The novel llama‐human chimeric antibody has potent effect in lowering LDL‐c levels in hPCSK9 transgenic rats

Abstract

BackgroundThe advent of proprotein convertase subtilisin/kexin type 9 (PCSK9)–inhibiting drugs have provided an effective, but extremely expensive treatment for the management of low density lipoprotein (LDL). Our aim was to explore a cost‐effective application of camelid anti‐PCSK9 single domain antibodies (sdAbs), which are high variable regions of the camelid heavy chain antibodies (VHHs), as a human PCSK9 (hPCSK9) inhibitor. One female llama was immunized with hPCSK9. Screening of high affinity anti‐PCSK9 VHHs was carried out based on surface plasmon resonance (SPR) technology. We reported a lysate kinetic analysis method improving the screening efficiency. To increase the serum half‐life and targeting properties, the constant region fragment of the human immunoglobulin gamma sub‐type 4 (IgG4 Fc) was incorporated to form a novel llama‐human chimeric molecule (VHH‐hFc). ResultsThe PCSK9 inhibiting effects of the VHH proteins were analyzed in two human liver hepatocellular cells (HepG2 and Huh7) and in the hPCSK9 transgenic Sprague–Dawley (SD) rat model. The hPCSK9 antagonistic potency of the bivalent VHH‐hFc exceeded the monovalent VHH (P < 0.001) in hepatocarcinoma cells. Furthermore, the llama‐human chimeric VHH‐Fc protein had a similar reduction (~ 40%) of the LDL‐c and total cholesterol when compared to the approved evolocumab in transgenic SD rat model, but with low cost. More surprisingly, the chimeric heavy chain antibodies could be persevered for 3 months at room temperature with little loss of the affinity. ConclusionsDue to the high yield and low cost of Pichia pastoris, lipid‐lowering effect and strong stability, the llama‐human chimeric antibody (VHH‐Fc) offers a potent therapeutic candidate for the control of the serum lipid level.

A novel nanobody-heavy chain antibody against Angiopoietin-like protein 3 reduces plasma lipids and relieves nonalcoholic fatty liver disease

“…Nanobody and its fusion protein, which are promising therapeutic agents, display excellent binding affinity and stability. Besides, they could be expressed in yeast with high yield and low cost [ 44 ]. Recently, a bivalent VHHs fusion protein, caplacizumab, had been approved by the FDA for thrombotic thrombocytopenic purpura treatment, indicating the druggability of nanobody and its fusion protein [ 35 , 45 , 46 ].…”

“…Meanwhile, previous studies revealed that bivalent nanobody had better affinity than monovalent modality. In particular, the bivalent form via Fc fusion showed above 10 times higher affinity and stronger therapeutic effects than monovalent in vitro and in vivo, also emerged longer half-life up to 15 days when compared with monovalent (~ 30 min) and tandem bivalent VHHs (~ 60 min) [ 44 , 49 ]. In present study, C44 was fused to human IgG1 Fc domain to construct the bivalent VHH-Fc fusion protein.…”

Research Progress and Applications of Multivalent, Multispecific and Modified Nanobodies for Disease Treatment

“…A nanobody inhibiting the proprotein convertase subtilisin/kexin type 9 (PCSK9) was expressed fused to an Fc domain and effectively reduced the production of low density lipoprotein (LDL) and cholesterol in a rat model ( 177 ). The results were comparable to those obtained with the approved monoclonal evolocumab but at extremely lower production costs.…”

Exploring cellular biochemistry with nanobodies

“…Nanobodies can be used to block a variety of biological processes, such as placental growth factor-induced angiogenesis in cancer (115); the action of inflammatory proteins, such as tumor necrosis factor, interleukin-23, granulocyte colonystimulating factor, and macrophage migration inhibitory factor (116)(117)(118)(119)(120); and the action of various toxins and venoms (121)(122)(123). A chimeric heavy chain-only antibody consisting of a proprotein convertase subtilisin/kexin type 9-binding nanobody and a portion of the human immunoglobulin heavy chain lowers low-density lipoprotein levels when administered in transgenic rats (124). Nanobodies against neuronal tau (125), human prion protein (126), and a-synuclein (127) provided insight into structural transitions that lead to amyloid formation (126) and served as sensors to differentiate between fibrils at characteristically different stages (127).…”