Immunogenicity Of Novel CHO- And HEK293- Produced Variants Of The Atheroprotective chP3R99 mAb In Distinct Animal Models
Creators: Araujo, Gala P
Publisher: name: University of Alberta Library
Year: 2024

<p>Cardiovascular diseases (CVDs) are the leading cause of death globally and exert a significant burden on healthcare systems and public governmental institutions. Atherosclerosis is a predominant form of CVD characterized by the accumulation of cholesterol-rich lipoproteins within the innermost wall of the artery, known as the tunica intima. This lipoprotein accumulation ultimately leads to fibrous-rich plaque formation, which reduces blood flow and causes major cardiovascular events, including myocardial infarctions, strokes, and disabling peripheral artery disease. Atherosclerosis is theorized to be linked to specialized sugar side chains, known as Glycosaminoglycans (GAGs), in the tunica intima that can specifically bind cholesterol-rich lipoproteins. Two main forms of cholesterol can interact with these GAGs (i) namely Low-Density Lipoprotein (LDL) (or ‘bad’) cholesterol and (ii) &#39;Remnant&#39; cholesterol. Despite the prevalence of atherosclerosis, therapeutic approaches for the treatment of atherosclerosis have been primarily limited to the management of atherosclerotic risk factors (e.g. lipid-lowering) rather than disease prevention per se. In this thesis, we introduce the development of a new class of monoclonal antibody (mAb) (called chP3R99) that has been demonstrated to compete with lipoprotein binding to GAGs within the extracellular matrix of the tunica intima. ChP3R99 is a novel human-murine chimeric mAb capable of binding specifically to proatherogenic GAGs with high specificity and has been shown to inhibit lipid retention and reduce atherosclerosis progression in a number of small animal models. Previous studies of chP3R99 have attributed these atheroprotective features to be associated with the induction of secondary (Ab2) and tertiary antibodies (Ab3) generated in the host due to the unique immunogenic idiotype of the P3R99. <br>
However, inefficiencies with the production of P3R99 via the murine myeloma, NS0, cell line has led to the development of two new P3R99 variants produced from more productive cell lines: the Chinese Hamster Ovary, CHO-P3R99, and Human Embryonic Kidney, HEK-P3R99. Despite the maintenance of the P3R99 structure postproduction and recognition of chondroitin sulfate, the CHO-P3R99 and HEK-P3R99 variants are yet to be tested for the conservation of the original NS0-P3R99 (chP3R99) immunogenic function.<br>
Thus, this thesis aimed to assess the immunogenicity and idiotypic strength of the variant P3R99s in distinct animal models compared to the original NS0-P3R99 and a negative isotype-matched control hR3. Immunogenicity was assessed through the induction of host-derived Ab2 and Ab3 antibodies.<br>
Heterozygous lean JCR:LA-cp rats, homozygous obese-insulin resistant JCR:LA-cp rats, and white-landrace piglets were used as models for immunogenicity assessment. Rodents received 6 doses of 200 ug (400 ug/mL) and underwent 5 blood draws during treatment, while piglets received 5 doses of 1.0 mg (0.33 ug/mL) and underwent 4 blood draws. Sera taken during blood sampling was separated via differential centrifugation and used for Enzyme-linked Immunosorbent Assay (ELISA) Ab2 and Ab3 antibody detection. Results in lean JCR:LA-cp rats and white-landrace piglets confirmed the immunogenicity of the CHO-P3R99, NS0-P3R99, and HEK-P3R99 variants, following a minimum of 4 and 5 doses, respectively. Furthermore, results in obese insulin-resistant JCR:LA-cp rats confirmed the immunogenicity of the CHO-P3R99 and NS0-P3R99 variants following a minimum of four doses. Additionally, both rodent studies confirmed the conserved immunogenic strength of the NS0-P3R99 in the HEK-P3R99 and CHO-P3R99 variants. Similarly, piglet studies demonstrated the conserved immunogenic strength of the NS0-P3R99 in the two variants in regard to Ab3 antibody induction. Although, this was independent of the Ab2 response for the HEK-P3R99. <br>
In conclusion, we demonstrate for the first time that the new P3R99 variants are not inferior in their capacity of inducing anti-CS anti-atherogenic Ab3 antibodies in rodents and swine, but due to some inconsistencies between animal models used for their anti-atherogenic properties, further testing will be required.</p>