US 2026/0297166 A1 · ASYMMETRIC CIRCULAR TANDEM REPEAT PROTEINS

Abstract

In certain embodiments, the present invention provides protomers and asymmetrical circular tandem repeat proteins (cTRPs). Asymmetric cTRPs are provided as assemblage of two or more protomers having amino acid substitutions with cysteine or with non-canonical amino acids at different amino acid residue positions on respective terminal α-helical structural units, thereby facilitating disulfide bonding or click conjugation in between adjacent protomers so as to form hetero-oligomer cTRPs with interface bonding/conjugation. Preferably cargo molecules are operably linked to the protomers, so as to form an asymmetrical cTRPs with different cargo molecules.

Claims60

Dependent claims

A protomer comprising the following formula:

wherein
R1 is an N-terminal linker or a covalent bond,
R2 is a C-terminal linker or a covalent bond,
X is an α-helical peptide repeat unit comprising 30-35 amino acid residues and forming an outer α-helix and an inner α-helix joined by a helix-linker,
Y is a repeat unit having an identical amino acid sequence to X except for the substitution of one residue with a cysteine or with a non-canonical amino acid, or Y is a repeat unit having at least 90% sequence identity to X except for the substitution of the one residue with a cysteine or with a non-canonical amino acid and further except for C-terminal deletion or substitution of an amino acid residue outside of the outer α-helix—helix linker—inner α-helix,
Z is a repeat unit having an identical amino acid sequence identity to X except for the substitution of one residue with a cysteine or with a non-canonical amino acid, or Z is a repeat unit having at least 90% sequence identity to X except for the substitution of the one residue with a cysteine or with a non-canonical amino acid and further except for C-terminal deletion or substitution of an amino acid residue outside of the outer α-helix—helix linker—inner α-helix,
wherein the cysteine or the non-canonical amino acid in Z is at a different position than in Y, wherein if the substitution in Y is with the cysteine then the substitution in Z is with the cysteine, and wherein if the substitution in Y is with the non-canonical amino acid then the substitution in Z is with the non-canonical amino acid,
m is 0 or 1, wherein if m is 0 then R1 is absent,
p is 1,
q is 1,
t is 0 or 1, wherein if t is 0 then R2 is absent, and
n is a repeat number of the α-helical peptide repeat unit being 4, 1, 2, 6, 10 or 0.

9 more elements

An asymmetrical circular tandem repeat protein (cTRP) comprising:

(a) four protomers, associated in tandem in a closed circular structure, wherein each protomer independently comprises the following formula:
wherein
R1 is an N-terminal linker or a covalent bond,
R2 is a C-terminal linker or a covalent bond,
X is an α-helical peptide repeat unit comprising 30-35 amino acid residues and forming an outer α-helix and an inner α-helix joined by a helix-linker, and X being devoid of cysteines,
Y is a repeat unit having an identical amino acid sequence or at least 90% sequence identity to X except for the substitution of one residue with a cysteine or a non-canonical amino acid, and
Z is a repeat unit having an identical amino acid sequence or at least 90% sequence identity to X except for the substitution of one residue with a cysteine or a non-canonical amino acid, wherein the cysteine or the non-canonical amino acid in Z is at a different position than in Y, wherein if the substitution in Y is with the cysteine then the substitution in Z is with the cysteine, and wherein if the substitution in Y is with the non-canonical amino acid then the substitution in Z is with the non-canonical amino acid;
wherein the cysteine residue or the non-canonical amino acid residue in Z of each protomer forms a covalent bond with the cysteine residue or the non-canonical amino acid residue, respectively, in Y of a next protomer in tandem in the closed circular structure;
wherein the protein is left-handed;
and
wherein each pair of cysteine substitutions in Z of a protomer and Y of a next protomer is different in amino acid residue positions relative to respective protomers, and wherein each pair of non-canonical amino acid residue substitutions in Z of a protomer and Y of a next protomer is a mutually exclusive orthogonal pair of reactive groups for click conjugation;
(b) two protomers, associated in tandem in a closed circular structure, wherein each protomer independently comprises the following formula:
wherein
R1 is an N-terminal linker or a covalent bond,
R2 is a C-terminal linker or a covalent bond,
X is an α-helical peptide repeat unit comprising 30-35 amino acid residues and forming an outer α-helix and an inner α-helix joined by a helix-linker, X being devoid of cysteines,
Y is a repeat unit having an identical amino acid sequence or at least 90% sequence identity to X except for the substitution of one residue with a cysteine or with a non-canonical amino acid, and
Z is a repeat unit having an identical amino acid sequence or at least 90% sequence identity to X except for the substitution of one residue with a cysteine or with a non-canonical amino acid, wherein the cysteine or the non-canonical amino acid in Z is at a different position than in Y, wherein if the substitution in Y is with the cysteine then the substitution in Z is with the cysteine, and wherein if the substitution in Y is with the non-canonical amino acid then the substitution in Z is with the non-canonical amino acid;
wherein the cysteine or the non-canonical amino acid residue in Z of each protomer forms a covalent bond with the cysteine or the non-canonical amino acid residue, respectively, in Y of a next protomer in tandem in the closed circular structure;
wherein the protein is left-handed;
and
wherein each pair of cysteine substitutions in Z of a protomer and Y of a next protomer is different in amino acid residue positions relative to respective protomers, and wherein each pair of non-canonical amino acid residue substitutions in Z of a protomer and Y of a next protomer is an orthogonal pair of reactive groups for click conjugation;
(c) three protomers, associated in tandem in a closed circular structure, wherein each protomer independently comprises the following formula:
wherein
R1 is an N-terminal linker or a covalent bond,
R2 is a C-terminal linker or a covalent bond,
X is an α-helical peptide repeat unit comprising 30-35 amino acid residues and forming an outer α-helix and an inner α-helix joined by a helix-linker, and X being devoid of cysteines,
Y is a repeat unit having an identical amino acid sequence or at least 90% sequence identity to X except for the substitution of one residue with a cysteine or with a non-canonical amino acid, and
Z is a repeat unit having an identical amino acid sequence or at least 90% sequence identity to X except for the substitution of one residue with a cysteine or with a non-canonical amino acid, respectively, wherein the cysteine or the non-canonical amino acid in Z is at a different position than in Y, wherein if the substitution in Y is with the cysteine then the substitution in Z is with the cysteine, and wherein if the substitution in Y is with the non-canonical amino acid then the substitution in Z is with the non-canonical amino acid;
wherein the cysteine residue or the non-canonical amino acid residue in Z of each protomer forms a covalent bond with the cysteine or the non-canonical amino acid residue, respectively, in Y of a next protomer in tandem in the closed circular structure;
wherein the protein is left-handed;
and
wherein each pair of cysteine substitutions in Z of a protomer and Y of a next protomer is different in amino acid residue positions relative to respective protomers, and wherein each pair of non-canonical amino acid residue substitutions in Z of a protomer and Y of a next protomer is an orthogonal pair of reactive groups for click conjugation;
or
(d) six protomers, associated in tandem in a closed circular structure, wherein each protomer independently comprises the following formula:
wherein
R1 is an N-terminal linker or a covalent bond,
R2 is a C-terminal linker or a covalent bond,
X is an α-helical peptide repeat unit comprising 30-35 amino acid residues and forming an outer α-helix and an inner α-helix joined by a helix-linker, X being devoid of cysteines,
Y is a repeat unit having an identical amino acid sequence or at least 90% sequence identity to X except for the substitution of one residue with a cysteine or with a non-canonical amino acid, and
Z is a repeat unit having an identical amino acid sequence or at least 90% sequence identity to X except for the substitution of one residue with a cysteine or with a non-canonical amino acid, wherein the cysteine or the non-canonical amino acid in Z is at a different position than in Y, wherein if the substitution in Y is with the cysteine then the substitution in Z is with the cysteine, and wherein if the substitution in Y is with the non-canonical amino acid then the substitution in Z is with the non-canonical amino acid;
wherein the cysteine or the non-canonical amino acid residue in Z of each protomer forms a covalent bond with the cysteine or the non-canonical amino acid residue, respectively, in Y of a next protomer in tandem in the closed circular structure;
wherein the protein is left-handed;
and
wherein each pair of cysteine substitutions in Z of a protomer and Y of a next protomer is different in amino acid residue positions relative to respective protomers, and wherein each pair of non-canonical amino acid residue substitutions in Z of a protomer and Y of a next protomer is an orthogonal pair of reactive groups for click conjugation;
or
(e) 12 protomers, associated in tandem in a closed circular structure, wherein each protomer independently comprises the following formula:
wherein
R1 is an N-terminal linker or a covalent bond,
R2 is a C-terminal linker or a covalent bond,
Y is an α-helical peptide repeat unit comprising 30-35 amino acid residues and comprises a single, cysteine or non-canonical amino acid, and Z is an α-helical peptide repeat unit comprising 30-35 amino acid residues and comprises a single, cysteine or non-canonical amino acid, respectively,
wherein the cysteine or the non-canonical amino acid residue in Z of each protomer forms a covalent bond with the cysteine or the non-canonical amino acid residue, respectively, in Y of a next protomer in tandem in the closed circular structure;
wherein the protein is left-handed;
and
wherein each pair of cysteine substitutions in Z of a protomer and Y of a next protomer is different in amino acid residue positions relative to respective protomers, and wherein each pair of non-canonical amino acid residue substitutions in Z of a protomer and Y of a next protomer is an orthogonal pair of reactive groups for click conjugation.

52 more elements

An asymmetrical circular tandem repeat protein (cTRP) comprising four protomers,

wherein each of the protomers comprise a different one of cTRP24(6)SS_aSym1 (SEQ ID NO:
1), cTRP24(6)SS_aSym2 (SEQ ID NO:
2), cTRP24(6)SS_aSym3 (SEQ ID NO:
3) and cTRP24(6)SS_aSym4 (SEQ ID NO:
4),
wherein the protomers self-assemble to form a toroid, and
wherein disulfide bonds form between the protomers.

4 more elements

Dependencies are inferred from the claim text.

Asymmetric circular tandem repeat proteins

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Asymmetric circular tandem repeat proteins

Life of this patent

Abstract

Claims60