WO2011114353A1 - Monovalent human anti-hepatitis a virus antibody and uses thereof - Google Patents

Monovalent human anti-hepatitis a virus antibody and uses thereof Download PDF

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Publication number
WO2011114353A1
WO2011114353A1 PCT/IN2011/000176 IN2011000176W WO2011114353A1 WO 2011114353 A1 WO2011114353 A1 WO 2011114353A1 IN 2011000176 W IN2011000176 W IN 2011000176W WO 2011114353 A1 WO2011114353 A1 WO 2011114353A1
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Prior art keywords
hepatitis
seq
monoclonal antibody
virus
antibody
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PCT/IN2011/000176
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French (fr)
Inventor
Nimmagadda Venkata Sridevi
Aavula Shukra Madhaha
Dev Chandran
Lingala Rajendra
Villuppanoor Alwar Srinivasan
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Indian Immunologicals Limited
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Publication of WO2011114353A1 publication Critical patent/WO2011114353A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1009—Picornaviridae, e.g. hepatitis A virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • the present invention relates to the field of immunology in particular to the field of production of antibodies.
  • the present invention specifically relates to production of single chain antibody against Hepatitis A virus.
  • Hepatitis A virus is the important pathogen responsible for human Hepatitis infections (Francki, R.I.B., Fauquet, CM., Knudson, DJ., Brown, F., 1991. Classification and nomenclature of viruses. Arch. Virol., Suppl. 2, 320-326) which is a self-limiting disease of liver , that is prevalent world wide, represents a substantial economic burden in countries with low and intermediate incidence rates. An estimated 1.5 million cases occurring worldwide each year. Several sero- epidemiological studies have shown the prevalence of Hepatitis A antibodies in populations to vary from 15 to 70% in various parts of the world.
  • the disease can induce long periods of debilitating illness which is normally transmitted through the faeco- oral route and is also acquired by the direct contact with an infected individual or by ingestion of contaminated drinking water and/ or food (Jones, A.E., Smith,J.L., Hindman, S.H., Fleissner, M.L., Judelsohn, R., E nglender, S.J., Tilson, H., Maynard, J.E., 1977. Foodborne hepatitis A infection : a report of two urban restaurant- associated outbreaks. Am. J. Epidemiol.
  • Single chain variable fragments consists of variable heavy (VH) and light (VL) chain domains tethered by a flexible peptide linker which retains the antigen binding site in a single linear molecule and their design, construction and expression in Escherichia coli demonstrated their structure-function relationship and antigen-antibody interactions makes ScFv useful in both clinical and medical application (Huston, J.S., D.Levinson, M.Mudgett-Hunter, M.S.Tai, J. Novotny.M.N. Margolies, R.J. Ridge, R.E. Bruccoleri, E. Haber, R.Crea, H. Oppermann.1988.
  • One aspect of the present invention provides a monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24 or a functional fragment or variant thereof; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25 or a functional fragment or variant thereof.
  • Another aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24 and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25 and a pharmaceutically acceptable carrier.
  • Another aspect of the present invention provides a method of detecting Hepatitis A virus in a biological sample, wherein the method comprises contacting the sample with a monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24 and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25; and detecting the presence of the immune complex formed between the antibody and Hepatitis A virus antigen, wherein presence of the immune complex confirm presence of Hepatitis A virus in the biological sample.
  • kits for detection of Hepatitis A virus comprising the monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24 and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25.
  • Further aspect of the present invention provides use of the monoclonal antibody as disclosed in the present invention for the preparation of anti-Hepatitis A virus medicament.
  • Still another aspect of the present invention provides a method of detecting an agents that inhibits Hepatitis A virus replication, wherein the method comprises contacting the monoclonal antibody a monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24 and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25 with Hepatitis A virus in presence of an agent of interest and assaying binding ability of the monoclonal antibody with Hepatitis A virus, wherein decrease in binding ability of the monoclonal antibody with Hepatitis A virus suggests interference of the agent in antibody -antigen interaction.
  • Figure 1 shows vector map showing cloned single chain variable fragment (scFv)
  • Figure 2 shows vector construct comprising single chain variable fragment (scFv)
  • Figure 3 shows electrophoretic analysis of PCR amplified variable heavy and light chains; M-Molecular weight markers, VH- Variable heavy chain, VL- Variable light chain and scFv - Assembled scFv product.
  • Figure 4 shows SDS-PAGE analysis of purified monovalent scFv.
  • the purified protein was detected by staining with coomassie brilliant blue.
  • the size of the corresponding protein molecular weight standards in KDa are mentioned in the left.
  • Figure 5 shows western blot analysis of purified scFv protein.
  • the blot was reacted with His probe specific for the histidine tag of the scFv.
  • a protein band of 30 kDa was detected.
  • Lanes Lanes: Lane 1-6, fractions obtained from column chromatography for purification of scFv; Lane M: Prestained molecular weight marker.
  • Figure 6 shows analysis of the antigen binding affinity by indirect ELISA to evaluate the activity of scFv to Hepatitis A virus
  • Figure 7 shows competitive ELISA using the scFv and the anti Hepatitis A murine polyclonal sera.
  • Figure 8 shows ELISA based comparison of scFv binding to unrelated virus.
  • FIG. 9 shows Immunofluorescence staining (FITC) FRhk-4 cells infected with
  • Hepatitis A virus ScFv is showing binding to Hepatitis A virus in live cells were observed under fluorescence microscope, (a) Detection with anti-mouse IgG-FITC conjugate, (b) Detection with mouse anti-His IgG-FITC conjugate, (c) Uninfected- cell control
  • Figure 10 shows detection of the hepatitis A virus antigen content in human vaccine preparation by immuno-capture ELISA.
  • NIBSC was used as standard control.
  • Oligonucleotide sequences of forward primers for amplification of Human heavy chain variable domain are as set forth in:
  • Oligonucleotide sequences of reverse primers for amplification of Human heavy chain variable domain are as set forth in:
  • Oligonucleotide sequences of forward primers for amplification of Human light chain variable domain are as set forth in:
  • Oligonucleotide sequences of reverse primers for amplification of Human light chain variable domain are as set forth in:
  • Nucleotide sequence of heavy chain variable domain (363 nts): SEQ ID NO: 21
  • Amino acid sequence of heavy chain variable domain SEQ ID NO: 24
  • Amino acid sequence of light chain variable domain SEQ ID NO: 25
  • Amino acid sequence of recombinant scFv (Light chain: amino acid position 1- 112; Linker: 113-127; Heavy chain: 128-248): SEQ ID NO: 27
  • Nucleotide sequence of recombinant scFv (744 nts; Light chain: 1-336 nts; Linker: 337-381 ; Heavy chain: 382-744): SEQ ID NO: 28
  • the present invention provides a human recombinant monoclonal anti-Hepatitis A antibody and a method for production of the antibody.
  • the present invention further provides a recombinant vector and host cells comprising the monoclonal antibody.
  • the present invention provides a pharmaceutical composition comprising the antibody for prophylactic and/or therapeutic treatment of Hepatitis A infection.
  • the present invention also provides a method of detection of Hepatitis A virus present in a sample using the human recombinant monoclonal anti-Hepatitis A antibody of the present invention.
  • the human anti-Hepatitis A antibody disclosed in the present invention recognizes Hepatitis A virus.
  • a human immunoglobulin ScFv library was generated from vaccinated human peripheral blood lymphocytes (PBL's) using phage display technology. ScFv's screened against Hepatitis A virus and expressed in prokaryotic system which showed strong binding to Hepatitis A. The isolated ScFv's may provide an opportunity to be used in place of the presently available IgG for the quantitation of Hepatitis A antigen in vaccine manufacture.
  • the recombinant monovalent human anti-Hepatitis A antibody as disclosed in the present invention comprises single chain variable (scFv) fragments and is generated from immunised antibody library using the phage display technology. Biopanning was performed for selection of Hepatitis A virus specific antibody fragments. The eluted phages obtained after third round of panning was used to infect Escherichia coli TGI and 96 individual clones were tested for binding to target antigen. As indicated by the phage ELISA, specific clones showed positive binding to antigen. Furthermore, PCR analysis and enzymatic analysis showed the presence of scFv antigen binding fragments.
  • scFv single chain variable
  • Selected scFv plasmids were transformed into Escherichia coli BL21 (DE3) for production of soluble scFv fragments.
  • the purified protein was checked for their antigen binding activity against the Hepatitis A virus was found to be active.
  • One embodiment of the present invention provides a human recombinant monoclonal anti-Hepatitis A antibody capable of recognizing Hepatitis A virus.
  • Another embodiment of the present invention provides a method of production of human recombinant monoclonal anti-Hepatitis A antibody.
  • Another embodiment of the present invention provides in-vitro ELISA based method for quantification of Hepatitis A virus antigen by using the human recombinant monoclonal anti-Hepatitis A antibody generated from human immunised antibody library.
  • nucleotide sequence of the heavy chain variable domain of human recombinant monoclonal anti-Hepatitis A antibody wherein the nucleotide sequence is as set forth in SEQ ID NO: 21.
  • nucleotide sequence of the light chain variable domain of human anti-Hepatitis A antibody wherein the nucleotide sequence is as set forth in SEQ ID NO: 22.
  • nucleotide sequence of a linker wherein the nucleotide sequence is as set forth in SEQ ID NO: 23.
  • the heavy chain variable domain having the amino acid sequence as set forth in SEQ ID NO: 24.
  • the light chain variable domain having the amino acid sequence as set forth in SEQ ID NO: 25.
  • linker having the amino acid sequence as set forth in SEQ ID NO: 26.
  • polynucleotide sequence having the nucleotide sequence as set forth in SEQ ID NO: 28, wherein the polynucleotide sequence encodes the human recombinant monoclonal anti- Hepatitis A antibody having amino acid sequence as set forth in SEQ ID NO: 27.
  • a recombinant DNA construct comprising a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 28, wherein the polynucleotide comprises the human heavy chain variable domain having the nucleotide sequence as set forth in SEQ ID NO: 21, the human light chain variable domain having the nucleotide sequence as set forth in SEQ ID NO: 22 and the linker having the nucleotide sequence as set forth in SEQ ID NO: 23.
  • a recombinant vector comprising the polynucleotide having nucleotide sequence as set forth in SEQ ID NO: 28, wherein the polynucleotide encodes a human recombinant monoclonal anti-Hepatitis A antibody which specifically binds to Hepatitis A antigen, wherein the amino acid sequence of the antibody is as set forth in SEQ ID NO: 27.
  • a host cell comprising the recombinant vector comprising the polynucleotide having nucleotide sequence as set forth in SEQ ID NO: 28, wherein the polynucleotide encodes a human recombinant monoclonal anti-Hepatitis A antibody which specifically binds to Hepatitis A antigen, wherein the amino acid sequence of the antibody is as set forth in SEQ ID NO: 27, wherein the host cell is capable of expressing the antibody.
  • a human recombinant monoclonal anti-Hepatitis A antibody having the amino acid sequence as set forth in SEQ ID NO: 27.
  • a human recombinant monoclonal anti-Hepatitis A antibody having the amino acid sequence as set forth in SEQ ID NO: 27 encoded by the nucleotide sequence as set forth in SEQ ID NO: 28.
  • a human recombinant monoclonal anti- Hepatitis A antibody containing a short linker sequence located between the heavy chain variable domain and light chain variable domain, wherein the nucleotide sequence of the linker is as set forth in SEQ ID NO:23.
  • a pharmaceutical composition comprising the human recombinant monoclonal anti-Hepatiitis A antibody as disclosed in the present invention together with one or more pharmaceutically acceptable excipients, diluents or carriers.
  • composition comprising the human recombinant monoclonal anti-Hepatiitis A antibody having the amino acid sequence as set forth in SEQ ID NO: 27.
  • a pharmaceutical composition comprising the human recombinant monoclonal anti-Hepatiitis A antibody having the amino acid sequence as set forth in SEQ ID NO: 27 together with one or more pharmaceutically acceptable excipients, diluents or carriers.
  • a monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25
  • a monoclonal antibody having the amino acid sequence of the monoclonal antibody is as set forth in SEQ ID NO: 27.
  • a monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25, wherein the heavy chain variable domain is encoded by the nucleotide sequence as set forth in SEQ ED NO: 21.
  • a monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25, wherein the light chain variable domain is encoded by the nucleotide sequence as set forth in SEQ ID NO: 22.
  • a monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25, wherein the antibody is a monovalent or divalent antibody.
  • a monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25, wherein the antibody is a human monoclonal antibody.
  • a monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25, wherein the monoclonal antibody binds a Hepatitis A virus antigen.
  • nucleic acid molecule encoding the monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25.
  • nucleic acid molecule encoding the monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25, wherein the nucleic acid comprises a nucleotide sequence as set forth in SEQ ID NO: 28.
  • a vector comprising the nucleic acid encoding the monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25.
  • a host cell comprising the nucleic acid encoding the monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25.
  • a pharmaceutical composition comprising the monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25 and a pharmaceutically acceptable carrier.
  • a method of detecting Hepatitis A virus in a biological sample comprises contacting the sample with the antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25 and detecting the presence of the immune complex formed between the antibody and Hepatitis A virus antigen, wherein presence of the immune complex confirm presence of Hepatitis A virus in the biological sample.
  • kits for detection of Hepatitis A virus comprising the monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25.
  • the present invention also provides use of the monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25 for the preparation of anti-Hepatitis A virus medicament.
  • the present invention further provides a prophylactic and/or therapeutic method of treatment of Hepatitis A virus infection in a subject, said method comprises administering an effective amount of the monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25 to a the subject.
  • a method of detecting an agents that inhibits Hepatitis A virus replication comprises contacting the monoclonal antibody as claimed in claim 1 with Hepatitis A virus in presence of an agent of interest and assaying binding ability of the monoclonal antibody with Hepatitis A virus, wherein decrease in binding ability of the monoclonal antibody with Hepatitis A virus suggests interference of the agent in antibody -antigen interaction.
  • the present invention also provides use of the monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25 for detecting Hepatitis A virus in a biological sample.
  • the monoclonal anti-Hepatitis A antibody as disclosed in the present invention binds to Hepatitis A virus and neutralizes Hepatitis A virus. Further, the antibody can be used as prophylactic, therapeutic and/or diagnostic agent.
  • nucleic and amino acid sequence as disclosed or described in the present invention are shown using the standard letter abbreviations for nucleotide bases and single letter codes for amino acid.
  • a monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25 to a the subject, wherein the antibody is encoded by polynucleotide sequence having the nucleotide sequence as set forth in SEQ ID NO: 28.
  • monoclonal antibody encoded by the nucleotide sequence of the light chain variable domain that is at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identical to the nucleotide sequence is as set forth in SEQ ID NO: 22, and the heavy chain variable domain that is at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identical to the nucleotide sequence as set forth in SEQ ID NO: 21.
  • monoclonal antibody encoded by the nucleotide sequence of the light chain variable domain as set forth in SEQ ID NO: 22, and the nucleotide sequence of the heavy chain variable domain as set forth in SEQ ID NO: 21.
  • RNA was isolated from peripheral blood lymphocytes of vaccinated donors and cDNA was synthesized by RT-PCR.
  • the RT-PCR amplified DNA was used as template for amplification of variable domains of an antibody with universal primers.
  • the amplified variable domains were assembled to form single chain variable fragment (scFv) by splicing by overlap extension PCR ( Figure 3) and cloned into phagemid vector to obtain recombinant phagemid vector.
  • the recombinant phagemid vector thus obtained was transformed into E. coli TGI .
  • the transformed E. coli cells comprising the recombinant phagemid vector were infected with M13K07 helper phage to yield recombinant phages.
  • Human phage displayed scFv library was constructed from immunised peripheral blood lymphocytes.
  • the library comprises over 5 x 10 7 different scFv fragments cloned in an ampicillin- resistant: phagemid vector pCANTAB5E and transformed into TGI E. coli cells.
  • scFv fragment comprises one variable heavy chain and one variable light chain linked by a peptide linker.
  • Phages displaying individual human scFv antibody fragments were selected by three rounds of biopanning on immunotubes coated with a decreasing amount of purified Hepatitis A antigen for the first, second and third round of panning. Eluted phage titers showed an increase after each of the three rounds of panning, indicating successful enrichment (Table.l). Eluted phage from round 3 was used to infect E. coli TGI and randomly selected 96 individual clones were tested by phage ELISA against Hepatitis A antigen. Of the 96 clones tested, forty five clones showed positive binding to Hepatitis A virus. Out of forty clones selected few clones based on their high binding affinity for further study.
  • the schematic representations as depicted in Figure 1 and figure 2 show that the nucleotide sequence encoding monovalent human anti-Hepatitis A antibody comprising scFv was cloned between the EcoRl and Noil sites of pET 20b bacterial expression vector, wherein the size of the scFv is 744 bp in which a 45bp linker (Gly 4 Ser) 3 was used to connect variable regions of heavy (363 bp) and light (336 bp) chains of antibody.
  • the pET 20b vector carries ribosome binding site, signal peptide sequence of bacterial pectate lyase, variable fragment of light chain, heavy chain, C-terminal His tag sequence and T7 promoter and T7 terminator. Unique site are shown on the circle map. ⁇ ' denotes the ampicillin resistance gene.
  • the vector pET 20b and insert referred to monovalent antibody fragment of size 744 bps was digested with EcoRl and Notl, respectively by incubating at 37°C for 12 hours.
  • the digested products was purified using the kit provided by QIAGEN and kept for various ratios of vector to insert (i.e., 1 :3 and 1 :6) blunt end ligation and incubated at 22°C for 2 hours.
  • the ligated product was incubated for further 20 minutes at 65°C in order to inactive the enzyme.
  • the final product was chemically transformed into XL-Blue strain of E. coli cells.
  • Overnight grown XL-Blue strain were sub-cultured and grown at 37°C, shaking until the OD of the culture reaches to 0.6 at 600nm.
  • the culture was harvested by centrifuging at 5000 x rpm for 10 minutes at 4°C and resuspended in ice-cold 0.1 mM CaCl 2 and incubated overnight on the ice, before proceeding for transformation.
  • the chemically competent XL-Blue cells were incubated with plasmid DNA for 30 minutes on ice.
  • the cells were given heat shock at 42°C for 90 seconds and immediately placed the tubes on ice for 2 minutes before the media was added to cells.
  • the cells were incubated for one hour at 37°C for recovery and plated on containing lOOmg/ml of ampicillin.
  • the plates were incubated for overnight and screened for positive clones by isolating the plasmids and subjected to digestion with EcoRl and Notl.
  • the positive clones were sequence verified before the plasmid was transformed into BL21 (DE3) cells of E.coli for soluble expression of the antibody gene.
  • the competitive ELISA was performed using commercial kit to check for competition between scFv and Hepatitis A kit Mab. Competition was done by incubating the Kit Mab with varying dilutions of scFv. No increase of optical density values following the dilution of scFv indicating the scFv is not competing with kit Mab for the same site on Hepatitis A virus ( Figure 7) was observed.
  • Example 5
  • Sandwich ELISA was performed to determine the binding specificity of the scFv to Hepatitis A virus.
  • the presence of reactivity of the scFv against Hepatitis A and not to other viruses (rabies, hepatitis B, and Chikungunya viruses) indicates specificity of the scFv against Hepatitis A ( Figure 8).
  • Immunocapture ELISA was performed to detect the hepatitis A virus antigen in vaccine preparation, wherein the scFv was used as detection reagent. Titration of the vaccine against a constant dilution of scFv revealed a concentration dependent reduction of the binding signal.
  • NIBSC sample was used as reference standard and the kit MAb used as a positive control. Linear graph was plotted for NIBSC standard and estimated the antigen content in vaccine batch. (Fig.10)
  • SEQ ID NO: 21 Nucleotide sequence of heavy chain variable domain (363 nts) CAGGTCACCTTGAGGAGTCTGGTCCTACGCTGGGGAAACCCACACAGA CCCTCACGCGGACCTGCACCTTCTCTGGGTTCTCACTCAGCACTAGTGG
  • SEQ ID NO: 27 Amino acid sequence of recombinant scFv (Light chain: amino acid position 1-112; Linker: 113-127; Heavy chain: 128-248)
  • SEQ ID NO: 28 Nucleotide sequence of recombinant scFv (744 nts; Light chain: 1- 336 nts; Linker: 337-381; Heavy chain: 382-744)

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Abstract

The present invention relates to generation of monovalent antibody fragment, single chain variable fragment (ScFv) from human immunised library having one antigen binding site that is reactive against the Hepatitis A virus which can be used as a diagnostic tool. The present invention particularly provides monovalent human anti-Hepatitis A antibody fragments and a process of preparation thereof.

Description

MONOVALENT HUMAN ANTI-HEPATITIS A VIRUS ANTIBODY
AND USES THEREOF
FIELD OF INVENTION
The present invention relates to the field of immunology in particular to the field of production of antibodies. The present invention specifically relates to production of single chain antibody against Hepatitis A virus.
BACK GROUND OF THE INVENTION
Hepatitis A virus (HAV) is the important pathogen responsible for human Hepatitis infections (Francki, R.I.B., Fauquet, CM., Knudson, DJ., Brown, F., 1991. Classification and nomenclature of viruses. Arch. Virol., Suppl. 2, 320-326) which is a self-limiting disease of liver , that is prevalent world wide, represents a substantial economic burden in countries with low and intermediate incidence rates. An estimated 1.5 million cases occurring worldwide each year. Several sero- epidemiological studies have shown the prevalence of Hepatitis A antibodies in populations to vary from 15 to 70% in various parts of the world. The disease can induce long periods of debilitating illness which is normally transmitted through the faeco- oral route and is also acquired by the direct contact with an infected individual or by ingestion of contaminated drinking water and/ or food (Jones, A.E., Smith,J.L., Hindman, S.H., Fleissner, M.L., Judelsohn, R., E nglender, S.J., Tilson, H., Maynard, J.E., 1977. Foodborne hepatitis A infection : a report of two urban restaurant- associated outbreaks. Am. J. Epidemiol. 105 : 156-162, Mahoney, F.J., Farley, T.A., Kelso, K.Y., Wilson, S.A., Horan, J.M., McFarland, L.M., 1992. An outbreak of hepatitis A associated with swimming in a public pool. J.Infect. Dis. 165 : 613-618).
Currently used inactivated virus vaccines and pooled human immune globulin (Ig) which is used for active and passive immunization for prevention of HAV infection have various limitations, such as difference of HAV antibody levels in various batches and decrease of anti- HAV antibodies in donor populations which gives lower titres than naturally infected people (Stapleton, J.T., Hepatitis A : biology, pathogenesis,epidemiology,cIinical description and diagnosis. In: Spector, S. (Ed.), Viral Hepatitis :Diagnosis, Therapy and Prevention. Humana Press, Totowa, NJ, pp. 7-33, Winokur, P.L and Stapleton, J.T. 1992. Immunoglobulin prophylaxis for hepatitis A. Clin. Infect. Dis. 14(2), 580-586).
Use of monoclonal antibodies for estimation of Hepatitis A virus involves production of antibodies from conventional technologies, a process which is expensive and requires complex bio-processes. The use of recombinant antibody fragments such as monovalent antibodies will overcome these limitations and provide homogenous, pure and large scale reagents necessary for Hepatitis A virus.
The field of antibody engineering has opened new avenues and gaining attention of large number of researchers as compared to conventional antibody technologies, Human antibody phage technology has more advantages in producing large number of antibodies which mimics the key features of the humoral immune system invitro than when produced in animals without prior immunization. This leads to the production of high affinity antibodies. The display of Fab and scFv antibody fragments on the surface of bacteriophage for generation of libraries provides an effective platform for isolation of diverse set of antibodies (Brekke OH, Loset GA.New technologies in therapeutic antibody development. Curr Opin Pharmacol, 2003 Oct;3(5):544-50, Thullier P, P. Lafaye, Megret F, Deubel V, Jouan A, Mazie JC. A recombinant Fab neutralizes dengue virus in vitro. J Biotechnol. 1999 69(2-3): 183-90) against various disease targets from immune or naive samples which can be over expressed in bacterial systems (Barbas III, C.F., Crowe Jr., Cababa,D., Jones, T.M, Zebedee, S.L., Murphy, B.R., Chanock, R.M., Burton, D.R., 1992. Human monoclonal Fab fragments derived from a combinatorial library bind to respiratory syncytial virus F glycoprotein and neutralize infectivity. Proc. Natl. Acad. Sci. U.S.A. 89 (21), 10164-10168, Burton, D.R., Barbas III, C.F., Persson, M.A., Koenig, S., Chanock, R.M., Lerner, R.A., 1991. A large array of human monoclonal antibodies to type I human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals. Proc. NatLAcad. Sci. U.S.A. 88 (22), 10134-10137, Marks, J. D., H. R. Hoogenboom, T. P. Bonnert, J. McCafferty, A. D. Griffiths and G. Winter. 1991. By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J. Mol. Biol. 222:581, Hoogenboom, H.R. and G. Winter.
1992. By-passing immunization. Human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro. J.MoLBiol. 227 : 381-388).
Single chain variable fragments (ScFv) consists of variable heavy (VH) and light (VL) chain domains tethered by a flexible peptide linker which retains the antigen binding site in a single linear molecule and their design, construction and expression in Escherichia coli demonstrated their structure-function relationship and antigen-antibody interactions makes ScFv useful in both clinical and medical application (Huston, J.S., D.Levinson, M.Mudgett-Hunter, M.S.Tai, J. Novotny.M.N. Margolies, R.J. Ridge, R.E. Bruccoleri, E. Haber, R.Crea, H. Oppermann.1988. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia col. Proc. Natl. Acad. Sci.USA. 85: 5879-5883, Bird, R. E., K.D. Hardman, J.W. Jacobson, S. Johnson, B.M. Kaufman,S.M. Lee, T. Lee, S.H. Pope, G.S. Riordan, M. Whitlow. 1988. Single-chain antigen-binding proteins, Science. 242 : 423^126, Condra JH, Sardana VV, Tomassini JE, Schlabach AJ, Davies ME, Lineberger DW, Graham DJ, Gotlib L, Colonno RJ. Bacterial expression of antibody fragments that block human rhinovirus infection of cultured cells. J Biol Chem. 1990 Feb 5;265(4):2292-2295).
SUMMARY OF THE INVENTION
One aspect of the present invention provides a monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24 or a functional fragment or variant thereof; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25 or a functional fragment or variant thereof.
Another aspect of the present invention provides a pharmaceutical composition comprising a monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24 and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25 and a pharmaceutically acceptable carrier.
Another aspect of the present invention provides a method of detecting Hepatitis A virus in a biological sample, wherein the method comprises contacting the sample with a monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24 and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25; and detecting the presence of the immune complex formed between the antibody and Hepatitis A virus antigen, wherein presence of the immune complex confirm presence of Hepatitis A virus in the biological sample.
Yet another aspect of the present invention provides a kit for detection of Hepatitis A virus, comprising the monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24 and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25.
Further aspect of the present invention provides use of the monoclonal antibody as disclosed in the present invention for the preparation of anti-Hepatitis A virus medicament.
Still another aspect of the present invention provides a method of detecting an agents that inhibits Hepatitis A virus replication, wherein the method comprises contacting the monoclonal antibody a monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24 and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25 with Hepatitis A virus in presence of an agent of interest and assaying binding ability of the monoclonal antibody with Hepatitis A virus, wherein decrease in binding ability of the monoclonal antibody with Hepatitis A virus suggests interference of the agent in antibody -antigen interaction.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
It is to be noted, however, that the appended drawings and tables illustrated only typical embodiments of this invention and are therefore not to be considered for limiting of its scope, for the invention may admit to other equally effective embodiments.
Figure 1 shows vector map showing cloned single chain variable fragment (scFv) Figure 2 shows vector construct comprising single chain variable fragment (scFv) Figure 3 shows electrophoretic analysis of PCR amplified variable heavy and light chains; M-Molecular weight markers, VH- Variable heavy chain, VL- Variable light chain and scFv - Assembled scFv product.
Figure 4 shows SDS-PAGE analysis of purified monovalent scFv. The purified protein was detected by staining with coomassie brilliant blue. The size of the corresponding protein molecular weight standards in KDa are mentioned in the left.
Figure 5 shows western blot analysis of purified scFv protein. The blot was reacted with His probe specific for the histidine tag of the scFv. A protein band of 30 kDa was detected.
Lanes: Lane 1-6, fractions obtained from column chromatography for purification of scFv; Lane M: Prestained molecular weight marker.
Figure 6 shows analysis of the antigen binding affinity by indirect ELISA to evaluate the activity of scFv to Hepatitis A virus
Figure 7 shows competitive ELISA using the scFv and the anti Hepatitis A murine polyclonal sera.
A -> Precoated Ag + (scFv + Kit Mab) + Anti Mouse HRP
B -» Precoated Ag + (scFv + Kit Mab)+ Anti His HRP
C -> Precoated Ag + (E.coli Lysate + Kit Mab) + Anti Mouse HRP
Figure 8 shows ELISA based comparison of scFv binding to unrelated virus.
Figure 9 shows Immunofluorescence staining (FITC) FRhk-4 cells infected with
Hepatitis A virus. ScFv is showing binding to Hepatitis A virus in live cells were observed under fluorescence microscope, (a) Detection with anti-mouse IgG-FITC conjugate, (b) Detection with mouse anti-His IgG-FITC conjugate, (c) Uninfected- cell control
Figure 10 shows detection of the hepatitis A virus antigen content in human vaccine preparation by immuno-capture ELISA. NIBSC was used as standard control. SEQUENCE LISTING
Oligonucleotide sequences of forward primers for amplification of Human heavy chain variable domain are as set forth in:
SEQ ID NO: 1 HuVHla
SEQ ID NO: 2 HuVH2a
SEQ ID NO: 3 HuVH3a
SEQ ID NO: 4 HuVH4a
SEQ ID NO: 5 HuVH5a
SEQ ID NO: 6 HuVH6a
Oligonucleotide sequences of reverse primers for amplification of Human heavy chain variable domain are as set forth in:
SEQ ID NO: 7 HuJHl-2
SEQ ID NO: 8 HuJH3
SEQ ID NO: 9 HuJH4-5
SEQ ID NO: 10 HuJH6
Oligonucleotide sequences of forward primers for amplification of Human light chain variable domain are as set forth in:
SEQ ID NO: 1 1 HuLAMl
SEQ ID NO: 12 HuLAM2
SEQ ID NO: 13 HuLAM3a
SEQ ID NO: 14 HuLAM3b
SEQ ID NO: 15 HuLAM4
SEQ ID NO: 16 HuLAM5
SEQ ID NO: 17 HuLAM6
Oligonucleotide sequences of reverse primers for amplification of Human light chain variable domain are as set forth in:
SEQ ID NO: 18 HuJLAMl
SEQ ID NO: 19 HuJLAM2-3
SEQ ID NO: 20 HuJLAM4-5
Nucleotide sequence of heavy chain variable domain (363 nts): SEQ ID NO: 21
Nucleotide sequence of light chain variable domain (336 nts): SEQ ID NO: 22
Nucleotide sequence of linker (45 nts): SEQ ID NO: 23
Amino acid sequence of heavy chain variable domain: SEQ ID NO: 24
Amino acid sequence of light chain variable domain: SEQ ID NO: 25
Amino acid sequence of linker: SEQ ID NO: 26
Amino acid sequence of recombinant scFv (Light chain: amino acid position 1- 112; Linker: 113-127; Heavy chain: 128-248): SEQ ID NO: 27
Nucleotide sequence of recombinant scFv (744 nts; Light chain: 1-336 nts; Linker: 337-381 ; Heavy chain: 382-744): SEQ ID NO: 28
DETAILED DESCRD?TION OF THE INVENTION
The present invention provides a human recombinant monoclonal anti-Hepatitis A antibody and a method for production of the antibody. The present invention further provides a recombinant vector and host cells comprising the monoclonal antibody. Further, the present invention provides a pharmaceutical composition comprising the antibody for prophylactic and/or therapeutic treatment of Hepatitis A infection.
The present invention also provides a method of detection of Hepatitis A virus present in a sample using the human recombinant monoclonal anti-Hepatitis A antibody of the present invention. The human anti-Hepatitis A antibody disclosed in the present invention recognizes Hepatitis A virus.
In the present invention, a human immunoglobulin ScFv library was generated from vaccinated human peripheral blood lymphocytes (PBL's) using phage display technology. ScFv's screened against Hepatitis A virus and expressed in prokaryotic system which showed strong binding to Hepatitis A. The isolated ScFv's may provide an opportunity to be used in place of the presently available IgG for the quantitation of Hepatitis A antigen in vaccine manufacture.
The recombinant monovalent human anti-Hepatitis A antibody as disclosed in the present invention comprises single chain variable (scFv) fragments and is generated from immunised antibody library using the phage display technology. Biopanning was performed for selection of Hepatitis A virus specific antibody fragments. The eluted phages obtained after third round of panning was used to infect Escherichia coli TGI and 96 individual clones were tested for binding to target antigen. As indicated by the phage ELISA, specific clones showed positive binding to antigen. Furthermore, PCR analysis and enzymatic analysis showed the presence of scFv antigen binding fragments. Selected scFv plasmids were transformed into Escherichia coli BL21 (DE3) for production of soluble scFv fragments. The purified protein was checked for their antigen binding activity against the Hepatitis A virus was found to be active.
One embodiment of the present invention provides a human recombinant monoclonal anti-Hepatitis A antibody capable of recognizing Hepatitis A virus.
Another embodiment of the present invention provides a method of production of human recombinant monoclonal anti-Hepatitis A antibody.
Another embodiment of the present invention provides in-vitro ELISA based method for quantification of Hepatitis A virus antigen by using the human recombinant monoclonal anti-Hepatitis A antibody generated from human immunised antibody library.
In one embodiment of the present invention there is provided the nucleotide sequence of the heavy chain variable domain of human recombinant monoclonal anti-Hepatitis A antibody, wherein the nucleotide sequence is as set forth in SEQ ID NO: 21.
In another embodiment of the present invention there is provided the nucleotide sequence of the light chain variable domain of human anti-Hepatitis A antibody, wherein the nucleotide sequence is as set forth in SEQ ID NO: 22.
In another embodiment of the present invention there is provided the nucleotide sequence of a linker, wherein the nucleotide sequence is as set forth in SEQ ID NO: 23.
In one embodiment of the present invention there is provided the heavy chain variable domain having the amino acid sequence as set forth in SEQ ID NO: 24.
In another embodiment of the present invention there is provided the light chain variable domain having the amino acid sequence as set forth in SEQ ID NO: 25.
In another embodiment of the present invention there is provided a linker having the amino acid sequence as set forth in SEQ ID NO: 26.
In another embodiment of the present invention there is provided a polynucleotide sequence having the nucleotide sequence as set forth in SEQ ID NO: 28, wherein the polynucleotide sequence encodes the human recombinant monoclonal anti- Hepatitis A antibody having amino acid sequence as set forth in SEQ ID NO: 27.
In another embodiment of the present invention there is provided a recombinant DNA construct comprising a polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 28, wherein the polynucleotide comprises the human heavy chain variable domain having the nucleotide sequence as set forth in SEQ ID NO: 21, the human light chain variable domain having the nucleotide sequence as set forth in SEQ ID NO: 22 and the linker having the nucleotide sequence as set forth in SEQ ID NO: 23.
In another embodiment of the present invention there is provided a recombinant vector comprising the polynucleotide having nucleotide sequence as set forth in SEQ ID NO: 28, wherein the polynucleotide encodes a human recombinant monoclonal anti-Hepatitis A antibody which specifically binds to Hepatitis A antigen, wherein the amino acid sequence of the antibody is as set forth in SEQ ID NO: 27.
In another embodiment of the present invention there is provided a host cell comprising the recombinant vector comprising the polynucleotide having nucleotide sequence as set forth in SEQ ID NO: 28, wherein the polynucleotide encodes a human recombinant monoclonal anti-Hepatitis A antibody which specifically binds to Hepatitis A antigen, wherein the amino acid sequence of the antibody is as set forth in SEQ ID NO: 27, wherein the host cell is capable of expressing the antibody.
In another embodiment of the present invention there is provided a human recombinant monoclonal anti-Hepatitis A antibody having the amino acid sequence as set forth in SEQ ID NO: 27.
In another embodiment of the present invention there is provided a human recombinant monoclonal anti-Hepatitis A antibody having the amino acid sequence as set forth in SEQ ID NO: 27 encoded by the nucleotide sequence as set forth in SEQ ID NO: 28.
In another embodiments there is provided a human recombinant monoclonal anti- Hepatitis A antibody containing a short linker sequence located between the heavy chain variable domain and light chain variable domain, wherein the nucleotide sequence of the linker is as set forth in SEQ ID NO:23.
In one embodiment of the present invention there is provided a pharmaceutical composition comprising the human recombinant monoclonal anti-Hepatiitis A antibody as disclosed in the present invention together with one or more pharmaceutically acceptable excipients, diluents or carriers.
In one embodiment of the present invention there is provided a pharmaceutical composition comprising the human recombinant monoclonal anti-Hepatiitis A antibody having the amino acid sequence as set forth in SEQ ID NO: 27.
In one embodiment of the present invention there is provided a pharmaceutical composition comprising the human recombinant monoclonal anti-Hepatiitis A antibody having the amino acid sequence as set forth in SEQ ID NO: 27 together with one or more pharmaceutically acceptable excipients, diluents or carriers.
In one embodiment of the present invention there is provided use of the human recombinant monoclonal anti-Hepatitis A antibody for the preparation of medicament for the treatment of Hepatitis A virus infection.
In accordance with ithe present invention in one embodiment of the present invention there is provided a monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25
In another embodiment of the present invention there is provided a monoclonal antibody having the amino acid sequence of the monoclonal antibody is as set forth in SEQ ID NO: 27.
In another embodiment of the present invention there is provided a monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25, wherein the heavy chain variable domain is encoded by the nucleotide sequence as set forth in SEQ ED NO: 21.
In another embodiment of the present invention there is provided a monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25, wherein the light chain variable domain is encoded by the nucleotide sequence as set forth in SEQ ID NO: 22.
In another embodiment of the present invention there is provided a monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25, wherein the antibody is a monovalent or divalent antibody.
In another embodiment of the present invention there is provided a monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25, wherein the antibody is a human monoclonal antibody.
In another embodiment of the present invention there is provided a monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25, wherein the monoclonal antibody binds a Hepatitis A virus antigen.
In another embodiment of the present invention there is provided a recombinant nucleic acid molecule encoding the monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25.
In another embodiment of the present invention there is provided a recombinant nucleic acid molecule encoding the monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25, wherein the nucleic acid comprises a nucleotide sequence as set forth in SEQ ID NO: 28.
In another embodiment there is provided a vector comprising the nucleic acid encoding the monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25.
In another embodiment there is provided a host cell comprising the nucleic acid encoding the monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25.
In one embodiment there is provided a pharmaceutical composition comprising the monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25 and a pharmaceutically acceptable carrier.
In yet another embodiment there is provided a method of detecting Hepatitis A virus in a biological sample, said method comprises contacting the sample with the antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25 and detecting the presence of the immune complex formed between the antibody and Hepatitis A virus antigen, wherein presence of the immune complex confirm presence of Hepatitis A virus in the biological sample.
In further embodiment of the present invention there is provided a kit for detection of Hepatitis A virus, comprising the monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25.
The present invention also provides use of the monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25 for the preparation of anti-Hepatitis A virus medicament.
The present invention further provides a prophylactic and/or therapeutic method of treatment of Hepatitis A virus infection in a subject, said method comprises administering an effective amount of the monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25 to a the subject.
In yet another embodiment there is provided a method of detecting an agents that inhibits Hepatitis A virus replication, said method comprises contacting the monoclonal antibody as claimed in claim 1 with Hepatitis A virus in presence of an agent of interest and assaying binding ability of the monoclonal antibody with Hepatitis A virus, wherein decrease in binding ability of the monoclonal antibody with Hepatitis A virus suggests interference of the agent in antibody -antigen interaction.
The present invention also provides use of the monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25 for detecting Hepatitis A virus in a biological sample.
The monoclonal anti-Hepatitis A antibody as disclosed in the present invention binds to Hepatitis A virus and neutralizes Hepatitis A virus. Further, the antibody can be used as prophylactic, therapeutic and/or diagnostic agent.
The nucleic and amino acid sequence as disclosed or described in the present invention are shown using the standard letter abbreviations for nucleotide bases and single letter codes for amino acid.
In one embodiment there is provided a monoclonal antibody comprising a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25 to a the subject, wherein the antibody is encoded by polynucleotide sequence having the nucleotide sequence as set forth in SEQ ID NO: 28.
In another embodiment of the present invention there is provided monoclonal antibody encoded by the nucleotide sequence of the light chain variable domain that is at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identical to the nucleotide sequence is as set forth in SEQ ID NO: 22, and the heavy chain variable domain that is at least 80%, at least 85%, at least 90%, at least 95% or at least 98% identical to the nucleotide sequence as set forth in SEQ ID NO: 21.
In another embodiment of the present invention there is provided monoclonal antibody encoded by the nucleotide sequence of the light chain variable domain as set forth in SEQ ID NO: 22, and the nucleotide sequence of the heavy chain variable domain as set forth in SEQ ID NO: 21.
EXAMPLES
The invention is described in detail with reference to the example given below. The example is provided just to illustrate the invention and therefore, should not be construed to limit the scope of the invention.
Example 1
Preparation of antibody fragment
RNA extraction and Amplification of Antibody Variable domain sequences
Total RNA was isolated from peripheral blood lymphocytes of vaccinated donors and cDNA was synthesized by RT-PCR. The RT-PCR amplified DNA was used as template for amplification of variable domains of an antibody with universal primers. The amplified variable domains were assembled to form single chain variable fragment (scFv) by splicing by overlap extension PCR (Figure 3) and cloned into phagemid vector to obtain recombinant phagemid vector. The recombinant phagemid vector thus obtained was transformed into E. coli TGI . The transformed E. coli cells comprising the recombinant phagemid vector were infected with M13K07 helper phage to yield recombinant phages.
Phage display scFv library
Human phage displayed scFv library was constructed from immunised peripheral blood lymphocytes. The library comprises over 5 x 107 different scFv fragments cloned in an ampicillin- resistant: phagemid vector pCANTAB5E and transformed into TGI E. coli cells. scFv fragment comprises one variable heavy chain and one variable light chain linked by a peptide linker.
Production of the phage librar
Production of the phage was performed essentially as described (Marks, J.D., Hoogenboom, H.R., Bonnert, T P., McCafferty, J., Griffiths, A.D., Winter, G.s 1991. By-passing immunization, Human antibodies from V-gene libraries displayed on phage. J.Mol. Biol. 222: 581-597). Briefly, E. coli TGI cultures in the exponential phase were infected with helper phage M13K07 at multiplicity of infection of 10 to the bacteria. The bacteria infected with helper phage were grown over night for phage production. The phages were PEG precipitated and used for Biopanning.
Selection of recombinant antibodies against Hepatitis A virus from a human immunised antibody library by biopanning
Phages displaying individual human scFv antibody fragments were selected by three rounds of biopanning on immunotubes coated with a decreasing amount of purified Hepatitis A antigen for the first, second and third round of panning. Eluted phage titers showed an increase after each of the three rounds of panning, indicating successful enrichment (Table.l). Eluted phage from round 3 was used to infect E. coli TGI and randomly selected 96 individual clones were tested by phage ELISA against Hepatitis A antigen. Of the 96 clones tested, forty five clones showed positive binding to Hepatitis A virus. Out of forty clones selected few clones based on their high binding affinity for further study.
Analysis of scFv sequences
Twelve clones from each round of Biopanning were selected for sequencing analysis. Phagemid DNA was purified according to the manufacturer protocol and sequenced at MWG Ltd, India.The sequencing primers, T7 promotor and T7 terminator sequences were used respectively for variable heavy and light gene analysis. The entire sequences was submitted to IMGT and verified as the variable domains of the antibody. The polynucleotide sequence (744 base pairs) comprising heavy and light chain regions and linker is as set forth in SEQ ID NO: 28.
Cloning of scFv into pET vector
The schematic representations as depicted in Figure 1 and figure 2 show that the nucleotide sequence encoding monovalent human anti-Hepatitis A antibody comprising scFv was cloned between the EcoRl and Noil sites of pET 20b bacterial expression vector, wherein the size of the scFv is 744 bp in which a 45bp linker (Gly4Ser)3 was used to connect variable regions of heavy (363 bp) and light (336 bp) chains of antibody. The pET 20b vector carries ribosome binding site, signal peptide sequence of bacterial pectate lyase, variable fragment of light chain, heavy chain, C-terminal His tag sequence and T7 promoter and T7 terminator. Unique site are shown on the circle map. Άρ' denotes the ampicillin resistance gene.
The vector pET 20b and insert referred to monovalent antibody fragment of size 744 bps was digested with EcoRl and Notl, respectively by incubating at 37°C for 12 hours. The digested products was purified using the kit provided by QIAGEN and kept for various ratios of vector to insert (i.e., 1 :3 and 1 :6) blunt end ligation and incubated at 22°C for 2 hours. The ligated product was incubated for further 20 minutes at 65°C in order to inactive the enzyme. The final product was chemically transformed into XL-Blue strain of E. coli cells.
E. coli transformation
Preparation of Chemical competent XL-Blue strain of E.coli cells
Overnight grown XL-Blue strain were sub-cultured and grown at 37°C, shaking until the OD of the culture reaches to 0.6 at 600nm. The culture was harvested by centrifuging at 5000 x rpm for 10 minutes at 4°C and resuspended in ice-cold 0.1 mM CaCl2 and incubated overnight on the ice, before proceeding for transformation.
Transformation
The chemically competent XL-Blue cells were incubated with plasmid DNA for 30 minutes on ice. The cells were given heat shock at 42°C for 90 seconds and immediately placed the tubes on ice for 2 minutes before the media was added to cells. The cells were incubated for one hour at 37°C for recovery and plated on containing lOOmg/ml of ampicillin. The plates were incubated for overnight and screened for positive clones by isolating the plasmids and subjected to digestion with EcoRl and Notl. The positive clones were sequence verified before the plasmid was transformed into BL21 (DE3) cells of E.coli for soluble expression of the antibody gene.
Example 2
Production of soluble scFv fragments and reactivity with Hepatitis A virus antigen
Positive scFv clones identified by phage ELISA were transformed into non- suppressor Escherichia coli strain BL21 (DE3) for production of soluble scFv's. Individually selected clones were grown and production of scFv was induced by addition of ImM isopropyl-p-D-thiogalactopyranoside (IPTG). The purified protein eluted fractions of scFv fragments were analyzed by SDS-PAGE analysis under reducing conditions after staining with coomassie brilliant blue (Figure 4) and immunoblot analysis under reducing conditions revealed a major band with an apparent molecular weight of about 30 kDa (Figure 5 ) which was probed with 6x Histidine tag HRP. The eluted fractions were screened for antigen binding by ELISA using precoated Hepatitis A antigen commercial kit.
Example 3
Determination of binding activity of scFv by Indirect ELISA
Indirect ELISA was done to determine the binding activity of the scFv. Titration of scFv against the precoated ELISA plate, revealed concentration dependent reduction of the optical density values (Figure 6). The control showed no increase in ELISA values indicating the specificity of scFv.
Example 4
Competitive ELISA
The competitive ELISA was performed using commercial kit to check for competition between scFv and Hepatitis A kit Mab. Competition was done by incubating the Kit Mab with varying dilutions of scFv. No increase of optical density values following the dilution of scFv indicating the scFv is not competing with kit Mab for the same site on Hepatitis A virus (Figure 7) was observed. Example 5
Specificity of scFv
Sandwich ELISA was performed to determine the binding specificity of the scFv to Hepatitis A virus. The presence of reactivity of the scFv against Hepatitis A and not to other viruses (rabies, hepatitis B, and Chikungunya viruses) indicates specificity of the scFv against Hepatitis A (Figure 8).
Example 6
Specificity of single chain variable fragment to Hepatitis A virus: Indirect Fluorescent Antibody Technique
The scFv and positive control immunised mouse serum were added to Hepatitis A virus infected and uninfected live FRhk-4 cells in suspension and incubated with anti-mouse and anti-His IgG FITC in the respective wells and observed under an epi-fluorescence microscope. Single chain variable fragment bound specifically to the membranes of Hepatitis A virus infected FRhk-4 cells (Figure 9).
Example 7
Immuno-capture ELISA for quantification of antigen in human Hepatitis A virus vaccine batches.
Immunocapture ELISA was performed to detect the hepatitis A virus antigen in vaccine preparation, wherein the scFv was used as detection reagent. Titration of the vaccine against a constant dilution of scFv revealed a concentration dependent reduction of the binding signal. NIBSC sample was used as reference standard and the kit MAb used as a positive control. Linear graph was plotted for NIBSC standard and estimated the antigen content in vaccine batch. (Fig.10)
Table 1 : Enrichment of Hepatitis A virus specific scFv clones in three rounds of Biopanning
Figure imgf000020_0001
Forward Primers for amplification of Human heavy chain variable domain:
SEQ ID NO: l : HuVHla
GGCGGCGGCGGCTCCGGTGGTGGTCAGGTGCAGCTGGTGCAGTCTGG SEQ ID NO: 2 HuVH2a
GGCGGCGGCGGCTCCGGTGGTGGTCAGGTCAACTTAAGGGAGTCTGG SEQ ID NO: 3 HuVH3a
GGCGGCGGCGCCTCCGGTGGTGGTGAGGTGCAGCTGGTGGAGTCTGG SEQ ID NO: 4 HuVH4a
GGCGGCGGCGGCTCCGGTGGTGGTCAGGTGCAGCTGCAGGAGTCGGG SEQ ID NO: 5 HuVH5a
GGCGGCGGCGGCTCCGGTGGTGGTGAGGTGCAGCTGTTGCAGTCTGC SEQ ID NO: 6 ; HuVH6a
GGCGGCGGCGGCTCCGGTGGTGGTCAGGTACAGCTGCAGCAGTCAGG
Reverse Primers for amplification of Human heavy chain variable domain
SEQ ID NO: 7 HuJHl-2
GGAATTCTGAGGAGACGGTGACCAGGGTGCC
SEQ ID NO: 8 HuJH3
GGAATTCTGAGGAGACGGTGACCATTGTCCC
SEQ ID NO: 9 HuJH4-5
GGAATTCTGAGGAGACGGTGACCAGGGTTCC
SEQ ID NO: 10 HuJH6
GGAATTCTGAGGAGACGGTGACCGTGGTTCC
Forward Primers for amplification of Human light chain variable domain
SEQ ID NO: 11 HuLAMl GCCATGGCGCAGTCTGTGTTGACGCAGCCGCC
SEQ ID NO: 12 HuLAM2
GCCATGGCGCAGTCTGCCCTGACTCAGCCTGC
SEQ ID NO: 13 HuLAM3a
GCCATGGCGTCCTATGTGCTGACTCAGCCACC
SEQ ID NO: 14 HuLAM3b
GCCATGGCGTCTTCTGAGCTGACTCAGGACCC
SEQ ID NO: 15 HuLAM4
GCCATGGCGCACGTTATACTGACTCAACCGCC
SEQ ID NO: 16 HuLAM5
GCCATGGCGCAGGCTGTGCTCACTCAGCCGTC
SEQ ID NO: 17 HuLAM6
GCCATGGCGAATTTTATGCTGACTCAGCCCCA
Reverse Primers for amplification of Human light chain variable domain
SEQ ID NO: 18 HuJLAM 1
GGAGCCGCCGCCGCCAGAACCACCACCACCAGAACCACCACCACCACC TAGGACGGTGACCTTGGTCCC
SEQ ID NO: 19 HuJLAM2-3
GGAGCCGCCGCCGCCAGAACCACCACCACCAGAACCACCACCACCACC TAGGACGGTCAGCTTGGTCCC
SEQ ID NO: 20 HuJLAM4-5
GGAGCCGCCGCCGCCAGAACCACCACCACCAGAACCACCACCACCACC TAAAACGGTGAGCTGGGTCCC
SEQ ID NO: 21 Nucleotide sequence of heavy chain variable domain (363 nts) CAGGTCACCTTGAGGAGTCTGGTCCTACGCTGGGGAAACCCACACAGA CCCTCACGCGGACCTGCACCTTCTCTGGGTTCTCACTCAGCACTAGTGG
AGGGGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTGGAGT
GGCTTGCACTCATTTATTGGAATGATGATAAGCGCTACAGCCCATCTCT
GAAGAGCAGGCTCACCATCACCAAGGACACCTCCAAAAACCAGGTGGT
CCTTACAATGACCAACATGGACCCTGTGGACACAGCCACATATTACTGT
GCACACAGGAGACTACCAGCAGCTGTCCGAATCTTTAATATCGGCCAA
GGGACTCTGGTCACTGTCTCTTCT
SEQ ID NO: 22 Nucleotide sequence of light chain variable domain (336 nts)
ATGGGGAAGGAGGTTGTTTTAGAGAGAAAGTTGTTAATAGACGTAGGG
GTGGTCTTAAACCGGGCGAGGCCATTTCTTGCTTGGAGCGCTAACATCG
GAGGAACGGGAACTGGTACTGCAGCTCCCAGGAACGGCCCAAAAGTA
ATCATCTATAGGAATGCTCAGCGGCCCTCAGGGGTCCCTGACCGATTCT
CCGGCTCCAAGTCTGGTACTTCAGCCTCCCTGGCCGTCAGTGGGCTCCA
GTCTGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGTCT
GAATGCTTGGGTGTTCGGCGGAGGGACCAAGGTGACCGTCCTAGGT
SEQ ID NO: 23 Nucleotide sequence of linker (45 nts)
GGTGGTGGTGGTTCAGGTGGTGGTGGTTCTGGTGGTGGTGGTTCT
SEQ ID NO: 24 Amino acid sequence of heavy chain variable domain
QVTLRSLVLRWGNPHRPSRGPAPSLGSHSALVEGGVGWIRQPPGKALEWL
ALIYWNDDKRYSPSLKSRLTIT DTSK^QVVLTTViTNMDPVDTATYYCAH
RRLPAAVRIFNIGQGTLVTVSS
SEQ ID NO: 25 Amino acid sequence of light chain variable domain
MGKEWLERKLLIDVGVYLNRARPFLAWSANIGGTGTGTAAPRNGPKVII YRNAQRPSGVPDRFSGSKSGTSASLAVSGLQSEDEADYYCAAWDDSLNA
WVFGGGT VTVLG
SEQ ID NO: 26 Amino acid sequence of linker
GGGGSGGGGSGGGGS
SEQ ID NO: 27 Amino acid sequence of recombinant scFv (Light chain: amino acid position 1-112; Linker: 113-127; Heavy chain: 128-248)
MGKEVVLERKLLIPVGWLNRARPFLAWSA IGGTGTGTAAPRNGPKVII YR AQRPSGVPDRFSGSKSGTSASLAVSGLQSEDEADYYCAAWDDSLNA
WVFGGGTKVTVLGGGGGSGGGGSGGGGSQVTLRSLVLRWGNPHRPSRGP APSLGSHSALVEGGVGWIRQPPG ALEWLALIYWNDD RYSPSLKSRLTIT KDTSKNQVVLTMTNMDPVDTATYYCAHRRLPAAVRIF IGQGTLVTVSS
SEQ ID NO: 28 Nucleotide sequence of recombinant scFv (744 nts; Light chain: 1- 336 nts; Linker: 337-381; Heavy chain: 382-744)
ATGGGGAAGGAGGT GTTTTAGAGAGAAAGTTGTTAATAGACGTAGGG
GTGGTCTTAAACCGGGCGAGGCCATTTCTTGCTTGGAGCGCTAACATCG
GAGGAACGGGAACTGGTACTGCAGCTCCCAGGAACGGCCCAAAAGTA
ATCATCTATAGGAATGCTCAGCGGCCCTCAGGGGTCCCTGACCGATTCT
CCGGCTCCAAGTCTGGTACTTCAGCCTCCCTGGCCGTCAGTGGGCTCCA
GTCTGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGTCT
GAATGCTTGGGTGTTCGGCGGAGGGACCAAGGTGACCGTCCTAGGTGG
TGGTGGTGGTTCAGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTCAGGTC
ACCTTGAGGAGTCTGGTCCTACGCTGGGGAAACCCACACAGACCCTCA
CGCGGACCTGCACCTTCTCTGGGTTCTCACTCAGCACTAGTGGAGGGGG
GTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTGGAGTGGCTTG
CACTCATTTATTGGAATGATGATAAGCGCTACAGCCCATCTCTGAAGAG
CAGGCTCACCATCACCAAGGACACCTCCAAAAACCAGGTGGTCCTTAC
AATGACCAACATGGACCCTGTGGACACAGCCACATATTACTGTGCACA
CAGGAGACTACCAGCAGCTGTCCGAATCTTTAATATCGGCCAAGGGAC
TCTGGTCACTGTCTCTTCT

Claims

WE CLAIM:
1. A monoclonal antibody comprising
a. a heavy chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 24; or a functional fragment or a variant thereof; and
b. a light chain variable domain comprising amino acid sequence as set forth in SEQ ID NO: 25 or a functional fragment or variant thereof.
2. The monoclonal antibody as claimed in claim 1, wherein the amino acid sequence of the monoclonal antibody is as set forth in SEQ ID NO: 27.
3. The monoclonal antibody as claimed in claim 1, wherein the heavy chain variable domain is encoded by the nucleotide sequence as set forth in SEQ ID NO: 21.
4. The monoclonal antibody as claimed in claim 1, wherein the light chain variable domain is encoded by the nucleotide sequence as set forth in SEQ ID NO: 22.
5. The monoclonal antibody as claimed in claim 1 is a monovalent or divalent antibody.
6. The monoclonal antibody as claimed in claim 1 is a human monoclonal antibody.
7. The monoclonal antibody as claimed in claim 1, wherein the monoclonal antibody binds a Hepatitis A virus antigen.
8. A recombinant nucleic acid molecule encoding the monoclonal antibody as claimed in claim 1.
9. The recombinant nucleic acid as claimed in claim 8, wherein the nucleic acid comprises a nucleotide sequence as set forth in SEQ ID NO: 28.
10. A vector comprising the nucleic acid as claimed in claim 8 or 9.
11. A host cell comprising the nucleic acid as claimed in claim 8 or 9.
12. A pharmaceutical composition comprising the antibody as claimed in claim 1 and a pharmaceutically acceptable carrier.
13. A method of detecting Hepatitis A virus in a biological sample, said method comprises
a) contacting the sample with the antibody as claimed in claim 1 ; and
b) detecting the presence of the immune complex formed between the antibody and Hepatitis A virus antigen, wherein presence of the immune complex confirm presence of Hepatitis A virus in the biological sample.
14. A kit for detection of Hepatitis A virus, comprising the monoclonal antibody as claimed in claim 1.
15. A method of detecting an agents that inhibits Hepatitis A virus replication, said method comprises
a. contacting the monoclonal antibody as claimed in claim 1 with Hepatitis A virus in presence of an agent of interest; and b. assaying binding ability of the monoclonal antibody with Hepatitis A virus
wherein decrease in binding ability of the monoclonal antibody with Hepatitis A virus suggests interference of the agent in antibody-antigen interaction.
16. Use of the monoclonal antibody as claimed in claim 1 for detecting Hepatitis A virus in a biological sample.
PCT/IN2011/000176 2010-03-19 2011-03-16 Monovalent human anti-hepatitis a virus antibody and uses thereof WO2011114353A1 (en)

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CN111138533A (en) * 2019-12-30 2020-05-12 南京融捷康生物科技有限公司 Single domain antibody against hepatitis A virus and derived protein thereof
CN116514965A (en) * 2023-06-15 2023-08-01 上海精翰生物科技有限公司 Hepatitis A virus antibody and application thereof

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Publication number Priority date Publication date Assignee Title
CN111138532A (en) * 2019-12-30 2020-05-12 南京融捷康生物科技有限公司 Use of single domain antibodies against hepatitis a virus
CN111138533A (en) * 2019-12-30 2020-05-12 南京融捷康生物科技有限公司 Single domain antibody against hepatitis A virus and derived protein thereof
CN111138532B (en) * 2019-12-30 2022-05-20 南京融捷康生物科技有限公司 Use of single domain antibodies against hepatitis a virus
CN111138533B (en) * 2019-12-30 2022-06-10 南京融捷康生物科技有限公司 Single domain antibody against hepatitis A virus and derived protein thereof
CN116514965A (en) * 2023-06-15 2023-08-01 上海精翰生物科技有限公司 Hepatitis A virus antibody and application thereof
CN116514965B (en) * 2023-06-15 2023-11-10 上海精翰生物科技有限公司 Hepatitis A virus antibody and application thereof

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