Multivalent heteromultimer scaffold design and constructs

Patent metadata

Field	Value
Publication number	US-11248037-B2
Application number	US-201916417969-A
Country	US
Kind code	B2
Filing date	May 21, 2019
Priority date	Jul 13, 2012
Publication date	Feb 15, 2022
Grant date	Feb 15, 2022

Abstract

Provided herein are multifunctional heteromultimer proteins. In specific embodiments is a heteromultimer comprising: at least two polypeptide constructs, each polypeptide construct comprising at least one cargo polypeptide attached to a transporter polypeptide, said transporter polypeptides derived from a monomeric native protein such that said monomeric constructs associate to form the heteromultimer and said transporter polypeptides associate to form a quasi-native structure of the monomeric native protein or analog thereof. These therapeutically novel molecules encompass heteromultimers comprising constructs that function as scaffolds for the conjugation or fusion of therapeutic molecular entities (cargo polypeptides) resulting in the creation of bispecific or multivalent molecular species. Provided herein is a method for creation of bispecific or multivalent molecular species.

First claim

What is claimed:

1. A heteromultimer comprising:
a first polypeptide construct comprising (i) a first transporter polypeptide comprising a first segment of albumin, and (ii) at least one first cargo molecule; and
a second polypeptide construct comprising (iii) a second transporter polypeptide comprising a second segment of albumin,
wherein a) said first segment of albumin and said second segment of albumin are obtained by segmentation of an albumin polypeptide at a segmentation site to delete zero to 3 amino acid residues at the segmentation site, b) said first transporter polypeptide and said second transporter polypeptide are different from each other, and c) said first transporter polypeptide and said second transporter polypeptide self-assemble to form a quasi-native structure of albumin.

2. The heteromultimer of claim 1 , wherein a) the segmentation site resides on a loop of the albumin polypeptide that has a high solvent accessible surface area (SASA) and limited contact with the rest of the albumin polypeptide, orb) the segmentation results in a complementary interface between the first segment of albumin and the second segment of albumin wherein the complementary interface is apolar, extensive and interdigitate.

3. The heteromultimer of claim 2 , wherein the segmentation deletes zero to 2 amino acid residues at the segmentation site, or deletes zero or 1 amino acid residues at the segmentation site.

4. The heteromultimer of claim 2 , wherein at least one naturally occurring disulphide bond is formed between the first segment of albumin and the second segment of albumin.

5. The heteromultimer of claim 2 , wherein at least one of said first and second transporter polypeptides comprises at least one mutation of an amino acid residue to cysteine such that said cysteine forms a disulfide bond with a cysteine residue on the other transporter polypeptide.

6. The heteromultimer of claim 2 , wherein the thermal stability of the quasi-native structure is within approximately 20° C. of the thermal stability of albumin, or wherein the quasi-native structure of albumin has a melting temperature greater than 65° C., 70° C., or 75° C., wherein the thermal stability is measured by differential scanning calorimetry.

7. The heteromultimer of claim 6 wherein the C-terminus and N-terminus introduced at the segmentation site are each fused to a linker.

8. The heteromultimer of claim 7 , wherein the linker is a GGGGS (SEQ ID NO:179) peptide sequence.

9. The heteromultimer of claim 2 , wherein
a) at least one of said first segment of albumin and the second segment of albumin is a segment of an alloalbumin polypeptide;
b) the first segment of albumin and the second segment of albumin are segments of an alloalbumin polypeptide;
c) the first segment of albumin and the second segment of albumin are segments of the same type of albumin polypeptide; or
d) the first segment of albumin and the second segment of albumin are segments of different types of albumin polypeptides.

10. The heteromultimer of claim 2 , wherein said first segment of albumin and said second segment of albumin are from a mammalian albumin.

11. The heteromultimer of claim 2 , wherein said first segment of albumin and/or said second segment of albumin each comprise an amino acid sequence with at least 95% sequence identity to human albumin.

12. The heteromultimer of claim 2 , wherein said albumin polypeptide is human serum albumin having a sequence of SEQ ID NO: 1 without the signal peptide sequence.

13. The heteromultimer of claim 12 , wherein the location of the segmentation site is between residues 339 and 340, between residues 300 and 301, between residues 364 and 365, between residues 441 and 442, between residues 171 and 172, between residues 281 and 282, or between residues 114 and 115 of the albumin polypeptide sequence of SEQ ID NO:1, wherein the numbering of residues begins with the first residue after the signal sequence.

14. The heteromultimer of claim 12 , wherein the segmentation site deletes a residue at a location corresponding to residue 84 of the albumin polypeptide sequence of SEQ ID NO:1, wherein the numbering of residues begins with the first residue after the signal sequence.

15. The heteromultimer of claim 1 , wherein
a) said first transporter polypeptide comprises the sequence set forth in SEQ ID NO:39 without the signal peptide sequence, and wherein said second transporter polypeptide comprises the sequence set forth in SEQ ID NO:40 without the signal peptide sequence;
b) said first transporter polypeptide comprises the sequence set forth in SEQ ID NO:41 without the signal peptide sequence, and wherein said second transporter polypeptide comprises the sequence set forth in SEQ ID NO:42 without the signal peptide sequence;
c) said first transporter polypeptide comprises the sequence set forth in SEQ ID NO:43 without the signal peptide sequence, and wherein said second transporter polypeptide comprises the sequence set forth in SEQ ID NO:44 without the signal peptide sequence;
d) said first transporter polypeptide comprises the sequence set forth in SEQ ID NO:45 without the signal peptide sequence, and wherein said second transporter polypeptide comprises the sequence set forth in SEQ ID NO:46 without the signal peptide sequence;
e) said first transporter polypeptide comprises the sequence set forth in SEQ ID NO:47 without the signal peptide sequence, and wherein said second transporter polypeptide comprises the sequence set forth in SEQ ID NO:48 without the signal peptide sequence;
f) said first transporter polypeptide comprises the sequence set forth in SEQ ID NO:49 without the signal peptide sequence, and wherein said second transporter polypeptide comprises the sequence set forth in SEQ ID NO:50 without the signal peptide sequence;
g) said first transporter polypeptide comprises the sequence set forth in SEQ ID NO:51 without the signal peptide sequence, and wherein said second transporter polypeptide comprises the sequence set forth in SEQ ID NO:52 without the signal peptide sequence; or
h) said first transporter polypeptide comprises the sequence set forth in SEQ ID NO:53 without the signal peptide sequence, and wherein said second transporter polypeptide comprises the sequence set forth in SEQ ID NO:54 without the signal peptide sequence.

16. The heteromultimer of claim 2 , wherein said second polypeptide construct further comprises at least one second cargo molecule.

17. The heteromultimer of claim 2 , wherein the activity of the cargo molecule is altered on fusion to the transporter polypeptide relative to its unfused format, or wherein the activity of the cargo molecule is attenuated on fusion to the transporter polypeptide relative to its unfused format.

18. The heteromultimer of claim 16 , wherein the at least one first cargo molecule or the at least one second cargo molecule is a therapeutic agent, a toxin, a natural product or prodrug, a cargo polypeptide, or a nucleic acid.

19. The heteromultimer of claim 18 , wherein the first polypeptide construct comprises one first cargo polypeptide or two first cargo polypeptides, and/or the second polypeptide construct comprises one second cargo polypeptide or two second cargo polypeptides.

20. The heteromultimer of claim 19 , wherein at least one of the first cargo polypeptides or at least one of the second cargo polypeptides is an antigen-binding polypeptide construct.

21. The heteromultimer of claim 20 , wherein the antigen binding polypeptide construct binds CD3,

Assignees

• Zymeworks Inc

Inventors

• Dixit Surjit Bhimarao
• D'Angelo Igor Edmondo Paolo
• Sanches Mario
• Ng Gordon Yiu Kon

Classifications

• C07K14/76 Primary

  Albumins · CPC title

• C07K14/765 Primary

  Serum albumin, e.g. HSA · CPC title

• C07K16/2803

  against the immunoglobulin superfamily · CPC title

• C07K2319/31

  fusions, other than Fc, for prolonged plasma life, e.g. albumin · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title