Influenza A virus specific antibodies
09718874 · 2017-08-01
Assignee
Inventors
- Hergen Spits (Amsterdam, NL)
- Tim Beaumont (Amsterdam, NL)
- Mark Jeroen Kwakkenbos (Amsterdam, NL)
- Arjen Quirinus Bakker (Amsterdam, NL)
Cpc classification
C07K2317/76
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
C07K2317/33
CHEMISTRY; METALLURGY
International classification
Abstract
The invention relates to isolated, synthetic or recombinant antibodies and functional parts thereof specific for multiple influenza A virus subtypes. The invention further relates to the use of such antibodies for diagnosis of an influenza A virus infection and as a medicament and/or prophylactic agent for at least in part treating or alleviating symptoms of an influenza A virus infection.
Claims
1. A synthetic or recombinant multimeric antibody, multimeric immunoglobulin or antigen binding fragment thereof, specifically binding to the HA protein of influenza A and neutralizing influenza A virus, comprising: i) a first antibody comprising heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 sequences of an antibody selected from the group consisting of AT10_001, AT10_002, AT10_003, AT10_004 and AT10_005; and ii) a second antibody comprising heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 sequences of an antibody selected from the group consisting of AT10_001, AT10_002, AT10_003, AT10_004 and AT10_005, wherein said antibody selected in i) is different from said antibody selected in ii); and wherein the AT10_001 heavy and light chain CDRs1-3 are defined by SEQ ID NO: 4, 9, 14, 19, 24 and 29, respectively; the AT10_002 heavy and light chain CDRs1-3 are defined by SEQ ID NO: 3, 8, 13, 18, 23 and 28, respectively; the AT10_003 heavy and light chain CDRs1-3 are defined by SEQ ID NO: 2, 7, 12, 17, 22 and 27, respectively; the AT10_004 heavy and light chain CDRs1-3 are defined by SEQ ID NO: 1, 6, 11, 16, 21 and 28, respectively; and the AT10_005 heavy and light chain CDRs1-3 are defined by SEQ ID NO: 5, 10, 15, 20, 25 and 30, respectively.
2. The synthetic or recombinant multimeric antibody, multimeric immunoglobulin or antigen binding fragment thereof according to claim 1, comprising: i) at least two different heavy chain CDR sequences and at least two different light chain CDR sequences of antibody AT10_002; and ii) at least two different heavy chain CDR sequences and at least two different light chain CDR sequences of antibody AT10_005.
3. The synthetic or recombinant multimeric antibody, multimeric immunoglobulin or antigen binding fragment thereof according to claim 1, comprising: iii) heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 sequences of antibody AT10_002; and iv) heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 sequences of antibody AT10_005.
4. The synthetic or recombinant multimeric antibody, multimeric immunoglobulin or antigen binding fragment thereof according to claim 1, comprising: i) the heavy chain sequence and the light chain sequence of antibody AT10_002; and ii) the heavy chain sequence and the light chain sequence of antibody AT10_005.
5. The synthetic or recombinant multimeric antibody, multimeric immunoglobulin or antigen binding fragment thereof according to claim 1, which is a dimeric antibody.
6. An isolated or recombinant cell or a pharmaceutical composition comprising the synthetic or recombinant multimeric antibody, multimeric immunoglobulin or antigen binding fragment thereof according to claim 1.
7. A method for treating and/or inhibiting an influenza A virus infection, comprising administering to an individual in need thereof a therapeutically effective amount of the synthetic or recombinant multimeric antibody, multimeric immunoglobulin or thereof according to claim 1.
8. A method for neutralizing a H1N1 influenza A virus and/or an H3N2 influenza A virus, comprising contacting said H1N1 influenza A virus and/or said H3N2 influenza A virus with the synthetic or recombinant multimeric antibody, multimeric immunoglobulin or antigen binding fragment thereof according to claim 1, resulting in neutralization of said virus.
9. A method for determining whether an influenza A virus is present in a sample comprising: contacting said sample with the synthetic or recombinant multimeric antibody, multimeric immunoglobulin or antigen binding fragment thereof according to claim 1, allowing said multimeric antibody, multimeric immunoglobulin or antigen binding fragment thereof to bind said influenza A virus, if present, and determining whether influenza A virus is bound to said multimeric antibody, multimeric immunoglobulin or antigen binding fragment thereof, thereby determining whether an influenza A virus is present in said sample.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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EXAMPLES
(15) Generation of Immortalized B Cells
(16) Human memory B cells were immortalized using the BCL6/Bcl-xL technology described by Kwakkenbos et al. (Generation of stable monoclonal antibody-producing B cell receptor-positive human memory B cells by genetic programming. Nature Medicine (2010) vol. 16 (1) pp. 123-8 and patent application WO 2007/067046). In brief, human memory B cells from Influenza vaccinated donors were transduced with a retroviral vector containing BCL6 and Bcl-xL. Transduced B cells can be maintained in culture with CD40Ligand expressing L-cells and interleukin (IL)-21 (R&D systems).
(17) Selection of Heterosubtypic mAbs
(18) To identify B cells that secrete heterosubtypic cross-binding mAbs two approaches were used.
(19) i) The Influenza H3 (A/Wyoming/03/2003) and H7 (A/Netherlands/219/2003) HA proteins (Protein Sciences) were labeled with Alexa Fluor 647 (Molecular Probes) and incubated with Bcl6 and Bcl-xL transduced polyclonal cultured B cells. HA binding B cells were sorted single cell per well by FACSAria (
ii) Cells were seeded in small pools, e.g. 40 cells per well and maintained in culture for 2-3 weeks. The supernatant of these pools was screened for binding to H7 transfected HEK cells. The B cells of the double positive tested wells were seeded 1 cell per well. The culture supernatant of these monoclonal B cell lines was used to screen for HA binding by ELISA.
(20) B cells that showed reactivity to more than 1 HA type were further cultured and characterized for HA recognition by ELISA (Table 2) and binding to HA expressing HEK cells (
(21) HA ELISA
(22) The B cell supernatant of cross-reactive B cell clones was tested for binding to different HA antigens by ELBA. Recombinant HA of H1 (A/New Caledonia/20/1999), H3 (A/Wyoming/03/2003), H5 (A/Vietnam/1203/2004) and H7 (A/Netherlands/219/2003) (Protein Sciences) were coated to ELISA plates at 1 μg/ml. After coating, the plates were washed 1× with PBS and 350 μl blocking buffer, PBS/4% Protivar, was added and incubated 1 hr at RT. The plates were then washed 3× with PBST (PBS/0.05% Tween20) and the antibodies/culture supernatants were added to the wells. Incubation was allowed to proceed for 1 hr at RT, then the plates were washed 3× with PBST. Samples were then incubated with a goat anti-human IgG-HRP antibody (Jackson) for 1 hr at RT. Bound antibodies were detected using TMB (3,3′,5,5′tetramethyl benzidine) substrate buffer, the reaction was stopped using H.sub.2SO.sub.4. OD 450 nm was measured on an Envision (PerkinElmer). AT10_001 and AT10_002 recognized both H3 and H7 proteins but not the HA proteins of H1 and H5. AT10_003 recognized H3, H5 and H7 protein while AT10_004 recognized H1, H3 and H7 proteins (Table 2).
(23) Antibody Binding HA Transfected 293T Cells
(24) To test heterosubtypic binding of the AT10 mAbs to cell surface expressed HA, 293T cells were transfected with different full length HA constructs. Using Fugene (Roche) 293T cells were transfected with DNA encoding the HA of H1 (A/New Caledonia/20/1999), H3 (A/Wisconsin/67/2005), H5 (A/Thailand/Vietnam Consensus/2004) and H7 (A/Netherlands/219/2003). The transfected cells were incubated with B cell supernatant containing IgG antibodies for 30 minutes at 4° C. and then washed 2× with 150 μl PPBS/2% FCS. Antibody binding was detected with anti-human IgG-PE (Southern Biotech) and analyzed on a FACScanto (Becton, Dickinson and Company) (
(25) Cloning of Selected Antibodies.
(26) We isolated total RNA with the RNeasy® mini kit (Qiagen), generated cDNA, performed PCR and cloned the heavy and light chain variable regions into the pCR2.1 TA cloning vector (Invitrogen). To rule out reverse transcriptase or DNA polymerase induced mutations, we performed several independent cloning experiments. To produce recombinant mAb we cloned the heavy and light variable regions in frame with human IgG1 and Kappa constant regions into a pcDNA3.1 (Invitrogen) based vector and transiently transfected 293T cells. We purified recombinant mAb from the culture supernatant with an ÄKTA (GE healthcare).
(27) Cross Binding Specificity of AT10 Antibodies
(28) Eleven different recombinant HA proteins (Sino Biological Inc and Protein Sciences) were used to test the potential of the antibodies to bind different HA subtypes. Reactivity to these HA proteins (table 3) was tested in an ELISA, as described above. None of the mAbs showed reactivity with Influenza B. AT10_001, AT10_002, AT10_003 and AT10_004 showed binding to all human group 2 HA proteins. AT10_001, AT10_003 and AT10_004 also showed reactivity to Swine H4N6 (A/Swine/Ontario/01911-1/1999). AT10_002 and AT10_003 recognized Duck H10N3 (A/duck/Hong Kong/786/1979) and Duck H15N8 (A/duck/AUS/341/1983), AT10_004 also showed some activity to H15N8 (A/duck/AUS/341/1983). AT10_003 recognized the group 1 HA molecules from H9N2 (A/Hong Kong/1073/1999) and H5N1 (A/Vietnam/1203/2004) while AT10_004 also showed binding to the HAs of H1N1 (A/California/07/2009) and H9N2 (A/Hong Kong/1073/1999). AT10_005 bound exclusively to the group 1 HA proteins tested.
(29) Antibody Binding to Virus Infected Cells
(30) To test the binding capacity of the AT10 antibodies AT10_001, AT10_002, AT10_003 and AT10_004 to virus infected cells we performed FACS analysis on Influenza H1N1 (A/Hawaii/31/2007) and H3N2 (A/Netherlands/177/2008) infected cells (both virus strains were obtained from the Department of Medical Microbiology, AMC, Amsterdam). MDCK-SIAT cells were grown in a T175 culture flask to 80-100% confluency in DMEM/FCS/PS/G418. The cell layer was washed 2× with 10 ml PBS after which 15 ml of Optimem/PS/G418/Trypsin was added. Subsequently 0.5 ml of 100.000 TCID50 Influenza virus (H1N1 or H3N2) was added to the flask and cells were cultured at 37° C. After 24-48 hr the cells were washed 2× with 10 ml PBS and detached from the plastic using Trypsin-EDTA. Cells were counted and frozen at −150° C. until use. The infected cells were defrosted and incubated with IgG antibodies/B cell supernatant for 30 minutes at 4° C. and then washed 2× with 150 μl PBS/2% FCS. Antibody binding was detected with anti-human IgG-PE and analyzed on a FACScanto (Becton, Dickinson and Company). As a control non-infected cells were used (
(31) Similar experiments were performed for the AT10 antibodies AT10_001, AT10_002, AT10_003, AT10_004 and AT10_005 with Influenza H1N1 (A/Neth/602/2009), H3N2 (A/Swine/St. Oedenrode/1996), high pathogenic H5N1 (A/Turkey/Turkey/2004), high pathogenic H7N7 (A/Ch/Neth/621557/03) and low pathogenic H7N1 (A/Ch/Italy/1067/1999) infected cells (Central Veterinary Institute, Lelystad). MDCK cells were infected with virus as described above, only the cells were fixated with 4% paraformaldehyde for 20 minutes at 4° C., washed 1× with PBS and then frozen. As a control non-infected cells were used. FACS staining and analysis was done a described above (
(32) Virus Neutralization
(33) To determine whether the obtained antibodies were capable of blocking Influenza A virus infection, an in vitro neutralization assay was performed. The assay was performed on MDCK-SIAT cells (Journal of Virology August 2003; pp. 8418-25). MDCK-SIAT cells were grown in DMEM/8% FCS/PS/G418 in an 96 well plate (CellCarrier Plate, PerkinElmer) to 80-100% confluency. Neutralization assays are performed in Optimem/PS/G418/Trypsin medium without FCS or BSA. Fifty μl of recombinant mAb was mixed with 50 μl of virus suspension (100TCID50/50 μl) of H3N2 (A/Ned/177/2008) or H1N1 (A/Hawaii/31/2007) Influenza and incubated for 1 hr at 37° C. The suspension was then transferred in multiply into 96-well plates containing MDCK-SIAT cells in 100 μl Optimem/PS/G418/Trypsin. Prior to use the MDCK-SIAT cells were washed twice with 150 μl PBS. The plates were then centrifuged for 15 minutes at RT at 2500 rpm and placed at 37° C./5% CO2. After 24 hr cells were washed twice with PBS, fixed with Formalin (37% formaldehyde in water) for 10 minutes at RT, washed twice with 150 μl PBS and stained with DAPI and an antibody against the nuclear protein of the Influenza virus (NP-FITC, Abcam) at RT. After 30 minutes cells were washed twice with 150 μl PBS and 100 μl of PBS/50% Glycerol was added to the wells. Viral infection of the MDCK-SIAT cells was measured and analyzed on the Operetta (PerkinElmer) using an 20× objective. To quantify neutralizing capacity of the mAbs the number of infected cells was counted (positive for DAPI and NP-FITC) (Table 5). IC50 values were calculated in Prism, values are from 1 representative experiment, assay points performed in quadruplicate. AT10_001, AT10_002 and AT10_004 showed potent inhibition of H3N2 (A/Ned/177/2008) and H3N2 HKX-31 Influenza virus infection in vitro. Neutralization of H1N1 (A/Hawaii/31/2007) was not observed for AT10_001, AT10_002, AT10_003 and AT10_004.
(34) To determine whether the obtained AT10 antibodies were capable of blocking multiple Influenza A virus strains, additional in vitro neutralization assays were performed. Influenza viruses A/swine/Neth/St. Oedenrode/96 (H3N2; de Jong et al. 1999), A/ck/Neth/621557/03 (H7N7; van der Goa et al. 2005), A/ck/Italy/1067/99 (H7N1), A/turkey/Turkey/05 (H5N1; Löndt et al. 2008) and A/Neth/602/2009 (swine-origin H1N1; Munster et al. 2009) were used in this assay. Madin-Darby canine kidney (MDCK) cells were cultured in Optimem (Gibco BRL Life Technologies) containing 5% FBS (Integro) and 1% Pen Streptomycine (Gibco BRL Life Technologies). Cells were seeded at a density of 3×10.sup.4 cells per well in 96-well plates and incubated 0/N at 37° C. Three-fold serial dilutions of the mAbs were made in PBS starting with a concentration of 15 μg/ml. Rituximab mAb was taken along as negative control. Virus dilutions were prepared in virus infection medium consisting of Optimem supplemented with antibiotics and, in case of LPAI viruses, 1 μg/ml trypsin/TPCK (Sigma). Each mAb dilution was mixed with an equal volume of virus followed by 1 hour incubation at 37° C. After washing of the cells with PBS, the mAb/virus mixture (˜100-1000 TCID50) was inoculated onto the cell monolayers. Cells were incubated for 24-32 hours at 37° C., after which they were washed twice with PBS, fixed with 4% formalin for 20 min and then washed again with PBS. Cells incubated with medium only were included as negative control and cells incubated with virus only as positive control. The assay was performed in quadruplicate. Cells were stained with 1 hour with DAPI and an antibody against the nuclear protein of the Influenza virus (NP-FITC, Abcam or HB65 followed by Goat-anti-mouse IgG Alexa-647, Invitrogen) at RT. Cells were washed twice with 150 μl PBS after staining and 100 μl of PBS/50% Glycerol was added to the wells. Viral infection of the MDCK cells was measured and analysed on the Operetta (PerkinElmer) using an 10× objective. To quantify neutralizing capacity of the mAbs the number of infected cells was counted (positive for DAPI and NP-FITC/HB65-Alexa-647). IC50 values were calculated in Prism, values are from 1 representative experiment. The results are shown in Table 7. AT10_002 and AT10_004 showed potent inhibition of the group 2 influenza virus infection in vitro but did not prevent infection with group 1 viruses. Antibody AT10_005 prevents infection with group 1 Influenza A viruses but has no effect on group 2 viruses. Antibody CR8020 (WO 2010 130636) does not show any neutralizing capacity for H3N2 A/Swine/Neth/St. Oedenrode/96 and H7N1 A/A/Italy/1067/99 at 15 μg/ml while AT10_002 and AT10_004 show IC50 values below 4 μg/ml.
(35) Antibody Competition
(36) Antibody AT10_001 and AT10_003 were labeled with Alexa Fluor 555 (Molecular Probes) and antibody AT10_002 and AT10_004 were labeled with Alexa Fluor 647 (Molecular Probes). Labeled antibodies were tested for binding to H3N2 (A/Netherlands/177/2008) infected MDCK-SIAT cells to determine if they maintained their binding capacity. For competition experiments H3N2 (A/Netherlands/177/2008) infected cells were incubated with increasing amounts of non-labeled competitor antibody for 10 minutes at 4° C. before the addition of Alexa Fluor-labeled antibody. Cell-antibody mix was incubated for another 15 minutes at 4° C. and washed 2× with PBS/2% FCS before analysis on the Guava easyCyte 8 (Millipore). AT10_001, AT10_002 and AT10_004 all bind to a similar region on the HA protein as they all block each other's binding (
(37) HA1 Subunit ELISA
(38) To test whether the HA1 subunit is essential for the binding of the antibodies to the HA protein an HA1 subunit specific ELISA was done. Recombinant HA of full length H3 (A/Aichi/2/1968, full length) and H3 HA1 subunit (A/Aichi/2/1968, HA1 subunit, Met-1-Arg 345) were coated to ELISA plates at 1 μg/ml. After coating, the plates were washed 1× with PBS and 300 μl blocking buffer, PBS/4% Protivar, was added and incubated 1 hr at RT. The plates were then washed 3× with PBST (PBS/0.05% Tween20) and the recombinant antibodies were added to the wells. Incubation was allowed to proceed for 1 hr at RT, then the plates were washed 3× with PBST. Samples were then incubated with a goat anti-human IgG-HRP antibody (Jackson) for 1 hr at RT. Bound antibodies were detected using TMB substrate buffer, the reaction was stopped using H.sub.2SO.sub.4. OD 450 nm was measured on an Envision (PerkinElmer) (Table 6). AT10_001, AT10_002 and AT10_004 recognized full-length H3 HA protein but not the HA1 subunit of this protein indicating their binding epitope is, at least partly, located on the HA2 subunit of the protein. AT10_003 recognized both the full-length HA protein and the HA1 subunit indicating that the AT10_003 epitope is located on the HA1 subunit of the HA protein.
(39) Binding of AT10 Antibodies to Different HA Conformations
(40) Upon endocytic uptake of virions, the acidic environment of the endosome triggers HA-driven fusion of the viral and the endosomal membrane. This fusion is mediated by a conformational change of the HA protein (triggered by the low pH) from a pre-fusion state to a post-fusion state. We performed an in vitro pH-shift experiment to test to which conformational configuration of HA the antibodies can bind. Using Fugene (Roche) 293T cells were transfected with DNA encoding the HA of H3 (A/Wisconsin/67/2005). 48 hours post transfection the cells were harvested using trypsin-EDTA and stored at −150° C. until further use. For the pH-shift experiment, cells were washed 2× with PBS and then incubated for 30 minutes at room temperature with 10 μg/ml trypsin-EDTA in PBS. Cells were washed 2× with PBS and a fraction was set aside as trypsin condition. Remaining cells were split to two tubes and treated with either 500 mM Dithiothreitol (DTT) for 20 minutes at room temperature or incubated for 5 minutes at 37° C. with PBS pH5. Cells were washed 2× with PBS and incubated with recombinant AT10_001, AT10_002, AT10_003 or AT10_004. Antibody binding was detected using anti-human-IgG-PE (southern Biotech) antibody and analyzed on a Guava easyCyte 8 (Millipore) (
(41) Prophylactic and Therapeutic Efficacy of AT10 Antibodies In Vivo
(42) The AT10 antibodies were tested in a mouse influenza challenge model to determine their efficacy. Male C57Bl/6J mice (4 per group) were intranasally challenged with increasing amounts of influenza A/HKx-31 (H3N2) and body weight changes were monitored twice a day for 14 days to determine the viral dose response. Twenty-five percent bodyweight loss was used as humane endpoint; mice loosing more than 25% of their body weight were removed from the study. In the highest dose group (20000 TCID50) all animals lost 25% of their bodyweight within 8 days while in the 2000 TCID50 group only 50% of the mice reached this bodyweight loss (
(43) Antibodies AT10_001, AT10_002, AT10_004 and a negative control antibody (Rituximab) were tested for prophylactic efficacy in the influenza model. Mice were intravenously injected with 1 or 5 mg/kg antibody one day before challenge with 10 LD50 influenza A/HKx31. Bodyweight was monitored for 10 days after which the experiment was terminated. All control mice lost 25% bodyweight within 8 days and were removed from the study, however none of the mice that received prophylactic AT10 antibody had to be removed from the study demonstrating a protective effect of the antibodies (P=<0.000.1, Mantel-Cox,
(44) The AT10 antibody that showed the best protective activity in the prophylactic Influenza experiment, AT10_002, was tested for therapeutic efficacy in the influenza model. Mice were intravenously injected with 15 mg/kg antibody two, three, or four days post challenge with 10 LD50 influenza A/HKx31. As controls, mice were injected with 15 mg/kg AT10_002 or a negative control antibody (Rituximab, 15 mg/kg) one day before 10 LD50 influenza A/HKx31 challenge. Bodyweight was monitored for 10 days after which the experiment was terminated. The results are shown in
(45) Generation of Pan-Specific Anti-Influenza a lgG Multimeric Antibody
(46) To generate a multimeric antibody complex that recognises most Influenza A viruses we coupled AT10_002 and AT10_005 together (BiFlu) using the sortase technology, described in detail in WO 2010/087994. To be able to link AT10_002 and AT10_005 a tag (named ST) containing a sortase recognition site plus a His6 tag, with sequence GGGGSLPETGGGHHHHHH (SEQ ID NO:83), is attached to the C-terminus of the heavy chain of the antibodies via genetic fusion.
(47) To be able to link AT10_002 and AT10_005 a tag (named ST) containing a sortase recognition site plus a His6 tag, with sequence GGGGSLPETGGGHHHHHH, is attached to the C-terminus of the heavy chain of the antibodies via genetic fusion.
(48) The sortase reaction was performed by mixing 10.0 mg AT10-002 ST antibody in 2000 reaction buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 10 mM CaCl.sub.2) containing 60 μM sortase and 500 μM GGG-Dibenzo-azacyclo-octyn (DIBAC). Similarly, 10.0 mg AT10-005 ST antibody was mixed with 2000 μl sortase-buffer containing 90 04 sortase and 1 mM GGG-azide.
(49) Both samples were incubated 16 h at 37° C. After incubation sortase was de-activated by the addition of 50 mM EDTA. Before loading the sample on a gel filtration column, the reaction mixture was centrifuged (3 min, 13.200 rpm) to pellet any aggregates. Gel filtration chromatography of the two sortase-tagged antibodies was performed on a HiLoad Superdex 200 16/60 column (GE Healthcare, Piscataway, N.J., United States) in coupling buffer (25 mM Tris, pH 7.5+150 mM NaCl). Before loading samples, the column was equilibrated with 1.0 CV (column volumes) coupling buffer. After loading, the column was run with 1.5 CV equilibration buffer.
(50) Next, the purified antibodies were subjected to click-chemistry coupling. 3.0 mg AT10-002-DIBAC was mixed with 2.9 mg AT10-005-azide and incubated at 25° C. in 3.0 ml coupling buffer (25 mM Tris, pH 7.5, 150 mM NaCl). After 16 h the sample was subjected to gel filtration (as above) in PBS (Fresenius Kabi, Bad Homburg, Germany). Fractions containing the IgG dimers were collected, pooled and concentrated with 50 kDa cut-off membranes AMICON centrifugal filter devices (Millipore, Billerica, Mass., United States).
(51) Qualitative SPR Analysis of the BiFlu Preparation
(52) Surface plasma resonance (SPR) analysis was performed on the BiFlu preparation to determine if dimeric BiFlu was formed (e.g. dimers consisting of both AT10_002 (lambda light chain) and AT10_005 (kappa light chain)) and if the preparation consists of AT10_002 AT10_005 heterodimers
(53) SPR analysis was performed on an IBIS MX96 SPR imaging system (IBIS Technologies BV., Enschede. The Netherlands) as described (Lokate et al., 2007, J. Am. Chem. Soc. 129:14013-140318). In short, one SPR analysis cycle consists of one or more incubation steps in which analytes are flushed over a coated sensor. This is followed by a regeneration step in which any bound analyte is removed from the sensor. Multiple cycles can be performed in one experiment. In our SPR capture-binding assay the antibodies of interest are first captured on an isotype-specific antibody (i.e. anti-human IgG, anti-human kappa light chain or anti-human lambda light chain), which is immobilized on the SPR sensor and then incubated with analytes. Obtained data was analyzed using Sprint software (version 1.6.8.0, IBIS Technologies BV, Enschede, The Netherlands).
(54) The SPR sensor was coated by immobilization of isotype specific antibodies anti-human IgG (Jackson Immunoresearch, West Grove, Pa., USA), anti-human kappa light chain (Dako, Glostrup, Denmark) and anti-human lambda light chain (Dako, Glostrup, Denmark) on an amine-specific EasySpot gold-film gel-type SPR-chip (Ssens BV, Enschede, The Netherlands) by spotting them on the sensor surface using a continuous flow microspotter device (Wasatch Microfluidics, Salt Lake City, Utah, USA) in coupling buffer (10 mM NaAc, pH 4.5, 0.03% Tween20).
(55) After spotting for 45 minutes the sensor is deactivated with 0.1 M ethanolamine, pH 8.5 and washed three times with system buffer (PBS+0.03% Tween20+0.05% NaN.sub.3). Before starting the analysis, the coupled sensor was incubated for two minutes with regeneration buffer (10 mM glycine-HCl, pH 2), followed by three wash steps with system buffer.
(56) Then the coated SPR chip is injected either with AT10_002, AT10_005 (2 μg/ml in system buffer) or BiFlu (4 μg/ml in system buffer) and incubated for 30 min. Subsequently, non-captured IgG is removed by a 5 minute incubation with system buffer. Next, the sensor is injected with influenza H3-hemagglutinin protein (H3N2, Wyoming, 03/2003, Sino Biological inc., Beijing, P.R. China, 0.25 to 2.0 μg/ml) in system buffer and incubated for 30 min to measure association. To measure complex dissociation the sensor is washed with system buffer and incubated for 40 min. The injection of H3 is followed by injections with influenza H1-hemagglutinin (H1N1, New Caledonia, 20/1999, Sin Biological inc., Beijing, P.R. China, 1.0 ug/ml) and anti-human light chain antibody (anti-kappa or anti-lambda) in a similar fashion as described above.
(57) When the single antibodies and BiFlu are captured on anti-human IgG (
(58) Antibody Binding to Virus Infected Cells
(59) To test if the binding capacity of the BiFlu antibodies is maintained and if the BiFlu has the combined binding properties of AT10_002 and AT10_005 we performed FACS analysis on Influenza H1N1 (A/Hawaii/31/2007) and H3N2 (A/Netherlands/177/2008) infected cells. Influenza A infected MDCK-SIAT cells were generated as described above. The infected cells were defrosted and incubated with different concentrations of AT10_002, AT10_005 or BiFlu antibodies for 30 minutes at 4° C. and then washed 2× with 150 d IMDM/2% FCS. Antibody binding was detected with anti-human IgG-APC and analyzed on a Guava easyCyte 8HT (Millepore). The results are shown in
(60) Virus Neutralization
(61) To determine whether BiFlu is also capable of blocking Influenza A virus infection, an in vitro neutralization assay was performed. The assay was performed on MDCK-SIAT cells as described above. To quantify neutralizing capacity of the mAbs the number of infected cells was counted (positive for DAPI and NP-FITC). Shown in
(62) Prophylactic Efficacy of BiFlu Antibodies In Vivo (
(63) Antibodies AT10_002, AT10_005, BiFlu (AT10_002 AT10_005 dimer), AT10_002/AT10_005 mix and a negative control antibody (Rituximab) were tested for prophylactic efficacy in the influenza model. Male C57Bl/6J mice (6 per group) were intranasally challenged with 10 LD50 influenza A/HKx31 or 10 LD50 H1N1 Influenza A/PR/8/34 and body weight changes were monitored for 10 days. Twenty-five percent bodyweight loss was used as humane endpoint; mice loosing more than 25% of their body weight were removed from the study.
(64) Mice were intravenously injected with 1 mg/kg AT10_002, 1 mg/kg AT10_005, a mix of AT10_002 and AT10_005 1 mg/kg each, 2 mg/kg BiFlu or 1 mg/kg Rituximab antibody one day before viral challenge. All control mice (Rituximab) lost 25% bodyweight within 8 days and were removed from the study. In the H1N1 challenge model AT10_005 antibody showed a protective effect e.g. none of the mice had to be removed from the study. In addition, the mice that received the BiFlu preparation and the AT10_002/AT10_005 antibody mix were also protected (
(65) Protein Modelling to Determine the Amino Acids Involved in the Antibody Hemagglutinin Interaction. (Table 8, 9 and 10)
(66) The multiple sequence alignments were done by ClustalΩ and further processed by showalign, part of EMBOSS. All the structural work was done with Pymol. Minimisation was done using the software NAMD with the force field CHARMM.
(67) The first step to build a 3D model of the antibody is to select the best 3D template. This is done by using a global alignment (Needleman and Wunsch) of the query sequence against a databank of all sequences of antibodies present in the protein database (PDB). Then one structure is chosen amongst the structure with the highest percentage of identity in the sequence.
(68) The next step is to highlight the regions where substitutions occurred and modify the sequence and the structure in such a way that the final model resembles the antibody to analyse. Two techniques are applied: 1) Substitution of amino acid, this method keeps the main chain in place and only replaces the side chain. 2) Grafting of loop, this method modifies the main chain and is necessary when there are insertion or deletion in a loop, when the sequence is too far or when substitutions may affect the main chain conformation, e.g. substitution of Glycine or Proline.
(69) To generate the complex antibody-hemagglutinin with the antibodies AT10_005 and AT10_004 the structure of experimentally determined complexes were used as template. The model of the antibody is superimposed on the antibody of the crystal determined structure, the hemagglutinin is kept intact. For AT10_002 the docking procedure was to: (i) analyse the stem of hemagglutinin to restrict the area where actual binding were tested, (ii) manual positioning of the antibody in the remaining area of point (i), (iii) evaluation of the quality of the complex by checking the structure for short contacts, hydrogen bond capable groups missing hydrogen bonds in the complex, size of the contact area.
(70) AT10_005:
(71) The amino acids of influenza A virus group 1 haemagglutinin (H1/H5) in contact with AT10_005 are: A38, A40, A41, A42, A291, A292, A293, A318, B18, B19, B20, B21, B38, B41, B42, B45, B46, B48, B49, B52, B53, B56.
(72) AT10_004:
(73) The amino acids of influenza A virus group 2 haemagglutinin (H3/H7) in contact with AT10_004 are: A21, A324, A325, A327, B12, B14, B15, B16, B17, B18, B19, B25, B26, B30, B31, B32, B33, B34, B35, B36, B38, B146, B150, 13153, B154.
(74) AT10_002:
(75) The amino acids of influenza A virus group 2 haemagglutinin (H3/H7) in contact with AT10_002 are: A38, A48, A275, A276, A277, A278, A289, A291, A318, B19, B20, B21, B36, B38, B39, B41, B42, B45, B46, B48, B49, B50, B52, B53, B56, B57, B58, B150.
(76) Amino acid numbering for the HA molecule was done according to: Wilson et al. 1981 Nature 289, 366-373 and Nobusawa et al. 1991 Virology 182, 475-485.
(77) Interactions Antibody-Haemagglutinin
(78) AT10_005 interacts with the conserved hydrophobic pocket demonstrated by the crystal of the complex of CR6261 or F10 antibodies with haemagglutinin. The interaction is mainly hydrophobic as for all antibodies binding this pocket. AT10_004 interacts with the same beta strand as CR8020 in its crystal complex with haemagglutinin but AT10_004 binds in a stronger way by, among other interactions, continuing the beta sheet of haemagglutinin. This interaction is mediated via the main chain and thus it allows cross-reactivity between H1 and H3 even in the absence of conservation (because the main chain is conserved between amino acids). AT10_002 interacts with the conserved hydrophobic patch in a new way since except for the CDR3 of VH, all interactions come from the VL domain.
REFERENCES
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(80) Nobusawa E, Aoyama T, Kato H, Suzuki Y, Tateno Y, Nakajima K: Comparison of complete amino acid sequences and receptor-binding properties among 13 serotypes of hemagglutinins of influenza A viruses. Virology. 1991 June; 182(2):475-85. Subbarao K. and Joseph T. Nature Reviews Immunology 2007: 7, 267-278 Sui J, Hwang W C, Perez S, Wei G, Aird D, Chen L M, Santelli E, Stec B, Cadwell G, Ali M, Wan H, Murakami A, Yammanuru A, Han T, Cox N J, Bankston L A, Donis R O, Liddington R C, Marasco W A: Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses. Nat Struct Mol Biol. 2009 March; 16(3):265-73, Epub 2009 Feb. 22. Throsby M, van den Brink E, Jongeneelen M, Poon L L, Alard P, Cornelissen L, Bakker A, Cox F, van Deventer E, Guan Y, Cinatl J, ter Meulen J, Lasters I, Carsetti R, Peiris M, de Kruif J, Goudsmit J; Heterosubtypic neutralizing monoclonal antibodies cross-protective against H5N1 and H1N1 recovered from human IgM+ memory B cells. PLoS One. 2008; 3(12):e3942. Epub 2008 Dec. 16. Wilson I A, Skehel J J, Wiley D C: Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution, Nature. 1981 Jan. 29; 289(5796):366-73. Yoshida R, Igarashi M, Ozaki H, Kishida N, Tomabechi D, Kida H, Ito K, Takada A: Cross-protective potential of a novel monoclonal antibody directed against antigenic site B of the hemagglutinin of influenza A viruses. PLoS Pathog. 2009 March; 5(3):e1000350. Epub 2009 Mar. 20. WO 2007/067046 WO 2009/115972 WO 2010/010466 WO 2010/130636 WO 2010/087994 WO 2007/067046
(81) TABLE-US-00001 TABLE 1 Preferred influenza A virus specific antibodies according to the invention SEQ ID NO Antibody Identity Sequence 1 AT10-004 Heavy chain CDR1 RHGIS 2 AT10-003 Heavy chain CDR1 ELSIH 3 AT10-002 Heavy chain CDR1 SSNYY 4 AT10-001 Heavy chain CDR1 TYAMS 5 AT10-005 Heavy chain CDR1 NYAIS 6 AT10-004 Heavy chain CDR2 WISAYTGDTDYAQKFQG 7 AT10-003 Heavy chain CDR2 SFDPEDGETIYAQKFQG 8 AT10-002 Heavy chain CDR2 TIYHSGSTYYNPSLKS 9 AT10-001 Heavy chain CDR2 GISGSGESTYYADSVKG 10 AT10-005 Heavy chain CDR2 GIIPIFGTTNYAQKFQG 11 AT10-004 Heavy chain CDR3 LRLQGEVVVPPSQSNWFDP 12 AT10-003 Heavy chain CDR3 GWGAVTSPFDF 13 AT10-002 Heavy chain CDR3 GGGFGWSQTYFGY 14 AT10-001 Heavy chain CDR3 QGDHIAWLLRGINFDY 15 AT10-005 Heavy chain CDR3 HGGVYYYGSASSGWFDP 16 AT10-004 Light chain CDR1 RASQSVSRYLA 17 AT10-003 Light chain CDR1 RSSQSLLHSNGHIYFD 18 AT10-002 Light chain CDR1 TGTSSDVGAYNYVS 19 AT10-001 Light chain CDR1 RASQSVSSSYLA 20 AT10-005 Light chain CDR1 RASQSVSSSYLA 21 AT10-004 Light chain CDR2 DASNRAT 22 AT10-003 Light chain CDR2 LVSKRAS 23 AT10-002 Light chain CDR2 DVTYRPS 24 AT10-001 Light chain CDR2 GASTRAT 25 AT10-005 Light chain CDR2 GASTRAT 26 AT10-004 Light chain CDR3 QQRSNWLK 27 AT10-003 Light chain CDR3 MQALETP 28 AT10-002 Light chain CDR3 SSQSRSST 29 AT10-001 Light chain CDR3 QNYGSPF 30 AT10-005 Light chain CDR3 QQYGSLP 31 AT10-004 Heavy chain QVQLVQSGAEVRKPGASVKVSCKASGYTFTRHGISWVRQAPGQGLEW MGWISAYTGDTDYAQKFQGRVTMTTDTSTNTAYMELRSLRSDDAAVY YCARLRLQGEVVVPPSQSNWFDPWGQGTLVTVSS 32 AT10-003 Heavy chain QVHLVQSGAEVRKPGASVKVSCKVSGYTLNELSIHWLRQAPGRGLEW MGSFDPEDGETIYAQKFQGRVTMTGDTSTDTAYLELTSLRSEDTALY YCARGWGAVTSPFDFWGQGTLVTVSS 33 AT10-002 Heavy chain QLQLQESGPRLVKPSETLSLTCSVSGVSISSSNYYWGWIRQPPGKGL EWIGTIYHSGSTYYNPSLKSRLIISVDTSKNQFYLQLTSLTAADSAV YYCATGGGFGWSQTYFGYWGQGTLVTASS 34 AT10-001 Heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFSFSTYAMSWVRQAPGKGLEW VSGISGSGESTYYADSVKGRFTVSRDNSKNTLYLQMNSLRAEDTAVY YCAKQGDHIAWLLRGINFDYWGQGVLVTVSS 35 AT10-005 Heavy chain QVQLVQSGAEVKKPGSSVKVSCKASGGAFSNYAISQAPGQGLEWMGG IIPIFGTTNYAQKFQGRVTITADKFTTIAYMELRSLRSEDTAVYYCA RHGGVYYYGSASSGWFDPWGQGTLVTVSS 36 AT10-004 Light chain EIVLTQSPATLSLATGERATLSCRASQSVSRYLAWYNKPGQAPRLLI YDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCCDR3 QQ RSNWLKITFGQGTRLEIKGTV 37 AT10-003 Light chain DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGHIYFDWYLQKPGQ SPQLLIYLVSKRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCM QALETPFTFGPGTKVHIKRTV 38 AT10-002 Light chain QSALTQPASVSGSPGQSITISCTGTSSDVGAYNYVSWYQHHPGKAPK LMIYDVTYRPSGVSTRFSGSKSGNTASLTISGLQAEDEADYYCSSQS RSSTLVIFGGGTKLTVLGQPK 39 AT10-001 Light chain EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRL LIYGASTRATGIPDRFSGRGSGTDFTLTISSLEPEDFAVYYCQNYGS PFLFTFGPGTKVDIKRTV 40 AT10-005 Light chain EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWIQQKPGQAPRL LIFGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVFYCQQYGS LPLTFGGGTKVEIKGTV 41 AT10-004 Heavy chain CDR1 agg cat ggt atc agc 42 AT10-003 Heavy chain CDR1 gaa tta tcc att cac 43 AT10-002 Heavy chain CDR1 agt agt aat tat tac 44 AT10-001 Heavy chain CDR1 acc tat gcc atg agc 45 AT10-005 Heavy chain CDR1 aac tat gct atc agc 46 AT10-004 Heavy chain CDR2 tgg atc agc gct tac act ggt gac aca gac tat gca cag aaa ttc cag ggg 47 AT10-003 Heavy chain CDR2 agt ttt gat cct gaa gat ggt gaa aca atc tac gcg cag aag ttc cag ggc 48 AT10-002 Heavy chain CDR2 act atc tat cac agt ggc agc acc tac tac aac ccg tcc ctc aag agt 49 AT10-001 Heavy chain CDR2 ggt att agt ggt agt ggt gag agc aca tac tac gca gac tcc gtg aag ggc 50 AT10-005 Heavy chain CDR2 ggg atc atc cct atc ttt gga aca aca aac tac gca cag aag ttc cag ggc 51 AT10-004 Heavy chain CDR3 ctt cgt ttg cag ggt gaa gtg gtg gtc cct cct agt caa tcc aat tgg ttc gac ccc 52 AT10-003 Heavy chain CDR3 ggt tgg ggg gcg gtg act tca ccc ttt gac ttc 53 AT10-002 Heavy chain CDR3 ggg ggg ggg ttt ggc tgg tct caa acc tac ttt ggc tac 54 AT10-001 Heavy chain CDR3 caa ggg gat cat att gcc tgg tta tta agg ggg att aac ttt gac tac 55 AT10-005 Heavy chain CDR3 cat ggg gga gtg tat tat tat ggg tcg gcg agt tcg gga tgg ttc gac ccc 56 AT10-004 Light chain CDR1 agg gcc agt cag agt gtt agc agg tac tta gcc 57 AT10-003 Light chain CDR1 agg tct agt cag agc ctc ctg cat agt aat ggg cac atc tat ttc gat 58 AT10-002 Light chain CDR1 act gga acc agc agt gac gtt ggt gct tat aac tat gtt tct 59 AT10-001 Light chain CDR1 agg gcc agt cag agt gtt agc agc agt tac tta gcc 60 AT10-005 Light chain CDR1 agg gcc agt cag agt gtt agt agc agc tac tta 61 AT10-004 Light chain CDR2 gat gca tcc aac agg gcc act 62 AT10-003 Light chain CDR2 ttg gtt tct aag cgg gcc tcc 63 AT10-002 Light chain CDR2 gat gtc act tat cgg ccc tca 64 AT10-001 Light chain CDR2 ggt gca tcc acc agg gcc act 65 AT10-005 Light chain CDR2 ggt gca tcc acc agg gcc act 66 AT10-004 Light chain CDR3 cag cag cgt agc aac tgg ctt aag 67 AT10-003 Light chain CDR3 atg caa gct cta gaa act cca 68 AT10-002 Light chain CDR3 agt tca cag tca cgc agc agc act 69 AT10-001 Light chain CDR3 cag aac tat ggt agt cca ttt 70 AT10-005 Light chain CDR3 cag cag tat ggt agc tta cct 71 AT10-004 Heavy chain cag gtt cag ctg gtg cag tct gga gct gag gtg agg aag cct ggg gcc tca gtg aag gtc tcc tgc aag gct tcc ggt tac acg ttt acc agg cat ggt atc agc tgg gtg cga cag gcc cct gga caa ggg ctt gag tgg atg gga tgg atc agc gct tac act ggt gac aca gac tat gca cag aaa ttc cag ggg cga gtc acc atg acc aca gat aca tcc acg aac aca gcc tac atg gaa ctg agg agc ctg aga tct gac gac gcg gcc gta tat tac tgt gcg aga ctt cgt ttg cag ggt gaa gtg gtg gtc cct cct agt caa tcc aat tgg ttc gac ccc tgg ggc cag gga acc ctg gtc acc gtc tcc tca 72 AT10-003 Heavy chain cag gtc cac ctg gta cag tct ggg gct gag gtg agg aag cct ggg gcc tca gtg aag gtc tcc tgc aaa gtt tcc gga tac aca ctc aat gaa tta tcc att cac tgg ctg cga cag gct cct gga aga ggg ctt gag tgg atg gga agt ttt gat cct gaa gat ggt gaa aca atc tac gcg cag aag ttc cag ggc aga gtc acc atg acc ggg gac aca tct aca gac aca gcc tac ctg gag ctg acc agc ctg aga tct gag gac acg gcc ctc tat tac tgt gca aga ggt tgg ggg gcg gtg act tca ccc ttt gac ttc tgg ggc cag gga aca ctg gtc acc gtc tcc tca 73 AT10-002 Heavy chain cag ctg cag ctg cag gag tcg ggc cca cga ctg gtg aag ccc tcg gag acc ctg tcc ctc acc tgc tct gtc tcc ggt gtc tcc atc agc agt agt aat tat tac tgg ggc tgg atc cgc cag ccc cca ggg aag ggg ctg gag tgg att ggg act atc tat cac agt ggc agc acc tac tac aac ccg tcc ctc aag agt cga ctc atc atc tcc gtc gac acg tcc aag aat cag ttc tac ctg cag ttg acc tct ctg acc gcc gca gac tcg gct gtc tat tac tgt gcg acc ggg ggg ggg ttt ggc tgg tct caa acc tac ttt ggc tac tgg ggc cag gga acc ctg gtc acc gcc tcc tca 74 AT10-001 Heavy chain gag gtg cag ctg ttg gag tct ggg gga ggc ttg gta cag cct ggg ggg tcc ctg aga ctc tcc tgt gca gcc tcc gga ttc agc ttt agc acc tat gcc atg agc tgg gtc cgc cag gct cca gga aag ggg ctg gag tgg gtc tca ggt att agt ggt agt ggt gag agc aca tac tac gca gac tcc gtg aag ggc cgg ttc acc gtc tcc aga gac aat tcc aag aac acc ctg tat ctg caa atg aac agc ctg aga gcc gag gac acg gcc gtc tat tac tgt gcg aaa caa ggg gat cat att gcc tgg tta tta agg ggg att aac ttt gac tac tgg ggc cag gga gtc ctt gtc acc gtc tcc tca 75 AT10-005 Heavy chain cag gtg cag ctg gtg cag tct ggg gct gaa gtg aag aag cct ggg tcc tcg gtg aag gtc tcc tgc aag gct tct gga ggc gcc ttc agc aac tat gct atc agc tgg gtg cga cag gcc cct gga caa ggg ctt gag tgg atg gga ggg atc atc cct atc ttt gga aca aca aac tac gca cag aag ttc cag ggc aga gtc acg att acc gcg gac aaa ttc acg acc ata gcc tac atg gag ttg cgc agc ctg aga tct gag gac acg gcc gtt tat tac tgt gcg agg cat ggg gga gtg tat tat tat ggg tcg gcg agt tcg gga tgg ttc gac ccc tgg ggc cag gga acc ctg gtc acc gtc tcc tca 76 AT10-004 Light chain gaa att gtg ttg aca cag tct cca gcc acc ctg tct ttg tat cca ggg gaa aga gcc acc ctc tct tgc agg gcc agt cag agt gtt agc agg tac tta gcc tgg tac caa cag aaa cct ggc cag gct ccc agg ctc ctc atc tat gat gca tcc aac agg gcc act ggc atc cca gcc agg ttc agt ggc agt ggg tct ggg aca gac ttc acc ctc acc atc agc agc cta gag cct gaa gat ttt gca gtt tat tac tgt cag cag cgt agc aac tgg ctt aag atc acc ttc ggc caa ggg aca cga ctg gaa att aaa gga act gtg 77 AT10-003 Light chain gat att gtg atg act cag tct cca ctc tcc ctg ccc gtc acc cct gga gag ccg gcc tcc atc tcc tgc agg tct agt cag agc ctc ctg cat agt aat ggg cac atc tat ttc gat tgg tac ctg cag aag cca ggg cag tct cca cag ctc ctg atc tat ttg gtt tct aag cgg gcc tcc ggg gtc cct gac agg ttc agt ggc agt gga tca ggc aca gat ttt aca ctg aaa atc agc aga gtg gag gct gag gat gtt ggg gtt tat tac tgc atg caa gct cta gaa act cca ttc act ttc ggc cct ggg acc aaa gtg cat atc aaa cga act gtg 78 AT10-002 Light chain cag tct gcc ctg act cag cct gcc tcc gtg tct ggg tct cct ggc cag tcg atc acc atc tcc tgc act gga acc agc agt gac gtt ggt gct tat aac tat gtt tct tgg tac caa cac cac cca ggc aaa gcc ccc aaa ctc atg att tat gat gtc act tat cgg ccc tca ggg gtt tct act cgc ttc tct ggc tcc aag tct ggc aac acg gcc tcc ctg acc atc tct ggg ctc cag gct gag gac gag gct gat tat tat tgc agt tca cag tca cgc agc agc act ctc gtg att ttc ggc ggg ggg acc aag ttg acc gtc cta ggt cag ccc aag 79 AT10-001 Light chain gaa att gtg ttg acg cag tct cca ggc acc ctg tct ttg tct cca ggt gaa aga gcc acc ctc tcc tgc agg gcc agt cag agt gtt agc agc agt tac tta gcc tgg tac cag cag aaa cct ggc cag gct ccc agg ctc ctc atc tat ggt gca tcc acc agg gcc act ggc atc cca gac agg ttc agt ggc cgt ggg tct ggg aca gac ttc act ctc acc atc agc agc ctg gag cct gaa gat ttt gca gtg tat tac tgt cag aac tat ggt agt cca ttt tta ttc act ttc ggc cct ggg acc aaa gtg gat atc aaa cga act gtg 80 AT10-005 Light chain gaa att gtg ttg acg cag tct cca ggc acc ctg tct ttg tct cca ggg gaa aga gcc acc ctc tcc tgc agg gcc agt cag agt gtt agt agc agc tac tta gcc tgg tac cag cag aaa cct ggc cag gct ccc agg ctc ctc atc ttt ggt gca tcc acc agg gcc act ggc atc cca gac agg ttc agc ggc agt ggg tct ggg aca gac ttc act ctc acc atc agc aga ctg gag cct gaa gat ttt gca gtg ttt tac tgt cag cag tat ggt agc tta cct ctc act ttc ggc gga ggg acc aag gtg gag atc aaa gga act gtg
(82) TABLE-US-00002 TABLE 2 Recombinant human HA recognition by B cells that secrete heterosubtypic cross-binding mAbs. Group Host Virus Strain AT10_001 AT10_002 AT10_003 AT10_004 1 Human H1N1 A/New Caledonia/20/1999 Negative Negative Negative Positive 2 Human H3N2 A/Wyoming/03/2003 Positive Positive Positive Positive 1 Human H5N1 A/Vietnam/1203/2004 Negative Negative Positive Negative 2 Human H7N7 A/Netherlands/219/2003 Positive Positive Positive Positive
(83) TABLE-US-00003 TABLE 3 Recombinant human, swine and duck infecting Influenza HA protein recognition by heterosubtypic cross-binding mAbs. Neg Group Host Virus Strain AT10_001 AT10_002 AT10_003 AT10_004 AT10_005 ctrl mAb 1 Human H1N1 A/California/07/2009 Negative Negative Negative Negative Positive Negative 1 Human H1N1 A/New Caledonia/20/1999 Negative Negative Negative Positive Positive Negative 1 Human H5N1 A/Vietnam/1203/2004 Negative Negative Positive Negative Positive Negative 1 Human H9N2 A/Hong Kong/1073/1999 Negative Negative Positive Positive Positive Negative 2 Human H3N2 A/Aichi/2/1968 Positive Positive Positive Positive Negative Negative 2 Human H3N2 A/Wyoming/03/2003 Positive Positive Positive Positive Negative Negative 2 Swine H4N6 A/Swine/Ontario/01911-1/1999 Low Positive Negative Positive Low Positive Negative Negative 2 Human H7N7 A/Netherlands/219/2003 Positive Positive Positive Positive Negative Negative 2 Duck H10N3 A/duck/Hong Kong/786/1979 Negative Positive Positive Negative Negative Negative 2 Duck H15N8 A/duck/AUS/341/1983 Negative Positive Positive Low Positive Negative Negative Human Influenza B B/Florida/4/2006 Negative Negative Negative Negative Negative Negative
(84) TABLE-US-00004 TABLE 4 Antibody binding to virus infected MDCK cells. Group Host Virus Strain AT10_001 AT10_002 AT10_003 AT10_004 AT10_005 Neg ctrl mAb 1 Human H1N1 A/Neth/602/2009 Low positive Negative Negative Negative Positive Negative 1 Turkey H5N1 A/Turkey/Turkey/2004 (HPAI) Negative Negative Negative Low positive Positive Negative 2 Swine H3N2 A/swine/St.oedenrode/1996 Negative Positive Low positive Positive Negative Negative (LPAI) 2 Chicken H7N1 A/Ch/Italy/1067/1999 (LPAI) Positive Positive Negative Positive Negative Negative 2 Chicken H7N7 A/Ch/Neth/621557/2003 (HPAI) Positive Positive Low positive Positive Negative Negative
(85) TABLE-US-00005 TABLE 5 In vitro influenza A virus neutralization of virus infected MDCK-SIAT cells by recombinant antibodies. AT10_001 AT10_002 AT10_003 AT10_004 AT10_005 H3N2 A/Ned/177/2008 0.64 0.18 >50 0.17 ND H3N2 HKX-31 2.1 0.25 >15 0.017 ND H1N1 A/Hawaii/31/2007 >15 >15 >15 >50 0.24 ND = Not done IC50 values displayed in μg/ml
(86) TABLE-US-00006 TABLE 6 Recombinant HA and HA1 subunit recognition by recombinant antibodies. AT10-001 AT10-002 AT10-003 AT10-004 AT10_005 H3N2 A/Aichi/2/1968 Full length 0.953 0.920 1.319 0.491 −0.003 H3N2 A/Aichi/2/1968 HA1 subunit 0.010 −0.006 1.277 0.096 −0.007
(87) TABLE-US-00007 TABLE 7 Table 7. In vitro influenza A neutralization of virus infected MDCK cells by recombinant antibodies TCID50 tested Virus AT10_001 AT10_002 AT10_003 AT10_004 AT10_005 CR8020 917 H1N1 A/Neth/602/2009 (swine-origin) >15 >15 >15 >15 2.7 >15 41 H5N1 A/turkey/Turkey/05 >15 >15 >15 >15 1.3 >15 355 H3N2 A/swine/Neth/St. Oedenrode/96 14 0.3 >15 2.3 >15 >15 100 H7N1 A/ck/Italy/1067/99 >15 3.6 >15 0.6 >15 >15 40 H7N7 A/ck/Neth/621557/03 0.4 0.1 >15 0.2 >15 0.6 IC50 values displayed in μg/ml
(88) TABLE-US-00008 TABLE 8 Table 8. Selection of sequences to replace segments where substitutions occur between the target sequence (AT10_002) and the template (2XZA and 3TNN). The numbering follows the rules of Kabat. AT10_002 3D template: VH 2XZA 77.2% identity VL 3TNN 85.6% identity Region Amino Acids Alignment (PDB nr) VH 2 2J6EH, 3Q6GH, 3TJEH, 3THMH, 2XZAH 10 2XZAH, 2XZAH, 3B2UH, 3B2VH, 2J6EH 23-33 3B2UH, 3B2VH, 2J6EH, 1MCOH, 2VXQH 50 2XZAH, 2XZCH, 2VXQH, 3B2UH, 3B2VH 67-68 2XZAH, 2XZCH, 2J6EH, 2JIXD, 2EKSB 79-82c 1U6AH, 3HI1H, 2XZAH, 2XZCH, 2J6EH 87 3GO1H, 2XZAH, 2XZCH, 3B2UH, 3B2VH 94-102 1BZ7B, 1R24B, 1XIWD, 3IVKH, 4DKEH 109-111 3B2UH, 3B2VH, 2JIXD, 2YK1H, 2YKLH VL 29-38 3TNML, 3KDML, 3TNNL, 2JB5L, 2JB6A 50-60 3KDML, 2OLDA, 2OMBA, 2OMNA, 1NL0L 91-97 1JVKA, 1LGVA, 1LHZA, 2OLDA, 2OMBA 107 1JVKA, 1LGVA, 1LHZA, 2JB5L, 2JB6A
(89) TABLE-US-00009 TABLE 9 Table 9. Selection of sequences to replace segments where substitutions occur between the target sequence (AT10_004) and the template (3SDY). The numbering follows the rules of Kabat. AT10_004 3D template: VH 3SDYH 77.3% identity VL 3EYQ 96.3% identity Region Amino Acids Alignment (PDB nr) VH 5 2XQBH, 4FQJH, 4FQKE, 3IYWH, 3N9GH 12-13 3IYWH, 3N9GH, 3QEHA, 2CMRH, 3LMJH 31-37 3GRWH, 2XQBH, 3SDYH, 4FQJH, 4FQKE 48 4FQJH, 4FQKE, 2XQBH, 3SDYH, 3SM5H 54-58 3SDYH, 4FQJH, 4FQKE, 1RMFH, 2XQBH 65 2D7TH, 2XQBH, 3SDYH, 4FQJH, 4FQKE 76 3C08H, 3C09H, 3LMJH, 3LQAH, 3NTCH 82 2XQBH, 4FQJH, 4FQKE, 1HZHH, 1N0XH 87 2XQBH, 3SDYH, 2D7TH, 1WT5A, 1IQDB 95-103 3BN9D, 3MLXH, 3MLYH, 3MLZH, 1KXTB VL CDR3 2XQY
(90) TABLE-US-00010 TABLE 10 Table 10. Selection of sequences to replace segments where substitutions occur between the target sequence (AT10_005) and the template (3QOT). The numbering follows the rules of Kabat. AT10_005 3D template: VH 3QOTH 83.3% identity VL 4FQL 93.5% identity Region Amino Acids Alignment (PDB nr) VH 28-31 3NPSB, 2JB5H, 2JB6B, 1RZIB 57 3MA9H, 3NPSB, 2CMRH, 1RHHB 73-82A 3PP3H, 3PP4H, 3HC4H, 3HC0H, 3HC3H 95-102 4FQIH, 3P30H, 2FB4H, 2IG2H, 3NPSB, 3MLWH VL CDR3 1DN0