Prioritization of immune therapy targets leads to the identification of SEMA4A as a potential ADC target in myeloma. (A) Expression of SEMA4A by plasma membrane profiling (PMP) in myeloma cell lines and primary myeloma cells. Log relative expression is standardized to the range 0 to 1 for each sample. The median expressions of SLAMF7 (the target of elotuzumab), CD38 (the target of daratumumab), and BCMA (the target of CAR T cells) are also indicated. (B) Comparison of SEMA4A expression by flow cytometry and by PMP. Relative protein abundance by tandem mass tag labeling and mass spectrometry in arbitrary units (AU; x-axis) is compared with protein abundance by median fluorescence intensity (MFI) as determined by flow cytometry with the SEMA4A 5E3 clone antibody relative to isotype control (y-axis). Error bars are ±SD of biological replicates; n = 3. (C) Representative images of healthy (non–multiple myeloma [MM]) and MM bone marrow after duplex immunohistochemistry staining with CD138 (yellow) and SEMA4A (purple). CD138⁺ cells in the non-MM bone marrow are indicated by arrows. (D) Comparison of SEMA4A expression by flow cytometry in CD138⁺ cells in non-MM (n = 3) and in MM (n = 5) as described in panel B. (E) Representative histograms of flow cytometric profiling of SEMA4A expression in hematopoietic cells from a single patient. Markers used are CD66b (granulocytes), CD14 (monocytes), CD138 (myeloma cells), CD3 (T cells), CD19 (B cells), and CD34 (primitive hematopoietic cells). Black lines show intensity of isotype control, and blue lines show the intensity of SEMA4A. (F) Summary of the flow cytometric profiling of hematopoietic cells as shown in panel E. SEMA4A expression for each patient was calculated as the MFI of the 5E3 clone relative to the isotype control and then normalized to SEMA4A expression on CD19⁺ cells; n = 12. (G) Expression of SEMA4A in normal healthy tissues by whole-cell proteomic profiling (Human Proteome Map, www.humanproteomemap.org; left) and by tissue microarray (Human Protein Atlas, proteinatlas.org; right). NK, natural killer. ***P < .001.