Method for selecting polynucleotides encoding antigen-specific immunoglobulin subunit

Patent metadata

Field	Value
Publication number	US-2017306318-A1
Application number	US-201715619790-A
Country	US
Kind code	A1
Filing date	Jun 12, 2017
Priority date	Apr 26, 2012
Publication date	Oct 26, 2017
Grant date	—

Abstract

The present invention relates to a high efficiency method of expressing immunoglobulin molecules in eukaryotic cells. The invention is further drawn to a method of producing immunoglobulin heavy and light chain libraries, particularly using the trimolecular recombination method, for expression in eukaryotic cells. The invention further provides methods of selecting and screening for antigen-specific immunoglobulin molecules, and antigen-specific fragments thereof. The invention also provides kits for producing, screening and selecting antigen-specific immunoglobulin molecules. Finally, the invention provides immunoglobulin molecules, and antigen-specific fragments thereof, produced by the methods provided herein.

First claim

1 . A method for selecting polynucleotides that encode an antigen-specific immunoglobulin heavy chain variable region or antigen-binding fragment thereof, comprising:
(a) introducing a first library of polynucleotides, each polynucleotide comprising a nucleic acid molecule encoding a heavy chain fusion protein, into a population of host cells permissive for vaccinia virus infectivity, wherein the first library of polynucleotides is constructed in a vaccinia virus vector, and wherein each heavy chain fusion protein comprises:
i. a first polypeptide segment comprising an immunoglobulin heavy chain variable region or fragment thereof;
ii. a second polypeptide segment comprising an immunoglobulin heavy chain constant region or portion thereof situated downstream of the first polypeptide segment, and
iii. a third polypeptide segment comprising the transmembrane domain of a vaccinia virus extracellular enveloped virus (EEV)-specific membrane protein situated downstream of the second polypeptide segment;
(b) introducing one or more polynucleotides, each encoding an immunoglobulin light chain, into the population of host cells;
wherein a heavy chain fusion protein and an immunoglobulin light chain expressed in the population of host cells are capable of combining within a host cell to form an antigen-binding domain of an immunoglobulin molecule that can be expressed on the surface of an EEV;
(c) permitting release of EEV from the host cells;
(d) contacting the released EEV with an antigen; and
(e) recovering the polynucleotides of the first library from EEV that bind to the antigen.

2 . The method of claim 1 , further comprising:
introducing the polynucleotides recovered in (e) into a second population of host cells permissive for vaccinia virus infectivity; (g) introducing one or more polynucleotides, each encoding an immunoglobulin light chain, into the population of host cells; (h) permitting release of EEV from the host cells; (i) contacting the released EEV with an antigen; and (j) recovering the polynucleotides of the first library from EEV that bind to the antigen.

3 . The method of claim 2 , further comprising repeating steps (f)-(j) one or more times, thereby enriching for polynucleotides of the first library that encode heavy chain fusion proteins that specifically bind the antigen.

4 . The method of claim 1 , further comprising isolating the polynucleotides recovered in (e).

5 . The method of claim 1 , wherein the second polypeptide segment comprises a heavy chain CH1 domain, or portion thereof.

6 . The method of claim 5 , wherein the second polypeptide segment further comprises a heavy chain CH2 domain, a heavy chain CH3 domain, or a combination thereof.

7 . The method of claim 5 , wherein the second polypeptide segment comprises a human IgG constant region, or portion thereof.

8 . The method of claim 7 , wherein the second polypeptide segment comprises amino acids 117 to 214 of SEQ ID NO: 11 or amino acids 117 to 446 of SEQ ID NO: 30.

9 . The method of claim 1 , wherein the third polypeptide segment comprises the transmembrane domain of the vaccinia EEV-specific membrane protein A33R, A34R, A56R, A36R, or B5R.

10 . The method of claim 9 , wherein the vaccinia EEV-specific membrane protein is A56R.

11 . The method of claim 10 , wherein the third polypeptide segment comprises the stalk region, the transmembrane domain, and the intracellular domain of the A56R protein.

12 . The method of claim 1 , wherein each polynucleotide of the first library further comprises a nucleic acid molecule encoding a drug resistance marker.

13 . The method of claim 12 , wherein the nucleic acid molecule encoding the drug resistance marker is linked to the nucleic acid molecule encoding the heavy chain fusion protein via an internal ribosome entry site (IRES).

14 . The method of claim 1 , wherein each heavy chain fusion protein further comprises a signal peptide for facilitating expression of the heavy chain fusion protein on the surface of an EEV.

15 . The method of claim 1 , wherein the one or more polynucleotides encoding an immunoglobulin light chain of (b) are each contained on a vaccinia virus vector.

16 . The method of claim 1 , wherein the one or more polynucleotides encoding an immunoglobulin light chain of (b) are contained on a plasmid vector.

17 . The method of claim 1 , wherein the third segment comprises polynucleotide sequences 108 to 314 of SEQ ID NO: 10.

18 . The method claim 1 , wherein the third segment comprises polynucleotide sequences 240 to 314 of SEQ ID NO: 10.

19 . A recombinant vaccinia library comprising a library of polynucleotides constructed in a vaccinia virus vector encoding a plurality of immunoglobulin fusion polypeptides, wherein the vaccinia virus vector comprises (a) a first polynucleotide encoding a first polypeptide segment comprising a heavy chain CH1 domain (b) a second polynucleotide encoding a second polypeptide segment comprising the transmembrane domain of a vaccinia virus EEV-specific membrane protein situated downstream of the CH1 domain, and (c) a third polynucleotide encoding an immunoglobulin heavy chain variable region or fragment thereof situated upstream of the CH1 domain.

20 . The recombinant vaccinia library of claim 19 , wherein each immunoglobulin fusion polypeptide further comprises a signal peptide for facilitating expression of the plurality of immunoglobulin fusion polypeptides on the surface of EEV.

21 . The recombinant vaccinia library of claim 19 , wherein the first polypeptide segment further comprises a heavy chain CH2 domain, a heavy chain CH3 domain, or a combination thereof.

22 . The recombinant vaccinia library of claim 21 , wherein the first polypeptide segment comprises a human IgG constant region, or portion thereof.

23 . The recombinant vaccinia library of claim 19 , wherein the second polypeptide segment comprises the transmembrane domain of the vaccinia EEV-specific membrane protein A33R, A34R, A56R, A36R, or BSR.

24 . The recombinant vaccinia library of claim 23 , wherein the vaccinia EEV-specific membrane protein is A56R.

25 . The recombinant vaccinia library of claim 24 , wherein the second polypeptide segment comprises the stalk region, the transmembrane domain, and the intracellular domain of the A56R protein.

26 . The recombinant vaccinia library of claim 25 , wherein the third polypeptide segment comprises amino acids 215 to 421 of SEQ ID NO: 11 or amino acids 447 to 653 of SEQ ID NO: 30.

Assignees

• Vaccinex Inc

Inventors

• Smith Ernest S
• Pandina Tracy
• Croy Leslie A
• Paris Mark
• Zauderer Maurice
• Moksa Angelica
• Kirk Renee

Classifications

• C40B40/08

  Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries · CPC title

• C07K16/32

  against translation products of oncogenes · CPC title

• C07K16/00 Primary

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title

• C07K2319/03

  containing a transmembrane segment · CPC title

• C07K2317/56

  variable (Fv) region, i.e. VH and/or VL · CPC title