# US 2019/0376979 A1 · Protein combination-based fv library, and preparation method therefor

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- Number: US 2019/0376979 A1
- Document ID: US20190376979A1
- Status: Published application
- Kind: A1
- Application number: 16/385,867
- Filed: 2019-04-16
- Published: 2019-12-12
- Earliest claimed: 2013-01-24
- Assignees: AbTLAS CO., LTD.
- Applicants: AbTLAS CO., LTD.
- Inventors: Byeong Doo SONG; Jee Sun YUN; Song Yi LEE; Hye In KIM; Hyo Jung CHOI; Jong Rip CHOI
- Web page: https://patentgrep.com/patent/US20190376979A1

## CPC

- G01N 33/6845: {Methods of identifying protein-protein interactions in protein mixtures}
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/46: Hybrid immunoglobulins (hybrids of an immunoglobulin with a peptide not being an immunoglobulin C07K19/00)
- G01N 33/6857: {Antibody fragments}
- C07K 2317/56: variable (Fv) region, i.e. VH and/or VL
- C07K 2317/565: Complementarity determining region [CDR]
- C07K 2317/62: comprising only variable region components
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- 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)

## Abstract

The present invention relates to a method for constructing an Fv library based on a combination of proteins, a method of screening a desired antibody using the constructed Fv library, an Fv antibody screened by the screening method, and an Fv library constructed by the Fv library construction method. The Fv library of the present invention is based on a combination of proteins so that members thereof can be individually analyzed for their function. Moreover, the Fv library enables a desired Fv antibody to be screened without needing a target antigen preparation. In addition, the protein combination based Fv library makes it possible to significantly reduce the number of protein purification processes to thereby reduce costs and time, compared to conventional DNA-based libraries.

## Claims

### Claim at position 1

1-21. (canceled)

### Claim 22

A method for constructing an Fv library comprising:
(a) expressing and purifying individual heavy-chain variable region (VH) domain proteins from cells;
(b) expressing and purifying light-chain variable region (VL) domain proteins from cells separate from the cells in (a); and
(c) pairing the purified VH domain proteins and the purified VL domain proteins to one another.

### Claim 23 (depends on 22)

A method for screening a desired VH domain protein and VL domain protein pair, the method comprising the steps of:
(a) performing functional analysis on individual members of a Fv library constructed according to the method of claim 22; and
(b) identifying a VH domain protein and VL domain protein pair that exhibits a desired property, characteristic, or activity.

### Claim 24

An Fv library comprising:
(a) individual heavy-chain variable region (VH) domain proteins expressed and purified from cells;
(b) individual light-chain variable region (VL) domain proteins expressed and purified from cells separate from the cells in (a); and
wherein the purified VH domain proteins and the purified VL domain proteins are paired to one another.

### Claim 25 (depends on 24)

The Fv library of claim 24, wherein the VH domain proteins comprise amino acid sequences that differ from each other.

### Claim 26 (depends on 24)

The Fv library of claim 24, wherein the VL domain proteins comprise amino acid sequences that differ from each other.

### Claim 27 (depends on 24)

The Fv library of claim 24, wherein VH domain proteins comprise amino acid sequences that differ from each other and the VL domain proteins comprise amino acid sequences that differ from each other.

### Claim 28 (depends on 24)

The Fv library of claim 24, wherein the VH domain proteins derive from the same original VH domain and the VH domain proteins comprise amino acid sequences that differ from each other; or wherein the VL domain proteins derive from the same original VL domain and the VL domain proteins comprise amino acid sequences that differ from each other.

### Claim 29 (depends on 24)

The Fv library of claim 24, wherein the VH domain proteins derive from the same original VH domain and the VH domain proteins comprise amino acid sequences that differ from each other; and wherein the VL domain proteins derive from the same original VL domain and the VL domain proteins comprise amino acid sequences that differ from each other.

### Claim 30 (depends on 24)

The Fv library of claim 24, wherein the VH domain proteins comprise complementarity determining regions (CDRs) and framework regions (FRs) and the VL domain proteins comprise complementarity determining regions (CDRs) and framework regions (FRs).

### Claim 31 (depends on 30)

The Fv library of claim 30, wherein CDRs of the VH domain proteins comprise amino acid sequences that differ from each other; CDRs of the VL domain proteins comprise amino acid sequences that differ from each other; FRs of the VH domain proteins comprise amino acid sequences that differ from each other; or FRs of the VL domain proteins comprise amino acid sequences that differ from each other.

### Claim 32 (depends on 31)

The Fv library of claim 31, wherein CDRs of the VH domain proteins comprise amino acid sequences that differ from each other; and CDRs of the VL domain proteins comprise amino acid sequences that differ from each other.

### Claim 33 (depends on 31)

The Fv library of claim 31, wherein FRs of the VH domain proteins comprise amino acid sequences that differ from each other; and FRs of the VL domain proteins comprise amino acid sequences that differ from each other.

### Claim 34 (depends on 32)

The Fv library of claim 32, wherein FRs of the VH domain proteins comprise amino acid sequences that differ from each other; and FRs of the VL domain proteins comprise amino acid sequences that differ from each other.

### Claim 35 (depends on 24)

The Fv library of claim 24, wherein the VH domain proteins and the VL domain proteins are paired by random pairing or target pairing.

### Claim 36 (depends on 24)

The Fv library of claim 24, wherein the pairing of the VH domain proteins with the VL domain proteins is performed by a method comprising: (i) pairing between wild-type VH domain proteins and the VL domain proteins; (ii) pairing by disulfide bonds between cysteine residues introduced into each of the VH domain proteins and the VL domain proteins; (iii) pairing by fusion between coiled-coil domains introduced into each of the VH domain proteins and the VL domain proteins; or (iv) pairing by protein-protein interaction between proteins fused to each of the VH domain proteins and the VL domain proteins.

### Claim 37 (depends on 24)

The Fv library of claim 24, wherein the paired VH domain proteins and the VL domain proteins are stored in individual compartments.

### Claim 38 (depends on 37)

The Fv library of claim 37, wherein the individual compartments are assigned a unique identification (ID) number.

### Claim 39 (depends on 37)

The Fv library of claim 37, wherein the compartments comprise plate wells, test tubes, microfluidic channels, or chips.

### Claim 40 (depends on 24)

The Fv library of claim 24, wherein the Fv library enables functional analysis of individual members thereof.

### Claim 41 (depends on 40)

The Fv library of claim 40, wherein the functional analysis does not comprise a pre-screening step based on binding to a target.

### Claim 42 (depends on 40)

The Fv library of claim 40, wherein the desired property, characteristic, or activity is cell proliferation, differentiation, or cell death.