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Title: Plasma membrane profiling of multiple myeloma and the identification of novel monoclonal antibody targets
Author: Anderson, Georgina
ISNI:       0000 0004 7968 3931
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
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Plasma membrane proteins are ideal therapeutic targets, being both easily accessible and involved in mediating numerous cellular processes. However, the development of therapeutic agents targeting these proteins has been hindered by the difficulties associated with their identification and characterisation. Transcriptomic approaches are unreliable and previous proteomic efforts have failed to sufficiently enrich for plasma membrane (PM) proteins or were not quantitative. Plasma membrane profiling (PMP) is a novel proteomic technique that overcomes previous limitations and enables the identification and quantification of hundreds to thousands of PM proteins across multiple samples. We have adopted this technique to characterise the whole cell surface proteome in myeloma and to identify a novel antibodydrug conjugate (ADC) target. Eight primary samples and ten human myeloma cell lines were profiled by mass spectrometry using PMP. A total of 2,077 proteins were identified with high confidence across all samples of which at least 1,319 were PM proteins. This represents a substantial improvement over other reported datasets, both in terms of total PM proteins identified and in the number of primary samples quantitated. This dataset was validated by repeat profiling and by comparing the relative PMP expression values against flow cytometry expression for six different antigens. To identify a novel antibody-drug conjugate target, proteins were ranked according to a combination of a) presence of a targetable extracellular domain b) high and ubiquitous on-tumour expression and c) low off-tumour expression. High-ranking candidates were screened for internalisation by flow cytometry and microscopy. One target, SEMA4A, which was rapidly internalised and exhibited restricted healthy tissue expression, was taken forward to test for in vitro killing activity. An anti-SEMA4A ADC induced cell death exclusively in high SEMA4A-expressing cell lines in vitro and demonstrated potent activity in an in vivo xenograft model of myeloma. We also observed that the knock-down of SEMA4A using RNA-interference causes a competitive disadvantage, suggesting that target-downregulation would not be a viable mechanism of tumour escape.
Supervisor: Chapman, Michael Sponsor: Cancer Research UK (CRUK)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Biological science ; Haematology ; Multiple myeloma ; target identification