Plasma kallikrein (PK) is a coagulation factor that activates Factor XII in the contact pathway and generates vasoactive bradykinin from kininogen. C1-esterase inhibitor (C1INH) regulates PK levels. C1INH deficiency manifests as potentially life-threatening hereditary angioedema (HAE). Several drugs are licensed for HAE treatment and/or prophylaxis, including C1INH concentrates and recombinant Ecallantide (rEcall). rEcall is a small protein comprised of the first Kunitz domain of Tissue Factor Pathway Inhibitor and was substituted at seven residues making it a PK-specific inhibitor. rEcall is licensed for HAE treatment, but its short half-life prevents prophylactic application. This project focused on comparing the inhibition of PK and the in vivo clearance of hexahistidine-tagged rEcall (H6-rEcall) and H6-rEcall genetically fused to human serum albumin (H6-rEcall-HSA), or an albumin binding domain (H6-rEcall-ABD). To determine if the orientation of the constructs contributed to activity and half-life, proteins were reoriented, and HSA was moved from the C- to N-terminus.
Proteins produced were H6-rEcall (64 amino acids), H6-rEcall-ABD (110 amino acids), H6-rEcall-HSA (666 amino acids), H6-rEcall-HSA, rEcall-H6 (64 amino acids), and HSA-rEcall- H6 (666 amino acids). All proteins were expressed in Pichia pastoris, secreted, and purified from conditioned media via nickel-chelate chromatography. PK activity was assessed via amidolysis of S2302, and the inhibitory constant (Ki) was determined. Purified proteins were radiolabeled with 125I and injected intravenously into CD1 mice. Acid-precipitable radioactivity in timed blood samples was expressed as a percentage of the peak post-injection value. Values were means ± SD, n = 6.
Next, the role of Lipoprotein related receptor protein 1 (LRPI) or neonatal Fc receptor (FcRn) in rEcall proteins’ clearance was examined by performing a competition experiment. In
this experiment, rEcall associated with albumin was co-administered with a competitor ligand that blocked FcRn or LRP1. To further investigate the role of these receptors and the organs responsible for rEcall proteins’ clearance, organ distribution was conducted by cannulating mice via the right jugular vein and delivering 125I-rEcall or 125I-HSA-rEcall. The organs analyzed were the kidneys, liver, spleen, and heart.
In vitro, rEcall and its modified proteins had a Ki of ~ 2 – 3 nM. In vivo, fusion proteins behaved similarly and presented some pharmacokinetic enhancements. Two hours post-injection, 50 ± 10% of H6-rEcall-HSA and 36 ± 6 % of HSA-rEcall-H6 remained in the circulation vs. 6 ± 2 % of H6-rEcall and 7 ± 1% of H6-rEcall-ABD (p <0.0001), while 75 ± 5% of HSA-H6 was recovered in the circulation.
In the competition experiment, the presence of GST-RAP elevated the recovery of 125I- H6-rEcall-HSA 2.2-fold, from 37 ± 3 % for GST + H6-rEcall-HSA to 81 ± 3 % for GST-RAP +
H6-rEcall-HSA. Competition with IVIg also significantly increased the recovery of 125I- H6- rEcall-HSA, but to a lesser degree, by 1.13-fold (to 42 ± 5% for IVIg + H6-rEcall-HSA). Similar significant changes were also observed 30 mins after injection, of 2.9- and 1.9-fold, respectively.
Finally, results indicate that albumin changed rEcall’s organ distribution. Of the four organs extracted 30 minutes after injection, rEcall-H6 was predominantly found in the kidneys, and fusion to albumin largely redirected rEcall to the liver. 3.6-fold more HSA-rEcall-H6 was found in the liver in comparison to rEcall (p<0.0001). In contrast, 3.3-fold more rEcall was observed in the kidneys vs. rEcall-HSA (p<0.0001).
In conclusion, fusing HSA to either the N- or C-terminus of rEcall extended its plasma residency time and did not impact its function towards PK. / Thesis / Master of Health Sciences (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/28937 |
| Date | January 2023 |
| Creators | Al-Adimi, Ghofran |
| Contributors | Sheffield, William, Medical Sciences |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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