The accessibility of cell surface proteins makes them tractable for targeting by cancer immunotherapy, but identifying suitable targets remains challenging. Here we describe plasma membrane profiling of primary human myeloma cells to identify an unprecedented number of cell surface proteins of a primary cancer. We employed a novel approach to prioritize immunotherapy targets and this identified a cell surface protein not previously implicated in myeloma, SEMA4A. Using knock-down by shRNA and CRISPR/dCas9, we demonstrate that expression of SEMA4A is essential for normal myeloma cell growth in vitro, indicating that myeloma cells cannot downregulate the protein to avoid detection. We further show that SEMA4A would not be identified as a myeloma therapeutic target by standard CRISPR/Cas9 knockout screens because of exon skipping. Finally, we potently and selectively targeted SEMA4A with a novel antibody-drug conjugate in vitro and in vivo.
Secreted proteins fulfill a vast array of functions, including immunity, signaling, and extracellular matrix remodeling. In the trans-Golgi network, proteins destined for constitutive secretion are sorted into post-Golgi carriers which fuse with the plasma membrane. The molecular machinery involved is poorly understood. Here, we have used kinetic trafficking assays and transient CRISPR-KO to study biosynthetic sorting from the Golgi to the plasma membrane. Depletion of all canonical exocyst subunits causes cargo accumulation in post-Golgi carriers. Exocyst subunits are recruited to and co-localize with carriers. Exocyst abrogation followed by kinetic trafficking assays of soluble cargoes results in intracellular cargo accumulation. Unbiased secretomics reveals impairment of soluble protein secretion after exocyst subunit knockout. Importantly, in specialized cell types, the loss of exocyst prevents constitutive secretion of antibodies in lymphocytes and of leptin in adipocytes. These data identify exocyst as the functional tether of secretory post-Golgi carriers at the plasma membrane and an essential component of the mammalian constitutive secretory pathway.
Sensitive cell surface proteomics studies have shown that the number of completely tumour-specific targets for adoptive cellular immunotherapy is extremely low. Even approved CAR T-cell targets appear to have expression in the central nervous system, leading to long-term neurological complications. We propose that this toxicity could be significantly improved by adoption of NOT-gates, which have been shown to limit CAR T-cell activity against healthy tissue expressing a second target that is absent on the tumour. Furthermore, the approach could also target essential, but non-specific proteins on tumour cells. The use of a NOT gate confers the specificity, whilst targeting the essential protein limits antigen escape. Here we explore the feasibility of such an approach for CAR T-cell targeting of primary myeloma. We show that none of the 45 most essential proteins are unique to the myeloma cell. However, whilst widely expressed, one of the most important proteins for myeloma cell survival, the transferrin receptor, could safely be targeted by a NOT-gate approach. Exploring co-expression patterns demonstrate 26 proteins that are not expressed on myeloma cells, but which are co-expressed with the transferrin receptor in all healthy tissues. We also describe a web app, NOTATER, which can be used by scientists with no bioinformatic capabilities to explore potential NOT-gate combinations in myeloma.
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