# US 10,501,558 B2 · Modified Fc proteins comprising site-specific non-natural amino acid residues, conjugates of the same, methods of their preparation and methods of their use

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- Number: US 10,501,558 B2
- Document ID: US10501558B2
- Status: Granted
- Kind: B2
- Application number: 15/632,196
- Filed: 2017-06-23
- Published: 2017-12-21
- Granted: 2019-12-10
- Earliest claimed: 2012-06-26
- Assignees: Sutro Biopharma, Inc.
- Applicants: Sutro Biopharma, Inc.
- Inventors: Christopher D. Thanos; Leslie McEvoy; Gang Yin; Kalyani Penta; Ramesh Baliga; Sunil Bajad; Sonia Pollitt; Chris Murray; Alex Steiner; Avinash Gill
- Web page: https://patentgrep.com/patent/US10501558B2

## CPC

- C07K 16/46: Hybrid immunoglobulins (hybrids of an immunoglobulin with a peptide not being an immunoglobulin C07K19/00)
- A61K 47/68: the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K 47/6817: {Toxins}
- A61K 47/6851: {the antibody targeting a determinant of a tumour cell}
- C07K 16/2878: {against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95}
- C07K 16/32: against translation products of oncogenes
- A61K 2039/505: {comprising antibodies}
- C07K 2317/41: Glycosylation, sialylation, or fucosylation
- C07K 2317/52: Constant or Fc region; Isotype
- 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)

## Abstract

Provided herein are modified Fc proteins comprising non-natural amino acid residues at site-specific positions, conjugates of the modified Fc proteins for therapy or diagnosis, compositions comprising the modified Fc proteins and conjugates thereof, methods of their production and methods of their use. The modified Fc proteins and conjugates are useful for methods of treatment and prevention, methods of detection and methods of diagnosis.

## Claims

### Claim 1

An antibody fragment of the IgG class comprising an Fc protein, wherein the Fc protein comprises a polypeptide chain having one or more non-natural amino acid residues at specific sites selected from the group consisting of heavy chain residues H241 and H222 according to the EU index of Kabat in the polypeptide chain, or a post-translationally modified variant or an aglycosylated variant thereof,
wherein the non-natural amino acid residue is selected from the group consisting of: ortho-substituted tyrosine, meta-substituted tyrosine, para-substituted phenylalanine, ortho-substituted phenylalanine, and meta-substituted phenylalanine.

### Claim 2

A antibody fragment of the IgG class comprising an Fc protein, wherein the Fc protein comprises a polypeptide chain having one or more non-natural amino acid residues at specific sites selected from the group consisting of heavy chain residues H241 and H222 according to the EU index of Kabat, wherein each non-natural amino acid residue is according to the formula
wherein each L is independently a divalent linker;
each R is a reactive group; and
wherein the non-natural amino acid residue is selected from the group consisting of: ortho-substituted tyrosine, meta-substituted tyrosine, para-substituted phenylalanine, ortho-substituted phenylalanine, and meta-substituted phenylalanine.

### Claim 3 (depends on 2)

The antibody fragment of claim 2, wherein each R is a reactive group selected from the group consisting of amino, carboxy, acetyl, hydrazino, hydrazido, semicarbazido, sulfanyl, azido and alkynyl.

### Claim 4 (depends on 2)

The antibody fragment of claim 2, wherein each L is a divalent linker selected from the group consisting of a bond, alkylene, substituted alkylene, heteroalkylene, substituted heteroalkylene, arylene, substituted arylene, heteroarylene and substituted heteroarylene.

### Claim 5 (depends on 1)

The antibody fragment of claim 1, wherein the Fc protein is aglycosylated.

### Claim 6 (depends on 5)

The antibody fragment of claim 5, wherein the Fc protein has a higher thermal stability (Tm1) compared to the corresponding wild-type Fc protein.

### Claim 7 (depends on 5)

A composition comprising the antibody fragment of claim 5, wherein said composition is substantially pure.

### Claim 8 (depends on 5)

A composition comprising the antibody fragment of claim 5, wherein said antibody fragment is at least 95% by mass of the total protein mass of said composition.

### Claim 9 (depends on 5)

The antibody fragment of claim 5, wherein the Fc protein does not comprise a variable domain or a light chain.

### Claim 10 (depends on 5)

The antibody fragment of claim 5, wherein the Fc protein corresponds to a subclass selected from the group consisting of Ig1, IgG2, IgG3, and IgG4.

### Claim 11 (depends on 5)

The antibody fragment of claim 5, wherein said non-natural amino acid residue comprises a moiety selected from the group consisting of amino, carboxy, acetyl, hydrazino, hydrazido, semicarbazido, sulfanyl, azido, and alkynyl.

### Claim 12 (depends on 5)

The antibody fragment of claim 5, wherein each non-natural amino acid residue is according to the formula
wherein each L is independently a phenylene; and
each R is independently a functional group selected from the group consisting of a therapeutic moiety, a labeling moiety, amino, carboxy, acetyl, hydrazino, hydrazido, semicarbazido, sulfanyl, azido, and alkynyl.

### Claim 13 (depends on 5)

The antibody fragment of claim 5, wherein the non-natural amino acid residue is selected from the group consisting of: p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, p-propargyloxy-phenylalanine, and p-azidomethyl-L-phenylalanine.

### Claim 14 (depends on 5)

The antibody fragment of claim 5, wherein the non-natural amino acid residue is p-azido-L-phenylalanine.

### Claim 15 (depends on 5)

The antibody fragment of claim 5, wherein the non-natural amino acid residue is p-azidomethyl-L-phenylalanine.

### Claim 16 (depends on 5)

An Fc protein conjugate comprising the antibody fragment of claim 5, wherein the Fc protein is linked to one or more therapeutic moieties or labeling moieties.

### Claim 17 (depends on 16)

The Fc protein conjugate of claim 16, wherein the Fc protein is linked to one or more drugs or polymers.

### Claim 18 (depends on 16)

The Fc protein conjugate of claim 16, wherein the Fc protein is linked to one or more labeling moieties.

### Claim 19 (depends on 16)

The Fc protein conjugate of claim 16, wherein said Fc protein is linked to one or more single chain binding domains (scFv).

### Claim 20 (depends on 16)

The Fc protein conjugate of claim 16, wherein said one or more therapeutic moieties or labeling moieties is linked to the non-natural amino acid, or a residue thereof, via one or more linkers.