# Patent

## Title
H1N1 flu virus neutralizing antibodies

## Abstract

An antibody, or a binding fragment of the antibody, against H1N1 virus, includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains complementarity determining regions (CDR) that have the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7; and wherein the light chain variable region contains complementarity determining regions that have the amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. A method for treating or preventing H1N1 infection in a subject includes administering to the subject the antibody or the binding fragment of the antibody.

## Background
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates generally to the fields of treatment and prevention of influenza virus infections using antibodies or fragments thereof. More particularly, it relates to the development of monoclonal antibodies for use in the diagnosis, prevention and treatment of H1N1 infections.
Background
When an infection breaks out, finding an effective treatment or prevention in a timely manner is critical in order to control the spread of the disease. The urgency of finding effective treatments is highlighted by recent outbreaks, such as the H1N1 outbreak of 2009.
The 2009 H1N1 influenza outbreak inflicted more than 60 million patients and more than a quarter million required hospitalization. The 2009 H1N1 pandemic influenza virus, which is designated as A(H1N1)pdm09 by WHO, disproportionally caused severe syndromes in young children and the elderly.
The highly infectious nature of H1N1 prompted an unusually swift and intense effort to find a vaccine to prevent further spread of this illness. Due to this intense effort, an effective H1N1 vaccine, PANFLU.1, became available five months after the outbreak. This vaccine was effective in inducing immune responses in more than 90% of people aged between 12 and 60 years old. However, this vaccine was less effective in children and elderly and was ineffective in people with compromised immune system.
To have an effective counter measure during an outbreak, it is important to have passive immunotherapy to provide immediate protection. In addition, passive immunotherapy can also be used to treat infected individuals. Passive immunotherapies require antibodies that are effective in the preventing infection by the infectious agents. Such antibodies may be developed using various technologies, typically using a virus-derived antigen or an inactivated virus to induce antibodies in a suitable subject. In addition, such antibodies may be isolated from patients who had been infected by the virus and have since recovered from the illness.
For example, Hao Wang et al. (Cellular & Molecular Immunology, (2013) 10, 403-412) discloses an approach for screening viral neutralizing monoclonal antibodies (mAbs) from individuals vaccinated with the 2009 pandemic H1N1 vaccine PANFLU.1. Specifically, B cells from immunized individuals were isolated and immortalized by fusion with Epstein-Barr virus (EBV). The Epstein-Barr virus (EBV)-immortalized memory B cells were screened. From this screening, seven mAbs were identified with both high viral neutralizing capacities and hemagglutination inhibition (HAI) activities against the 2009 pandemic H1N1 viruses. These mAbs may be used for passive immunotherapy of infected patients.
While screening from memory B cells derived from donors vaccinated with a specific antigen can provide useful monoclonal antibodies against H1N1, there is still a need for better antibodies for the prevention and treatment of influenza infections (e.g., H1N1 infection).
SUMMARY OF THE INVENTION
Embodiments of the present invention relate to antibodies or binding fragments thereof against H1N1. The binding fragments of such antibodies may include Fab, scFv, F(ab′)2, etc. Some embodiments of the invention relate to vectors or host cells for expressing such antibodies or fragments. Some embodiments of the invention relate to methods for preventing or treating H1N1 infection.
One aspect of the invention relates to antibodies or binding fragments thereof, against H1N1 virus. An antibody or a binding fragment thereof in accordance with one embodiment of the invention includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains complementarity determining regions (CDR) that have the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7; and wherein the light chain variable region contains complementarity determining regions that have the amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.
In accordance with any of the above embodiments, the antibody may comprise a heavy chain having the amino acid sequence of SEQ ID NO: 2 and a light chain having the amino acid sequence of SEQ ID NO: 4. In accordance with any of the above embodiments, the H1N1 virus is a 2009 pandemic strain or a post 2009 strain.
Another aspect of the invention relates to expression vectors for expressing an antibody in accordance with embodiments of the invention. An expression vector in accordance with one embodiment of the invention comprises a polynucleotide sequence encoding an antibody, or a binding fragment thereof, wherein a heavy chain variable region of the antibody comprises complementarity determining regions (CDR) having the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7; and a light chain variable region of the antibody comprises complementarity determining regions having the amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.
In accordance with any of the above expression vectors, the polynucleotide sequence comprises a first polynucleotide region encoding for a heavy chain of the antibody that has the amino acid sequence of SEQ ID NO:2 and a second polynucleotide region encoding a light chain of the antibody that has the amino acid sequence of SEQ ID NO:4.
In accordance with any of the above expression vectors, the first polynucleotide region comprises the sequence of SEQ ID NO:1 and the second polynucleotide region comprises the sequence of SEQ ID NO:3.
In accordance with any of the above expression vectors, the polynucleotide sequence is linked with a glutamine synthase (GS) gene that encodes glutamine synthase.
Another aspect of the invention relates to host cells. A host cell of the invention may comprise any of the above expression vectors. The host cells may be a mammalian cell, a yeast cell, or an insect cell, such as CHO cells, Pichia cells, or sf9 cells.
Another aspect of the invention relates to methods for treating or preventing H1N1 infection. A method for treating or preventing H1N1 infection in accordance with one embodiment of the invention comprises administering to a subject in need of such treatments any antibody of the invention or a binding fragment thereof.
Another aspect of the invention relates to methods for detecting H1N1. A method for detecting H1N1 in accordance with embodiments of the invention comprises contacting a test sample with any of the above-described antibodies, or a binding fragment thereof; and determining presence or absence of binding with the antibody, or the binding fragment thereof, wherein presence of the binding indicates existence of H1N1 virus in the test sample.
Other aspects of the invention would be apparent to one skilled in the art in view of the drawings and the detailed description.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a flow chart illustrating one method for identifying an antibody from memory B cells in accordance with one embodiment of the invention.
FIG. 2(A) shows SDS PAGE and FIG. 2(B) shows a Western blot of isolated 32D6 antibody in accordance with embodiments of the invention. FIG. 2(C) shows the binding of 32D6 and HA using Biacore assay in accordance with embodiments of the invention.
FIG. 3(A) shows control cells, FIG. 3(B) shows cells infected with H1N1, and FIG. 3(C) shows the ability of 32D6 to neutralize the virus infection of the cells in accordance with embodiments of the invention.
FIG. 4(A) shows concentration dependence of the 32D6 activities in neutralizing H1N1 infection of the cells in accordance with embodiments of the invention. FIG. 4(B) shows a comparison of the activity of 32D6 in neutralizing virus infection of cells, as compared with another antibody 43G3, in accordance with embodiments of the invention. FIG. 4(C) shows the selectivity for H1N1 by 32D6. FIG. 4(D) shows that 32D6 cannot neutralize the infection abilities of H5N1 and H7N9 viruses. FIG. 4(E) shows that 32D6 is effective in neutralizing post pandemic (2009) strains of H1N1 viruses. FIG. 4(F) shows that 32D6 is not effective in neutralizing pre-pandemic (2009) strains of H1N1 viruses.
FIG. 5(A) shows the heavy chain nucleic acid sequences with CDR sequences underlined for 32D6. FIG. 5(B) shows the heavy chain amino acid sequences with CDR sequences underlined for 32D6. FIG. 5(C) shows the light chain nucleic acid sequences with CDR sequences underlined for 32D6. FIG. 5(D) shows the light chain amino acid sequences with CDR sequences underlined for 32D6. FIG. 5(E) shows the CDR sequences for 32D6.
FIG. 6 shows a map of an expression vector, pXC-17.4, in accordance with one embodiment of the invention.
FIG. 7(A) shows prophylactic effects of 32D6 in preventing H1N1 virus infection in animal models in accordance with one embodiment of the invention. FIG. 7(B) shows the treatment effects of 32D6 in preventing H1N1 virus infection in animal models in accordance with one embodiment of the invention.



DEFINITION
Unless otherwise defined, scientific and technical terms used herein shall have the meanings that are commonly understood by those of ordinary skill in the art. Furthermore, unless otherwise required by context, singular terms may include pluralities and plural terms may include the singular. A list of abbreviations used herein is as follows: Ab: antibody; CDRs: complementarity determining regions; CH: heavy-chain constant domain; CL: light-chain constant domain; PBS: phosphate buffered saline; PCR: polymerase chain reaction; scFv: single-chain variable fragment; VL: light-chain variable domain; VH: heavy-chain variable domain; HA: hemagglutinin; HAI: inhibition of hemagglutinination; mAb: monoclonal antibody; hmAb: human monoclonal antibody.
Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. These techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001).
As used herein, the term “antibody” refers generally and broadly to immunoglobulins, monoclonal antibodies, and polyclonal antibodies, as well as active fragments thereof. The fragment may be active in that it binds to the cognate antigen (e.g., HA or a virus).
As used herein, the term “human antibody” refers to an antibody in which substantially every part of the protein is substantially non-immunogenic in humans, with no or only minor sequence changes or variations, as compared with native human antibodies.
An antigen binding site is generally formed by the heavy chain variable region (VH) and the light chain variable region (VL) immunoglobulin domains, with the antigen-binding interface formed by six surface polypeptide loops, termed complimentarity determining regions (CDRs). There are three CDRs each in VH (HCDR1, HCDR2, HCDR3) and VL (LCDR1, LCDR2, LCDR3), together with framework regions (FRs) between or surrounding the CDRs.
The term “CDR region” or “CDR” means the hypervariable regions of the heavy or light chains of the immunoglobulin as defined by Kabat et al., 1991 (Kabat, E. A. et al., (1991) Sequences of Proteins of Immunological Interest, 5th Edition. US Department of Health and Human Services, Public Service, NIH, Washington), and later editions. An antibody typically contains 3 heavy chain CDRs and 3 light chain CDRs.
A binding fragment of an antibody of the invention may be a Fab, a Fab′, a F(ab′)2, or a scFv fragment. The binding fragments of an antibody can be obtained by a variety of methods, including, digestion of an intact antibody with an enzyme, such as pepsin (to generate (Fab′)2 fragments) or papain (to generate Fab fragments); or de novo synthesis. Antibody fragments can also be synthesized using recombinant DNA methodology. See, e.g., Fundamental Immunology (Paul ed., 4d ed. 1999); Bird, et al., Science 242:423 (1988); and Huston, et al., Proc. Natl. Acad. Sci. USA 85:5879 (1988).
DETAILED DESCRIPTION OF THE INVENTION
Embodiments of the invention relate to anti-H1N1 antibodies or fragments thereof. The antibodies of the invention may be generated using molecular biology techniques by screening immortalized memory B cells from healthy donors without the need for prior vaccination. Fragments of these antibodies may include Fab, scFv, F(ab′)2, etc. Antibodies of the invention have high viral neutralizing efficacies.
In accordance with embodiments of the invention, neutralizing antibodies against H1N1 viruses may be identified with a process shown in FIG. 1, which is similar to the process used in Hao Wang et al mentioned above. However, the memory B cells may be from healthy donors who need not have been vaccinated with PANFLU.1. As shown, the first step 11 involves isolating memory B cells from donors. The donors are healthy donors with or without prior exposure to the H1N1 virus or vaccines. The isolated memory B cells are then immortalized with EBV (step 12). Then, the EBV-immortalized B cells are screened for their abilities to secret antibodies that can bind to hemagglutinin (HA) and/or inhibit HA activity (i.e., hemagglutination) (step 13). Finally, the memory B cells identified in step 13 are further screened for their abilities to secret antibodies that can neutralize the viruses (step 14).
Using the protocols described above, a large number (20160 single B cell clones) of immortalized memory B-cell clones were screened. As result of this screening, several clones that can bind HA and/or inhibit HA activities were identified. Among these, one hmAb (32D6) was identified that has good binding affinity to hemagglutin (HA), viral neutralizing capacities and hemagglutination inhibition (HAI) activities against the 2009 pandemic H1N1 virus. The standard name for the H1N1 2009 pandemic strain is A(H1N1)pdm09, according to the recommendation of WHO.
The hmAb 32D6 secreted by the isolated clone of immortalized memory B cells was purified and the purity of the hmAb 32D6 was checked by SDS-PAGE. As shown in FIG. 2(A), Coomassie staining revealed that the recombinant antibody is relatively pure and its relative molecular weight is similar to that of human IgG (hIgG). FIG. 2(B) shows Western blot analysis of the purity of mAb 32D6. The Western blot analysis was performed using a goat anti-human IgG antibody fragment (F(ab′)2 fragment from Jackson Lab), which was affinity purified. This F(ab′)2 fragment is specific for IgG and is conjugated with horseradish peroxidase.
The HA binding affinity of hmAb 32D6 was assessed using surface plasmon resonance (Biacore T200). Briefly, hmAb 32D6 antibody was immobilized on CM5 chips at a density that allowed one to achieve Rmax in the range of 0-500 Response Units (RU). In this example, the kinetic assay parameters were as follows: data collection rate 1 Hz; dual detection mode; temperature: 25° C.; and concentration unit: nM. Serial dilutions of hemagglutinin (HA) in the running buffer were flowed into the system to bind with the hmAb 32D6 antibody. These afforded binding kinetic parameters. Then, the HA-antibody complex was washed by flowing buffer into the system to derive the dissociation kinetic parameters.
The results were evaluated with the BIAcoreT200 evaluation software. The binding responses were corrected for buffer effects by subtracting responses from a blank flow cell. A 1:1 Langmuir fitting model was used to estimate the ka or kon (association rate or on-rate) and kd or koff (dissociation rate or off-rate). The KD (or Kd) values may be determined from the ratios of koff and kon (i.e., KD=koff/kon).
As shown in FIG. 2(C), 32D6 is found to have a very high affinity for HA. In this example, the KD of 32D6 is found to be in the range of 10−10 M or better. Other clones were also identified, but were found to have lower affinities.
The hmAb 32D6 is effective in neutralizing H1N1 virus activity. As shown in FIGS. 3(A)-3(C), at 2,000 PFU per well (1×104 MDCK cells per well in 384-well format plate, greiner, Germany), H1N1 virus infects cells with high efficiency (FIG. 3(B)). However, in the presence of 0.01 μg/well hmAb 32D6, infection of the cells by 2,000 PFU/well H1N1 virus was completely prevented (FIG. 3(C)). FIG. 3(A) shows the cells without the virus or mAb as a control. These results indicate that hmAb 32D6 is very effective in neutralizing the activity of H1N1, even at a low concentration (0.01 μg/well).
To quantify the efficacy of hmAb 32D6 in neutralizing H1N1, the neutralizing assays were further performed using various concentrations of hmAb 32D6 and 2,000 PFU/well H1N1 in a cell invasion assay using MDCK cells as described above. A virus control (without adding hmAb 32D6) is shown as a control. As shown in FIG. 4(A), hmAb 32D6 is found to completely neutralize the activity of H1N1 at a concentration of 0.003 μg/per well. Based on the results from the extents of neutralization as a function of hmAb 32D6 concentrations, the IC50 value is estimated to be around 0.001 μg/well. In contrast, another clone mAb 43G3 is barely effective in neutralizing H1N1 viruses even at high concentrations (FIG. 4(B)).
This antibody, hmAb 32D6, is specific for H1N1. As shown in FIGS. 4(C) and 4(D), 32D6 is very effective in neutralizing the activities of H1N1. However, it is not effective in neutralizing H3N2 (FIG. 4(C)), H5N1 or H7N9 (FIG. 4(D)). This specificity indicates that the antibody likely recognize epitope(s) on HA that are unique to the H1N1 strain.
In addition to being highly effective in neutralizing H1N1 virus of the 2009 pandemic strain, hmAb 32D6 was also found to be highly specific for H1N1 strains post 2009 pandemic (FIG. 4(E)). In contrast, hmAb 32D6 is not effective against the pre-2009 H1N1 strains (FIG. 4(F)). This impressive selectivity is not expected even though the recombinant HA used for the screening was derived from the ectodomain DNA fragment of A/Sichuan/1/2009 (H1N1) HA gene. This unexpected selectivity likely arises from a new epitope that did not exist in H1N1 viruses prior to 2009. Preliminary data from our attempts to locate the epitope suggest that the epitope may be located from around residue 189 to around residue 272 of HA.
The native human Ab gene (8B4) corresponding to hmAb 32D6 was cloned and sequenced. The nucleotide sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of the heavy chain of 8B4 gene are shown in FIG. 5(A) and FIG. 5(B), respectively. The nucleotide sequence (SEQ ID NO: 3) and amino acid sequence (SEQ ID NO: 4) of the light chain of 8B4 gene are shown in FIG. 5(C) and FIG. 5(D), respectively. In these sequences, the CDR sequences are underscored. The six CDR sequences of 32D6 are shown in FIG. 5(E): SEQ ID NOS: 5-10.
Using the 8B4 gene sequence, expression vectors may be constructed. One skilled in the art would appreciate that any suitable expression vectors may be used. For example, the Lonza GS System pXC-17.4 plasmid (Lonza Group, Basel, Switzerland) may be used in conjunction with a glutamine synthase (GS) knockout host cell. Using the pXC-17.4 plasmid and a GS Chinese Hamster Ovary (CHO) cell line from Lonza, a recombinant human monoclonal antibody (rhmAb) (8B4) has been obtained.
For example, FIG. 6 shows an example of a pXC-17.4 vector that may be used to expression an antibody of the invention. In addition, any expression vector of the invention may be transfected into a suitable host cell, such as a mammalian cell, a yeast cell or an insect cell.
This rhmAb was found to have identical antibody properties as those of the native hmAb (32D6), with respect to the binding affinity, virus neutralizing activities, and HAI activities. These results indicate that the viral binding and neutralizing function of antibody 32D6 are not affected by glycosylation (or different glycosylation) in CHO cells.
One skilled in the art would appreciate that as long as the CDR sequences are retained, the antibody activities can be preserved. Thus, in addition to hmAb 32D6, any antibodies (e.g., recombinant antibodies) or binding fragments thereof would have the same binding specificity and similar affinities. That is, useful antibodies are not limited to hmAb 32D6 itself, but can also include other antibodies or binding fragments thereof (e.g., Fab fragment, F(ab′)2 fragment, single-chain Fv (scFv), etc.) that retain the same binding. For examples, such fragments containing the same CDR sequence are likely to retain the binding. The CDR sequences of 32D6 are summarized in the table shown in FIG. 5(E) with their SEQ ID NOs: 5-10 are also shown.
The hmAb 32D6 has tight binding to HA and high activity in neutralizing H1N1 virus. Therefore, this hmAb should be useful in preventing or treating H1N1 infections. The effectiveness of 32D6 in preventing and treating H1N1 infections is assessed with the in vivo tests.
In pharmacodynamic (PD) evaluation using an animal model, passive immunization with 32D6 displayed anti-H1N1 efficacy in in vivo protection, both prophylactically and therapeutically, against a lethal viral challenge in mice. Therefore, hmAb 32D6 may have important clinical implications for passive immune therapy of infected patients with severe respiratory syndrome, especially children, the elderly and immunodeficient individuals.
Embodiments of the invention will be further illustrated with the following examples with specific experimental procedures. One skilled in the art would appreciate that these examples are for illustration only and are not meant to limit the scope of the protection.
EXAMPLES
Cell Cultures and Virus Preparation
The marmoset B-lymphoblastoid cell line B95-8 and 293T cells were cultured in RPMI 1640 medium containing L-glutamine (Invitrogen, Carlsbad, Calif., USA) supplemented with 10% fetal calf serum (FCS) and penicillin-streptomycin at 37° C. in a humidified incubator with 5% CO2. Madin-Darby canine kidney (MDCK) cells were cultured in DMEM medium (Invitrogen) supplemented with 10% FCS. Spodoptera frugiperda (Sf9) cells were maintained in Sf-900 II SFM (Invitrogen).
H1N1 virus was generated by Medigen Vaccine Biologics Co. (MVC, Taiwan). Influenza A virus A/California/7/2009 NYMCX-179A (H1N1), this strain is a pandemic vaccine strain recommended for use in vaccine development. Other influenza virus clinical strains are provided by Taiwan CDC and used for neutralizing assay in this patent application, 2013-80813 (A/Taiwan/80813/2013 (H1N1)), 2013-INF-152(2013-00586; A/California/07/2009), 2013-INF-153 (2013-01141; A/California/07/2009), 2014-INF-36(2014-03006; A/California/07/2009), 2014-INF-37(2014-04001; A/California/07/2009), 2006-INF-00923 (925; A/Solomon_Islands/3e/2006), 2007-INF-00125 (A/Brisbane/59/2007), 2007-INF-01180 (A/NEW_CALEDONIA/20/99), 2009-INF-00480(A/Brisbane/59/2007), 2007-INF-00481 (180; B/Malaysia/2506/2004), 2009-INF-00479(A/Brisbane/59/2007), 2009-INF-00447(A/Brisbane/59/2007). Other influenza viruses used for neutralization assay are: the vaccine strain NIBRG-14M6 (H5N1), the vaccine strain NIBRG-268 (H7N9) and the clinical strain provided by Taiwan CDC 2013-INF-00723 (2013-02803; A/Victoria/361/2011 (H3N2)). Propagation of those influenza virus clinical strains, and determination of the 50% tissue culture infectious dose (TCID50) were performed in Madin-Darby canine kidney (MDCK) cells according to Reed-Muench method.
Purification of Human Memory B Cells
Peripheral blood memory B cells were isolated using a magnetic isolation kit (Miltenyi, Bergisch Gladbach, Germany) as described in A. Henn et al., “Modulation of single-cell IgG secretion frequency and rates in human memory B cells by CpG DNA, CD40L, IL-21, and cell division,” J. Immunol., (2009), 183: 3177-3187. Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors, including vaccinated donors and unvaccinated healthy donors. Non-memory B cells in PBMCs (such as T cells, natural killer cells, monocytes, dendritic cells, granulocytes, platelets and erythroid cells) were removed using biotinylated antibodies against CD2, CD14, CD16, CD36, CD43 and CD235a (glycophorin A), followed by binding with anti-biotin microbeads. After removal of the non-memory B cells, memory B cells may be isolated by magnetic labeling of memory B cells with CD27 microbeads, followed by isolation of the magnetically labeled cells.
The identity and purity of these memory B cells may be assessed using any methods known in the art, such as flow cytometry. For example, the isolated memory B cells, which are CD19+ and CD27+, may be labeled with a surface-staining antibody, such as FITC-conjugated CD27 or PE-conjugated CD19 (Miltenyi Biotech). The stained cells are then analyzed with a flow cytometer. The data may be analyzed using any suitable program, such as CellQuest software (BD, USA).
EBV Preparation and Immortalization of Peripheral Blood Memory B Cells
EBV for immortalization of human B lymphocytes was prepared from B95-8 cells, as described in E. James et al., “Growth of B95-8 cells and expression of Epstein-Barr Virus lytic phase in serum-free medium,” J. Virol. (1987) 61: 4033-4037. For example, B95-8 cells were cultured at a density of 106 cells/ml in RPMI 1640 with 10% (v/v) FCS at 37° C. in 5% CO2. The supernatant was collected and filtered with a sterile filter (0.45 μm; Millipore, Billerica, Mass., USA). The cells may be stored in aliquots in liquid nitrogen.
Peripheral blood memory B cells were EBV-transformed according to the protocol, as described in E. Traggiai et al., “An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus,” Nat. Med. (2004) 10: 871-875.
Briefly, allogenic mononuclear cells (5×105 cells/ml) in RPMI 1640 medium without sera were irradiated by 35 Grey Co60 and plated in 96-well plates (50 000 cells/100 μl per well) in 30% FCS. These irradiated cells were used as feeding cells. Memory B cells were then sorted into a single cell per well into 96-well plates with feeding cells, before the addition of 50 μl mixture per well containing 48 μl of EBV suspension, 1 μl of cyclosporine A solution (at the final concentration of 400 ng/ml) and 1 μl of CpG 2006 (at final concentration of 2.5 μg/ml). After EBV transformation, immortalized memory B-cell clones were expanded for 28 days with medium changed every 3-5 days. The supernatant of each clone (100 μl) was used to screen for positive clones specific for 2009 pandemic (H1N1) HA.
Expression of Recombinant HA Proteins (rHAs)
Recombinant hemagglutinins (rHAs) were expressed and purified using the Baculovirus/insect cell system (BD Biosciences, San Diego, Calif., USA), as described in Z. Li et al., “Serologic cross-reactivity among humans and birds infected with highly pathogenic avian influenza A subtype H5N1 viruses in China,” Immunol. Lett. (2011) 135: 59-63. Briefly, the HA ectodomain DNA fragment of A/Sichuan/1/2009 (H1N1) HA gene was cloned into the transfer vector FastBac (Invitrogen). Recombinant baculovirus genomes were transfected into Sf9 cells for the generation of recombinant baculoviruses. Viral supernatant was collected at 72 h post-infection. rHAs were expressed after three rounds of infection and purified using Ni-NTA chromatography (GE Healthcare, Pittsburgh, Pa., USA). Western blotting confirmed the expression and purification of rHAs with anti-His or anti-HA antibodies.
Enzyme-Linked Immunosorbent Assay (ELISA)
ELISAs were performed using a 96-well plate (Maxisorb, Nunc) to measure the HA-binding activities of sera from donors or hmAbs from immortalized memory B cells, as described in Z. Li et al., “Serologic cross-reactivity among humans and birds infected with highly pathogenic avian influenza A subtype H5N1 viruses in China,” Immunol. Lett. (2011) 135: 59-63. Briefly, ELISA was performed with reagents consisting of: (1) inactivated whole H1N1 virus (diluted in 10 μg/ml), manufactured by MVC; (2) serial dilutions of monoclonal antibodies; (3) goat anti-mouse IgG Ab (γ-chain specific,) conjugated with horseradish peroxidase (Jackson ImmunoResearch Laboratories, INC. USA); and (4) TMB substrates (3,3′,5,5′-Tetramethylbenzidine, Sigma). Inactivated H1N1 virus were coated on polystyrene plates at 4° C. overnight followed by blocking with 5% bovine serum albumin (Invitrogen) at 37° C. for 2 h. The sera/supernatants from donors/b cells at serial of dilution were incubated in wells at 37° C. for 2 h. After complete wash, detection antibody, anti-human IgG with horseradish peroxidase-labeled (dilution in 1:5000) was added at 37° C. for 1 h. The TMB/H2O2 was added to develop color, and the reaction was stopped with 50 μl of H2SO4. The amount of chromogen produced was measured based on absorbance at 410 nm and 630 nm using an ELISA reader (SpectraMax, MD, US). The clones that bind to the inactivated H1N1 viruses in the above ELISA assays were further confirmed using recombinant HA (rHA) as the ligand in a second ELISA assays.
Neutralization Assays—Activity Determined by High Content Image System
Neutralization assays of sera or hmAbs were performed with methods known in the art and modified according to procedures known in the art, see Z. Li Z et al., “Identification of amino acids in highly pathogenic avian influenza H5N1 virus hemagglutinin that determine avian influenza species specificity,” Arch. Virol. (2011), 156: 1803-1812. Briefly, MDCK cells were pre-cultured in DMEM-base serum free medium containing 1% P/S, 1% BSA, 25 mM HEPES and 2 ug/ml TPCK-trypsin before neutralizing assay. Serial dilutions of sera, cell culture supernatant, or purified 32D6/8B4 hmAbs were incubated with H1N1 (A/Taiwan/80813/2013 (H1N1)) viruses for 2 hour at 37° C. before the mixture was added to MDCK cells 1×104 per well in 384-well plates (Greiner, Germany). Following 60 mins co-culture, MDCK cells were washed with PBS to remove supernatant containing free antibodies and virus, and then replaced with 10% FBS DMEM-base complete medium for another 16 hours culture at 37° C. Consequentially, MDCK cell were fixed with 4% Paraformaldehyde (PFA) and performed intracelluar and nucleus co-staining with anti-NP-FITC labeled antibody (Millipore MAB8257F) and DAPI (SIGMA-B2261). Infection efficiency was quantified and analyzed with a high content image system (ImageXpress Micro, Molecular Device, Sunnyvale, Calif., USA). The neutralization activity of hmAbs was measured according to the equation: (A−B)/A×100%, wherein ‘A’ represents the cell counts of positive wells that contained only H1N1 or other flu virus strains, and ‘B’ represents the value of wells that contained the mixture of testing serum samples and H1N1 or other flu virus strains.
Construction of Expression Vectors
The nucleotide sequences of SEQ ID NOs: 1 and 3, which code for the amino acid sequences of the heavy chain and light chain, respectively, of hmAb 32D6, were cloned from the isolated memory B cell clone. These nucleotide sequences were inserted into a suitable cloning vector, having restriction sites for cloning these genes into an expression vector.
The cloning vector containing antibody gene was digested with restriction enzymes (e.g., HindIII and EcoRI) for cloning into an expression vector for the expression of the antibody. Any suitable expression vectors may be used, such as the Lonza GS System pXC-17.4 plasmid (Lonza Group, Basel, Switzerland). Suitable vectors may include a promoter (e.g., a viral promoter, mCMV) and contain gene-optimized antibody constant regions. In addition, a suitable may contain a selectable marker. For example, the expression plasmid, pXC-17.4, links the expression of an exogenous protein to the glutamine synthetase (GS) gene. The pXC-17.4 vector may be used with a GS knock-out host cells to facilitate selection of the transfected cells.
Once the antibody gene insert was cloned into the pXC-17.4 vector, individual bacterial colonies were selected for sequence analysis. The plasmids are verified for proper sequence and proper orientation within the plasmid using primers that are situated outside the ORF of the antibody. Based on this information, one single clone is selected for scale-up and generation of a sufficient quantity of plasmid for transfection of CHO cells.
The GS Chinese Hamster Ovary (CHO) cell lines are used, which have mutations in both copies of the GS gene, so that they require the expression of GS on the pXC-17.4 expression plasmid for survival in the absence of glutamine, an amino acid that is essential for growth. For plasmid transfection, GS CHO cells were thawed into appropriate media formulation under no selection, i.e., containing L-glutamine. CHO cells were maintained at 5% CO2 in a 37° C. incubator.
Transfection of GS CHO cells with the pXC-17.4 expression vector was performed using electroporation. For example, cells may be electroporated by delivering a single pulse of 300V, 900 uF with resistance set to infinity Immediately after electroporation, each batch of cells was added to a flask containing the appropriate media. Cells were gently mixed and incubated overnight in a cell culture incubator. The next day, cells were centrifuged to remove media containing plasmid.
When cell numbers reach about 0.6×106 cells per mL, cells were expanded into selection media. After two weeks in selection, individual colonies of CHO cells were selected based on growth on transwell plates. Individual colonies were assessed based on growth and productivity characteristics, through scale-up.
The quantity and integrity of product may be assayed by protein A affinity chromatography and monitored with absorbance at 280 nm, by ELISA, or by SDS-PAGE analysis.
Host Cells
Expression vectors in accordance with embodiments of the invention may be expressed in any suitable cells. As noted above, when using pXC-17.4 expression plasmids, it is convenient to use a GS knockout host cells to facilitate selection of transfected cells. CHOK1SV GS Knockout (KO) host cell line (from Lonza) is a derivative of Lonza's suspension adapted CHOK1SV host cell line. In this new GS-KO cell line, both alleles of the endogenous glutamine synthetase gene have been “knocked out,” leading to a requirement for exogenous glutamine.
Purification of 32D6/8B4 and Western Blot
rHAs and purified 32D6/8B4 monoclonal Ab were resolved using SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to a nitrocellulose membrane. After blocking with 5% BSA, the blots containing rHA were probed with anti-His-tag mouse MAbs at room temperature for 1 h. After a complete wash, horseradish peroxidase-conjugated anti-mouse IgG was added and incubated at room temperature for 0.5 h before rHA protein detection with the West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, Ill., USA). The blots containing 32D6/8B4 antibodies were directly probed with goat anti-mouse IgG Ab (γ-chain specific, USA) conjugated with horseradish peroxidase (Jackson ImmunoResearch Laboratories, INC.) and detected with West Pico Chemiluminescent Substrate.
In Vivo Protection Experiments
All animal experiments were conducted in accordance with ethical procedures and policies approved by an appropriate Institutional Animal Care and Use Committee. DBA/1 mice (average body weight 20 g) were randomly separated into different groups: the control group (5 mice) and three treatment groups (7 mice each). Mice were anesthetized by intraperitoneal administration of isoflurane and challenged with viruses (H1N1-TW80813).
To evaluate the prophylactic efficacy of the mAb 32D6, mice were injected intravenously with PBS (control) or the mAb 32D6. Each mouse in the control group received an equal volume (e.g., 50 μl/mouse) of PBS, and the mice in the three treatment groups each received 10 mpk (mg/Kg body weight) 32D6, 2.5 mpk 32D6, or 1 mpk 32D6, respectively. After 1 hour, the mice were anesthetized with isoflurane. Then, the mice were challenged with the virus (H1N1-TW80813; 1.3×104 PFU in 50 μl/mouse) via intranasal injection or inhalation. After the virus challenge, animals were observed and recorded daily for mortality, morbidity, body weight and facial expression for up to 14 days after infection.
As shown in FIG. 7(A), all mice in the control group died within 3 days after the virus challenge. In contrast, all mice in the treatment groups receiving 10 mpk and 2.5 mkp antibodies survived beyond 14 days. One mouse in the treatment group with 1 mpk antibody died in 3 days and the other died in 4 days, while the remaining 5 mice survived beyond 14 days. These results clearly show that mAb 32D6 of the invention is effective in preventing H1N1 infection.
To determine the therapeutic efficacy of the mAbs, mice were anesthetized with isoflurane, followed by intranasal injection (or inhalation) of the virus (H1N1-TW80813; 1.3×104 PFU in 50 μl/mouse). After 24 hours, each mouse in the control group received an equal volume (e.g., 50 μl/mouse) of PBS, and the mice in the three treatment groups each received 25 mpk (mg/Kg body weight) 32D6, 10 mpk 32D6, or 2.5 mpk 32D6, respectively. Animals were observed and recorded daily for mortality, morbidity, body weight and facial expression for up to 14 days after infection.
As shown in FIG. 7(B), all mice in the control group died within 6 days. In contrast, 2 mice each in the treatment groups receiving 25 mpk and 10 mkp antibodies survived beyond 14 days, and one mouse in the treatment group with 1 mpk antibody survived beyond 14 days. These results clearly show that mAb 32D6 of the invention is effective in treating H1N1 infection.
Statistical Analysis
For survival, probability was calculated using Fisher's exact test, comparing the rate of survival of mice treated with specific mAbs with those of the control groups.
Advantages of embodiments of the invention include one or more of the following. Embodiments of the invention can use memory B cells from any donor, with or without prior infection or vaccination. By not limiting to donors who had prior vaccination or donors who have been infected by the viruses and have since recovered, the donor population for use with embodiments of the invention is more diverse. Starting from a more diverse population of B cells, embodiments of the invention are more likely to identify antibodies that represent the best unbiased sequences in the naïve immune repertoire. In addition, because there is no need to wait for the development of a vaccine or infected donors, embodiments of the invention can be used to identify antibodies for passive immunotherapy at the moment of an outbreak of any virus.
In contrast, prior art methods rely on having a vaccine to vaccinate a donor or rely on patients who had been infected by the virus. These prior art methods cannot be used immediately after an outbreak. In addition, relying on donors who had been vaccinated or infected with the virus would result in a more limited population of the memory B cells. The limited population of memory B cells might be biased due to individual variations in the immune responses of the particular donors.
While the invention has been described with respect to a limited number of embodiments, those skilled in the art, having benefit of this disclosure, will appreciate that other embodiments can be devised which do not depart from the scope of the invention as disclosed herein. Accordingly, the scope of the invention should be limited only by the attached claims.

## Claims

1. A monoclonal antibody, or an antigen binding fragment thereof specifically binding influenza virus H1N1 protein, comprising a heavy chain variable region and a light chain variable region,
wherein the heavy chain variable region comprises complementarity determining regions (CDR) having the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7; and
wherein the light chain variable region comprises complementarity determining regions having the amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.

2. The monoclonal antibody, or the antigen binding fragment thereof, according to claim 1, wherein the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 2 and a light chain having the amino acid sequence of SEQ ID NO: 4.

3. The monoclonal antibody, or the antigen binding fragment thereof, according to claim 1, wherein the H1N1 virus is a 2009 strain or a post-2009 strain.

4. The monoclonal antibody, or the antigen binding fragment thereof, according to claim 1, wherein the binding fragment is an Fab, an Fab′, an F(ab′)2, or a scFv fragment.

5. A method for treating or preventing H1N1 infection, comprising administering to a subject in need thereof the antibody, or the antigen binding fragment thereof, according to claim 1.

6. The method according to claim 5, wherein the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 2 and a light chain having the amino acid sequence of SEQ ID NO: 4.

7. The method according to claim 5, wherein the H1N1 is a 2009 strain or a post-2009 strain.

8. The method according to claim 5, wherein the antigen binding fragment is an Fab, an Fab′, an F(ab′)2, or a scFv fragment.

9. A method for detecting H1N1 virus, comprising:
contacting a test sample with the antibody, or the antigen binding fragment thereof, according to claim 1; and
determining presence or absence of binding with the antibody, or the antigen binding fragment thereof, wherein presence of the binding indicates existence of H1N1virus in the test sample.