# US 10,870,699 B2 · Anti-CD47 antibodies and uses thereof

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- Number: US 10,870,699 B2
- Document ID: US10870699B2
- Status: Granted
- Kind: B2
- Application number: 15/541,003
- Filed: 2015-12-28
- Published: 2017-12-28
- Granted: 2020-12-22
- Earliest claimed: 2014-12-30
- Assignees: Celgene Corporation
- Applicants: Celgene Corporation
- Inventors: Aaron Sato; Ryan Stafford; Junhao Yang
- Web page: https://patentgrep.com/patent/US10870699B2

## CPC

- C07K 16/2803: {against the immunoglobulin superfamily}
- A61K 39/39541: {against normal tissues, cells}
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61P 35/00: Antineoplastic agents
- A61P 35/02: specific for leukemia
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- A61K 2039/505: {comprising antibodies}
- C07K 2317/14: Specific host cells or culture conditions, e.g. components, pH or temperature
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- 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 2317/734: Complement-dependent cytotoxicity [CDC]
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- 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)
- 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
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

Provided herein are compositions, methods and uses involving antibodies that specifically bind to human CD47. Also provided are uses and methods, such as therapeutic methods, diagnostic methods, and methods of making such antibodies.

## Claims

### Claim 1

A monoclonal anti-CD47 antibody which specifically binds to human CD47, wherein the anti-CD47 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence:
(SEQ ID NO: 20)
X1QX2QLVQSGAEVKKX3GX4SVKVSCKASGFNIKDYYLHWVRQAPGQX5
LEWMGWIDPDQGDTEYAQKX6QX7RVTX8TX9DX10SX11STAYMELX12
SLRSX13DTAX14YYCNAAYGSSSYPMDYWGQGTTVTV,
wherein X1 is M or there is no amino acid at position X1, X2 is M or V, X3 is P, X4 is S or A, X5 is A or G, X6 is F or L, X7 is G, X8 is I or M, X9 is R or T, X10 is R or T, X11 is T, X12 is R, X13 is E or D, and X14 is V; and
wherein the anti-CD47 antibody comprises a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 comprising amino acid sequences KASQDIHRYLS (SEQ ID NO:17), RANRLVS (SEQ ID NO: 18), and LQYDEFPYT (SEQ ID NO:19), respectively.

### Claim 2 (depends on 1)

The anti-CD47 antibody of claim 1, wherein the antibody expression titer or yield is higher by at least 1 fold, at least 2 fold, or at least 3 fold compared to the parental antibody.

### Claim 3 (depends on 1)

The anti-CD47 antibody of claim 1, which is an IgG1 antibody or an IgG4 antibody.

### Claim 4 (depends on 1)

The anti-CD47 antibody of claim 1, which is an IgG4 antibody comprising a S228P amino acid substitution or comprising S228P and L235E amino acid substitutions according to the EU numbering index.

### Claim 5 (depends on 1)

The anti-CD47 antibody of claim 1, wherein the antibody comprises 13 or 14 amino acid modifications in the framework region of the heavy chain variable region of the amino acid sequence of SEQ ID NO:1.

### Claim 6 (depends on 1)

The anti-CD47 antibody of claim 1, wherein the antibody comprises 10 amino acid modifications in the heavy chain variable region of the amino acid sequence of SEQ ID NO:1.

### Claim 7 (depends on 1)

The anti-CD47 antibody of claim 1, wherein the VH comprises the amino acid sequence of SEQ ID NO:21 or SEQ ID NO:22.

### Claim 8 (depends on 1)

The anti-CD47 antibody of claim 1, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 SEQ ID NO:10, or SEQ ID NO:11.

### Claim 9 (depends on 1)

The anti-CD47 antibody of claim 1, which comprises a light chain comprising the amino acid sequence of SEQ ID NO:13 or SEQ ID NO:13 without amino acid M at the N-terminus.

### Claim 10 (depends on 1)

The anti-CD47 antibody of claim 1, wherein the antibody:
(i) does not cause substantial red blood cell depletion, anemia, or both red blood cell depletion and anemia after administration;
(ii) inhibits CD47 from interacting with signal-regulatory-protein α (SIRPα);
(iii) promotes macrophage-mediated phagocytosis of a CD47-expressing cell;
(iv) does not cause a significant level of effector function; or
(v) does not cause or promote a significant level of ADCC or CDC.

### Claim 11 (depends on 10)

The anti-CD47 antibody of claim 10, wherein the antibody does not cause substantial red blood cell depletion, anemia, or both red blood cell depletion and anemia after administration, and wherein the antibody does not cause substantial platelet depletion, agglutination of cells, or hemagglutination of red blood cells after administration.

### Claim 12 (depends on 1)

The anti-CD47 antibody of claim 1, wherein the antibody:
(i) when expressed using a cell-free system, exhibits lower binding affinity, or does not bind, to an FcγR than when expressed using CHO cells; or
(ii) is aglycosylated or has less glycosylation when expressed using the cell free system compared to when expressed in CHO cells.

### Claim 13 (depends on 12)

The anti-CD47 antibody of claim 12, wherein the antibody when expressed using a cell-free system, exhibits lower binding affinity, or does not bind, to an FcγR than when expressed using CHO cells, and wherein the lower binding affinity is at least 1 log lower or at least 2 log lower.

### Claim 14 (depends on 13)

The anti-CD47 antibody of claim 13, wherein the FcγR is FcγRI, FcγRIIA R131, FcγRIIA H131, FcγRIIB, or FcγRIIIA V158.

### Claim 15 (depends on 1)

The anti-CD47 antibody of claim 1, which is a bispecific antibody.

### Claim 16 (depends on 1)

The anti-CD47 antibody of claim 1, wherein the anti-CD47 antibody is conjugated to an agent, and wherein the agent is a label or a toxin.

### Claim 17 (depends on 1)

The anti-CD47 antibody of claim 1, wherein the anti-CD47 antibody has an expression titer or yield when produced using a cell-free system that is higher than an expression titer or yield of a parental anti-CD47 antibody produced using the cell-free system, wherein the parental anti-CD47 antibody comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 1, 2, 3 or 4.

### Claim 18 (depends on 17)

The anti-CD47 antibody of claim 17, wherein the parental antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1.

### Claim 19 (depends on 18)

The anti-CD47 antibody of claim 18, wherein the parental antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12.

### Claim 20 (depends on 1)

The anti-CD47 antibody of claim 1, wherein X1 is M or there is no amino acid at position X1, X2 is M, X3 is P, X4 is S, X5 is G, X6 is F, X7 is G, X8 is I, X9 is R, X10 is R, X11 is T, X12 is R, X13 is E, and X14 is V.

### Claim 21 (depends on 1)

The anti-CD47 antibody of claim 1, wherein the anti-CD47 antibody comprises the VH within HC SEQ ID NO: 7.

### Claim 22 (depends on 1)

The anti-CD47 antibody of claim 1, wherein X1 is M or there is no amino acid at position X1, X2 is M, X3 is P, X4 is 5, X5 is A, X6 is F, X7 is G, X8 is I, X9 is R, X10 is R, X11 is T, X12 is R, X13 is E, and X14 is V.

### Claim 23 (depends on 1)

A pharmaceutical composition comprising a therapeutically effective amount of the anti-CD47 antibody according to claim 1 or an antigen-binding fragment thereof.

### Claim 24 (depends on 23)

The pharmaceutical composition of claim 23, further comprising a pharmaceutically acceptable carrier.