Polypeptides comprising a modified bacteriophage G3P amino acid sequence lacking a glycosylation signal
Inventors
Krishnan, Rajaraman • Asp, Eva • Proschitsky, Ming • Fisher, Richard • Carr, Francis J. • Holgate, Robert G. E. • Jones, Timothy D.
Assignees
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Publication Number
US-11723951-B2
Publication Date
2023-08-15
Expiration Date
Abstract
The invention relates to polypeptides that comprise a portion of filamentous bacteriophage gene 3 protein (g3p) sufficient to bind to and/or disaggregate amyloid, e.g., the N1-N2 portion of g3p and mutants and fragments thereof, wherein that g3p amino acid sequence has been modified through amino acid deletion, insertion or substitution to remove a putative glycosylation signal. The invention further relates to such polypeptides that are also modified through additional amino acid substitution to be substantially less immunogenic than the corresponding wild-type g3p amino acid sequence when used in vivo. The polypeptides of the invention retain their ability to bind and/or disaggregate amyloid. The invention further relates to the use of these g3p-modified polypeptides in the treatment and/or prevention of diseases associated with misfolding or aggregation of amyloid.
Core Innovation
The invention relates to a polypeptide variant of a starting amino acid sequence, wherein the variant binds to and/or disaggregates amyloid. The starting amino acid sequence is amino acids 1-217 of SEQ ID NO:1, and the variant removes a putative glycosylation signal at amino acids 39-41. The described polypeptides include variants with engineered sequence changes that eliminate the putative N-linked glycosylation signal while retaining amyloid binding/disaggregation activity for β-amyloid, tau, and prion proteins.
The variant optionally and additionally comprises specific sequence modifications selected from substitutions, deletions, or an N-terminal methionine addition. In particular, the variant is stated to have decreased immunogenicity resulting from substitution of V215A and G220E as compared to SEQ ID NO:1 and from deletion of an amino acid corresponding to amino acids 258 and 259 of SEQ ID NO:1. The described immunogenicity reduction is tied to CD4+ T-cell epitope mapping and deimmunizing amino-acid substitutions within mapped CD4+ T-cell epitopes.
Embodiments include N1–N2–human IgG Fc fusion proteins, and the disclosure further covers nucleic acids, vectors, host cells, and pharmaceutical compositions containing the variants. Therapeutic and diagnostic use is described for protein misfolding and amyloid diseases, including use directed to biomarker-defined patients, such as florbetapir-positive patients assessed by PET imaging.
Claims Coverage
The independent claim is clm-00001. It covers a variant polypeptide defined by amyloid binding and/or disaggregation, elimination of a putative glycosylation signal in amino acids 39-41, and decreased immunogenicity through specified immunogenicity-related substitutions/deletions.
Amyloid binding or disaggregation variant of SEQ ID NO:1
A polypeptide comprising a variant of amino acids 1-217 of SEQ ID NO:1, wherein the variant binds to and/or disaggregates amyloid.
Removal of the putative glycosylation signal at amino acids 39-41
The variant differs from the starting amino acid sequence by removal of the putative glycosylation signal at amino acids 39-41.
Decreased immunogenicity via V215A and G220E and deletion of amino acids corresponding to 258-259
The variant has decreased immunogenicity resulting from substitution of V215A and G220E as compared to SEQ ID NO:1 and from deletion of amino acid corresponding to amino acids 258 and 259 of SEQ ID NO:1.
Optionally add amino-acid substitutions, deletions, and N-terminal methionine
The variant optionally additionally differs by one or more modifications selected from VVV→AAA substitutions at amino acids 43-45; substitution C53W; deletion of amino acids 96-103; QPP→AGA substitutions at amino acids 212-214; W181A, F190A and F194A substitutions; deletion of amino acid 1; deletion of amino acids 1 and 2; and addition of an N-terminal methionine residue.
Human or humanized immunoglobulin Fc fusion architecture
A polypeptide wherein the variant of amino acids 1-217 is fused to a human or humanized immunoglobulin Fc polypeptide via a peptide linker or at the C-terminus.
Florbetapir-positive patient selection for imaging-defined therapy use
In patient use, the patient is florbetapir-positive when florbetapir is used as an imaging agent in positron emission tomography.
Overall, the claim coverage centers on an amyloid-binding/disaggregating polypeptide variant of SEQ ID NO:1 with engineered removal of a putative glycosylation signal at amino acids 39-41, and decreased immunogenicity via V215A and G220E and a deletion corresponding to amino acids 258-259, with optional additional specified sequence edits and embodiments including human/humanized Fc fusions and biomarker-defined (florbetapir-positive) patient use.
Stated Advantages
Decreased immunogenicity resulting from substitution of V215A and G220E and deletion of an amino acid corresponding to amino acids 258 and 259 of SEQ ID NO:1.
Documented Applications
Therapeutic and diagnostic use for protein misfolding/amyloid diseases, including Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, with biomarker-defined patients (e.g., florbetapir-positive by PET).
Use of florbetapir as an imaging agent in positron emission tomography for defining florbetapir-positive patients.
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