Subtilase cytotoxin B subunit mutant
Inventors
Jennings, Michael Paul • Day, Christopher • Paton, Adrienne Webster • Paton, James Cleland
Assignees
University of Adelaide • Griffith University
Publication Number
US-11371033-B2
Publication Date
2022-06-28
Expiration Date
2037-11-09
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Abstract
A mutant subtilase cytotoxin B subunit protein is provided which can bind glycans having α2-3-linked N-glycolylneuraminic acid and glycans having α2-6-linked N-glycolylneuraminic acid. The mutant SubB protein has deletions of one or more of the amino acid sequence TTSTE and has a previously undescribed ability to bind glycans having α2-6-linked N-glycolylneuraminic acid, while not losing the ability to bind glycans having α2-3-linked N-glycolylneuraminic acid.
Core Innovation
The invention relates to a mutant subtilase cytotoxin B subunit protein (SubB) that can bind glycans having both α2-3-linked and α2-6-linked N-glycolylneuraminic acid (Neu5Gc). The mutant SubB protein features deletions of one or more amino acid residues in the sequence TTSTE, imparting a previously undescribed ability to bind glycans with α2-6-linked Neu5Gc while retaining binding capacity to α2-3-linked Neu5Gc-containing glycans.
The problem addressed by the invention arises from the limited binding specificity of the wild-type SubB protein, which binds strongly to α2-3-linked Neu5Gc but binds poorly to α2-6-linked Neu5Gc. Since α2-6-linked sialic acids are common markers associated with cancer prognosis, this limited affinity reduces the utility of SubB as a tool for detecting a broader spectrum of Neu5Gc-containing glycans. The invention overcomes this by engineering mutations, such as deletion of specific residues in the T104-E108 loop, that reduce steric hindrance and enhance binding affinity specifically toward α2-6-linked Neu5Gc glycans.
Claims Coverage
The claims disclose five main inventive features focusing on the mutant SubB protein, its binding capabilities, compositions, molecular complexes, and methods involving detection and isolation of glycans and cells.
Mutant subtilase cytotoxin B subunit protein with modified TTSTE sequence
An isolated SubB protein comprising SEQ ID NO:2 or fragments/variants/derivatives thereof that include deletions and/or substitutions of one or more of the amino acid residues of the sequence TTSTE (SEQ ID NO:3), where the protein is capable of binding both α2-3-linked and α2-6-linked N-glycolylneuraminic acid.
Specific mutations within the TTSTE sequence
The protein includes a substitution or deletion of at least one of the third or fourth amino acid residues of TTSTE, optionally with deletion of the fifth residue, and specifically a deletion of the fourth and fifth amino acid residues.
SubB protein comprising the amino acid sequence of SEQ ID NO:1
An isolated SubB protein comprising the specific amino acid sequence as set forth in SEQ ID NO:1 representing a deletion mutant.
Isolated molecular complex with glycans comprising Neu5Gc linkages
An isolated molecular complex comprising the mutant SubB protein bound to a glycan containing α2-3-linked and/or α2-6-linked N-glycolylneuraminic acid.
Methods using the isolated SubB protein for detection and isolation
Methods of detecting α2-3- and/or α2-6-linked Neu5Gc by combining the isolated protein or composition with a sample to form a detectable complex, and methods of isolating glycans or glycan-expressing cells by forming a complex with the isolated protein and isolating the complex.
The claims cover the isolated mutant SubB protein with specific mutations in the TTSTE region enabling binding to α2-3- and α2-6-linked Neu5Gc glycans, compositions and complexes comprising this protein, and methods of detection and isolation leveraging this binding specificity.
Stated Advantages
The mutant SubB protein exhibits enhanced and previously undescribed ability to bind α2-6-linked Neu5Gc glycans while retaining binding to α2-3-linked Neu5Gc, significantly improving detection range.
This improved binding specificity allows discrimination between Neu5Gc and Neu5Ac glycans with high affinity and specificity.
The mutant protein can detect Neu5Gc-containing glycans with enhanced sensitivity in biological samples such as serum, and discriminate between glycans in different species (e.g., human vs bovine alpha-1 acid glycoprotein).
The mutant SubB protein enables broader detection and targeting of Neu5Gc glycans, including potential applications in cancer diagnostics and therapeutics due to Neu5Gc enrichment in tumour cells.
Documented Applications
Detection of α2-3-linked and α2-6-linked Neu5Gc-containing glycans expressed by tumour cells for diagnostic or prognostic use in various human carcinomas including breast, ovarian, prostate, colon, and lung cancers.
Detection of Neu5Gc-containing glycans on feline blood cells for blood typing.
Identification and removal of Neu5Gc-containing glycan contaminants in preparations of recombinant glycosylated drugs, antibodies, and other therapeutic biomolecules to improve efficacy and reduce immunogenicity in human administration.
Isolation or depletion of glycans or cells expressing α2-3- and/or α2-6-linked Neu5Gc glycans using the mutant SubB protein coupled to labels or substrates.
Therapeutic targeting of tumour cells expressing α2-3-linked and/or α2-6-linked Neu5Gc glycans by administering the mutant protein, optionally coupled to cytotoxic agents, for targeted cancer treatment.
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