Date Published: January 26, 2017
Publisher: Public Library of Science
Author(s): Amelie Vallet-Courbin, Mélusine Larivière, Agnès Hocquellet, Audrey Hemadou, Sarjapura-Nagaraja Parimala, Jeanny Laroche-Traineau, Xavier Santarelli, Gisèle Clofent-Sanchez, Marie-Josée Jacobin-Valat, Abdelmajid Noubhani, Pablo Garcia de Frutos.
Cells of the innate and adaptive immune system are key factors in the progression of atherosclerotic plaque, leading to plaque instability and rupture, potentially resulting in acute atherothrombotic events such as coronary artery disease, cerebrovascular disease and peripheral arterial disease. Here, we describe the cloning, expression, purification, and immunoreactivity assessment of a recombinant single-chain variable fragment (scFv) derived from a human anti-αIIbβ3 antibody (HuAb) selected to target atheromatous lesions for the presence of platelets. Indeed, platelets within atheroma plaques have been shown to play a role in inflammation, in platelet-leucocyte aggregates and in thrombi formation and might thus be considered relevant biomarkers of atherosclerotic progression. The DNA sequence that encodes the anti-αIIbβ3 TEG4 scFv previously obtained from a phage-display selection on activated platelets, was inserted into the eukaryote vector (pPICZαA) in fusion with a tag sequence encoding 2 cysteines useable for specific probes grafting experiments. The recombinant protein was expressed at high yields in Pichia pastoris (30 mg/L culture). The advantage of P. pastoris as an expression system is the production and secretion of recombinant proteins in the supernatant, ruling out the difficulties encountered when scFv are produced in the cytoplasm of bacteria (low yield, low solubility and reduced affinity). The improved conditions allowed for the recovery of highly purified and biologically active scFv fragments ready to be grafted in a site-directed way to nanoparticles for the imaging of atherosclerotic plaques involving inflammatory processes and thus at high risk of instability.
Atherosclerosis is an inflammatory disease associated with the formation of unstable thrombosis-prone atheroma plaques made of large lipid cores, thin fibrous cap and inflammatory cell infiltrates within the walls of arteries. Atherosclerotic plaque rupture is the mechanistic cause of about 75% of all sudden and often fatal heart attacks. As the risk of plaque rupture is more related to the plaque contents than to the plaque size, molecular imaging modalities have risen as a new imperative. Current studies tend towards the development of non-invasive targeted methods to assess the cellular components that underlie the risk of rupture.[3,4] Molecular imaging requires highly sensitive and specific probes made of a signal detection compound and an affinity ligand for targeting. The affinity ligand should be able to recognize molecules and cells over-expressed during the course of atherogenesis. Inflammation is a well-recognized pathophysiological process involving both innate and adaptive immune cells. Recruitment of monocytes in the vascular wall and macrophage differentiation and proliferation represent a hallmark in the pathology of atherosclerotic lesions. They contribute to the processes that underlie atherogenesis such as lipid accumulation, secretion of pro-inflammatory cytokines, extracellular matrix remodelling. Moreover, the observation of activation and oligoclonal expansion of T cells has suggested the presence of inciting antigens (Ags) that sustain T cell recruitment within coronary lesions. B cells also play a pro or anti-atherogenic role depending on the subtypes (B1(a) or B2), and in atherosclerosis they accumulate both in the atherosclerotic intima and associated adventitia.[8–10] More recently, platelets have come to the forefront as partners of macrophages, T cells and B cells in inflammation and immune responses. They are now recognized as key players in innate and adaptive immune responses [11,12] and notably shown to modulate the T-effector/T-regulator balance via the CD40 ligand.[13,14] Platelet-derived CD40 ligand has also been reported to support B-cell differentiation and immunoglobulin class switching in mice. Several cytokines released by activated platelets have been demonstrated to modulate monocyte and macrophage function. Moreover platelet—leukocyte interactions largely contribute to OxLDL uptake and foam cell formation. A recent study has underlined the presence of platelets not only in thrombi and intraplaque hemorrhage but also in atheroma burden, around necrotic areas and neovessels, shedding light on the rationale for targeting platelets within atherosclerotic lesions.
In the present study, TEG4-2c scFv was expressed at high-level in Pichia pastoris using a fed-batch fermentation system monitored by pO2 level. We produced the TEG4 scFv with cysteine tags at the end of the C-terminal sequence for site-specific conjugation to contrast agents, precluding the loss of reactivity potentially occurring when the grafting process affects antigen-recognition sites. TEG4 scFv had been previously expressed in E coli. Unfortunately, despite optimization tests leading to high yields of cytoplasmic production, proteins also frequently accumulated into inclusion bodies (data not shown). In bacterial systems, many scFv can be produced into the periplasmic space but they are obtained with a very low yield. Higher levels of production can be achieved in inclusion bodies, with the limitation of the presence of insoluble scFv aggregates and the need for subsequent in vitro folding that make the use of this bacterial system not attractive for the large scale production of scFv. In addition, many authors have described that the final yield of scFv was only a small percentage of produced proteins with a low specificity for targets.[33,34]