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NZ746657B2 - Integral membrane protein display on poxvirus extracellular enveloped virions - Google Patents

Integral membrane protein display on poxvirus extracellular enveloped virions Download PDF

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Publication number
NZ746657B2
NZ746657B2 NZ746657A NZ74665717A NZ746657B2 NZ 746657 B2 NZ746657 B2 NZ 746657B2 NZ 746657 A NZ746657 A NZ 746657A NZ 74665717 A NZ74665717 A NZ 74665717A NZ 746657 B2 NZ746657 B2 NZ 746657B2
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New Zealand
Prior art keywords
imp
fragment
protein
membrane
polynucleotide
Prior art date
Application number
NZ746657A
Other versions
NZ746657A (en
Inventor
Angelica A Cornelison
Renee A Kirk
Mark Paris
Maria G M Scrivens
Ernest S Smith
Original Assignee
Vaccinex Inc
Filing date
Publication date
Application filed by Vaccinex Inc filed Critical Vaccinex Inc
Priority to NZ786461A priority Critical patent/NZ786461A/en
Priority claimed from PCT/US2017/028787 external-priority patent/WO2017184951A1/en
Publication of NZ746657A publication Critical patent/NZ746657A/en
Publication of NZ746657B2 publication Critical patent/NZ746657B2/en

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Abstract

This disclosure provides compositions and methods for expressing and displaying isolated integral membrane proteins (IMPs) or fragments thereof in a native conformation for use in the screening, selecting, and identifying of antibodies or antibody-like molecules that bind to a target IMP of interest.

Claims (28)

WHAT IS CLAIMED IS:
1. An isolated polynucleotide comprising: (a) a first nucleic acid fragment that encodes an integral membrane protein (IMP) or fragment thereof, wherein the IMP or fragment thereof comprises at least one extra-membrane region and at least one intra-membrane region, wherein the IMP is a multi-pass membrane protein comprising at least two, at least three, at least four, at least five, at least six or at least seven transmembrane domains, and wherein a portion of the first nucleic acid fragment encoding at least one intra-membrane region is situated at the 5’ or 3’ end of the first nucleic acid fragment; and (b) a second nucleic acid fragment that encodes a vaccinia virus F13L protein or functional fragment thereof, wherein the second nucleic acid fragment is fused in frame to a portion of the first nucleic acid fragment that encodes an intra-membrane region of the IMP; wherein a poxvirus infected isolated cell comprising the polynucleotide can express an IMP- F13L fusion protein as part of the outer envelope membrane of an extracellular enveloped virion (EEV).
2. The polynucleotide of claim 1, wherein the IMP has an odd number of transmembrane domains, wherein the 5’ end of the first nucleic acid fragment encodes an extra- membrane region, wherein the 3’ end of the first nucleic acid fragment encodes an intra- membrane region, and wherein the 5’ end of the second polynucleotide is fused to the 3’ end of the first nucleic acid fragment.
3. The polynucleotide of claim 2, wherein the IMP comprises a G-protein coupled receptor (GPCR).
4. The polynucleotide of claim 3, wherein the IMP is the human frizzled-4 protein (FZD4), or a fragment thereof.
5. The polynucleotide of claim 1, which is operably associated with a poxvirus promoter.
6. The polynucleotide of claim 3, wherein the IMP is the CXC chemokine receptor CXCR4 or a fragment thereof.
7. The polynucleotide of claim 1, wherein the IMP has an even number of transmembrane domains, and wherein both the 5’ and 3’ ends of the first nucleic acid fragment encode intra-membrane regions, and wherein the second nucleic acid fragment is fused to the 3’ end of the first nucleic acid fragment.
8. The polynucleotide of claim 7, wherein the IMP is human CD20 protein, or a fragment thereof.
9. A F13L fusion protein encoded by the polynucleotide of any one of claims 1 to 8.
10. A poxvirus genome comprising the polynucleotide of any one of claims 1 to 8.
11. The poxvirus genome of claim 10, which is a vaccinia virus genome.
12. A method of producing a recombinant vaccinia virus EEV, comprising: (a) infecting an isolated host cell permissive for vaccinia virus infectivity with a vaccinia virus comprising the poxvirus genome of claim 11, and (b) recovering EEV released from the isolated host cell.
13. A method to display an integral membrane protein (IMP) or fragment thereof in a native conformation comprising: (a) infecting isolated host cells permissive for poxvirus infectivity with a recombinant poxvirus that expresses the IMP or fragment thereof as a fusion protein with Vaccinia virus F13L protein or functional fragment thereof, wherein the IMP is a multi-pass membrane protein comprising at least two, at least three, at least four, at least five, at least six, or at least seven transmembrane domains, and wherein EEV produced by the infected host cell comprise the IMP fusion protein as part of the EEV outer envelope membrane; (b) recovering EEV released from the isolated host cell wherein the IMP or fragment thereof displays on the surface of the EEV in a native conformation.
14. The method of claim 13, (a) wherein the IMP comprises a G protein coupled receptor (GPCR) comprising seven transmembrane domains selected from the group consisting of (i) the human fizzled-4 protein (FZD4), or a fragment thereof; (ii) a CXC chemokine receptor; or (iii) the CXC chemokine receptor CXCR4, or a fragment thereof; wherein the F13L is fused to the C-terminus of the IMP; or (b) wherein the IMP is human CD20, or a fragment thereof.
15. An IMP-F13L fusion protein, which comprises (a) an integral membrane protein (IMP) or fragment thereof, which comprises at least one extra-membrane region, at least two transmembrane domains, and at least one intra-membrane region; and (b) a vaccinia virus F13L protein or functional fragment thereof.
16. The IMP-F13L fusion protein of claim 15 comprising: (a) amino acids 20 to 892 of SEQ ID NO: 2; (b) SEQ ID NO: 3; or (c) SEQ ID NO: 4.
17. The IMP-F13L fusion protein of claim 15, wherein the IMP or fragment thereof is fused at its C-terminus to the F13L protein.
18. The IMP-F13L fusion protein of claim 15, wherein the IMP or fragment thereof is fused at its N-terminus to the F13L protein.
19. The IMP-F13L fusion protein of claim 15, wherein the IMP has an odd number of transmembrane domains, wherein the N-terminus is an extra-membrane region, and wherein the C-terminus is an intra-membrane region; or the IMP has an even number of transmembrane domains, and wherein both the N-terminus and C-terminus are intra-membrane regions.
20. A recombinant poxvirus EEV comprising the fusion protein of any one of claims 15 to 19.
21. A recombinant poxvirus EEV, comprising the IMP-F13L fusion protein encoded by the polynucleotide of any one of claims 1 to 8, wherein the IMP or fragment thereof is heterologous to the F13L protein fused thereto, wherein the IMP-F13L fusion protein is situated in the outer envelope membrane, and the IMP or fragment thereof is displayed on the surface of the outer membrane in its native conformation.
22. The recombinant poxvirus EEV of claim 21, wherein the poxvirus EEV is a vaccinia virus EEV.
23. A method of selecting antibodies that bind to a multi-pass membrane protein, which comprises: (a) attaching a vaccinia virus that expresses the IMP-F13L fusion protein of claim 19, wherein the IMP or fragment thereof is expressed on the surface of its outer membrane protein, to a solid support; (b) providing an antibody display library, wherein the library comprises display packages displaying a plurality of antigen binding domains; (c) contacting the display library with the vaccinia virus such that display packages displaying antigen binding domains that specifically bind to the IMP can bind thereto; (d) removing unbound display packages; and (e) recovering display packages having antigen binding domains bound to the IMP or fragment thereof.
24. The method of claim 23 wherein the vaccinia virus is attached to the solid support via reaction with tosyl groups on the solid support.
25. The method of claim 23, wherein the solid support is tosyl-activated magnetic beads.
26. The method of 23, wherein the vaccinia virus is biotinylated and attached to a streptavidin coated solid support.
27. The method of claim 23, wherein the solid support is streptavidin-coated magnetic beads.
28. A polynucleotide as claimed in any one of claims 1-8; a fusion protein as claimed in any one of claims 15-19; a poxvirus genome as claimed in claim 10; a method as claimed in any one of claims 13-14 and 23-27; or a recombinant poxvirus EEV as claimed in any one of claims 20-22, substantially as described herein and with reference to any example thereof.
NZ746657A 2016-04-22 2017-04-21 Integral membrane protein display on poxvirus extracellular enveloped virions NZ746657B2 (en)

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