# US 2018/0022807 A1 · Bispecific binding proteins and uses thereof

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- Number: US 2018/0022807 A1
- Document ID: US20180022807A1
- Status: Published application
- Kind: A1
- Application number: 15/588,271
- Filed: 2017-05-05
- Published: 2018-01-25
- Earliest claimed: 2016-05-06
- Assignees: MedImmune, LLC
- Applicants: Medimmune, LLC
- Inventors: Srinath KASTURIRANGAN; CHANGSHOU GAO; GODFREY RAINEY; MICHELLE MORROW; CLAIRE LOUISE DOBSON; STACEY DRABIC; DARREN SCHOFIELD; GIANLUCA CARLESSO; KRISTEN POLLIZZI; YARIV MAZOR; MICHAEL OBERST; SCOTT A. HAMMOND; BRIAN LOBO; PRAKASH MANIKWAR; JONATHAN SEAMAN
- Web page: https://patentgrep.com/patent/US20180022807A1

## CPC

- C07K 16/2818: {against CD28 or CD152}
- A61P 35/00: Antineoplastic agents
- C07K 16/2803: {against the immunoglobulin superfamily}
- C07K 16/2827: {against B7 molecules, e.g. CD80, CD86}
- C07K 16/2878: {against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95}
- A61K 2039/505: {comprising antibodies}
- C07K 2317/31: multispecific
- C07K 2317/33: Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
- C07K 2317/35: Valency
- C07K 2317/41: Glycosylation, sialylation, or fucosylation
- C07K 2317/51: Complete heavy chain or Fd fragment, i.e. VH + CH1
- C07K 2317/515: Complete light chain, i.e. VL + CL
- C07K 2317/52: Constant or Fc region; Isotype
- C07K 2317/524: CH2 domain
- C07K 2317/526: CH3 domain
- C07K 2317/53: Hinge
- C07K 2317/55: Fab or Fab'
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K 2317/732: Antibody-dependent cellular cytotoxicity [ADCC]
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- C07K 2317/77: Internalization into the cell
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C07K 2317/94: Stability, e.g. half-life, pH, temperature or enzyme-resistance
- 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 disclosure generally provides proteins that bind two epitopes (e.g., a first and a second epitope) and that are bivalent for binding to each of the first and second epitopes. The disclosure also provides for specific binding proteins, including antibodies, which bind to a target protein. The disclosure also provides compositions comprising such proteins, nucleic acid molecules encoding such proteins and methods of making such proteins. The disclosure provides methods of inducing an immune response in a subject as well as methods for treating or preventing cancer in a subject by administering the proteins, nucleic acid molecules and/or compositions to the subject.

## Claims

### Claim 1

A protein, comprising:
a first binding domain (BD1) that binds to a first epitope,
a second binding domain (BD2) that binds to a second epitope, and
an Fc region comprising CH2 and CH3 domains;
wherein the Fc region comprises BD2 at a solvent exposed loop in the CH2 domain, the CH3 domain, or at the interface of the CH2 and CH3 domains; and
wherein the protein is bivalent for binding to each of the first and second epitopes.

### Claim 2 (depends on 1)

The protein of claim 1, wherein the Fc region comprises BD2 at a solvent exposed loop in the amino acid sequence in the CH2 domain, the CH3 domain, or at the interface of the CH2 and CH3 domain.

### Claim 3 (depends on 2)

The protein of claim 2, wherein the solvent exposed loop comprises an amino acid sequence from the CH2 domain.

### Claim 4 (depends on 3)

The protein of claim 3, wherein the solvent exposed loop comprises the amino acid sequence ISRTP (SEQ ID NO:39).

### Claim 5 (depends on 2)

The protein of claim 2, wherein the solvent exposed loop comprises an amino acid sequence from the CH3 domain.

### Claim 6 (depends on 5)

The protein of claim 5, wherein the solvent exposed loop comprises the amino acid sequence SNG.

### Claim 7 (depends on 2)

The protein of claim 2, wherein the solvent exposed loop comprises an amino acid sequence from the interface of the CH2 domain and the CH3 domain.

### Claim 8 (depends on 7)

The protein of claim 7, wherein the solvent exposed loop comprises the amino acid sequence AKGQP (SEQ ID NO:40).

### Claim 9 (depends on 1)

The protein of claim 1 wherein BD2 comprises a single-chain variable fragment (scFv).

### Claim 10 (depends on 1)

The protein of claim 1 wherein BD1 comprises a binding domain selected from the group consisting of a Fab domain, an scFv, a single domain antibody, and an antibody variable domain.

### Claim 11 (depends on 1)

The protein of any claim 1, wherein BD1 comprises a Fab domain.

### Claim 12 (depends on 11)

The protein of claim 11, wherein the Fab domain is connected to the Fc region via an antibody hinge region.

### Claim 13 (depends on 1)

The protein of claim 1, wherein the Fc region comprises a domain selected from the group consisting of an Fc region from an IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

### Claim 14 (depends on 13)

The protein of claim 13, wherein the Fc region comprises a variant Fc region.

### Claim 15 (depends on 13)

The protein of claim 13, wherein the Fc region is aglycosylated.

### Claim 16 (depends on 13)

The protein of claim 13, wherein the Fc region is deglycosylated.

### Claim 17 (depends on 13)

The protein of claim 13, wherein the Fc region has reduced fucosylation or is afucosylated.

### Claim 18 (depends on 1)

The protein of claim 1 wherein the protein further comprises a protein linker L1 between BD2 and the Fc region.

### Claim 19 (depends on 1)

The protein of claim 1 wherein the protein further comprises a first protein linker, L1, and a second protein linker, L2, between BD2 and the Fc region.

### Claim 20 (depends on 1)

The protein of claim 1 wherein BD2 is associated with the Fc region via a protein linker L1.

### Claim 21 (depends on 1)

The protein of claim 1 wherein BD2 is associated with the Fc region via two protein linkers, L1 and L2.

### Claim 22 (depends on 19)

The protein of claim 19, wherein L1 and L2 are independently selected from (G4S)2 (SEQ ID NO:41), (G4S)3, (SEQ ID NO:42), and (G4S)4 (SEQ ID NO:43).

### Claim 23 (depends on 1)

The protein of claim 1, wherein the protein comprises a chimeric heavy chain comprising the following polypeptide domains, from N-terminus to C-terminus
VH1-CH1-CH2(N-term)-BD2-CH2(C-term)-CH3; and
BD1 comprises a Fab domain;
wherein VH1 comprises a heavy chain variable domain of the Fab domain and CH1 comprises the heavy chain constant domain 1 of the Fab.

### Claim 24 (depends on 1)

The protein of claim 1, wherein the protein comprises a chimeric heavy chain comprising the following polypeptide domains, from N-terminus to C-terminus
VH1-CH1-CH2-BD2-CH3; and
BD1 comprises a Fab domain;
wherein VH1 comprises a heavy chain variable domain of the Fab domain and CH1 comprises the heavy chain constant domain 1 of the Fab.

### Claim 25 (depends on 1)

The protein of claim 1, wherein the protein comprises a chimeric heavy chain comprising the following polypeptide domains, from N-terminus to C-terminus
VH1-CH1-CH2-CH3(N-term)-BD2-CH3(C-term); and
BD1 comprises a Fab domain;
wherein VH1 comprises a heavy chain variable domain of the Fab domain, CH1 comprises the heavy chain constant domain 1 of the Fab.

### Claim 26 (depends on 1)

The protein of claim 1, wherein BD2 comprises an scFv.

### Claim 27 (depends on 26)

The protein of claim 26, wherein the scFv comprises, from N-terminus to C-terminus,
VH2-polypeptide linker-VL2 or VL2-polypeptide linker-VH2;
wherein VH2 comprises the heavy chain variable domain of the scFv and VL2 comprises the light chain variable domain of the scFv.

### Claim 28 (depends on 1)

The protein of claim 1 wherein the protein further comprises a protein linker L1 between BD2 and the Fc region.

### Claim 29 (depends on 1)

The protein of claim 1 wherein the protein further comprises a first protein linker, L1, and a second protein linker, L2, between BD2 and the Fc region.

### Claim 30 (depends on 1)

The protein of claim 1, wherein BD2 is associated via a linker (L1) to the CH2 domain, the CH2 domain, or the interface of the CH2 and CH3 domains of the Fc region.

### Claim 31 (depends on 1)

The protein of claim 1, wherein BD2 is associated via two protein linkers, L1 and L2 to the CH2 domain, the CH2 domain, or the interface of the CH2 and CH3 domains of the Fc region.

### Claim 32 (depends on 29)

The protein of claim 29, wherein L1 and L2 are independently selected is from protein linkers having a length of 1-25 amino acids.

### Claim 33 (depends on 29)

The protein of claim 29, wherein L1 and L2 are independently selected from (G4S)2 (SEQ ID NO:41), (G4S)3, (SEQ ID NO:42), and (G4S)4 (SEQ ID NO:43).

### Claim 34 (depends on 1)

The protein of claim 1, wherein the first and second epitopes are different.

### Claim 35 (depends on 1)

The protein of claim 1, wherein the first and second epitopes are the same.

### Claim 36

A bispecific binding protein that binds to PD-1 and CTLA-4 comprising a first peptide comprising the amino acid sequence of SEQ ID NO:1, and a second peptide the amino acid sequence of SEQ ID NO:2.

### Claim 37

A bispecific binding protein that binds to PD-1 and CTLA-4 comprising a first peptide comprising the amino acid sequence of SEQ ID NO:3, and a second peptide comprising the amino acid sequence of SEQ ID NO:4.

### Claim 38

A bispecific binding protein that binds to PD-1 and CTLA-4 comprising, a first peptide comprising the amino acid sequence of SEQ ID NO:5, and a second peptide comprising the amino acid sequence of SEQ ID NO:6.

### Claim 39

A bispecific binding protein that binds to PD-1 and CTLA-4 comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 9, a first light chain comprising the amino acid sequence of SEQ ID NO: 7, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 12, and a second light chain comprising the amino acid sequence of SEQ ID NO: 4.

### Claim 40

A bispecific binding protein that binds to PD-L1 and CTLA-4 comprising a first peptide comprising the amino acid sequence of SEQ ID NO:14 and a second peptide comprising the amino acid sequence of SEQ ID NO:15.

### Claim 41

A bispecific binding protein that binds to PD-L1 and CTLA-4 comprising a first peptide comprising the amino acid sequence of SEQ ID NO:16, and a second peptide comprising the amino acid sequence of SEQ ID NO:17.

### Claim 42

A bispecific binding protein that binds to PD-L1 and CTLA-4 comprising a first peptide comprising the amino acid sequence of SEQ ID NO:18, and a second peptide comprising the amino acid sequence of SEQ ID NO:19.

### Claim at position 43

43-51. (canceled)

### Claim 52 (depends on 1)

A composition comprising the protein of claim 1 and a pharmaceutically acceptable carrier.

### Claim 53 (depends on 1)

A nucleic acid molecule comprising a nucleotide sequence encoding a protein of claim 1.

### Claim 54 (depends on 53)

A vector comprising the nucleic acid molecule of claim 53.

### Claim 55 (depends on 54)

A host cell comprising the vector of claim 54.

### Claim 56 (depends on 1)

A method of treating or preventing cancer in a subject, the method comprising administering the protein of claim 1 to the subject.

### Claim 57 (depends on 56)

The method of claim 56, wherein the cancer is one or more of ovarian cancer, breast cancer, colorectal cancer, prostate cancer, cervical cancer, uterine cancer, testicular cancer, bladder cancer, head and neck cancer, melanoma, pancreatic cancer, renal cell carcinoma, and lung cancer.

### Claim 58 (depends on 1)

A method of enhancing an immune response in a subject, the method comprising administering the protein of claim 1 to the subject.