PRINT: A Protein Bioconjugation Method with Exquisite N-terminal Specificity

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Unmet Need:
The use of proteins and peptides for therapeutic applications are often compromised by low biological stability, high renal clearance, and non-optimal bio-distribution.  Chemical attachment of poly-(ethylene glycol) (PEGylation) is often considered the most effective way to improve these pharmacologic properties by increasing circulation half-life and reducing both the immunogenicity of proteins and protease mediated degradation.  However, random conjugation results in heterogeneous derivatives with undefined composition and can substantially lower the bioactivity of the modified protein, leading to unpredictable in vivo behavior.  The same issues apply to conjugations for other purposes, such as the attachment of toxic small molecules to increase the therapeutic efficacy of antibodies.  Site-specific modification of proteins is therefore an attractive approach to circumvent the non-specificity resulting from random conjugation to amines, thiols, or other specific amino acids on proteins.  Currently used site-specific strategies exploit rare chemo-selective anchors present either naturally or introduced artificially into protein backbones.  
Technology Overview
All of the current bio-conjugation techniques can be usefully employed, but in view of the ubiquity of this problem and its importance, new ways to site-specifically modify proteins, regardless of the tag used for purification, with inexpensive, commercially available reagents, are still a high priority. JHU inventors have developed a novel technique named PRINT (PRotect, INcise Tag) for N-terminal specific bio-conjugation of proteins and peptides. In theory, PRINT can be used for selective attachment of any desired entity bearing a nitrogen-reactive functionality. The inventors have shown that through PRINT, they are able to engineer exclusive N-terminal conjugation of model proteins without altering their biological properties.
Stage of Development
In a recent study, JHU inventors have used TNF-α as an example to show that PRINT is a robust, reproducible and mild strategy which is able to target the α-amine and provide N-terminal specific protection to proteins or peptides that suffer from similar issues.  In principle, PRINT can be used to generate a variety of N-terminal conjugates using NHS ester chemistry on any recombinant protein or peptide bearing a cleavable purification tag.  We believe that this approach is strongly orthogonal to current methods and will be applicable to many bio-therapeutics and bio-probes that are currently being designed to treat cancer or other diseases.
Patent Information:
Title App Type Country Serial No. Patent No. File Date Issued Date Expire Date Patent Status
PROTEIN BIOCONJUGATION METHOD PCT: Patent Cooperation Treaty United States 15/768,053 4/13/2018     Pending
For Information, Contact:
Hassan Naqvi
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