# US 2014/0193408 A1 · Soluble proteins for use as therapeutics

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- Number: US 2014/0193408 A1
- Document ID: US20140193408A1
- Status: Published application
- Kind: A1
- Application number: 14/126,223
- Filed: 2012-06-15
- Published: 2014-07-10
- Earliest claimed: 2011-06-16
- Assignees: Novartis AG
- Inventors: Thomas Huber; Frank Kolbinger; Karl Welzenbach
- Web page: https://patentgrep.com/patent/US20140193408A1

## CPC

- A61P 1/04: for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
- C07K 16/2896: {against molecules with a "CD"-designation, not provided for elsewhere}
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- A61P 11/00: Drugs for disorders of the respiratory system
- A61P 11/06: Antiasthmatics
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 35/00: Antineoplastic agents
- A61P 35/02: specific for leukemia
- A61P 37/08: Antiallergic agents (antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14)
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 14/70596: {Molecules with a "CD"-designation not provided for elsewhere}
- C07K 16/241: {Tumor Necrosis Factors}
- C07K 16/2803: {against the immunoglobulin superfamily}
- C07K 16/44: against material not provided for elsewhere {, e.g. haptens, metals, DNA, RNA, amino acids}
- C07K 2317/35: Valency
- C07K 2317/51: Complete heavy chain or Fd fragment, i.e. VH + CH1
- C07K 2317/515: Complete light chain, i.e. VL + CL
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C07K 2319/32: fusions with soluble part of a cell surface receptor, "decoy receptors"
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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)

## Abstract

The present invention relates to improved binding proteins, for use as a medicament, in particular for the prevention or treatment of autoimmune and inflammatory disorders, for example allergic asthma and inflammatory bowel diseases. The invention more specifically relates to a soluble protein, comprising a complex of two heterodimers, wherein each heterodimer essentially consists of: (i) a first single chain polypeptide comprising: (a) an antibody heavy chain sequence having VH, CH1, CH2, and CH3 regions; and (b) a monovalent region of a mammalian binding molecule fused to the VH region; and (ii) a second single chain polypeptide comprising: (c) an antibody light chain sequence having a VL and CL region; and (d) a monovalent region of a mammalian binding molecule fused to the VL region; characterised in that each pair of VH and VL CDR sequences has specificity for an antigen, such that the total valency of said soluble protein is six. The invention further relates to soluble SIRPa-binding antibody-like proteins as shown in FIG. 1.

## Claims

### Claim 1

A soluble protein, comprising a complex of two heterodimers, wherein each heterodimer essentially consists of:
(i) a first single chain polypeptide comprising:
(a) an antibody heavy chain sequence having VH, CH1, CH2, and CH3 regions; and
(b) a monovalent region of a mammalian binding molecule fused to the VH region; and
(ii) a second single chain polypeptide comprising:
(c) an antibody light chain sequence having a VL and CL region; and
(d) a monovalent region of a mammalian binding molecule fused to the VL region;
characterised in that each pair of VH and VL CDR sequences has specificity for an antigen, such that the total valency of said soluble protein is six.

### Claim 2 (depends on 1)

The soluble protein as claimed in claim 1 wherein the protein has binding specificity for one, two or three antigens.

### Claim 3 (depends on 1)

The soluble protein as claimed in claim 1 wherein the regions of the mammalian binding molecule comprised within said first and second single chain polypeptides are the same.

### Claim 4 (depends on 1)

The soluble protein as claimed in claim 1, wherein each region of the mammalian binding molecule and each pair of VH and VL CDR sequences have binding specificity for the same single antigen.

### Claim 5 (depends on 1)

The soluble protein as claimed in claim 1, wherein the regions of the mammalian binding molecule can bind a first epitope on the antigen, and each pair of VH and VL CDR sequences can bind a second epitope on the same antigen.

### Claim 6 (depends on 1)

The soluble protein as claimed in claim 1, wherein the regions of the mammalian binding molecule and each pair of VH and VL CDR sequences can bind the same epitope on the same antigen.

### Claim 7 (depends on 1)

The soluble protein as claimed in claim 1, said protein having binding specificity for two antigens, wherein each region of the mammalian binding molecule has binding specificity for a first antigen, and each pair of VH and VL CDR sequences has binding specificity for a second antigen.

### Claim 8 (depends on 1)

The soluble protein as claimed in claim 1, wherein the mammalian binding molecule comprised within said first and second single chain polypeptides is different.

### Claim 9 (depends on 8)

The soluble protein as claimed in claim 8, said protein having binding specificity for two antigens, wherein the regions of the mammalian binding molecule comprised within the first single polypeptide chain have binding specificity for a first antigen, and the regions of the mammalian binding molecule comprised within the second single polypeptide chain have binding specificity for a second antigen, and each pair of VH and VL CDR sequences has binding specificity for either the first or second antigen.

### Claim 10 (depends on 1)

The soluble protein as claimed in claim 1, said protein having binding specificity for three antigens, wherein the regions of the mammalian binding molecule comprised within the first single polypeptide chain have binding specificity for a first antigen, the regions of the mammalian binding molecule comprised within the second single polypeptide chain have binding specificity for a second antigen, and each pair of VH and VL CDR sequences has binding specificity for a third antigen.

### Claim 11 (depends on 1)

The soluble protein as claimed in claim 1, wherein said mammalian binding molecule is a protein, cytokine, growth factor, hormone, signaling protein, inflammatory mediator, low molecular weight compound, ligand, cell surface receptor, or fragment thereof.

### Claim 12 (depends on 11)

The soluble protein as claimed in claim 11, wherein said mammalian binding molecule is an extracellular domain of a monomeric or homopolymeric cell surface receptor.

### Claim 13 (depends on 12)

The soluble protein as claimed in claim 12, wherein said mammalian monomeric or homopolymeric cell surface receptor comprises an IgSF domain.

### Claim 14 (depends on 12)

The soluble protein as claimed in claim 12, wherein said mammalian binding molecule comprises a SIRPalpha binding domain.

### Claim 15 (depends on 14)

The soluble protein as claimed in claim 14, wherein said SIRPα binding domain is selected from the group consisting of:
(i) an extracellular domain of the human cell surface receptor CD47;
(ii) an extracellular domain derived of SEQ ID NO:2;
(iii) a polypeptide of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:57 or a fragment thereof retaining SIRPα binding properties; and,
(iv) a variant polypeptide of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:57 or said fragment, having at least 60, 70, 80, 90, 95, 96, 97, 98, or 99 percent sequence identity, and retaining SIRPα binding properties.

### Claim 16 (depends on 14)

The soluble protein as claimed in claim 14, wherein two or more SIRPα binding domains comprised within said first and second single polypeptide chains share at least 60, 70, 80, 90, 95, 96, 97, 98, 99, or 99.5% percent sequence identity with each other.

### Claim 17 (depends on 14)

The soluble protein as claimed in claim 14 wherein two or more SIRPα binding domains have identical amino acid sequences.

### Claim 18 (depends on 14)

The soluble protein as claimed in claim 14, wherein the SIRPα binding domains within each heterodimer have identical amino acid sequences.

### Claim 19 (depends on 14)

The soluble protein as claimed in claim 14, wherein the SIRPα binding domain is an extracellular domain of the human cell surface receptor CD47 having an amino acid sequence selected from the group consisting of: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:57.

### Claim 20 (depends on 15)

The soluble protein as claimed in claim 15, comprising a complex of two heterodimers, wherein each heterodimer essentially consists of:
(i) a first single chain polypeptide comprising:
(a) an antibody heavy chain sequence having VH, CH1, CH2, and CH3 regions; and
(b) a monovalent region of an extracellular domain of CD47, the carboxyl-terminus of said CD47 region being fused to the N-terminus of the VH region; and
(ii) a second single chain polypeptide comprising:
(c) an antibody light chain sequence having a VL and CL region; and
(d) a monovalent region of an extracellular domain of CD47, the carboxyl-terminus of said CD47 region being fused to the N-terminus of the VL region.

### Claim 21 (depends on 20)

The soluble protein as claimed in claim 20, wherein said region of an extracellular domain of CD47 is SEQ ID NO:3 or SEQ ID NO:57.

### Claim 22 (depends on 1)

The soluble protein as claimed in claim 1, wherein the VH and VL CDR sequences have binding specificity for TNFalpha, cyclosporin A, or epitopes derived therefrom.

### Claim 23 (depends on 14)

The soluble protein as claimed in claim 14, which dissociates from binding to human SIRPalpha with a koff (kd1) of 0.05 [1/s] or less, as measured in a BiaCORE assay, applying a bivalent kinetic fitting model.

### Claim 24 (depends on 14)

The soluble protein as claimed in claim 14, which inhibits the Staphylococcus aureus Cowan strain particles stimulated release of proinflammatory cytokines of in vitro generated monocyte-derived dendritic cells.

### Claim 25 (depends on 24)

The soluble protein of claim 24, which inhibits the Staphylococcus aureus Cowan strain particle-stimulated release of proinflammatory cytokines in in vitro generated monocyte-derived dendritic cells dendritic cells, with an IC50 of 0.1 nM or less, as measured in a dendritic cell cytokine release assay.

### Claim 26 (depends on 1)

The soluble protein as claimed in claim 1, wherein said first and second single chain polypeptides of each heterodimer are covalently bound by a disulfide bridge.

### Claim 27 (depends on 1)

The soluble protein as claimed in claim 1, wherein said first single chain polypeptide of each heterodimer comprises the hinge region of an immunoglobulin constant part, and said two heterodimers are stably associated with each other by a disulfide bridge at said hinge region.

### Claim 28 (depends on 1)

The soluble protein as claimed in claim 1, wherein each region of said mammalian binding molecule is fused to its respective VH or VL sequence in the absence of peptide linkers.

### Claim 29 (depends on 1)

The soluble protein as claimed in claim 1, wherein each region of said mammalian binding molecule is fused to its respective VH or VL sequence via peptide linkers.

### Claim 30 (depends on 29)

The soluble protein as claimed in claim 29, wherein said peptide linker comprises 5 to 20 amino acids.

### Claim 31 (depends on 29)

The soluble protein as claimed in claim 29, wherein said peptide linker is a polymer of glycine and serine amino acids, preferably of (GGGGS)n, wherein n is any integer between 1 and 4, preferably 2.

### Claim 32 (depends on 1)

The soluble protein as claimed in claim 1 wherein the CH1, CH2 and CH3 regions of the antibody are derived from a silent mutant of human IgG1, IgG2, or IgG4 corresponding regions with reduced ADCC effector function.

### Claim 33 (depends on 1)

The soluble protein as claimed in claim 1, wherein said heterodimers comprise either:
(i) a first single chain polypeptide of SEQ ID NO:20 and a second single chain polypeptide of SEQ ID NO:21;
(ii) a first single chain polypeptide of SEQ ID NO:22 and a second single chain polypeptide of SEQ ID NO:23; or
(ii) a first single chain polypeptide of SEQ ID NO:40 and a second single chain polypeptide of SEQ ID NO:41.

### Claim 34 (depends on 1)

The soluble protein as claimed in claim 1, wherein said first and said second single chain polypeptides have at least 60, 70, 80, 90, 95, 96, 97, 98, or 99 percent sequence identity to the corresponding first and second single chain polypeptides of
(i) SEQ ID NO:20 and SEQ ID NO:21;
(ii) SEQ ID NO:22 and SEQ ID NO:23; or
(ii) SEQ ID NO:40 and SEQ ID NO:41.

### Claim 35 (depends on 1)

The soluble protein as claimed in claim 1 comprising:
(i) a heavy chain encoded by a nucleotide sequence of SEQ ID NO:77; and a light chain encoded by a nucleotide sequence of SEQ ID NO:78,
(ii) a heavy chain encoded by a nucleotide sequence of SEQ ID NO:79; and a light chain encoded by a nucleotide sequence of SEQ ID NO:80,
(iii) a heavy chain encoded by a nucleotide sequence of SEQ ID NO:97; and a light chain encoded by a nucleotide sequence of SEQ ID NO:98,

### Claim 36 (depends on 1)

A multivalent soluble protein complex comprising two or more soluble proteins as claimed in claim 1, wherein if the protein complex comprises N soluble proteins, the valency is N×6.

### Claim at position 37

-41. (canceled)

### Claim 42 (depends on 1)

A pharmaceutical composition comprising a soluble protein or protein complex as claimed in claim 1, in combination with one or more pharmaceutically acceptable vehicles.

### Claim 43 (depends on 42)

The pharmaceutical composition as claimed in claim 42, additionally comprising at least one other active ingredient.

### Claim 44 (depends on 1)

An isolated nucleic acid encoding at least one single chain polypeptide of one heterodimer of the soluble protein as claimed in claim 1.

### Claim 45 (depends on 44)

The isolated nucleic acid as claimed in claim 44, or a cloning or expression vector, comprising at least one nucleic acid selected from the group consisting of: SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:97, and SEQ ID NO:98.

### Claim 46 (depends on 1)

A recombinant host cell suitable for the production of a soluble protein or protein complex as claimed in claim 1, comprising the nucleic acids encoding said first and second single chain polypeptides of said heterodimers of said protein, and optionally, secretion signals.

### Claim 47 (depends on 46)

The recombinant host cell as claimed in claim 46, comprising the nucleic acids of SEQ ID NO:77 and SEQ ID NO:78; or SEQ ID NO:79 and SEQ ID NO:80; or SEQ ID NO:97 and SEQ ID NO:98 stably integrated in the genome.

### Claim 48 (depends on 46)

The recombinant host cell as claimed in claim 46, wherein said host cell is a mammalian cell line.

### Claim 49 (depends on 1)

A process for the production of a soluble protein or protein complex as claimed in claim 1, comprising culturing a recombinant host cell suitable for the production of said soluble protein or protein complex under appropriate conditions for the production of said soluble protein or protein complex, and isolating said protein.