Cardiovascular diseases (CVDs) represent the leading cause of death in industrialized countries. Lipoprotein(a) [Lp(a)] is an important, yet novel and poorly characterized risk factor for atherosclerotic CVDs in humans, as elevated serum levels (>30-50 mg/dl) of this lipoprotein are strongly and causally associated to increased CVD risk. This circulating LDL-like lipoprotein carries a peculiar glycoprotein, named apolipoprotein(a) [apo(a)]. The fingerprint of Lp(a) is the astonishingly high degree of apo(a) size heterogeneity, which mainly depends on the genetically determined copy number (1-40 units) of its Kringle IV type 2 (KIV-2) domain. Interestingly, this number is inversely associated with Lp(a) levels in serum and with CVD risk, but, at present, there is no clear explanation for the higher pathogenicity of the small apo(a) isoforms compared to their large counterparts. The research work of my thesis was conceived to address, by a multi-disciplinary approach, two open issues, strictly related one to the other, regarding Lp(a) heterogeneity: (1) the need for improved research, diagnostic and therapeutic tools specific for the study and clinical monitoring of Lp(a), and (2) the need to clarify the role of apo(a) size, as determined by the copy number of KIV-2, in atherosclerosis pathophysiology. For the first aim of the work, production of an anti-KIV-2 mAb in hybridoma culture and engineering of its scFv format were carried out, with a good expression of the latter obtained in the yeast P. pastoris. KIV-2 single domain and KIV-2-KIV-2 tandem domains were over-expressed in E. coli and P. pastoris hosts, resulting in high yields. Binding of the anti-KIV-2 antibodies to these target antigens was demonstrated through immunoblotting, immunoenzymatic assays and gel filtration. In surface plasmon resonance, the mAb demonstrated very promising affinity properties. Thus, the antibodies here developed will be evaluated as potential key tools in systems dedicated to the diagnosis and therapy of hyperlipoproteinemia(a). For the second aim of this research, a set of specialized biophysical analyses allowed to highlight some isoform-specific properties of Lp(a) that suggested the identification of a new, isoform-related, threshold of CVD risk. In parallel, some interesting Lp(a)- and isoform-dependent effects on model target cell types were detected using an innovative and sensitive real-time in vitro cell analysis. This system is relevant to determine anti-Lp(a) antibody interference on Lp(a)-mediated biological effects. Preliminary evidence was achieved both on the Lp(a) particle structure by negative staining electron microscopy and on crystallization of the KIV-2, as a background work for future high-resolution structural analyses. Finally, an efficient Golden Gate cloning platform was set-up to allow, upon expression in suitable hosts, the generation of a library of recombinant apo(a)/Lp(a) isoforms, which could constitute reliable starting material to accurately study intrinsic and specific properties of these variants. It is expected that these contributions will be useful towards a better diagnosis and therapy of hyperlipoproteinemia(a).

Development of antibodies for diagnosis and therapy of hyperlipoproteinemia(a): targeting apolipoprotein(a) isoforms

SANTONASTASO, ALICE
2017

Abstract

Cardiovascular diseases (CVDs) represent the leading cause of death in industrialized countries. Lipoprotein(a) [Lp(a)] is an important, yet novel and poorly characterized risk factor for atherosclerotic CVDs in humans, as elevated serum levels (>30-50 mg/dl) of this lipoprotein are strongly and causally associated to increased CVD risk. This circulating LDL-like lipoprotein carries a peculiar glycoprotein, named apolipoprotein(a) [apo(a)]. The fingerprint of Lp(a) is the astonishingly high degree of apo(a) size heterogeneity, which mainly depends on the genetically determined copy number (1-40 units) of its Kringle IV type 2 (KIV-2) domain. Interestingly, this number is inversely associated with Lp(a) levels in serum and with CVD risk, but, at present, there is no clear explanation for the higher pathogenicity of the small apo(a) isoforms compared to their large counterparts. The research work of my thesis was conceived to address, by a multi-disciplinary approach, two open issues, strictly related one to the other, regarding Lp(a) heterogeneity: (1) the need for improved research, diagnostic and therapeutic tools specific for the study and clinical monitoring of Lp(a), and (2) the need to clarify the role of apo(a) size, as determined by the copy number of KIV-2, in atherosclerosis pathophysiology. For the first aim of the work, production of an anti-KIV-2 mAb in hybridoma culture and engineering of its scFv format were carried out, with a good expression of the latter obtained in the yeast P. pastoris. KIV-2 single domain and KIV-2-KIV-2 tandem domains were over-expressed in E. coli and P. pastoris hosts, resulting in high yields. Binding of the anti-KIV-2 antibodies to these target antigens was demonstrated through immunoblotting, immunoenzymatic assays and gel filtration. In surface plasmon resonance, the mAb demonstrated very promising affinity properties. Thus, the antibodies here developed will be evaluated as potential key tools in systems dedicated to the diagnosis and therapy of hyperlipoproteinemia(a). For the second aim of this research, a set of specialized biophysical analyses allowed to highlight some isoform-specific properties of Lp(a) that suggested the identification of a new, isoform-related, threshold of CVD risk. In parallel, some interesting Lp(a)- and isoform-dependent effects on model target cell types were detected using an innovative and sensitive real-time in vitro cell analysis. This system is relevant to determine anti-Lp(a) antibody interference on Lp(a)-mediated biological effects. Preliminary evidence was achieved both on the Lp(a) particle structure by negative staining electron microscopy and on crystallization of the KIV-2, as a background work for future high-resolution structural analyses. Finally, an efficient Golden Gate cloning platform was set-up to allow, upon expression in suitable hosts, the generation of a library of recombinant apo(a)/Lp(a) isoforms, which could constitute reliable starting material to accurately study intrinsic and specific properties of these variants. It is expected that these contributions will be useful towards a better diagnosis and therapy of hyperlipoproteinemia(a).
Lipoprotein(a),; apolipoprotein(a),; isoforms,; atherosclerosis,;
File in questo prodotto:
File Dimensione Formato  
Thesis_Santonastaso Alice.pdf

accesso aperto

Descrizione: tesi di dottorato
Dimensione 20.17 MB
Formato Adobe PDF
20.17 MB Adobe PDF Visualizza/Apri

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11571/1203271
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact