Compositions and methods for producing bioactive fusion proteins
First Claim
1. A composition of matter of the formula
(F1)a—
- X2 and multimers thereof, wherein;
F1 is a half-life extending moiety, and a is 1;
X2 is D-(L)c-(P5)d—
(X3)e, (X4)f—
(P5)d-(L)c-D, or (X4)f—
(P5)d-(L)c-D-(L)g-(P6)h—
(X3)i, whereinc and g are each independently 0 or 1, d and h are 1, and e, f, and i are each independently 0, 1, 2, 3, or 4;
X3 is -(L)j-(P7), j is 0 or 1;
X4 is (P8)-(L)k-, k is 0 or 1;
D is a pharmacologically inactive protein domain of human origin, wherein said pharmacologically inactive protein domain is a SH3 domain that comprises a native human amino acid sequence that is an intracellular proline motif (PxxP) recognition and binding domain, and(i) has a mass of 3 kDa to 20 kDa, and(ii) characteristically forms protein aggregates of less than 10 percent of total mass of protein when suspended without other proteins in a pharmaceutically acceptable formulation buffer of interest not comprising a detergent or chaotropic agent;
P5, P6, P7 and P8 are each independently a selected pharmacologically active protein of interest 5 to 80 amino acid residues in length; and
L is in each instance a peptidyl linker.
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Abstract
Disclosed is a composition of matter involving a recombinant fusion protein comprising a a pharmacologically active protein partner, and a small pharmacologically inactive protein domain partner of human origin, such as but not limited to, a 10th fibronectin III domain, a SH3 domain, a SH2 domain, a CH2 domain of IgG1, a PDZ domain, a thrombospondin repeat domain, an ubiquitin domain, a leucine-rich repeat domain, a villin headpiece HP35 domain, a villin headpiece HP76 domain, or a fragment or modification of any of these. Also disclosed are nucleic acids (e.g., DNA constructs) encoding the fusion protein, expression vectors and recombinant host cells for expression of the fusion protein, and pharmaceutical compositions containing the recombinant fusion protein and a pharmaceutically acceptable carrier, and method of producing a pharmacologically active recombinant fusion protein.
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Citations
18 Claims
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1. A composition of matter of the formula
(F1)a—- X2
and multimers thereof, wherein; F1 is a half-life extending moiety, and a is 1; X2 is D-(L)c-(P5)d—
(X3)e, (X4)f—
(P5)d-(L)c-D, or (X4)f—
(P5)d-(L)c-D-(L)g-(P6)h—
(X3)i, whereinc and g are each independently 0 or 1, d and h are 1, and e, f, and i are each independently 0, 1, 2, 3, or 4; X3 is -(L)j-(P7), j is 0 or 1; X4 is (P8)-(L)k-, k is 0 or 1; D is a pharmacologically inactive protein domain of human origin, wherein said pharmacologically inactive protein domain is a SH3 domain that comprises a native human amino acid sequence that is an intracellular proline motif (PxxP) recognition and binding domain, and (i) has a mass of 3 kDa to 20 kDa, and (ii) characteristically forms protein aggregates of less than 10 percent of total mass of protein when suspended without other proteins in a pharmaceutically acceptable formulation buffer of interest not comprising a detergent or chaotropic agent; P5, P6, P7 and P8 are each independently a selected pharmacologically active protein of interest 5 to 80 amino acid residues in length; and L is in each instance a peptidyl linker. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
- X2
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9. A composition of matter comprising a recombinant fusion protein, wherein said fusion protein comprises:
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(a) a pharmacologically inactive protein domain of human origin comprising the amino acid sequence of SEQ ID NO;
94; and(b) at least one pharmacologically active toxin peptide 20 to 80 amino acid residues in length comprising a selected ShK peptide analog of interest, wherein the ShK peptide analog comprises a modification of the native ShK sequence by addition, deletion or substitution of one to nine amino acid residues, deletion of up to two amino acid residues at the N-terminus, or deletion of up to 5 amino acid residues at the C-terminus. - View Dependent Claims (10, 11, 12, 13, 14, 15, 16, 17, 18)
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Specification