Integral membrane protein display on poxvirus extracellular enveloped virions

A technology that integrates membrane proteins and poxviruses, and is used in viruses, viruses/phages, fusion polypeptides, etc.

Active Publication Date: 2018-12-21
VACCINEX
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The lack of properly folded target proteins in isolation makes the identification and selection of antibodies against these targets challenging

Method used

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  • Integral membrane protein display on poxvirus extracellular enveloped virions
  • Integral membrane protein display on poxvirus extracellular enveloped virions
  • Integral membrane protein display on poxvirus extracellular enveloped virions

Examples

Experimental program
Comparison scheme
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Embodiment approach

[0039] Furthermore, "and / or" as used herein should be construed as specifically disclosing each of the two features or components, with or without the other. Thus, the term "and / or" as used herein in the phrase "A and / or B" is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Likewise, the term "and / or" used in phrases such as "A, B, and / or C" is intended to include the various embodiments of: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0040] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. For example, "Concise Dictionary of Biomedicine and Molecular Biology" (Concise Dictionary of Biomedicine and Molecular Biology), Juo, Pei-Show, 2nd edition, 2002, CRC Press (CRCPress); "Dictionary of Cell and Molecular Biology" (The Dictionary of Cell and Molecula...

Embodiment 1

[0207] Example 1: Fusion protein construction

[0208] The IMP was introduced into the vaccinia virus EEV by using the EEV specific proteins F13L, A56R and B5R as follows. In general, the extracellular domains of HER2, CD100 (axon guidance factor 4D) and FZD4 were introduced as fusions with single-transmembrane EEV-specific membrane proteins A56R and B5R, as schematically shown in Figure 1A. The mature FZD4-ECD-A56R fusion protein comprises amino acids 20-370 of SEQ ID NO:6, the mature HER2-ECD-A56R fusion protein comprises amino acids 20-855 of SEQ ID NO:7, and the mature CD100-ECD-A56R fusion protein comprises SEQ ID NO:7 Amino acids 20-935 of ID NO:8. Figure 1B and Figure 1C It is schematically shown how multi-transmembrane proteins such as GPCR and CD20 can be introduced into EEV as multi-transmembrane proteins in the form of fusions with the EEV membrane-associated protein F13L.

[0209] Preparation of F13L fusion proteins (FZD4-F13L, CD20-F13L and CXCR4-F13L)

[0210...

Embodiment 2

[0220] Example 2: Expression of CD20-F13L fusion protein on EEV

[0221] BHK cells were infected with IMV encoding CD20-F13L fusion protein (SEQ ID NO:4) or control Western Reserve (WR) virus at a multiplicity of infection (MOI) of 1 virus / cell for two days, and then EEV-containing cells were harvested by low-speed centrifugation. supernatant and remove debris. Protein G (110 μL) was pulled down with a magnet, and 1 mL of PBS + 20 μg of purified anti-CD20 antibody was added to the beads. The solution was incubated at room temperature for 30-60 minutes with gentle shaking to allow antibody coupling to the protein G beads. 10 μg of purified mIgG1 isotype control was added to the solution to ensure complete blocking, and the solution was then incubated for another 10-30 minutes at room temperature with gentle shaking. The beads were pulled down with a magnet, washed once with 1 mL of PBS, and resuspended in 110 μL of PBS.

[0222] 50 μL of anti-CD20-Pro G Add to 1 mL of CD...

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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.

Description

[0001] Inventor: E·S·Smith (Ernest S.Smith) [0002] Mark Paris [0003] Maria G.M. Scrivens [0004] R. A. Kirk (Renee A. Kirk) [0005] A. A. Connelson (Angelica A. [0006] Moksa-Cornelison) Background technique [0007] Antibodies with established specificities are increasingly employed in a variety of therapeutic applications. There are many approaches to obtain useful antibodies for human therapeutic applications. These include chimeric and humanized antibodies, as well as fully human antibodies, selected from libraries, eg, phage display libraries, or from transgenic animals. Immunoglobulin libraries constructed in bacteriophage can be derived from antibody-producing cells of naive or specially immunized individuals and can, in principle, include novel and diverse pairs of human immunoglobulin heavy and light chains. Although this strategy is not subject to intrinsic library constraints, it requires that the complementarity determining regions (CDRs) of the expres...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/12A61K39/285C07K16/28C12N15/39C12N15/62C12N15/863
CPCC07K16/081C07K16/2866C12N2710/24122C12N2710/24123C12N2710/24131C07K14/70596C07K2319/00C12N15/86C07K16/32G01N33/6872G01N33/6854G01N2500/04G01N2333/07C07K14/705C07K14/71C07K14/7158C07K14/723C07K2319/02C07K2319/21C12N2710/24134C12N2710/24142C12N2710/24143C07K14/005C07K16/286C12N7/00C12N15/62C07K2319/03C12Q1/6806A61K39/12C07K14/70503C07K16/2869C07K16/2896C07K2319/01C12N2710/24121
Inventor E·S·史密斯M·帕里斯M·G·M·斯科瑞文斯R·A·柯克A·A·康尼尔森
Owner VACCINEX
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