# US 2022/0033481 A1 · Compositions and methods for increasing protein half-life in a serum

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- Number: US 2022/0033481 A1
- Document ID: US20220033481A1
- Status: Published application
- Kind: A1
- Application number: 16/755,268
- Filed: 2017-10-11
- Published: 2022-02-03
- Earliest claimed: 2017-10-11
- Assignees: Nanjing Legend Biotech Co., Ltd.
- Applicants: Nanjing Legend Biotech Co., Ltd.
- Inventors: Yafeng ZHANG; Shu WU; Fei SUN; Shuai YANG; Chuan-Chu CHOU
- Web page: https://patentgrep.com/patent/US20220033481A1

## CPC

- C07K 16/18: against material from animals or humans
- C07K 16/28: against receptors, cell surface antigens or cell surface determinants
- A61K 39/395: Antibodies (agglutinins A61K38/36 {; as drug carriers A61K47/50}); Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K 39/3955: {against proteinaceous materials, e.g. enzymes, hormones, lymphokines}
- A61K 47/6811: {the drug being a protein or peptide, e.g. transferrin or bleomycin}
- A61K 47/6813: {the drug being a peptidic cytokine, e.g. an interleukin or interferon}
- C07K 19/00: Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C07K 14/79: Transferrins, e.g. lactoferrins, ovotransferrins
- C07K 2317/565: Complementarity determining region [CDR]
- C07K 2317/569: Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
- C07K 2319/03: containing a transmembrane segment
- C07K 2319/31: fusions, other than Fc, for prolonged plasma life, e.g. albumin
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Novel antibodies, such as single domain antibodies (sdAbs), or antigen-binding fragments thereof that specifically bind a transferrin are described. Compositions, methods and systems for increasing the half-life of a target protein in a serum using an antibody or fragment thereof against a transferrin are also described.

## Claims

### Claim 1

A polypeptide comprising at least one immunoglobulin single domain antibody (sdAb) that specifically bind human and cynomolgus monkey serum transferrin protein and protein A resin, wherein the sdAb can be purified by a protein A column and used for half-life extension fragments for short half-life proteins or fragments.

### Claim 2 (depends on 1)

The isolated sdAb according to claim 1, the antibody or antigen-binding fragment thereof comprising:
(a) a complementarity determining region (CDR)1 having an amino acid sequence selected from the group consisting of
a. SEQ ID NO: 46-SEQ ID NO: 90; and
b. amino acid sequences that have 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequences of SEQ ID NOs: 46-SEQ ID NO: 90;
and/or
(b) a CDR2 having an amino acid sequence selected from the group consisting of
a. SEQ ID NO: 136-SEQ ID NO: 180; and
b. amino acid sequences that have 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequences of SEQ ID NOs: 136-SEQ ID NO: 180; and/or
(c) a CDR3 having an amino acid sequence selected from the group consisting of
a. SEQ ID NO: 226-SEQ ID NO: 270; and
b. amino acid sequences that have 4, 3, 2, or 1 amino acid(s) difference with the amino acid sequences of SEQ ID NOs: 226-SEQ ID NO: 270.

### Claim 3 (depends on 1)

The isolated antibody or antigen-binding fragment thereof of claim 1, being a single-domain antibody (sdAb).

### Claim 4 (depends on 3)

The isolated antibody or antigen-binding fragment thereof of claim 3, comprising an amino acid sequence at least 95% identical to a sequence selected from the group consisting of SEQ ID NO: 316-SEQ ID NO: 360.

### Claim 5 (depends on 4)

The isolated antibody or antigen-binding fragment thereof of claim 4, comprising an amino acid sequence at least 95% identical to the amino acid sequence of
AS01274
(QVQLVESGGGLVQPGGSLRLSCVASGSIASIATMAWYRQAPGQQRELVA
GITRGGSTKYADSVKGRFTISRDNAKNTLYLQMNSLKPDDTAVYYCTDYS
RKYYQDYWGQGTQVTVSS (SEQ ID NO: 316));
or
AS01299
(QVQLVESGGGLVQPGGSLRLSCAASGSIFSIATMAWYRQAPGKQRELVA
GITRSGSTNYRDSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCTDYS
SRYYHDYWGQGTQVTVSS (SEQ ID NO: 332));
or
AS01299VH3a
(EVQLVESGGGLVQPGGSLRLSCAASGSIFSIATMAWYRQAPGKGLELVA
GITRSGSTNYRDSVKGRFTISRDNSKNTMYLQMNSLRAEDTAVYYCTDYS
SRYYHDYWGQGTLVTVSS (SEQ ID NO: 354);
or
AS01299VH3a-A49
(EVQLVESGGGLVQPGGSLRLSCAASGSIFSIATMAWYRQAPGKGLELVS
GITRSGSTNYRDSVKGRFTISRDNSKNTMYLQMNSLRAEDTAVYYCTDYS
SRYYHDYWGQGTLVTVSS (SEQ ID NO: 356));
or
AS01299VH3a-L47
(EVQLVESGGGLVQPGGSLRLSCAASGSIFSIATMAWYRQAPGKGLEWVA
GITRSGSTNYRDSVKGRFTISRDNSKNTMYLQMNSLRAEDTAVYYCTDYS
SRYYHDYWGQGTLVTVSS (SEQ ID NO: 357));
or
AS01299VH3a-M78
(EVQLVESGGGLVQPGGSLRLSCAASGSIFSIATMAWYRQAPGKGLELVA
GITRSGSTNYRDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTDYS
SRYYHDYWGQGTLVTVSS (SEQ ID NO: 358));
or
AS01299VH4
(EVQLVESGGGLVQPGGSLRLSCAASGSIFSIATMAWYRQAPGKGLELVS
GITRSGSTNYRDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTDYS
SRYYHDYWGQGTLVTVSS (SEQ ID NO: 359));
or
AS01299VH4-L47
(EVQLVESGGGLVQPGGSLRLSCAASGSIFSIATMAWYRQAPGKGLEWVS
GITRSGSTNYRDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTDYS
SRYYHDYWGQGTLVTVSS (SEQ ID NO: 360)).

### Claim 6 (depends on 1)

The isolated antibody or antigen-binding fragment thereof of claim 1, having a dissociation constant (KD) of 10−7 M or less.

### Claim 7 (depends on 1)

The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the transferrin is a human transferrin.

### Claim 8 (depends on 1)

The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the transferrin is a cynomolgus monkey transferrin.

### Claim 9 (depends on 1)

The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the isolated antibody or antigen-binding fragment thereof binds to protein A resin.

### Claim 10 (depends on 1)

The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the isolated antibody or antigen-binding fragment thereof is produced recombinantly.

### Claim 11 (depends on 1)

A fusion protein comprising an antibody or antigen-binding fragment thereof of claim 1, a target protein, and optionally a linker, wherein the antibody or fragment thereof is fused to the carboxyl-terminus or amino-terminus of the target protein, and the linker optionally separates the antibody and the carboxyl-terminus or amino-terminus of the target protein.

### Claim 12 (depends on 11)

A composition comprising an effective amount of the fusion protein of claim 11 and transferrin.

### Claim 13 (depends on 1)

The fusion protein comprising an antibody or antigen-binding fragment thereof of claim 1, wherein the fusion protein is capable of being purified by protein A resin.

### Claim 14 (depends on 11)

A method of increasing the half-life of the target protein in the fusion protein, the method comprising exposing the fusion protein of claim 11 to transferrin in cynomolgus monkey serum to thereby increase the half-life of the target protein in the fusion protein.

### Claim 15 (depends on 11)

A method of producing the fusion protein of claim 11, comprising:
(a) obtaining an expression vector encoding the fusion protein;
(b) introducing the expression vector into a cell to obtain a recombinant cell;
(c) growing the recombinant cell under conditions to allow expression of the fusion protein; and
(d) obtaining the fusion protein from the recombinant cell or its supernatant.

### Claim 16 (depends on 1)

A method for increasing the half-life of a target protein, the method comprising:
(1) obtaining a fusion protein, wherein the fusion protein comprises an antibody or antigen-binding fragment thereof of claim 1, a target protein, and optionally a linker, wherein the antibody or antigen-binding fragment thereof is fused to the carboxyl-terminus or amino-terminus of the target protein, and the linker optionally separates the antibody and the carboxyl-terminus or amino-terminus of the target protein, and
(2) exposing the fusion protein to transferrin,
wherein the transferrin increases the half-life of the target protein in the fusion protein compared to the target protein alone.

### Claim 17 (depends on 16)

The method of claim 16, wherein the exposing step comprises administering the fusion protein to a serum comprising the transferrin of cynomolgus monkey.

### Claim 18 (depends on 1)

A system for increasing the half-life of a target protein, the system comprising:
(a) a first nucleotide sequence encoding an antibody or antigen-binding fragment thereof of claim 1, and optionally a second nucleotide sequence encoding a linker, wherein the first and second nucleotide sequences are operably linked;
(b) a host cell; and
(c) the transferrin.