Proctor, S. D. | Search bioRxiv

bioRxiv2024

Characterization and Antiatherogenic Potential of P3R99 Monoclonal Antibody Against Sulfated Glycosaminoglycans: Physicochemical and Functional Insights

Atherosclerosis is initiated by the retention of ApoB-containing lipoproteins in the arterial wall, mediated by glycosaminoglycan chains of proteoglycans. At the Center for Molecular Immunology, we are developing the P3R99 monoclonal antibody (mAb) to target this process. This study characterizes new P3R99 mAb variants expressed in CHO-K1 and HEK-293 cell lines. We compared these variants with the parental mAb from NS0 cells using SDS-PAGE, size exclusion and cation exchange chromatography, dynamic light scattering, peptide mapping, far-UV circular dichroism, and PNGase F deglycosylation. All variants exhibited a molecular size of [~]150 kDa, [~]99% purity, and similar average particle sizes (12.5-13.7 nm). They displayed a high {beta}-sheet content (>40%) and basic amino acids on the surface, with minor differences in peptide maps compared to the parental mAb. Notable differences were found in the content of acidic and basic species and glycosylation profiles. NS0-derived P3R99 had lower G0F content (10.39%), higher G1F (38.29%) and G2F (30.44%) levels, with more terminal galactose (83.07%) and sialylation (15.33%). In contrast, CHO-K1 and HEK-293 variants showed similar glycosylation patterns. Despite these differences, the antigen and atherosclerotic lesion recognition properties of the mAb were unaffected in vitro. Biodistribution studies in Sprague Dawley rats (1 mg, IV, n=3) revealed preferential accumulation of the new P3R99 variants in aortas and reduced LDL arterial retention (1 mg, IP). Passive administration of the mAbs (2 mg every three days, three IV doses, n=6-7) in a Lipofundin 20%-induced atherosclerosis NZW rabbit model also demonstrated preferential accumulation in aortas and reduced atherosclerosis, with 60% of treated rabbits not developing lesions. These results suggest that the P3R99 mAb derived from CHO-K1 and HEK-293 cells retains its antiatherogenic properties despite structural differences from the NS0-derived mAb associated with the different expression systems.

immunology↗

bioRxiv2023

Novel chP3R99 mAb reduces subendothelial retention of atherogenic lipoproteins in Insulin-Resistant rats: Acute treatment versus long-term protection as an idiotypic vaccine for atherosclerosis

BACKGROUNDAtherosclerosis is triggered by the retention of apolipoprotein B-containing lipoproteins by proteoglycans. In addition to LDL, remnant lipoproteins have emerged as pivotal contributors to this pathology, particularly in the context of insulin resistance and diabetes. We have previously reported anti-atherogenic properties of a monoclonal antibody (chP3R99) that recognizes sulfated glycosaminoglycans on arterial proteoglycans. METHODS AND RESULTSSolid-phase assays demonstrated that chP3R99 effectively blocked over 50% lipoprotein binding to chondroitin sulfate and vascular extracellular matrix in vitro. The pre-perfusion of chP3R99 (competitive effect) resulted in specific antibody-arterial accumulation and reduced fluorescent lipoprotein retention by [~]60% in insulin resistant JCR:LA-cp rats. This competitive reduction was dose-dependent (25 {micro}g/mL-250 {micro}g/mL), effectively decreasing deposition of cholesterol associated with lipoproteins. In a five-week vaccination study in insulin resistant rats with (200 {micro}g SC, once a week), chP3R99 reduced arterial lipoprotein retention, and was associated with the production of anti-chondroitin sulfate antibodies (Ab3) able to accumulate in the arteries (dot-blot). Neither the intravenous inoculation of chP3R99 (4.5 mg/kg), nor the immunization with this antibody displayed adverse effects on lipid or glucose metabolism, insulin resistance, liver function, blood cell indices, or inflammation pathways in JCR:LA-cp rats. CONCLUSIONSBoth acute (passive) and long-term administration (idiotypic cascade) of chP3R99 antibody reduced LDL and remnant lipoprotein interaction with proteoglycans in an insulin-resistant setting. These findings support the innovative approach of targeting pro-atherogenic lipoprotein retention by chP3R99 as a passive therapy or as an idiotypic vaccine for atherosclerosis. CLINICAL PERSPECTIVEO_ST_ABSWhat Is New?C_ST_ABSO_LIInnovative anti-atherosclerotic chP3R99 mAb interferes with proteoglycan binding of both LDL and remnant lipoproteins in vitro and in vivo. C_LIO_LIIn vivo kinetic studies that immunize with chP3R99 reveal a temporal induction of an anti-idiotypic antibody cascade in a model of insulin resistance (analogous to a vaccine). C_LIO_LIWe discovered that the idiotypic chP3R99 monoclonal antibody (Ab1) was able to induce protective anti-anti-idiotypic (Ab3) antibodies present in both sera as well as the aorta (target organ) in vivo. C_LI What Are The Clinical Implications?O_LIThe chP3R99 mAb has efficacy for reducing the arterial retention of both LDL and remnant-derived lipoproteins and may be relevant of those with Type-2 Diabetes and/or residual CVD risk. C_LIO_LIWe show efficacy of chP3R99 mAb under pro-inflammatory conditions and that it does not exacerbate other metabolic aberrations during insulin resistance. C_LIO_LIData support the targeting pro-atherogenic lipoprotein retention with chP3R99 as a passive therapy or as an idiotypic vaccine for atherosclerosis, complementary to lipid lowering approaches. C_LI

pathology↗