全面的表面蛋白质组学揭示CD70为自然杀伤/T细胞淋巴瘤(NKTL)的新型免疫治疗靶点

Natural killer/ T cell lymphoma (NKTL), a subtype of non-Hodgkin lymphoma, is an Epstein-Barr virus (EBV) associated lympho-proliferative disease prevalent in Asia and South America. Current treatments options are inadequate with treatments such as conventional radiotherapy or chemotherapy having limited efficacy. Moreover, efficacy of newer therapies such as targeted therapies or immunotherapies has been less than ideal in clinical trials likely due to heterogeneous nature of disease. Novel treatments which are more efficacious, such as immunotherapy with higher specificity and less cytotoxicity, will have to be formulated. Hence, this project aims to identify new targets and develop novel antibody-based therapies against NKTL. First step in antibody-based therapy development involves identification of suitable targets. Here, we combined aminooxy-biotin labeling followed by streptavidin enrichment with label-free quantitative mass spectrometry analysis to systematically profile the plasma membrane landscape of NKTL. Considering a lack of proper pipeline for plasma membrane annotation, we designed a unique membrane annotation pipeline based off publicly available databases, including UniProt, Gene Ontology and in silico surfaceome database, SURFY. Strategy for prioritization of targets with highest immunotherapeutic potential was employed by looking at target specificity and accessibility. Plasma membrane profiling via this method of analysis was successful with GO terms enrichment of the significant proteins in different cell lines pre-membrane protein annotation revealing molecular functions and biological processes associated with surface membrane proteins such as signalling receptor activity and adhesion. Our method of immunotherapeutic potential analysis revealed CD70, ITGA4 and CD48 as top targets with CD70 having highest potential. CD70 is a tightly regulated immune co-stimulatory ligand which is aberrantly expressed in different malignancies. Expression of CD70 was validated on multiple NKTL cell lines while PBMCs and primary NK cells from healthy donors were CD70 negative. Substantial internalization of CD70 was validated via FACS, immunofluorescence and Fab-ZAP assays, revealing potential as an antibody-drug conjugate (ADC) target for NKTL. Cusatuzumab, anti-CD70 monoclonal antibody currently in clinical trials for other malignancies, elicited strong antibody effector functions such as complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC) in NKTL. Lastly, in-house generated ADC through conjugation of Cusatuzumab with MMAE via VC-PAB linker showed efficacious response in NKTL. As such, our work characterized plasma membrane landscape of NKTL and shed light on targets with potential to become novel treatments for this dreadful disease.