# US 9,753,040 B2 · Polynucleotide construct capable of displaying fab in a cell-free translation system, and method for manufacturing and screening fab using same

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- Number: US 9,753,040 B2
- Document ID: US9753040B2
- Status: Granted
- Kind: B2
- Application number: 13/990,654
- Filed: 2011-11-30
- Published: 2013-10-31
- Granted: 2017-09-05
- Earliest claimed: 2010-12-01
- Assignees: Mitsubishi Tanabe Pharma Corporation; The University of Tokyo
- Applicants: Yasuhiro Fujino; Risako Fujita; Kouichi Wada; Kotomi Oda; Takuya Ueda; Yoshihiro Shimizu; Takashi Kanamori
- Inventors: Yasuhiro Fujino; Risako Fujita; Kouichi Wada; Kotomi Oda; Takuya Ueda; Yoshihiro Shimizu; Takashi Kanamori
- Web page: https://patentgrep.com/patent/US9753040B2

## CPC

- G01N 33/6854: {Immunoglobulins}
- C07K 16/468: {Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies}
- C12N 15/1041: {Ribosome/Polysome display, e.g. SPERT, ARM}
- C12N 15/1062: {mRNA-Display, e.g. polypeptide and encoding template are connected covalently}
- C12N 15/1075: {by coupling phenotype to genotype, not provided for in other groups of this subclass}
- G01N 33/6857: {Antibody fragments}
- C07K 2317/31: multispecific
- C07K 2317/55: Fab or Fab'
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The polynucleotide construct of (1) or (2) below is used to perform ribosome display, CIS display and/or mRNA display in order to screen a Fab against an antigen of interest: (1) a polynucleotide construct which monocistronically comprises a ribosome-binding sequence, Fab first chain-coding sequence, linker peptide-coding sequence, Fab second chain-coding sequence and scaffold-coding sequence in this order, and further comprises at its 3′-end a structure necessary for maintaining a complex with the Fab encoded by itself; and (2) a polynucleotide construct which comprises a Fab first chain-expressing cistron and a Fab second chain-expressing cistron each containing a ribosome-binding sequence, a Fab first chain-coding sequence or Fab second chain-coding sequence, and a scaffold-coding sequence in this order, the first Fab-expressing cistron further comprising at its 3′-end a ribosome stall sequence, said Fab second chain-expressing cistron further comprising at its 3′-end a structure necessary for maintaining a complex with the Fab encoded by itself.

## Claims

### Claim 1

A polynucleotide construct comprising a Fab first chain-coding sequence and a Fab second chain-coding sequence, wherein said polynucleotide construct expresses a Fab encoded by itself without dissociation, and maintains a complex with the Fab, when it is introduced into a cell-free translation system containing ribosomes.

### Claim 2 (depends on 1)

The polynucleotide construct according to claim 1, wherein said polynucleotide construct monocistronically comprises a ribosome-binding sequence, Fab first chain-coding sequence, linker peptide-coding sequence, Fab second chain-coding sequence and scaffold-coding sequence in this order, and further comprises at its 3′-end a structure necessary for maintaining a complex with the Fab encoded by itself.

### Claim 3 (depends on 1)

The polynucleotide construct according to claim 1, wherein said polynucleotide construct comprises a Fab first chain-expressing cistron and a Fab second chain-expressing cistron each containing a ribosome-binding sequence, a Fab first chain-coding sequence or Fab second chain-coding sequence, and a scaffold-coding sequence in this order, said first Fab-expressing cistron further comprising at its 3′-end a ribosome stall sequence, said Fab second chain-expressing cistron further comprising at its 3′-end a structure necessary for maintaining a complex with the Fab encoded by itself.

### Claim 4 (depends on 2)

The polynucleotide construct according to claim 2, wherein said structure necessary for maintaining a complex with the Fab encoded by itself is a ribosome stall sequence, puromycin or a derivative thereof, or a DNA-binding protein-coding sequence and a binding sequence for said DNA-binding protein.

### Claim 5 (depends on 4)

The polynucleotide construct according to claim 4, wherein said structure necessary for maintaining a complex with the Fab encoded by itself is a ribosome stall sequence.

### Claim 6 (depends on 5)

The polynucleotide construct according to claim 5, wherein said ribosome stall sequence is a SecM sequence, diproline sequence, or both of them.

### Claim 7 (depends on 5)

The polynucleotide construct according to claim 5, wherein said ribosome stall sequence is composed of 2 to 4 repeats of the SecM sequence.

### Claim 8 (depends on 4)

The polynucleotide construct according to claim 4, wherein a stop codon is present in the 3′-side of said ribosome stall sequence.

### Claim 9 (depends on 4)

The polynucleotide construct according to claim 4, wherein said structure necessary for maintaining a complex with the Fab encoded by itself is puromycin or a derivative thereof.

### Claim 10 (depends on 4)

The polynucleotide construct according to claim 4, wherein said structure necessary for maintaining a complex with the Fab encoded by itself is a DNA-binding protein-coding sequence and a binding sequence for said DNA-binding protein.

### Claim 11 (depends on 10)

The polynucleotide construct according to claim 10, wherein said DNA-binding protein is a cis-binding protein which is never dissociated during transcription/translation reaction from the DNA molecule used as a template for said transcription/translation and which binds to said binding sequence for the DNA-binding protein located in the same DNA molecule.

### Claim 12 (depends on 10)

The polynucleotide construct according to claim 10, wherein said DNA-binding protein is RepA encoded by Escherichia coli R1 plasmid and said binding sequence for the DNA-binding protein is a CIS-ori sequence located downstream of the RepA-coding sequence in the same polynucleotide.

### Claim 13 (depends on 1)

The polynucleotide construct according to claim 1, wherein said polynucleotide construct is a library wherein said Fab first chain-coding sequence and said Fab second chain-coding sequence comprise random sequences.

### Claim 14 (depends on 13)

The polynucleotide construct according to claim 13, wherein said library comprising random sequences is a (i) naive library or (ii) focused library.

### Claim 15 (depends on 13)

The polynucleotide construct according to claim 13, wherein said library comprising random sequences is a library comprising single amino acid substitutions in the complementarity-determining region(s) (CDR(s)) of the Fab first chain and/or the Fab second chain.

### Claim 16 (depends on 1)

A method for screening a Fab, said method comprising the steps of:
(i) introducing the polynucleotide construct according to claim 1 into a cell-free translation system to synthesize Fabs, and displaying said synthesized Fabs on the polynucleotides encoding said Fabs;
(ii) bringing said Fabs displayed on said polynucleotides into contact with an antigen;
(iii) selecting a Fab of interest that reacts with said antigen; and
(iv) amplifying the polynucleotide encoding said Fab of interest.

### Claim 17 (depends on 15, 16)

The method for screening a Fab according to claim 16, said method comprising the steps of:
(I) providing a plurality of types of the polynucleotide construct according to claim 15, in each of which the Fab first chain-coding sequence or the Fab second chain-coding sequence encodes an amino acid sequence comprising a single amino acid substitution at a single position in a CDR in the amino acid sequence of the Fab first chain or the Fab second chain of the parent antibody, such that single amino acid substitutions are contained for a plurality of positions in the CDRs of the Fab first chain and the Fab second chain;
(II) carrying out first screening wherein said steps (i) to (iv) are repeated using said plurality of types of the polynucleotide construct, to screen a plurality of high-affinity Fabs;
(III) analyzing single amino acid substitutions at respective positions in the CDRs of the Fab first chain and the Fab second chain in said plurality of Fabs selected in said first screening step;
(IV) providing the polynucleotide construct according to claim 15 wherein the Fab first chain-coding sequence and the Fab second chain-coding sequence encode amino acid sequences comprising combinations of the single amino acid substitutions identified in said first screening at said respective positions in the CDRs of the Fab first chain and Fab second chain sequences of the parent antibody; and
(V) carrying out second screening wherein said steps (i) to (iv) are repeated using said polynucleotide construct, to screen a high-affinity Fab.

### Claim 18 (depends on 16)

The method according to claim 16, wherein said in vitro translation system is composed of factors independently purified.

### Claim 19 (depends on 18)

The method according to claim 18, wherein said in vitro translation system contains at least one component selected from the group consisting of initiation factors, elongation factors, aminoacyl-tRNA synthetase and methionyl-tRNA transformylase.

### Claim 20 (depends on 18)

The method according to claim 18, wherein said in vitro translation system does not contain a releasing factor.

### Claim 21 (depends on 16)

The method according to claim 16, wherein said in vitro translation system is a cell extract containing ribosomes.

### Claim 22 (depends on 1)

A method for producing a Fab, said method comprising the step of introducing the polynucleotide construct according to claim 1 into an in vitro translation system to produce a Fab.

### Claim 23 (depends on 1)

A kit for producing or screening a Fab, said kit comprising the polynucleotide construct according to claim 1.