# US 2013/0156765 A1 · FUSION PROTEINS OF NATURAL HUMAN PROTEIN FRAGMENTS TO CREATE ORDERLY MULTIMERIZED IMMUNOGLOBULIN Fc COMPOSITIONS

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- Number: US 2013/0156765 A1
- Document ID: US20130156765A1
- Status: Published application
- Kind: A1
- Application number: 13/812,269
- Filed: 2011-07-28
- Published: 2013-06-20
- Earliest claimed: 2010-07-28
- Inventors: David S. Block; Henrik Olsen
- Web page: https://patentgrep.com/patent/US20130156765A1

## CPC

- C07K 14/47: from mammals
- A61K 31/573: substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
- A61K 39/395: Antibodies (agglutinins A61K38/36 {; as drug carriers A61K47/50}); Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61P 37/00: Drugs for immunological or allergic disorders
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- A61K 2039/505: {comprising antibodies}
- C07K 2317/35: Valency
- C07K 2317/52: Constant or Fc region; Isotype
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- C07K 2319/73: containing coiled-coiled motif (leucine zippers)
- C07K 2319/735: containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
- 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)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

The current invention involves a series of fully recombinant multimerized forms of immunoglobulin Fc which thereby present polyvalent immunoglobulin Fc to immune cell receptors. The fusion proteins exist as both homodimeric and highly ordered multimeric fractions, termed stradomers. In comparison to the homodimeric fraction, purified multimeric stradomers have higher affinity and avidity for Fc Rs with slower dissociation and are useful in the treatment and prevention of disease. The current invention demonstrates that directly linking IgG1 Fc regions to multimerization domains leads to enhanced multimerization and biological activity.

## Claims

### Claim at position 1

1-156. (canceled)

### Claim 157

A stradomer unit comprising:
(a) a leader sequence
(b) an IgG1 Fc domain; and
(c) a multimerization domain;
wherein the leader sequence is directly linked to the IgG1 Fc domain and wherein the IgG1 Fc domain is directly linked to the multimerization domain or wherein the leader sequence is directly linked to the multimerization domain and the multimerization domain is directly linked to the IgG1 Fc domain.

### Claim 158 (depends on 157)

The stradomer unit of claim 157 wherein the IgG1 Fc domain is about 90% homologous to SEQ ID NO:2.

### Claim 159 (depends on 157)

The stradomer unit of claim 157 wherein the IgG1 Fc domain comprises the CH2 and CH3 domains of IgG1.

### Claim 160 (depends on 159)

Stradomer unit of claim 159 wherein the IgG1 Fc domain is at least about 90% homologous to SEQ ID NO: 19.

### Claim 161 (depends on 157)

The stradomer unit of claim 157 wherein the multimerization domain is an IgG2 hinge.

### Claim 162 (depends on 161)

The stradomer unit of claim 161 wherein the amino acid sequence of the multimerization domain is about 90% homologous to SEQ ID NO: 3 and is capable of multimerizing said stradomer units.

### Claim 163 (depends on 157)

The stradomer unit of claim 157 wherein the multimerization domain is directly linked to the amino terminus of the IgG1 Fc domain or the carboxy terminus of the IgG1 Fc domain.

### Claim 164 (depends on 157)

The stradomer unit of claim 157 wherein the stradomer unit comprises: SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 18.

### Claim 165 (depends on 157)

The stradomer unit of claim 157 wherein the multimerization domain creates multimers of said stradomer units.

### Claim 166 (depends on 165)

The stradomer unit of claim 165 wherein the multimers of said stradomer units are high order multimers

### Claim 167 (depends on 157)

A stradomer composition comprising the stradomer units of claim 157 wherein the multimers are present at a percentage of about 45% or about 55% or about 65% or about 65% or about 70% or about 73% of the total stradomer composition.

### Claim 168 (depends on 157)

The stradomer unit of claim 157 wherein the IgG1 Fc domain is capable of binding FcRn, DC-SIGN, SIGN-R1 or an FcγR.

### Claim 169 (depends on 157)

A cluster stradomer comprising at least two stradomer units of claim 157.

### Claim 170 (depends on 169)

A method of modulating an immune response in a subject comprising administering to a subject an effective amount of the cluster stradomer of claim 169.

### Claim 171 (depends on 169)

A method for treating an inflammatory disease in a subject comprising administering to the subject an effective amount of the cluster stradomer of claim 169.

### Claim 172 (depends on 171)

The method of claim 171 wherein the inflammatory disease is a disease selected from, multifocal mortor neuropathy, Alzheimer's disease, sepsis, arthritis, multiple sclerosis, type I diabetes, autoimmune thyroiditis, idiopathic thrombocytopenic purpura, chronic inflammatory polyneuropathy, scleroderma, autoimmune uveitis, systemic lupus erythematosus, myasthenia gravis, and atopic dermatitis or a disease associated with the transplantation of an organ from a donor to a recipient; or
a infectious disease a bacterial infection or a viral infection.

### Claim 173 (depends on 169)

A method for blocking nonspecific binding of antibodies in an in vitro or ex vivo assay comprising incubating target tissue or target cells with a composition comprising an effective amount of the cluster stradomer of claim 169.

### Claim 174

A method for producing a cluster stradomer comprising expressing a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 18 in a host.

### Claim 175 (depends on 19)

The method of claim 19 further comprising
(a) cloning a DNA encoding the stradomer selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, or SEQ ID NO: 18 into an expression vector;
(b) transfecting the expression vector into a bacterial host;
(c) isolating the plasmid DNA containing cloned DNA from the bacterial culture;
(d) linearizing the plasmid DNA containing cloned DNA;
(e) transfecting the linearized DNA into mammalian cells;
(f) expanding the positively transfected cells to obtain a pool of stably transfected cells;
(g) harvesting the stradomer protein from the media; and
(h) purifying the stradomer protein, wherein the stradomer protein contains no extraneous sequences.