Generation of a Novel High-Affinity Antibody Binding to PCSK9 Catalytic Domain with Slow Dissociation Rate by CDR-Grafting, Alanine Scanning and Saturated Site-Directed Mutagenesis for Favorably Treating Hypercholesterolemia

Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) has become an attractive therapeutic strategy for lowering low-density lipoprotein cholesterol (LDL-C). In this study, a novel high affinity humanized IgG1 mAb (named h5E12-L230G) targeting the catalytic domain of human PCSK9 (hPCSK9) was generated by using CDR-grafting, alanine-scanning mutagenesis, and saturated site-directed mutagenesis. The heavy-chain constant region of h5E12-L230G was modified to eliminate the cytotoxic effector functions and mitigate the heterogeneity. The biolayer interferometry (BLI) binding assay and molecular docking study revealed that h5E12-L230G binds to the catalytic domain of hPCSK9 with nanomolar affinity (KD = 1.72 nM) and an extremely slow dissociation rate (koff, 4.84 × 10−5 s−1), which interprets its quite low binding energy (−54.97 kcal/mol) with hPCSK9. Additionally, h5E12-L230G elevated the levels of LDLR and enhanced the LDL-C uptake in HepG2 cells, as well as reducing the serum LDL-C and total cholesterol (TC) levels in hyperlipidemic mouse model with high potency comparable to the positive control alirocumab. Our data indicate that h5E12-L230G is a high-affinity anti-PCSK9 antibody candidate with an extremely slow dissociation rate for favorably treating hypercholesterolemia and relevant cardiovascular diseases.

Analysis of Hendra Virus Fusion Protein N-Terminal Transmembrane Residues

Hendra virus (HeV) is a zoonotic enveloped member of the family Paramyoxviridae. To successfully infect a host cell, HeV utilizes two surface glycoproteins: the attachment (G) protein to bind, and the trimeric fusion (F) protein to merge the viral envelope with the membrane of the host cell. The transmembrane (TM) region of HeV F has been shown to have roles in F protein stability and the overall trimeric association of F. Previously, alanine scanning mutagenesis has been performed on the C-terminal end of the protein, revealing the importance of β-branched residues in this region. Additionally, residues S490 and Y498 have been demonstrated to be important for F protein endocytosis, needed for the proteolytic processing of F required for fusion. To complete the analysis of the HeV F TM, we performed alanine scanning mutagenesis to explore the residues in the N-terminus of this region (residues 487–506). In addition to confirming the critical roles for S490 and Y498, we demonstrate that mutations at residues M491 and L492 alter F protein function, suggesting a role for these residues in the fusion process.

Vaccine-Induced Thrombocytopenia and Thrombosis (VITT) Antibodies Recognize Neutrophil-Activating Peptide 2 (NAP2) As Well As Platelet Factor 4 (PF4): Mechanistic and Clinical Implications

VITT is an immune-based complication of adenoviral-based vaccines used to immunize against SARS_CoV2. The antibodies in VITT have been described as directed at the platelet-specific chemokine PF4 (CXCL4). While the clinical course and target chemokine in VITT has much in common with the better-known thrombocytopenic/prothrombotic disorder, heparin-induced thrombocytopenia (HIT), which involves antibodies directed against PF4 bound to the polyanion heparin, the specific loci where VITT and PF4/polyanion HIT antibodies bind appear to differ in studies using alanine-scanning mutations of PF4 (Nature, 2021. DOI: 10.1038/s41586-021-03744-4). The VITT antigenic site localizes to a heparin-binding domain. Unlike the dominant HIT locus, the VITT locus is conserved not only between human and mouse PF4, but also between PF4 and the related platelet-specific chemokine NAP2 (CXCL7). NAP2 is also expressed and stored in platelet alpha-granules and is present in equimolar concentrations to PF4. Unlike PF4, NAP2 avidly binds the chemokine receptor CXCR2 and strongly activates neutrophils. We now show that antibodies from patients who developed VITT after both AstraZeneca (AZ) or Johnson and Johnson (JJ) adenoviral vaccines, unlike HIT antibodies, recognize mouse PF4 (Figure 1A). More importantly, both AZ and JJ VITT antibodies bound NAP2, while none of the HIT antibodies tested bound PF4 or NAP2 in the absence of heparin (Figure 1A). These results are consistent with the alanine-scanning studies that distinguish the HIT and VITT binding sites. Dynamic light scattering (DLS) showed that NAP2 and PF4 bind to the adenoviral vectors, including Ad5 and the AZ vector ChAdOx5, which leads to expression of SARS_CoV2 spike protein. ChAdOx2 vaccine and CsCl 2-purified ChAdOx2 bound to both proteins, but form larger complexes with NAP2 than with PF4 even at lower concentrations of this chemokine (Figure 1C). Removal of anti-PF4 antibodies by hPF4-Sepharose abrogated PF4-dependent binding, but did not significantly reduce binding to NAP2 (not shown), indicating that VITT plasma contains discrete pools of anti-PF4 and anti-NAP2 antibodies that may have distinct functional properties. Sandwich ELISA (not shown) and Western blot analysis of purified VITT IgG demonstrates the presence of hPF4-IgG and NAP2-IgG immune complexes in purified patient's IgG (Figure 2A). Functional studies show that both PF4 and NAP2 can activate platelets in the presence of VITT antibodies. Anti-PF4-depleted VITT IgG fraction retains the ability to activate platelets in the presence of NAP2 (Figure 2B). Thus, unlike HIT, VITT appears to target a shared antigenic site on the related chemokines PF4 and NAP2. This raises the question as to whether NAP2, as one the most abundant platelet chemokines released from activated platelets, is involved in the initiation and propagation of the immunothrombotic response. Additional studies are needed to see whether NAP2, which can potently and specifically activate neutrophils via CXCLR2, contributes to the specific thromboinflammatory phenotype seen in VITT. We propose using FcgammaRIIA+ mice that concurrently express human PF4 and NAP2 and specific knockout of each chemokine, available in our group, to further understand the pathogenesis of VITT and its thrombocytopenic/ prothrombotic phenotype. Figure 1 Figure 1. Disclosures Padmanabhan: Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees. Cines: Dova: Consultancy; Rigel: Consultancy; Treeline: Consultancy; Arch Oncol: Consultancy; Jannsen: Consultancy; Taventa: Consultancy; Principia: Other: Data Safety Monitoring Board.

Alanine-scanning mutagenesis of protein mannosyl-transferase from Streptomyces coelicolor reveals strong activity-stability correlation

In Actinobacteria , protein O-mannosyl transferase (Pmt)-mediated protein O-glycosylation has an important role in cell envelope physiology. In S. coelicolor, defective Pmt leads to increased susceptibility to cell wall-targeting antibiotics, including vancomycin and β-lactams, and resistance to phage ϕC31. The aim of this study was to gain a deeper understanding of the structure and function of S. coelicolor Pmt. Sequence alignments and structural bioinformatics were used to identify target sites for an alanine-scanning mutagenesis study. Mutant alleles were introduced into pmt-deficient S. coelicolor strains using an integrative plasmid and scored for their ability to complement phage resistance and antibiotic hypersusceptibility phenotypes. Twenty-three highly conserved Pmt residues were each substituted for alanine. Six mutant alleles failed to complement the pmt ▬ strains in either assay. Mapping the six corresponding residues onto a homology model of the three-dimensional structure of Pmt, indicated that five are positioned close to the predicted catalytic DE motif. Further mutagenesis to produce more conservative substitutions at these six residues produced Pmts that invariably failed to complement the DT1025 pmt ▬ strain, indicating that strict residue conservation was necessary to preserve function. Cell fractionation and Western blotting of strains with the non-complementing pmt alleles revealed undetectable levels of the enzyme in either the membrane fractions or whole cell lysates. Meanwhile for all of the strains that complemented the antibiotic hypersusceptibility and phage resistance phenotypes, Pmt was readily detected in the membrane fraction. These data indicate a tight correlation between the activity of Pmt and its stability or ability to localize to the membrane.

Generation of a Novel High-Affinity Antibody Binding to PCSK9 Catalytic Domain with Slow Dissociation Rate by CDR-Grafting, Alanine Scanning and Saturated Site-Directed Mutagenesis

Inhibition of Proprotein convertase subtilisin/kexin type 9 (PCSK9) has become an attractive therapeutic strategy for lowering low-density lipoprotein cholesterol (LDL-C). In this study, a novel high affinity humanized IgG1 mAb (named h5E12-L230G) targeting the catalytic domain of human PCSK9 (hPCSK9) was generated by using CDR-grafting, alanine-scanning mutagenesis, and saturated site-directed mutagenesis. To eliminate the cytotoxic effector functions and mitigate the heterogeneity, the heavy-chain constant region of h5E12-L230G was modified with L234A/L235A/N297G mutations and C-terminal lysine deletion. The biolayer interferometry (BLI) binding assay and molecular docking study revealed that h5E12-L230G binds to the catalytic domain of hPCSK9 with nanomolar affinity (KD =1.72 nM) and an extremely slow dissociation rate (koff, 4.84 × 10−5 s−1), which interprets its quite low binding energy (-54.97 kcal/mol) with hPCSK9. Additionally, h5E12-L230G elevated the levels of LDLR and enhanced the LDL-C uptake in HepG2 cells, as well as reduced the serum LDL-C and total cholesterol (TC) levels in hyperlipidemic mouse model with high potency comparable to Alirocumab. Our data suggest that h5E12-L230G is a highly potent antibody binding to PCSK9 catalytic domain with slow dissociation rate which may be utilized as a therapeutic candidate for treating hypercholesterolemia and relevant cardiovascular diseases.