Hemagglutinin-specific antibodies and uses thereof

a technology of hemagglutinin and specific antibodies, applied in the field of hemagglutinin-specific antibodies, fragments thereof, can solve the problems of inability to meet the needs of patients, so as to speed up or improve the accuracy of quantification methods, and optimise the bioprocess used to generate vaccines.

Active Publication Date: 2020-10-22
NAT RES COUNCIL OF CANADA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0044]Hemagglutinin is a protein expressed at the surface of the influenza virus. Due to antigenic drift and shift, each new strain of influenza generally requires a specific antibody for its recognition. Presently, three novel antibodies [11H12 (SEQ ID NO. 36), 10A9 (SEQ ID NO. 35) and 9D1] have been identified that specifically bind hemagglutinin. The novel antibodies described herein are able to detect and bind multiple strains encompassing 13 HA subtypes. They can be used for detection, quantification, and neutralization. Furthermore, these antibodies are monoclonal, and thus can be produced in a reproducible and scalable fashion.
[0045]The antibodies of the present invention may be use to speed up or improve the accuracy of quantification methods, which would ease the bottleneck in vaccine distribution. The antibodies could also be used with in-process and crude samples in order to optimise the bioprocess used to generate vaccines.

Problems solved by technology

Point mutations in the viral genome RNA of a given HA subtype already in circulation, or a new subtype of HA that arises through antigenic shift (Kang et al., 2011) can result in an influenza pandemic.
Antibody production can take from 3 up to 16 weeks, which causes significant delays for the vaccine industry.
Generally, quantification of new vaccine lots is the bottleneck in vaccine distribution.
However, this assay is lengthy, laborious, and highly variable depending on the operator.
However, the use of strain-specific antibodies can cause delays in releasing new vaccine lots.
However, its ability to recognize all HA subtypes remains untested.
Unfortunately, the main constraint with Uni-1 is that the antibodies are polyclonal rabbit antibodies, which result in high lot to lot variations.
Additionally, the peptide used to raise Uni-1 was unable to elicit an immune response in mice, which has prevented production of monoclonal antibodies.
Thus, while some success has been achieved in the influenza field to generate antibodies with broad specificity to influenza HA, it is limited and not without drawbacks.

Method used

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  • Hemagglutinin-specific antibodies and uses thereof
  • Hemagglutinin-specific antibodies and uses thereof
  • Hemagglutinin-specific antibodies and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

Production and Purification of Antigen

[0139]A fusion peptide was prepared to use for immunization of mice. The fusion protein comprised a conserved peptide sequence from the N-terminal region of HA2 (GLFGAIAGFIEGGW; SEQ ID NO:26), functional groups on the peptide sequence, and keyhole limpet hemocyanin (KLH).

[0140]Conjugate structure. The peptide conjugate shown in FIG. 1A, was designed by using a conserved peptide sequence at the N-terminus of HA2 that was previously identified: GLFGAIAGFIEGGW (SEQ ID NO:26). The peptide epitope was functionalized with lactose and conjugated to Keyhole Limpet Hemocyanin (KLH) to obtain a conjugate of formula (I):

[0141]The KLH portion of the conjugate also comprised multiple epitopes conjugated to its surface.

[0142]Peptide conjugate synthesis. The peptide conjugate was prepared according to FIG. 1B via a thio-ether bond between terminal Cys of the (glyco)peptide antigen and bromoacetyl KLH.

[0143]Bromoacetylation of KLH. Typically, 20 mg of KLH (Sigm...

example 2

Generation of Anti-Influenza Antibodies

[0146]To produce antibodies that target the influenza virus, mice were immunized with the peptide conjugate obtained in Example 1. Hybridomas (monoclonal antibodies) were also prepared and evaluated by ELISA.

[0147]Immunizations. 6-week old A / J mice were bled (pre-immune serum) and immunized i.p. and s.c. with 100 μg of antigen (Example 1) in Titermax adjuvant. Three weeks later, a second injection of 100 μg of antigen in Titermax adjuvant was performed and mice were bled 7-10 days later. The serum titer was measured by ELISA. Two months later, a final i.p. booster injection using 100 μg of antigen was performed 4 days prior to fusion experiment.

[0148]Fusion of the harvested spleen cells. All manipulations were carried out under sterile conditions. Spleen cells were harvested from immunized mice in IMDM (Hy-Clone) and fused to NSO myeloma cell line using PEG fusion protocol. To this end, spleen cells and myeloma cells were washed in IMDM, counte...

example 3

Biophysical Characterization of the Anti-HA mAb

[0154]The anti-HA mAb of Example 2 were characterized using Differential Scanning calorimetry (DSC) and surface plasmon resonance (SPR). MAb resistance to freeze-thaw cycles were also evaluated.

[0155]Thermal stability of the mAb measured by DSC: The mAb were analysed by DSC to determine at which temperature unfolding of the mAb is induced. Each purified mAb was diluted to 0.4 mg / mL in PBS, and a total of 400 uL was used for DSC analysis with a VP-Capillary DSC (Malvern Inc.). At the start of each DSC run, 5 buffer blank injections are performed to stabilize the baseline and a buffer injection precede each mAb injection for referencing. Each sample was scanned from 20 to 100° C. at a 60° C. / hr rate, with low feedback, 8 sec filter, 5 min preTstat, and 70 psi nitrogen pressure. The mAb thermograms were referenced and analyzed using Origin 7 software. The melting points of the mAb were found to be between 71.28 and 72.90° C. as reported in...

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Abstract

The present invention relates to hemagglutinin-specific antibodies, fragments thereof, and uses thereof. More specifically, these antibodies and fragments thereof are able to recognize antigen from multiple influenza strains.

Description

FIELD OF THE INVENTION[0001]The present invention relates to hemagglutinin-specific antibodies, fragments thereof, and uses thereof. More specifically, the present invention relates to hemagglutinin-specific antibodies and fragments thereof able to recognize antigen from multiple influenza strains.BACKGROUND OF THE INVENTION[0002]Influenza is an infectious disease caused by the influenza virus which belongs to the Orthomyxoviridae family. Based on their core proteins, influenza viruses are classified into types A, B, and C. The two main types of influenza virus responsible for seasonal flu epidemics are types A and B. Influenza A virus can be further characterized by serotype based on the hemagglutinin (HA) and neuraminidase (NA) proteins on the viral surface. Currently, there are 18 known subtypes of HA and 11 subtypes of NA. Based on HA subtypes, influenza A viruses are further divided into two phylogenetic groups: group 1 (H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17 and H18) ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K16/10G01N33/569A61P31/16
CPCC07K2317/92G01N33/56983C07K16/1018A61P31/16A61K2039/505G01N2333/11C07K2317/76A61K47/643A61K47/646A61K2039/5256C07K2317/33C07K2317/94
Inventor MANCEUR, AZIZAMARCIL, ANNEZOU, WEIKAMEN, AMINEGADOURY, CHRISTINE
Owner NAT RES COUNCIL OF CANADA
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