# US 10,865,230 B2 · Construction and characterization of multimeric IL-15-based molecules with CD3 binding domains

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- Number: US 10,865,230 B2
- Document ID: US10865230B2
- Status: Granted
- Kind: B2
- Application number: 15/606,552
- Filed: 2017-05-26
- Published: 2017-11-30
- Granted: 2020-12-15
- Earliest claimed: 2016-05-27
- Assignees: Altor BioScience Corporation
- Applicants: Altor Bioscience Corporation
- Inventors: Bai Liu; Peter Rhode; Hing C. Wong
- Web page: https://patentgrep.com/patent/US10865230B2

## CPC

- C07K 14/5443: {IL-15}
- A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P 35/00: Antineoplastic agents
- A61P 35/02: specific for leukemia
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- C07K 14/7155: {for interleukins [IL]}
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/2809: {against the T-cell receptor (TcR)-CD3 complex}
- C07K 16/2887: {against CD20}
- A61K 2039/505: {comprising antibodies}
- C07K 2317/31: multispecific
- C07K 2317/524: CH2 domain
- C07K 2317/526: CH3 domain
- C07K 2317/56: variable (Fv) region, i.e. VH and/or VL
- C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K 2317/732: Antibody-dependent cellular cytotoxicity [ADCC]
- C07K 2318/20: Antigen-binding scaffold molecules wherein the scaffold is not an immunoglobulin variable region or antibody mimetics
- C07K 2319/00: Fusion polypeptide
- C07K 2319/02: containing a signal sequence
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C07K 2319/75: containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
- 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
- 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)

## Abstract

The invention features soluble fusion protein complexes comprising at least two soluble fusion proteins. For example, the first fusion protein is an anti-CD3 antibody covalently linked to an interleukin-15 (IL-15) polypeptide or functional fragment thereof. The second fusion protein comprises a binding domain that recognizes disease antigens, wherein this domain is covalently linked to a soluble interleukin-15 receptor alpha (IL-15Rα) polypeptide or a functional fragment thereof. One or both of the first and second fusion proteins further includes an immunoglobulin Fc domain or a functional fragment thereof, and the IL-15 domain of first fusion protein binds to the soluble IL-15Rα domain of the second fusion protein to form a soluble fusion protein complex. The invention further provides methods for making and using the complexes of the invention.

## Claims

### Claim 1

An isolated soluble fusion protein complex comprising at least two soluble fusion proteins, wherein
the first fusion protein comprises (a) a first binding domain covalently linked to (b) a interleukin-15 (IL-15) polypeptide domain; and the second fusion protein comprises (c) a second binding domain covalently linked to (d) a soluble IL-15 receptor alpha sushi-binding domain (IL-15RαSu) fused to an immunoglobulin Fc domain,
wherein the first binding domain comprises an anti-CD3 antibody and the second binding domain comprises a means for binding an antigen on a tumor cell, and
wherein the IL-15 domain of the first fusion protein binds to the soluble IL-15RαSu domain of the second fusion protein to form a soluble fusion protein complex, and wherein the first fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 2.

### Claim 2 (depends on 1)

The soluble fusion protein complex of claim 1, wherein the IL-15 polypeptide is an IL-15 variant comprising an N72D mutation (IL-15N72D).

### Claim 3 (depends on 1)

The soluble fusion protein complex of claim 1, wherein the anti-CD3 antibody comprises an immunoglobulin light chain variable domain covalently linked to an immunoglobulin heavy chain variable domain by a polypeptide linker sequence.

### Claim 4 (depends on 1)

The soluble fusion protein of claim 1, wherein the means for binding an antigen on a tumor cell binds an antigen on a tumor cell and comprises an anti-CD20 antibody.

### Claim 5 (depends on 4)

The soluble fusion protein complex of claim 4, wherein the anti-CD20 antibody comprises an immunoglobulin light chain variable domain covalently linked to an immunoglobulin heavy chain variable domain by a polypeptide linker sequence.

### Claim 6 (depends on 4)

The soluble fusion protein complex of claim 4, wherein the second fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 4.

### Claim 7 (depends on 1)

A soluble fusion protein complex comprising a first soluble fusion protein complex of claim 1 covalently linked to a second soluble fusion protein complex of claim 1.

### Claim 8 (depends on 7)

The soluble fusion protein complex of claim 7, wherein the first soluble fusion protein complex is covalently linked to the second soluble fusion protein complex by a disulfide bond linking the Fc domain of the first soluble fusion protein complex to the Fc domain of the second soluble fusion protein complex.

### Claim 9 (depends on 1)

A nucleic acid sequence encoding the first fusion protein of claim 1, wherein said nucleic acid sequence comprises the sequence set forth in SEQ ID NO: 1.

### Claim 10 (depends on 9)

The nucleic acid sequence of claim 9, wherein the nucleic acid sequence further comprises a promoter, translation initiation signal, and leader sequence operably linked to the sequence encoding the fusion protein.

### Claim 11 (depends on 1)

The soluble fusion protein complex of claim 1, wherein a nucleic acid sequence encoding the second fusion protein comprises the sequence set forth in SEQ ID NO: 3.

### Claim 12 (depends on 11)

The soluble fusion protein complex claim 11, wherein the nucleic acid sequence further comprises a promoter, translation initiation signal, and leader sequence operably linked to the sequence encoding the fusion protein.

### Claim 13 (depends on 1)

The soluble fusion protein complex of claim 1, wherein the antigen-specific binding domain recognizes disease antigens.

### Claim 14 (depends on 13)

The soluble fusion protein complex of claim 13, wherein the disease antigens are associated with neoplasia, infectious disease or autoimmune disease.

### Claim 15 (depends on 1)

The soluble fusion protein complex of claim 1, wherein the second binding domain comprises a tumor cell antigen-specific binding domain.