# US 2019/0375823 A1 · Multivalent heteromultimer scaffold design and constructs

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- Number: US 2019/0375823 A1
- Document ID: US20190375823A1
- Status: Published application
- Kind: A1
- Application number: 16/417,969
- Filed: 2019-05-21
- Published: 2019-12-12
- Earliest claimed: 2012-07-13
- Assignees: Zymeworks Inc.
- Applicants: Zymeworks Inc.
- Inventors: Surjit Bhimarao Dixit; Igor Edmondo Paolo D'Angelo; Mario Sanches; Gordon Yiu Kon Ng
- Web page: https://patentgrep.com/patent/US20190375823A1

## CPC

- C07K 14/765: Serum albumin, e.g. HSA
- A61P 35/00: Antineoplastic agents
- C07K 14/485: Epidermal growth factor [EGF], i.e. urogastrone
- C07K 14/605: Glucagons
- C07K 14/76: Albumins
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/2803: {against the immunoglobulin superfamily}
- C07K 16/2809: {against the T-cell receptor (TcR)-CD3 complex}
- C07K 16/283: {against Fc-receptors, e.g. CD16, CD32, CD64 (CD23 C07K16/2851)}
- C07K 16/2896: {against molecules with a "CD"-designation, not provided for elsewhere}
- C07K 16/40: against enzymes
- C07K 2317/35: Valency
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- C07K 2319/00: Fusion polypeptide
- C07K 2319/31: fusions, other than Fc, for prolonged plasma life, e.g. albumin
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## 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.

## Claims

### Claim at position 1

-100. (canceled)

### Claim 101

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.

### Claim 102 (depends on 101)

The heteromultimer of claim 101, 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, or b) 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.

### Claim 103 (depends on 102)

The heteromultimer of claim 102, 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.

### Claim 104 (depends on 102)

The heteromultimer of claim 102, wherein at least one naturally occurring disulphide bond is formed between the first segment of albumin and the second segment of albumin.

### Claim 105 (depends on 102)

The heteromultimer of claim 102, 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.

### Claim 106 (depends on 102)

The heteromultimer of claim 102, wherein the thermal stability of the quasi-native monomer 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.

### Claim 107 (depends on 106)

The heteromultimer of claim 106 wherein the C-terminus and N-terminus introduced at the segmentation site are each fused to a linker.

### Claim 108 (depends on 107)

The heteromultimer of claim 107, wherein the linker is a GGGGS (SEQ ID NO:179) peptide sequence.

### Claim 109 (depends on 102)

The heteromultimer of claim 102, 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.

### Claim 110 (depends on 102)

The heteromultimer of claim 102, wherein said first segment of albumin and said second segment of albumin are from a mammalian albumin.

### Claim 111 (depends on 102)

The heteromultimer of claim 102, 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 albumin.

### Claim 112 (depends on 102)

The heteromultimer of claim 102, wherein said albumin polypeptide is human serum albumin having a sequence of SEQ ID NO: 1 without the signal peptide sequence.

### Claim 113 (depends on 112)

The heteromultimer of claim 112, 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.

### Claim 114 (depends on 112)

The heteromultimer of claim 112, 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.

### Claim 115 (depends on 101)

The heteromultimer of claim 101, 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.

### Claim 116 (depends on 102)

The heteromultimer of claim 102, wherein said second polypeptide construct further comprises at least one second cargo molecule.

### Claim 117 (depends on 102)

The heteromultimer of claim 102, wherein the activity of the cargo polypeptide is altered on fusion to the transporter polypeptide relative to its unfused format, or wherein the activity of the cargo polypeptide is attenuated on fusion to the transporter polypeptide relative to its unfused format.

### Claim 118 (depends on 116)

The heteromultimer of claim 116, 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.

### Claim 119 (depends on 116)

The heteromultimer of claim 116, 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.

### Claim 120 (depends on 119)

The heteromultimer of claim 119, wherein at least one of the first cargo polypeptides or at least one of the second cargo polypeptides is an antigen-binding polypeptide construct.

### Claim 121 (depends on 120)

The heteromultimer of claim 120, wherein the antigen binding polypeptide construct binds CD3, CD19, CD20, HER2, or HER3.

### Claim 122 (depends on 116)

The heteromultimer of claim 116, wherein the first cargo molecule is a cargo polypeptide, and the second cargo molecule is a toxin, and/or the first cargo molecule is a toxin, and the second cargo molecule is a cargo polypeptide.

### Claim 123 (depends on 116)

The heteromultimer of claim 116, wherein the first cargo molecule and/or the second cargo molecule bind a target antigen selected from at least one of alpha-chain (CD25) of IL-2R, Amyloid beta, anti-EpCAM, anti-CD3, CD16, CD20, CD22, CD23, CD3, CD4, CD52, CD80, CTLA-4, EGFR, EpCAM, F protein of RSV, G250, glycoprotein IIB/IIIa R, HER2, HER3, IGF1R, EGFR, HSP90, IgE antibody, IL-12, IL-23, IL-1 beta, IL-5, IL-6, RANKL, TNF alpha, TNFR, TRAIL, VEGF-A, glucagon receptor, GLP receptor, and LDL receptor.

### Claim 124 (depends on 101)

A composition comprising the heteromultimer of claim 101 and a pharmaceutically acceptable carrier.

### Claim 125 (depends on 101)

One or more nucleic acids encoding the heteromultimer according to claim 101.

### Claim 126 (depends on 125)

One or more vectors comprising the one or more nucleic acids according to claim 125.

### Claim 127 (depends on 126)

A host cell comprising the one or more vectors according to claim 126.

### Claim 128 (depends on 101)

A method of preparing the heteromultimer according to claim 101, comprising expressing one or more nucleic acids encoding the heteromultimer in a host cell.

### Claim 129

A method of preparing self-assembling polypeptides from an albumin comprising: segmenting said albumin at one or more segmentation sites to obtain at least two albumin segments that self-assemble to form a quasi-native albumin structure, the method comprising the steps of
a) selecting at least one loop of an albumin that has a high solvent accessible surface area and limited contact with the rest of the structure of said albumin, and
b) introducing one segmentation site deleting zero to three amino acid residues at the segmentation site per selected loop to obtain at least two albumin segments having a complementary interface between the at least two albumin segments, wherein the interface is apolar, extensive and interdigitate.

### Claim 130

A heteromultimer comprising:
a first polypeptide construct comprising (i) a first transporter polypeptide comprising a first segment of albumin, 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.

### Claim 131 (depends on 130)

A composition comprising the heteromultimer of claim 130 and a pharmaceutically acceptable carrier.

### Claim 132 (depends on 130)

One or more nucleic acids encoding the heteromultimer according to claim 130.

### Claim 133 (depends on 132)

One or more vectors comprising the one or more nucleic acids according to claim 132.

### Claim 134 (depends on 133)

A host cell comprising the one or more vectors according to claim 133.

### Claim 135 (depends on 130)

A method of preparing the heteromultimer according to claim 130, comprising expressing one or more nucleic acids encoding the heteromultimer in a host cell.