# US 11,993,643 B2 · Method for isolation of soluble polypeptides

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- Number: US 11,993,643 B2
- Document ID: US11993643B2
- Status: Granted
- Kind: B2
- Application number: 17/372,966
- Filed: 2021-07-12
- Published: 2022-01-20
- Granted: 2024-05-28
- Earliest claimed: 2005-03-25
- Assignees: National Research Council of Canada
- Applicants: National Research Council of Canada
- Inventors: Jamshid Tanha
- Web page: https://patentgrep.com/patent/US11993643B2

## CPC

- C07K 16/005: {constructed by phage libraries}
- A61P 15/02: for disorders of the vagina
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 31/04: Antibacterial agents
- A61P 31/12: Antivirals
- A61P 35/00: Antineoplastic agents
- A61P 37/02: Immunomodulators
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 16/1271: Micrococcaceae (F); Staphylococcaceae (F), e.g. Staphylococcus (G)
- C07K 16/1282: Clostridium (G)
- C12N 15/1037: {Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display}
- G01N 33/6845: {Methods of identifying protein-protein interactions in protein mixtures}
- C07K 2317/21: from primates, e.g. man
- C07K 2317/56: variable (Fv) region, i.e. VH and/or VL
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)

## Abstract

Polypeptides with biophysical properties such as solubility, stability, high expression, monomericity, binding specificity or non-aggregation, including monomeric human heavy and light chain variable domains (VHs and VLs), are identified using a high throughput method for screening polypeptides, comprising the steps of obtaining a phage display library, allowing infection of a bacterial lawn by the library phage, and identifying phage which form larger than average plaques on the bacterial lawn. Sequences of monomeric human VHs and VLs are identified, which may be useful for immunotherapy or as diagnostic agents. Multimer complexes of human VHs and VLs are also identified. The VHs and VLs identified may be used to create further libraries for identifying additional polypeptides. Further, the VHs and VLs may be subjected to DNA shuffling to select for improved biophysical properties.

## Claims

### Claim 1

An antigen-binding VL comprising a FR1 sequence of EIVMTQSPGTLSLSPGDRATLSC (amino acids 1-23 of SEQ ID NO:42), a FR2 sequence of WYQQKPGQAPRLLIY (amino acids 35-49 of SEQ ID NO:42), a FR3 sequence of GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC (amino acids 57-88 of SEQ ID NO:42), and a FR4 sequence of FGQGTKVTVL (amino acids 98-107 of SEQ ID NO:42).

### Claim 2

An antigen-binding VL comprising the FR1, FR2, FR3, and FR4 portion of SEQ ID NO:42 and one or more randomized CDR sequences, wherein one or more of CDR1, CDR2 and CDR3 of SEQ ID NO:42 is replaced, respectively, with a randomized CDR1, CDR2 and CDR3.

### Claim 3 (depends on 1)

The antigen-binding VL of claim 1, wherein the VL is in a multimeric form.

### Claim 4 (depends on 1)

The antigen-binding VL of claim 1, wherein the VL is in a dimeric form.

### Claim 5 (depends on 1)

The antigen-binding VL of claim 1, wherein VL is in a trimeric form.

### Claim 6 (depends on 1)

The antigen-binding VL of claim 1, wherein the VL is in a pentameric form.

### Claim 7 (depends on 1, 2)

A display library constructed by preparing nucleic acid sequences coding for the antigen-binding VL of claim 1 or 2 and expressing the VL so as to display the antigen-binding VL sequence of claim 1 or 2.

### Claim 8 (depends on 7)

The display library of claim 7, wherein the library is a phage display library.

### Claim 9 (depends on 7)

The display library of claim 7, wherein the library is a ribosome display, ARM ribosome display, yeast display, bacterial cell display, or in vitro compartmentalization library.

### Claim 10 (depends on 1)

A method for producing a VL library, comprising:
a) providing a nucleotide sequence encoding the antigen-binding VL of claim 1;
b) providing oligonucleotide sequences with randomized codons;
c) incorporating the randomized oligonucleotides into the nucleotide sequence encoding the VL, such that one or more than one of the complementary determining regions is randomized, wherein one or more of CDR1, CDR2 and CDR3 of SEQ ID NO.: 42 is replaced, respectively, with a randomized CDR1, CDR2 and CDR3;
d) expressing the nucleotide sequences produced in step c); and
e) screening the expressed sequences for binding to a target polypeptide.

### Claim 11 (depends on 10)

The method of claim 10, wherein the screening comprises panning against a target molecule.

### Claim 12 (depends on 7)

A method of screening the display library of claim 7, comprising panning the displayed VL against a target molecule.

### Claim 13 (depends on 2)

The antigen-binding VL of claim 2, wherein CDR3 of SEQ ID NO:42 is replaced with a randomized CDR3, and the randomized CDR3 comprises 9 amino acid residues.

### Claim 14 (depends on 1)

The antigen-binding VL of claim 1, which is aggregation resistant.