines were treated with increasing concentrations of mAb and antibody-drug conjugate (ADC). ATP was quantitated as a measure of viability. SAS1B surface expression of human normal and tumor cell lines were evaluated by flow cytometry after staining with SB2, either with viable cells to assess surface staining or permeabilized cells to assess intracellular plus surface staining, with and without SAS1B blocking peptide.
Results: The SB2/SB5 epitope is located between amino acids 32-39 of SAS1B. SB2/SB5 mAbs demonstrated Western immunoreactivity to rhSAS1B in E. coli/HEK293T cells. SAS1B serves as a target for Ab-mediated cytotoxicity by ADC which is blocked by the SAS1B-specific N-terminal peptide, as cell viability decreased with increasing ADC concentrations. Monoclonal antibodies SB2/SB5, complexed with a Fab-Duocarmycin DM, were cytotoxic to SAS1B+ uterine, lung, renal, and pancreatic tumor cells (Figure 1). SAS1B N-terminus surface and cytoplasmic expression of these tumor cell lines, ovary, breast, and melanoma were confirmed by flow cytometry and SAS1B[24-42] peptide blocking. Intracellular SAS1B was detected in normal aortic endothelium, skeletal muscle, pancreatic islets, kidney, peripheral blood mononuclear cells (PBMCs), and lymph node. However, surface SAS1B was not detected on normal cardiac myocytes (0.1% of cells), aortic endothelium (0.8%), skeletal muscle (0.8%), pancreatic islets (0.65%), kidney (0.7%), skin fibroblasts (1.1%), PBMCs (0.1%), lymph node (0.2%), and spleen (0.2%), compared to MDA-MB-468 (38%). Only staining of MDA-MB-468 was blocked by the SAS1B[24-42] peptide (96%).
Conclusion: SAS1B is expressed on surfaces of multiple tumor cell types, but not on those of normal cells. The SB2/SB5 epitope is between amino acids 32-39. The SB2/SB5 mAbs offer promise as candidate immunotherapeutic entities for targeting SAS1B+ cancer cells with defined epitopes in the SAS1B N-terminal domain suitable for immunotherapeutic targeting with ADC or an scFv/CAR-T approach.
