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Non-aggregating human VH domains

A domain, non-aggregation technology, applied in the field of non-aggregating human VH domains

Inactive Publication Date: 2011-01-05
NAT RES COUNCIL OF CANADA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method requires V H Multivalent display on the surface of phage; this approach has been shown to be effective for phage vector-based display libraries, but not in monovalent display formats provided by phagemid vector-based systems (regarding phage display systems and their features, see Winter et al., 1994; Bradbury and Marks, 2004)

Method used

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  • Non-aggregating human VH domains
  • Non-aggregating human VH domains
  • Non-aggregating human VH domains

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0139] Example 1: HVHP430V H library construction

[0140] Construction of fully synthetic phagemid-based human V HPhage display library.

[0141] When in the HVHP430 bracket ( Figure 2A , SEQ ID NO: 1) build V H When using a library, maintain 2 CDR3 Cys to promote the formation of disulfide bonds within the CDR, thereby increasing the enzyme inhibitory V in the library H Frequency of. Randomize the remaining 14 CDR3 sites, site 94, and 8 H1 / CDR1 sites ( Figure 2A ). CDR2 was left unchanged as it has been shown to be involved in protein A binding (Randen et al., 1993; Bond et al., 2003). In addition, camelid VNARs that lack CDR2 (Stanfield et al., 2004) or utilize their CDR1 and CDR3 (Decanniere et al., 1999) or only CDR3 (Desmyter et al., 2001) for antigen recognition H H shows nanomolar affinities. according to Figure 2B The protocol shown in uses a phagemid vector ( image 3 ) to construct the library.

[0142] General V H HVHP430 (which has 2 Cys residues ...

Embodiment 2

[0148] Example 2: Panning and phage ELISA

[0149] A. Panning in the form of unit price display

[0150] In the first panning attempt, helper phage M13KO7 was used for superinfection, resulting in V H Monovalent display on the surface of phage (O'Connell et al., 2002). The initial aim was to explore the feasibility of the library in generating enzyme inhibitors. Four rounds of panning were performed against alpha amylase, lysozyme and carbonic anhydrase as described below.

[0151] A total of 50 μg of antigen (lysozyme, alpha amylase and carbonic anhydrase) in 100 μL PBS will be used to coat Maxisorp TM wells (Nunc, Roskilde, Denmark) overnight at 4°C. The solution was then removed and the wells were blocked by adding 200 μL of 3% BSA in PBS and incubating the wells at 37°C for 2 hours. Remove the blocking reagent and add 10 12 library phage (input), the wells were incubated at 37°C for 2 hours. The supernatant was removed and the wells were washed 7 times with 0.1% PBS...

Embodiment 3

[0168] Example 3: Analysis of clones

[0169] Nine V H , huVHAm302, huVHAm304, huVHAm309, huVHAm315, huVHAm316, huVHAm416, huVHAm427, huVHAm428 and huVHAm431 were used for subcloning and further analysis. However, except for 1 V H (huVHAm431), all V H Both have an amber stop codon that prevents their expression even in amber suppressor strains such as TG1 to be used as expression hosts (in TG1 cells the amber stop codon is read as an amino acid but mostly as a stop codon) . Therefore, the amber codon was replaced with a non-stop codon encoding the same amino acid before expressing the resulting V H .

[0170] However, inconsistent information was found regarding the nature of the amino acid encoded by the amber codon in E. coli TG1 cells when choosing an appropriate replacement codon. Some reported Glu as an overwriting amino acid (Hoogenboom, H.R. et al., 1991) (Baek, H. et al., 2002), while others reported Gln as an overwriting amino acid (Soltes, G. et al., 2003) (Ma...

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Abstract

The present invention relates to non-aggregating VH domains or libraries thereof. The VH domains comprise at least one disulfide linkage-forming cysteine in at least one complementarity-determining region (CDR) and an acidic isoelectric point (pI). A method of increasing the power or efficiency of selection of non-aggregating VH domains comprises panning a phagemid-based VH domain phage-display library in combination with a step of selecting non-aggregating phage-VH domains. Compositions of matter comprising the non-aggregating VH domains, as well as methods of use are also provided.

Description

field of invention [0001] The present invention relates to antibody heavy chain variable domains. In particular, the invention relates to non-aggregative human V H Domains and methods of making and using them. Background of the invention [0002] Antibodies play an important role in the diagnosis and clinical application of identifying and neutralizing pathogens. Antibodies are constructed from pairs of heavy and light chain polypeptides. When an antibody is properly folded, the individual chains fold into many different globular domains connected by a more linear polypeptide sequence. For example, the light chain folds into a variable (V L ) and constant (C L ) domain. Heavy and light chain variable domains (V H and V L ) interaction leads to the formation of the antigen-binding domain (Fv). Usually, V H and V L Required for optimal antigen binding, although heavy chain dimers and amino-terminal fragments have been shown to retain activity in the absence of light...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/00A61K39/395A61P37/04C07K16/12C12N15/13C40B30/04C40B40/02C40B40/10G01N33/53G01N33/567C12Q1/68
CPCC07K16/005C07K2317/565C07K2317/21C07K16/40C07K2317/22C07K2317/624G01N33/6857A61P31/00A61P37/04
Inventor M·阿巴比-格赫劳迪J·坦纳
Owner NAT RES COUNCIL OF CANADA
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