WO2011019932A9 - Compositions et méthodes utilisables dans le cadre du traitement et du diagnostic de la grippe - Google Patents

Compositions et méthodes utilisables dans le cadre du traitement et du diagnostic de la grippe Download PDF

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WO2011019932A9
WO2011019932A9 PCT/US2010/045346 US2010045346W WO2011019932A9 WO 2011019932 A9 WO2011019932 A9 WO 2011019932A9 US 2010045346 W US2010045346 W US 2010045346W WO 2011019932 A9 WO2011019932 A9 WO 2011019932A9
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seq
antibody
antibodies
amino acid
domain
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PCT/US2010/045346
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English (en)
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WO2011019932A3 (fr
WO2011019932A2 (fr
Inventor
Andres G. Grandea, Iii
Gordon King
Thomas C. Cox
Ole Olsen
Jennifer Mitcham
Matthew Moyle
Phil Hammond
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Theraclone Sciences, Inc.
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Priority to JP2012524878A priority Critical patent/JP2013501807A/ja
Priority to CA2771105A priority patent/CA2771105A1/fr
Priority to EP10808769A priority patent/EP2464383A4/fr
Priority to AU2010282415A priority patent/AU2010282415A1/en
Publication of WO2011019932A2 publication Critical patent/WO2011019932A2/fr
Publication of WO2011019932A9 publication Critical patent/WO2011019932A9/fr
Publication of WO2011019932A3 publication Critical patent/WO2011019932A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1018—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/11—Orthomyxoviridae, e.g. influenza virus

Definitions

  • the present invention relates generally to prevention, diagnosis, therapy and monitoring of influenza infection.
  • the invention is more specifically related to compositions containing a combination of human antibodies raised against either the influenza hemagglutinin or matrix 2 protein. Such compositions are useful in pharmaceutical compositions for the prevention and treatment of influenza, and for the diagnosis and monitoring of influenza infection.
  • Influenza virus infects 5-20% of the population and results in 30,000-50,000 deaths each year in the U.S. Disease caused by influenza A viral infections is typified by its cyclical nature. Antigenic drift and shift allow for different A strains to emerge every year. Added to that, the threat of highly pathogenic strains entering into the general population has stressed the need for novel therapies for flu infections.
  • the predominant fraction of neutralizing antibodies is directed to the polymorphic regions of the hemagglutinin and neuraminidase proteins.
  • M2 matrix 2
  • M2 protein is found in a homotetramer that forms an ion channel and is thought to aid in the uncoating of the virus upon entering the cell. After infection, M2 can be found in abundance at the cell surface. It is subsequently incorporated into the virion coat, where it only comprises about 2% of total coat protein.
  • M2 extracellular domain (M2e) is short, with the aminoterminal 2-24 amino acids displayed outside of the cell. Anti-M2 MAbs to date have been directed towards this linear sequence. Thus, they may not exhibit desired binding properties to cellularly expressed M2, including conformational determinants on native M2.
  • the invention provides diagnostic, prophylactic, and therapeutic compositions including a human antibody raised against the Influenza hemagglutinin protein and a human monoclonal antibody raised against the Influenza M2 protein. Moreover, the invention provides diagnostic, prophylactic, and therapeutic compositions including a human antibody raised against an epitope of the Influenza hemagglutinin protein and a human monoclonal antibody raised against an epitope of the Influenza M2 protein. Furthermore, these compositions are pharmaceutical compositions that include a pharmaceutical carrier. These compositions address a long-felt need in the art for pharmaceutical compositions that both strongly neutralizes Influenza virus infection and recognizes constant regions within proteins common to all Influenza strains.
  • the invention provides a composition including: (a) a human antibody that specifically binds to an epitope of the hemagglutinin (HA) glycoprotein of an influenza virus; and (b) a human monoclonal antibody that specifically binds to an epitope in the extracellular domain of the matrix 2 ectodomain (M2e) polypeptide of an influenza virus.
  • a human antibody that specifically binds to an epitope of the hemagglutinin (HA) glycoprotein of an influenza virus
  • M2e matrix 2 ectodomain
  • the human monoclonal antibody that specifically binds an epitope of the M2e polypeptide is TCN-032 (8110), 21 B1 5, TCN-031 (23 12), 3241_G23, 3244J 10, 3243J07, 3259J21 , 3245_019, 3244_H04, 3136_G05, 3252_C 13, 3255J06, 3420J23, 3139_P23, 3248_P18, 3253_P10, 3260_D19, 3362_B1 1 , or 3242_P05.
  • the human antibody that specifically binds an epitope of the HA glycoprotein is optionally SC06-141 , SC06-255, SC06-257, SC06-260, SC06-261 , SC06-262, SC06-268, SC06-272, SC06-296, SC06-301 , SC06-307, SC06-310, SC06-314, SC06-323, SC06-325, SC06-327, SC06-328, SC06-329, SC06-331 , SC06-332, SC06-334, SC06-336, SC06-339, SC06-342, SC06-343, SC06-344, CR6141 , CR6255, CR6257, CR6260, CR6261 , CR6262, CR6268, CR6272, CR6296, CR6301 , CR6307, CR6310, CR6314, CR6323, CR6325, CR6327, CR6328, CR6329, CR6331
  • the epitope of the HA glycoprotein is optionally GVTN VNSIID (SEQ ID NO: 198), GVTNKVNSIINK (SEQ ID NO: 283), GVTNKENSIID (SEQ ID NO: 202), GVTN VNRIIDK (SEQ ID NO: 201 ), GITNKVNSVIE (SEQ ID NO: 281 ),
  • GITN ENSVIE SEQ ID NO: 257
  • GITNKVNSIID SEQ ID NO: 225
  • the influenza hemaglutinin (HA) glycoprotein includes an HA 1 and HA2 subunit.
  • exemplary epitopes of the HA glycoprotein include the HA 1 subunit, HA2 subunit, or both the HA 1 and HA2 subunits.
  • the epitope of the M2e polypeptide is a discontinuous epitope.
  • the epitope of the 2e polypeptide includes the amino acid at positions 2, 5, and 6 of
  • MSLLTEVETPTRNEWGCRCNDSSD (SEQ ID NO: 1 ).
  • the invention further provides a composition including: (a) an isolated human anti- HA antibody, or an antigen-binding fragment thereof, including a heavy chain variable region (VH) domain and a light chain variable (VL) domain, wherein the VH domain and the VL domain each contain three complementarity determining regions 1 to 3 (CDRl-3), and wherein each CDR includes the following amino acid sequences: VH CDR1 : SEQ ID NOs: 566, 571 , 586, 597, 603, 609, 615, 627, 633, 637, 643, 649, 658, 664, 670, 303, 251 , 242, or 222; VH CDR2: SEQ ID NOs: 567, 572, 587, 592, 598, 604, 610, 616, 628, 634, 638, 644, 650, 655, 659, 665, 671 , 306, 249, 307, or 221 ; VH CDR3: SEQ ID NOs
  • the invention provides a composition including: (a) an isolated human anti-HA antibody, or an antigen-binding fragment thereof, including a heavy chain variable region (VH) domain, wherein the VH domain includes the following amino acid sequences: SEQ ID NOs 309, 313, 317, 321 , 325, 329, 333, 337, 341 , 345, 349, 353, 357, 361 , 365, 369, 373, 377, 381 , 385, 389, 393, 397, 401 ; 405, 409, 199, 417, 423, 429, 435, 441 , 447, 453, 459, 465, 471 , 477, 483, 489, 495, 501 , 507, 513, 519, 525, 531 , 537, 543, 550, 556, or 562, and a light chain variable (VL) domain, wherein the VL domain includes the following amino acid sequences: SEQ ID NOs
  • the invention provides a multivalent vaccine composition including any of the compositionsdescribed herein containing an isolated human anti-HA antibody, or an antigen-binding fragment thereof and an isolated anti-matrix 2 ectodomain (M2e) antibody, or antigen-binding fragment thereof.
  • the multivalent vaccine includes antibodies that bind to the epitopes to which the antibodies of the invention bind.
  • MSLLTEVETPTRNEWGCRCNDSSD (SEQ ID NO: 1 ) provided in its native conformation.
  • the multivalent vaccine also includes a composition including: (a) a human antibody that specifically binds to an epitope of the hemagglutinin (HA) glycoprotein of an influenza virus; and (b) a human monoclonal antibody that specifically binds to an epitope in the extracellular domain of the matrix 2 ectodomain (M2e) polypeptide of an influenza virus.
  • a composition including: (a) a human antibody that specifically binds to an epitope of the hemagglutinin (HA) glycoprotein of an influenza virus; and (b) a human monoclonal antibody that specifically binds to an epitope in the extracellular domain of the matrix 2 ectodomain (M2e) polypeptide of an influenza virus.
  • HA hemagglutinin
  • M2e matrix 2 ectodomain
  • the invention provides a pharmaceutical composition including any one of the compositions described herein. Moreover, the pharmaceutical composition includes a pharmaceutical carrier.
  • the invention provides a method for stimulating an immune response in a subject, including administering to the subject the pharmaceutical composition described herein.
  • the pharmaceutical composition may administered prior to or after exposure of the subject to an Influenza virus.
  • the invention also provides a method for the treatment of an influenza virus infection in a subject in need thereof, including administering to the subject the pharmaceutical composition described herein.
  • the subjection may have been exposed to an influenza virus.
  • the subject has not been diagnosed with an influenza infection.
  • the pharmaceutical composition may administered prior to or after exposure of the subject to an Influenza virus.
  • the pharmaceutical composition is administered at a dose sufficient to promote viral clearance or eliminate influenza infected cells.
  • the invention further provides a method for the prevention of an influenza virus infection in a subject in need thereof, including administering to the subject a vaccine composition described herein, prior to exposure of the subject to an influenza virus.
  • the subject is at risk of contracting an influenza infection.
  • the pharmaceutical composition may administered prior to or after exposure of the subject to an Influenza virus.
  • the pharmaceutical composition is administered at a dose sufficient to promote viral clearance or eliminate influenza infected cells.
  • the treatment and prevention methods provided by the invention further include administering an anti-viral drug, a viral entry inhibitor or a viral attachment inhibitor.
  • anti-viral drugs include, but are not limited to, a neuraminidase inhibitor, a HA inhibitor, a sialic acid inhibitor, or an M2 ion channel inhibitor.
  • the M2 ion channel inhibitor is amantadine or rimantadine.
  • the neuraminidase inhibitor is zanamivir or oseltamivir phosphate.
  • the antiviral drug may administered prior to or after exposure of the subject to an Influenza virus.
  • the treatment and prevention methods provided by the invention further include administering a second anti-Influenza A antibody.
  • the second antibody is optionally an antibody described herein.
  • the second antibody may administered prior to or after exposure of the subject to an Influenza virus.
  • the invention provides a method for determining the presence of an Influenza virus infection in a subject, including the steps of: (a) contacting a biological sample obtained from the subject with any one of the antibodies or pharmaceutical compositions described herein; (b) detecting an amount of the antibody that binds to the biological sample; and (c) comparing the amount of antibody that binds to the biological sample to a control value, and therefrom determining the presence of the Influenza virus in the subject.
  • the control value is determined by contacting a control sample obtained from the subject with any one of the antibodies or pharmaceutical compositions described herein and detecting an amount of the antibody that binds to the control sample.
  • the invention also provides a diagnostic kit including any one of the antibodies, compositions, or pharmaceutical compositions described herein.
  • the invention further provides a prophylactic kit including a vaccine composition described herein.
  • the vaccine is a multivalent vaccine.
  • multivalent vaccine describes a single vaccine that elicits an immune response either to more than one infectious agent, e.g. the influenza HA glycoprotein and the influenza M2e polypeptide, or to several different epitopes of a molecule, e.g. HA epitopes shown in SEQ ID NOs 198, 283, 202, 201, 281, 257, 225, and 216.
  • the term multivalent vaccine is meant to describe the administration of a combination of human antibodies raised against more than one infectious agent, e.g. the influenza HA glycoprotein and the influenza M2e polypeptide.
  • Figure 1 shows the binding of three antibodies of the present invention and control hul4C2 antibody to 293-HE cells transfected with an M2 expression construct or control vector, in the presence or absence of free M2 peptide.
  • Figures 2A and B are graphs showing human monoclonal antibody binding to influenza A/Puerto Rico/8/32.
  • Figure 3 A is a chart showing amino acid sequences of extracellular domains of M2 variants (SEQ ID NOS 1-3, 679 &5-40, respectively, in order of appearance).
  • Figures 3B and C are bar charts showing binding of human monoclonal anti-influenza antibody binding to M2 variants shown in Figure 3A.
  • Figures 4 A and B are bar charts showing binding of human monoclonal anti- influenza antibody binding to M2 peptides subjected to alanine scanning mutagenesis.
  • Figure 5 is a series of bar charts showing binding of MAbs 8il0 and 23K12 to M2 protein representing influenza strain A/HK/483/1997 sequence that was stably expressed in the CHO cell line DG44.
  • Figure 6A is a chart showing cross reactivity binding of anti-M2 antibodies to variant M2 peptides (SEQ ID NOS 680-704, respectively, in order of appearance).
  • Figure 6B is a chart showing binding activity of M2 antibodies to truncated M2 peptides (SEQ ID NOS 680, 705-724 &19, respectively, in order of appearance).
  • Figure 7 is a graph showing survival of influenza infected mice treated with human anti-influenza monoclonal antibodies.
  • Figure 8 is an illustration showing the anti-M2 antibodies bind a highly conserved region in the N-Terminus of M2e (SEQ ID NO: 19).
  • Figure 9 is a graph showing anti-M2 rHMAb clones from crude supernatant bound to influenza on ELISA, whereas the control anti-M2e mAb 14C2 did not readily bind virus.
  • FIG. 10 is a series of photographs showing anti-M2 rHMAbs bound to cells infected with influenza. MDC cells were or were not infected with influencza A/PR/8/32 and Ab binding from crude supernatant was tested 24 hours later. Data were gathered from the FMAT plate scanner.
  • FIG 11 is a graph showing anti-M2 rHMAb clones from crude supernatant bound to cells transfected with the influenza subtypes H3N2, H 483, and VN1203 M2 proteins. Plasmids encoding full length M2 cDNAs corresponding to influenza strains H3N2, HK483, and VN1203, as well as a mock plasmid control, were transiently transfected into 293 cells. The 14C2, 8il0, 23 12, and 21B15 mABs were tested for binding to the transfectants, and were detected with an AF647-conjugated anti-human IgG secondary antibody. Shown are the mean fluorescence intensities of the specific mAB bound after FACS analysis.
  • Figures 12A-B are amino acid sequences of the variable regions of anti-M2e mAbs.
  • Framework regions 1-4 FR 1-4
  • complementarity determining regions 1-3 CDR 1-3
  • FR, CDR, and gene names are defined using the nomenclature in the IMGT database (IMGT®, the Internationa] ImMunoGeneTics Information system® http:/ www.imgt.org ' ).
  • Grey boxes denote identity with the germline sequence which is shown in light blue boxes, hyphens denote gaps, and white boxes are amino acid replacement mutations from the germline.
  • FIG. 13 is a graph depicting the results of a competition binding analysis of a panel of anti-M2e mAbs with TCN-032 Fab.
  • the indicated anti-M2e mAbs were used to bind to the stable CHO transfectant expressing M2 of A/Hong Kong/483/97 that had previously been treated with or without 10 ⁇ g/mL TCN-032 Fab fragment.
  • the anti-M2e mAb bound to the cell surface was detected with goat anti-huIgG FcAlexafluor488 FACS and analyzed by flow cytometry. The results are derived from one experiment.
  • Figure 14A is a graph depicting the ability of anti-M2e mAbs TCN-032 and TCN- 031 to bind virus particles and virus- infected cells but not M2e-derived synthetic peptide.
  • Purified influenza virus A Puerto Rico/8/34 was coated at 10 ⁇ g/ml on ELISA wells and binding of anti-M2e mAbs TCN-031 , TCN-032, ch l 4C2, and the HCMV mAbs 2N9 was evaluated using HRP-labeled goat anti-human Fc. Results shown are representative of 3 experiments.
  • Figure 14B is a graph depicting the ability of anti-M2e mAbs TCN-032 and TCN- 031 to bind virus particles and virus-infected cells but not M2e-derived synthetic peptide.
  • 23mer synthetic peptide of M2 derived from A/Fort Worth/1/50 was coated at 1 ⁇ g/ml on ELISA wells and binding of mAbs TCN-031 , TCN-032, ch l 4C2, and 2N9 were evaluated as in panel a. Results shown are representative of 3 experiments.
  • Figure 14C is a graph depicting the ability of anti-M2e mAbs TCN-032 and TCN- 031 to bind virus particles and virus-infected cells but not M2e-derived synthetic peptide.
  • MDCK cells were infected with A/Puerto Rico/8/34 (PR8) and subsequently stained with mAbs TCN-031 , TCN-032, ch 14C2 and the HCMV mAb 5J 12. Binding of antibodies was detected using Alexafluor 647-conjugated goat anti-Human IgG H&L antibody and quantified by flow cytometry. Results shown are representative of 3 experiments.
  • FIG 14D is a series of photographs depicting HEK 293 cells stably transfected with the M2 ectodomain of A/Fort Worth /1/50 (D20) were stained with transient transfection supernatant containing mAbs TCN-031 , TCN-032, or the control chl4C2 and analyzed by FMAT for binding to M2 in the presence or absence of 5 ug/ml M2e peptide.
  • Mock transfected cells are 293 cells stably transfected with vector alone. Results shown are representative of one experiment.
  • FIGS 15A-D are graphs depicting the Therapeutic efficacy of anti-M2 mAbs TCN-031 and TCN-032 in mice.
  • FIG 16 is a series of graphs depicting the viral titers in lung, liver, and brain of mice treated with anti-M2e mAbs TCN-031 and TCN-032 after challenge with H5N 1 A/Vietnam/ 1203/04.
  • Tissue viral titers were determined from 3 mice per group at 3 and 6 days post-infection in the lungs (as an indicator of local replication) and in liver and brain (as an indicator of the systemic spread which is characteristic of H5N1 infection).
  • FIG. 17 is a graph depicting the ability of TCN-031 and TCN-032 can potentiate cytolysis by NK cells.
  • MDCK cells were infected with A/Solomon Island/3/2006 (H I Nl ) virus, and were treated with mAbs TCN-031 , TCN-032, or the subclass-matched negative control mAb 2N9. The cells were then challenged with purified human NK cells, and the lactate dehydrogenase released as a result of cell lysis was measured through light absorbance. The results are representative of two separate experiments with two different normal human donors.
  • FIG 18 is a graph depicting complement-dependent cytolysis (CDC) of M2- expressing cells bound with anti-M2 mAb.
  • CDC complement-dependent cytolysis
  • Figures 19A-C are graphs depicting binding of anti-M2e mAbs TCN-031 and TCN- 032 to M2 mutants indicates the epitope is located in the highly conserved N-terminal of M2e. Mutants with alanine substituted at each position of the M2 ectodomain of A Fort Worth /1 /50 (D20)(A) or forty wild-type M2 mutants including
  • A/Vietnam/1203/04 (VN) and A/Hong Kong/483/97 (HK) (5) were transiently transfected into 293 cells.
  • the identity of each wild-type M2 mutant is listed in Table 6.
  • Transfected cells were stained with mAbs TCN-031 , TCN-032, or the control chl4C2 and analyzed by FACS for binding to M2 at 24 hours post-transfection.
  • mAbs TCN-031 and TCN-032 do not bind variants with amino acid substitutions at positions 1 , 4, or 5 of M2e.
  • FIG. 20 is a graph depicting mAbs TCN-031 and TCN-032 recognize the same region on M2e.
  • the CHO transfectant stably expressing M2 for A/Hong Kong/483/97 as stained with 10 ⁇ g/mL TCN-031 , TCN-032, or 2N9, followed by detection with AlexafIuor647-labeled TCN-031 (TCN-031 AF647) or TCN-032(TCN-032AF647) and analysis by flow cytometry.
  • TCN-031 AF647 AlexafIuor647-labeled TCN-031
  • TCN-032 TCN-032(TCN-032AF647)
  • Figure 21 is a graph depicting anti-M2e mAbs TCN-031 and TCN-032 bind cells that have been infected with H1N1 A/California/4/09. MDCK cells were infected with Influenza A strain H 1N1 A/Memphis/ 14/96, H1 1 A/California/4/09, or mock infected. Twenty four hours post-infection cells were stained with mAbs TCN-031 , TCN-032, or the control chl 4C2 and analyzed by FACS for binding to M2. Results shown are for one experiment.
  • Influenza viruses consist of three types, A, B and C. Influenza A viruses infect a wide variety of birds and mammals, including humans, horses, marine mammals, pigs, ferrets, and chickens. In animals most influenza A viruses cause mild localized infections of the respiratory and intestinal tract. However, highly pathogenic influenza A strains such as H5N1 exist that cause systemic infections in poultry in which mortality may reach 100%. Animals infected with influenza A often act as a reservoir for the influenza viruses and certain subtypes have been shown to cross the species barrier to humans.
  • Influenza A viruses can be classified into subtypes based on allelic variations in antigenic regions of two genes that encode surface glycoproteins, namely, hemagglutinin (HA) and neuraminidase (NA) which are required for viral attachment and cellular release.
  • Other major viral proteins include the nucleoprotein, the nucleocapsid structural protein, membrane proteins (Ml and M2), polymerases (PA, PB and PB2) and non-structural proteins (NSl and NS2).
  • Ml and M2 membrane proteins
  • PA, PB and PB2 polymerases
  • NSl and NS2 non-structural proteins
  • avian influenza A has been reported to cross the species barrier and infect humans as documented in Hong Kong in 1997 and 2003, leading to the death of several patients.
  • the avian influenza virus infects cells of the respiratory tract as well as the intestinal tract, liver, spleen, kidneys and other organs. Symptoms of avian influenza infection include fever, respiratory difficulties including shortness of breath and cough, lymphopenia, diarrhea and difficulties regulating blood sugar levels.
  • the group most at risk is healthy adults, which make up the bulk of the population.
  • compositions including human antibodies raised against two influenza proteins, hemagglutinin (HA) and matrix 2 ectodomain (M2e), and shows that these compositions can be used in medicine, in particular for diagnosis, prevention and treatment of influenza infections, including H5N 1 .
  • HA hemagglutinin
  • M2e matrix 2 ectodomain
  • the present invention provides fully human monoclonal antibodies specifically directed against M2e.
  • the antibody is isolated form a B-cell from a human donor.
  • Exemplary monoclonal antibodies include TCN-032 (8110), 21 B15, TCN-031 (23K12), 3241_G23, 3244J10, 3243J07, 3259J21 , 3245_019, 3244_H04, 3136_G05, 3252_C13, 3255J06, 3420J23, 3139_P23, 3248_P18, 3253_P10, 3260_D19, 3362_B1 1 , and
  • the monoclonal antibody is an antibody that binds to the same epitope as TCN-032 (8110), 21 B15, TCN-031 (23 12), 3241 _G23, 3244J10, 3243J07, 3259J21 , 3245_019, 3244_H04, 3136_G05, 3252_C 13, 3255J06, 3420J23, 3139 P23, 3248_P18, 3253_P10, 3260_D19, 3362 B 1 1 , and 3242_P05.
  • the antibodies respectively referred to herein are huM2e antibodies.
  • the huM2e antibody has one or more of the following characteristics: a) binds to an epitope in the extracellular domain of the matrix 2 ectodomain (M2e) polypeptide of an influenza virus; b) binds to influenza A infected cells; or c) binds to influenza A virus.
  • M2e matrix 2 ectodomain
  • the epitope that huM2e antibody binds to is a non-linear epitope of a M2 polypeptide.
  • the epitope includes the amino terminal region of the M2e polypeptide. More preferably the epitope wholly or partially includes the amino acid sequence SLLTEV (SEQ ID NO: 42).
  • the epitope includes the amino acid at position 2, 5 and 6 of the M2e polypeptide when numbered in accordance with SEQ ID NO: 1.
  • the amino acid at position 2 is a serine; at position 5 is a threonine; and at position 6 is a glutamic acid.
  • a huM2e antibody contains a heavy chain variable having the amino acid sequence of SEQ ID NOs: 44, 277, 276, 50, 236, 235, 1 16, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, or 176 and a light chain variable having the amino acid sequence of SEQ ID NOs: 46, 52, 1 1 8, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 1 74, or 178.
  • the three heavy chain CDRs include an amino acid sequence at least ⁇ 90%, 92%, 95%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NOs: 72, 74, 76, 103, 105, 107, 179, 180, 181 , 187, 188, 189, 197, 203, 204, 205, 21 , 212, 213, 228, 229, 230, 237, 238, 252, 253, 254, 260, 261 , 262, 268, 269, 270, 284, 285, 286, 293, 294, 295, and 301 (as determined by the Kabat method) or SEQ ID NOs: 109, 1 10, 76, 1 12, 1 13, 107, 182, 183, 181 , 190, 191 , 189, , 197, 206, 207, 205, 214, 215, 213, 232, 230, 239, 240, 238, 255, 256, 254, 263, 264
  • the heavy chain of a M2e antibody is derived from a germ line V (variable) gene such as, for example, the IgHV4 or the IgHV3 germline gene.
  • the M2e antibodies of the invention include a variable heavy chain (VH) region encoded by a human IgHV4 or the IgHV3 germline gene sequence.
  • VH variable heavy chain
  • a IgHV4 germline gene sequence are shown, e.g. , in Accession numbers L10088, M29812, M951 14, X56360 and M951 17.
  • IgHV3 germline gene sequence are shown, e.g. , in Accession numbers X92218, X70208, Z27504, M99679 and AB019437.
  • the M2e antibodies of the invention include a V H region that is encoded by a nucleic acid sequence that is at least 80% homologous to the IgHV4 or the IgHV3 germline gene sequence.
  • the nucleic acid sequence is at least 90%, 95%, 96%, 97% homologous to the IgHV4 or the IgHV3 germline gene sequence, and more preferably, at least 98%, 99% homologous to the IgHV4 or the IgHV3 germline gene sequence.
  • the VH region of the M2e antibody is at least 80% homologous to the amino acid sequence of the V H region encoded by the IgHV4 or the IgHV3 VH germline gene sequence.
  • the amino acid sequence of VH region of the M2e antibody is at least 90%, 95%, 96%, 97% homologous to the amino acid sequence encoded by the IgHV4 or the IgHV3 germiine gene sequence, and more preferably, at least 98%, 99% homologous to the sequence encoded by the IgHV4 or the IgHV3 germiine gene sequence.
  • the M2e antibodies of the invention also include a variable light chain (VL) region encoded by a human IgKV l germiine gene sequence.
  • VL variable light chain
  • a human IgKVl VL germiine gene sequence is shown, e.g. , Accession numbers X59315, X59312, X59318, J00248, and Y 14865.
  • the M2e antibodies include a V L region that is encoded by a nucleic acid sequence that is at least 80% homologous to the IgKVl germiine gene sequence.
  • the nucleic acid sequence is at least 90%, 95%, 96%, 97% homologous to the IgKV l germiine gene sequence, and more preferably, at least 98%, 99% homologous to the IgKV l germiine gene sequence.
  • the VL region of the M2e antibody is at least 80% homologous to the amino acid sequence of the VL region encoded the IgKVl germiine gene sequence.
  • the amino acid sequence of VL region of the M2e antibody is at least 90%, 95%, 96%, 97% homologous to the amino acid sequence encoded by the IgKVl germiine gene sequence, and more preferably, at least 98%, 99% homologous to the sequence encoded by e the IgKVl germiine gene sequence.
  • the invention provides a composition including an huM2e antibody according to the invention.
  • the composition further includes an anti-viral drug, a viral entry inhibitor or a viral attachment inhibitor.
  • the anti-viral drug is for example a neuraminidase inhibitor, a HA inhibitor, a sialic acid inhibitor or an M2 ion channel inhibitor.
  • the M2 ion channel inhibitor is for example amantadine or rimantadine.
  • the neuraminidase inhibitor for example zanamivir, or oseltamivir phosphate.
  • the composition further includes a second anti-influenza A antibody.
  • huM2e antibodies according to the invention are operably- I inked to a therapeutic agent or a detectable label.
  • the invention provides methods for stimulating an immune response, treating, preventing or alleviating a symptom of an influenza viral infection by administering an huM2e antibody to a subject
  • the subject is further administered with a second agent such as, but not limited to, an influenza virus antibody, an anti-viral drug such as a neuraminidase inhibitor, a HA inhibitor, a sialic acid inhibitor or an M2 ion channel inhibitor, a viral entry inhibitor or a viral attachment inhibitor.
  • a second agent such as, but not limited to, an influenza virus antibody, an anti-viral drug such as a neuraminidase inhibitor, a HA inhibitor, a sialic acid inhibitor or an M2 ion channel inhibitor, a viral entry inhibitor or a viral attachment inhibitor.
  • the M2 ion channel inhibitor is, for example, amantadine or rimantadine.
  • the neuraminidase inhibitor is, for example, zanamivir or oseltamivir phosphate.
  • the subject is suffering from or is predisposed to developing an influenza virus infection, such as, for example, an autoimmune disease or an inflammatory disorder.
  • the invention provides methods of administering the huM2e antibody of the invention to a subject prior to, and/or after exposure to an influenza virus.
  • the huM2e antibody of the invention is used to treat or prevent
  • the huM2e antibody is administered at a dose sufficient to promote viral clearance or eliminate influenza A infected cells.
  • Also included in the invention is a method for determining the presence of an influenza virus infection in a patient, by contacting a biological sample obtained from the patient with a humM2e antibody; detecting an amount of the antibody that binds to the biological sample; and comparing the amount of antibody that binds to the biological sample to a control value.
  • the invention further provides a diagnostic kit comprising a huM2e antibody.
  • the present invention provides fully human monoclonal antibodies specific against the extracellular domain of the matrix 2 (M2) polypeptide.
  • the antibodies are respectively referred to herein as huM2e antibodies.
  • M2 is a 96 amino acid transmembrane protein present as a homotetramer on the surface of influenza virus and virally infected cells.
  • M2 contains a 23 amino acid ectodomain (M2e) that is highly conserved across influenza A strains. Few amino acid changes have occurred since the 1918 pandemic strain thus M2e is an attractive target for influenza therapies.
  • monoclonal antibodies specific to the M2 ectodomain (M2e) were derived upon immunizations with a peptide corresponding to the linear sequence of M2e.
  • the present invention provides a novel process whereby full-length M2 is expressed in cell lines, which allows for the identification of human antibodies that bound this cell- expressed M2e.
  • the hu 2e antibodies have been shown to bind conformational determinants on the M2-transfected cells, as well as native M2, either on influenza infected cells, or on the virus itself.
  • the huM2e antibodies did not bind the linear M2e peptide, but they do bind several natural 2 variants, also expressed upon cDNA transfection into cell lines.
  • this invention has allowed for the identification and production of human monoclonal antibodies that exhibit novel specificity for a very broad range of influenza A virus strains. These antibodies may be used diagnostically to identify influenza A infection and therapeutically to treat influenza A infection.
  • the huM2e antibodies of the invention have one or more of the following characteristics: the huM2e antibody binds a) to an epitope in the extracellular domain of the matrix 2 (M2) polypeptide of an influenza virus; b) binds to influenza A infected cells; and/or c) binds to influenza A virus (i.e., virons).
  • the huM2e antibodies of the invention eliminate influenza infected cells through immune effector mechanisms, such as ADCC, and promote direct viral clearance by binding to influenza virons.
  • the huM2e antibodies of the invention bind to the amino-terminal region of the M2e polypeptide.
  • the huM2e antibodies of the invention bind to the amino-terminal region of the M2e polypeptide wherein the N-terminal methionine residue is absent.
  • Exemplary M2e sequences include those sequences listed on Table 1 below
  • the huM2e antibodies of the invention bind to a M2e that wholly or partially includes the amino acid residues from position 2 to position 7 of M2e when numbered in accordance with SEQ ID NO: 1.
  • the huM2e antibodies of the invention bind wholly or partially to the amino acid sequence SLLTEVET (SEQ ID NO: 41 )
  • the huM2e antibodies of the invention bind wholly or partially to the amino acid sequence SLLTEV (SEQ ID NO: 42)
  • the huM2e antibodies of the invention bind to non-linear epitope of the M2e protein.
  • the huM2e antibodies bind to an epitope comprising position 2, 5, and 6 of the M2e polypeptide when numbered in accordance to SEQ ID NO: 1 where the amino acid at a) position 2 is a serine;b) position 5 is a threonine; and c) position 6 is a glutamic acid.
  • Exemplary huM2e monoclonal antibodies that bind to this epitope are the TCN-032 (8110), 21 B15, TCN-031 (23K12), 3241_G23, 3244 110, 3243J07, 3259_J21 , 3245_019, 3244_H04, 3136_G05, 3252_C13, 3255_J06, 3420J23, 3139 P23, 3248_P18, 3253_P10, 3260_D19, 3362_B1 1 , and 3242 P05 antibodies described herein.
  • the TCN-032 (8110) antibody includes a heavy chain variable region (SEQ ID NO: 44) encoded by the nucleic acid sequence shown below in SEQ ID NO: 43, a short heavy chain variable region (SEQ ID NO: 277) encoded by the nucleic acid sequence shown below in SEQ ID NO: 278, a long heavy chain variable region (SEQ ID NO: 276) encoded by the nucleic acid sequence shown below in SEQ ID NO: 196, and a light chain variable region (SEQ ID NO: 46) encoded by the nucleic acid sequence shown in SEQ ID NO: 45.
  • the heavy chain CDRs of the TCN-032 (8110) antibody have the following sequences per Kabat definition: NYYWS (SEQ ID NO: 72), FIYYGGNTKYNPSLKS (SEQ ID NO: 74) and ASCSGGYCILD (SEQ ID NO: 76).
  • the light chain CDRs of the TCN-032 (8110) antibody have the following sequences per Kabat definition: RASQNIYKYLN (SEQ ID NO: 59), AA SGLQS (SEQ ID NO: 61 ) and QQSYSPPLT (SEQ ID NO: 63).
  • the heavy chain CDRs of the TCN-032 (8110) antibody have the following sequences per Chothia definition: GSSISN (SEQ ID NO: 109), FIYYGGNTK (SEQ ID NO: 1 10) and ASCSGGYCILD (SEQ ID NO: 76).
  • the light chain CDRs of the TCN-032 (8110) antibody have the following sequences per Chothia definition: RASQNIYKYLN (SEQ ID NO: 59), AASGLQS (SEQ ID NO: 61 ) and QQSYSPPLT (SEQ ID NO: 63).
  • TCN-032 (8110) VH nucleotide sequence: (SEQ ID NO: 43)
  • TCN-032 (8110) VH amino acid sequence: (SEQ ID NO: 44)
  • TCN-032 (8110) VH short nucleotide sequence: (SEQ ID NO: 278)
  • the 21 B 15 antibody includes a heavy chain variable region (SEQ ID NO: 44) encoded by the nucleic acid sequence shown below in SEQ ID NO: 47, a short heavy chain variable region (SEQ ID NO: 277) encoded by the nucleic acid sequence shown below in SEQ ID NO: 278, a long heavy chain variable region (SEQ ID NO: 44) encoded by the nucleic acid sequence shown below in SEQ ID NO: 47, a short heavy chain variable region (SEQ ID NO: 277) encoded by the nucleic acid sequence shown below in SEQ ID NO: 278, a long heavy chain variable region (SEQ ID NO: 44) encoded by the nucleic acid sequence shown below in SEQ ID NO: 47, a short heavy chain variable region (SEQ ID NO: 277) encoded by the nucleic acid sequence shown below in SEQ ID NO: 278, a long heavy chain variable region (SEQ ID NO: 44) encoded by the nucleic acid sequence shown below in SEQ ID NO: 47, a short heavy chain variable region (SEQ ID
  • the heavy chain CDRs of the 21 Bl 5 antibody have the following sequences per Kabat definition: NYYWS (SEQ ID NO: 72), FIYYGGNTKYNPSLKS (SEQ ID NO: 74) and ASCSGGYCILD (SEQ ID NO: 76).
  • the light chain CDRs of the 21B15 antibody have the following sequences per Kabat definition: RASQNIYKYLN (SEQ ID NO: 59),
  • AASGLQS (SEQ ID NO: 61 ) and QQSYSPPLT (SEQ ID NO: 63).
  • the heavy chain CDRs of the 21 B 15 antibody have the following sequences per Chothia definition: GSSISN (SEQ ID NO: 109), FIYYGGNTK (SEQ ID NO: 1 10) and ASCSGGYCILD (SEQ ID NO: 76).
  • the light chain CDRs of the 21 B15 antibody have the following sequences per Chothia definition: RASQNIYKYLN (SEQ ID NO: 59), AASGLQS (SEQ ID NO: 61 ) and QQSYSPPLT (SEQ ID NO: 63).
  • the TCN-031 (23 12) antibody includes a heavy chain variable region (SEQ ID NO: 50) encoded by the nucleic acid sequence shown below in SEQ ID NO: 49, a short heavy chain variable region (SEQ ID NO: 236) encoded by the nucleic acid sequence shown below in SEQ ID NO: 244, a long heavy chain variable region (SEQ ID NO: 195) encoded by the nucleic acid sequence shown below in SEQ ID NO: 235, and a light chain variable region (SEQ ID NO: 52) encoded by the nucleic acid sequence shown in SEQ ID NO: 51.
  • the heavy chain CDRs of the TCN-031 (23K 12) antibody have the following sequences per Kabat definition: SNYMS (SEQ ID NO: 103), VIYSGGSTYYADSVK (SEQ ID NO: 105) and CLSRMRGYGLDV (SEQ ID NO: 107).
  • the light chain CDRs of the TCN-031 (23K12) antibody have the following sequences per Kabat definition:
  • RTSQSISSYLN (SEQ ID NO: 92), AASSLQSGVPSRF (SEQ ID NO: 94) and QQSYSMPA (SEQ ID NO: 96).
  • the heavy chain CDRs of the TCN-031 (23K 12) antibody have the following sequences per Chothia definition: GFTVSSN (SEQ ID NO: 112), VIYSGGSTY (SEQ ID NO: 113) and CLSRMRGYGLDV (SEQ ID NO: 107).
  • the light chain CDRs of the TCN- 031 (23K12) antibody have the following sequences per Chothia definition: RTSQSISSYLN (SEQ ID NO: 92), AASSLQSGVPSRF (SEQ ID NO: 94) and QQSYSMPA (SEQ ID NO: 96).
  • the 3241 G23 antibody (also referred to herein as G23) includes a heavy chain variable region (SEQ ID NO: 1 16) encoded by the nucleic acid sequence shown below in SEQ ID NO: 1 15, and a I ight chain variable region (SEQ ID NO: 1 18) encoded by the nucleic acid sequence shown in SEQ ID NO: 1 17.
  • the heavy chain CDRs of the G23 antibody have the following sequences per Kabat definition: GGGYSWN (SEQ ID NO: 179), FMFHSGSPRYNPTLKS (SEQ ID NO: 180) and VGQMDKYYAMDV (SEQ ID NO: 181).
  • the light chain CDRs of the G23 antibody have the following sequences per Kabat definition: RASQSIGAYVN (SEQ ID NO: 184), GASNLQS (SEQ ID NO: 185) and QQTYSTPIT (SEQ ID NO: 186).
  • the heavy chain CDRs of the G23 antibody have the following sequences per Chothia definition: GGPVSGGG (SEQ ID NO: 182), FMFHSGSPR (SEQ ID NO: 183) and
  • VGQMDKYYAMDV (SEQ ID NO: 181 ).
  • the light chain CDRs of the G23 antibody have the following sequences per Chothia definition: RASQSIGAYVN (SEQ ID NO: 184), GASNLQS (SEQ ID NO: 185) and QQTYSTPIT (SEQ ID NO: 186).
  • the 3244 110 antibody (also referred to herein as 110) includes a heavy chain variable region (SEQ ID NO: 120) encoded by the nucleic acid sequence shown below in SEQ ID NO: 119, and a light chain variable region (SEQ ID NO: 122) encoded by the nucleic acid sequence shown in SEQ ID NO: 121.
  • the heavy chain CDRs of the 110 antibody have the following sequences per Kabat definition: SDYWS (SEQ ID NO: 187), FFYNGGSTKYNPSLKS (SEQ ID NO: 188) and HDAKFSGSYYVAS (SEQ ID NO: 189).
  • the light chain CDRs of the 110 antibody have the following sequences per Kabat definition: RASQSISTYLN (SEQ ID NO: 192), GATNLQS (SEQ ID NO: 193) and QQSYNTPLI (SEQ ID NO: 194).
  • the heavy chain CDRs of the 110 antibody have the following sequences per Chothia definition: GGSITS (SEQ ID NO: 190), FFYNGGSTK (SEQ ID NO: 191) and HDA FSGSYYVAS (SEQ ID NO: 189).
  • the light chain CDRs of the 110 antibody have the following sequences per Chothia definition: RASQSISTYLN (SEQ ID NO: 192), GATNLQS (SEQ ID NO: 193) and QQSYNTPLI (SEQ ID NO: 194).
  • the 3243_J07 antibody (also referred to herein as J07) includes a heavy chain variable region (SEQ ID NO: 124) encoded by the nucleic acid sequence shown below in SEQ ID NO: 123, and a light chain variable region (SEQ ID NO: 126) encoded by the nucleic acid sequence shown in SEQ ID NO: 125.
  • the heavy chain CDRs of the J07 antibody have the following sequences per Kabat definition: SDYWS (SEQ ID NO: 187), FFYNGGSTKYNPSLKS (SEQ ID NO: 188) and HDVKFSGSYYVAS (SEQ ID NO: 197).
  • the light chain CDRs of the J07 antibody have the following sequences per Kabat definition: RASQSISTYLN (SEQ ID NO: 192), GATNLQS (SEQ ID NO: 193) and QQSYNTPLI (SEQ ID NO: 194).
  • the heavy chain CDRs of the J07 antibody have the following sequences per Chothia definition: GGSITS (SEQ ID NO: 190), FFYNGGSTK (SEQ ID NO: 191 ) and
  • HDVKFSGSYYVAS (SEQ ID NO: 197).
  • the light chain CDRs of the J07 antibody have the following sequences per Chothia definition: RASQSISTYLN (SEQ ID NO: 192), GATNLQS (SEQ ID NO: 193) and QQSYNTPLI (SEQ ID NO: 194).
  • the 3259_J21 antibody (also referred to herein as J21) includes a heavy chain variable region (SEQ ID NO: 128) encoded by the nucleic acid sequence shown below in SEQ ID NO:, 127, and a light chain variable region (SEQ ID NO: 130) encoded by the nucleic acid sequence shown in SEQ ID NO: 129.
  • the amino acids encompassing the CDRs as defined by Chothia et al., 1989 are underlined and those defined by Kabat et al., 1991 are highlighted in bold in the sequences below.
  • the heavy chain CDRs of the J21 antibody have the following sequences per abat definition: SYNWI (SEQ ID NO: 203), HIYDYGRTFYNSSLQS (SEQ ID NO: 204) and PLGILHYYAMDL (SEQ ID NO: 205).
  • the light chain CDRs of the J21 antibody have the following sequences per Kabat definition: RASQSIDKFLN (SEQ ID NO: 208), GASNLHS (SEQ ID NO: 209) and QQSFSVPA (SEQ ID NO: 210).
  • the heavy chain CDRs of the J21 antibody have the following sequences per Chothia definition: GGSISS (SEQ ID NO: 206), HIYDYGRTF (SEQ ID NO: 207) and
  • PLGILHYYAMDL (SEQ ID NO: 205).
  • the light chain CDRs of the J21 antibody have the following sequences per Chothia definition: RASQSIDKFLN (SEQ ID NO: 208),
  • GASNLHS SEQ ID NO: 209
  • QQSFSVPA SEQ ID NO: 210
  • the 3245 019 antibody (also referred to herein as 019) includes a heavy chain variable region (SEQ ID NO: 132) encoded by the nucleic acid sequence shown below in SEQ ID NO: 131 , and a light chain variable region (SEQ ID NO: 134) encoded by the nucleic acid sequence shown in SEQ ID NO: 133.
  • SEQ ID NO: 132 The amino acids encompassing the CDRs as defined by Chothia et al., 1989 are underlined and those defined by abat et al., 1991 are highlighted in bold in the sequences below.
  • the heavy chain CDRs of the 019 antibody have the following sequences per Kabat definition: STYMN (SEQ ID NO: 21 1 ), VFYSETRTYYADSVKG (SEQ ID NO: 212) and VQRLSYGMDV (SEQ ID NO: 213).
  • the light chain CDRs of the Ol 9 antibody have the following sequences per Kabat definition: RASQSISTYLN (SEQ ID NO: 192), GASTLQS (SEQ ID NO: 217) and QQTYSIPL (SEQ ID NO: 218).
  • the heavy chain CDRs of the 019 antibody have the following sequences per Chothia definition: GLSVSS (SEQ ID NO: 214), VFYSETRTY (SEQ ID NO: 215) and
  • VQRLSYGMDV (SEQ ID NO: 213).
  • the light chain CDRs of the 019 antibody have the following sequences per Chothia definition: RASQSISTYLN (SEQ ID NO: 192), GASTLQS (SEQ ID NO: 217) and QQTYSIPL (SEQ ID NO: 218).
  • the 3244_H04 antibody (also referred to herein as H04) includes a heavy chain variable region (SEQ ID NO: 136) encoded by the nucleic acid sequence shown below in SEQ ID NO: 135, and a light chain variable region (SEQ ID NO: 138) encoded by the nucleic acid sequence shown in SEQ ID NO: 137.
  • the heavy chain CDRs of the H04 antibody have the following sequences per Kabat definition: STYM (SEQ ID NO: 21 1 ), VFYSETRTYYADSVKG (SEQ ID NO: 212) and VQRLSYGMDV (SEQ ID NO: 213).
  • the light chain CDRs of the H04 antibody have the following sequences per Kabat definition: RASQSISTYLN (SEQ ID NO: 192), GASSLQS (SEQ ID NO: 226) and QQTYSIPL (SEQ ID NO: 218).
  • the heavy chain CDRs of the H04 antibody have the following sequences per Chothia definition: GLSVSS (SEQ ID NO: 214), VFYSETRTY (SEQ ID NO: 215) and
  • VQRLSYGMDV (SEQ ID NO: 213).
  • the light chain CDRs of the H04 antibody have the following sequences per Chothia definition: RASQSISTYLN (SEQ ID NO: 192), GASSLQS (SEQ ID NO: 226) and QQTYSIPL (SEQ ID NO: 218).
  • the 3136 G05 antibody (also referred to herein as G05) includes a heavy chain variable region (SEQ ID NO: 140) encoded by the nucleic acid sequence shown below in SEQ ID NO: 139, and a light chain variable region (SEQ ID NO: 142) encoded by the nucleic acid sequence shown in SEQ ID NO: 141.
  • the heavy chain CDRs of the G05 antibody have the following sequences per Kabat definition: SDFWS (SEQ ID NO: 228), YVYNRGSTKYSPSLKS (SEQ ID NO: 229) and NGRSSTSWGIDV (SEQ ID NO: 230).
  • the light chain CDRs of the 3136_G05 antibody have the following sequences per Kabat definition: RASQSISTYLH (SEQ ID NO: 233), AASSLQS (SEQ ID NO: 234) and QQSYSPPLT (SEQ ID NO: 63).
  • the heavy chain CDRs of the 3136 G05 antibody have the following sequences per Chothia definition: GGSISS (SEQ ID NO: 206), YVYNRGSTK (SEQ ID NO: 232) and NGRSSTSWGIDV (SEQ ID NO: 230).
  • the light chain CDRs of the 3136_G05 antibody have the following sequences per Chothia definition: RASQSISTYLH (SEQ ID NO: 233), AASSLQS (SEQ ID NO: 234) and QQSYSPPLT (SEQ ID NO: 63).
  • the 3252_C 13 antibody (also referred to herein as C13) includes a heavy chain variable region (SEQ ID NO: 144) encoded by the nucleic acid sequence shown below in SEQ ID NO: 143, and a light chain variable region (SEQ ID NO: 146) encoded by the nucleic acid sequence shown in SEQ ID NO: 145.
  • the heavy chain CDRs of the C13 antibody have the following sequences per Kabat definition: SDYWS (SEQ ID NO: 187), YIYNRGSTKYTPSLKS (SEQ ID NO: 237) and HVGGHTYGIDY (SEQ ID NO: 238).
  • the light chain CDRs of the C13 antibody have the following sequences per Kabat definition: RASQSISNYLN (SEQ ID NO: 241), AASSLQS (SEQ ID NO: 234) and QQSYNTPIT (SEQ ID NO: 243).
  • the heavy chain CDRs of the CI 3 antibody have the following sequences per Chothia definition: GASISS (SEQ ID NO: 239), YIYNRGSTK (SEQ ID NO: 240) and
  • HVGGHTYGIDY SEQ ID NO: 2378.
  • the light chain CDRs of the CI 3 antibody have the following sequences per Chothia definition: RASQSISNYLN (SEQ ID NO: 241 ), AASSLQS (SEQ ID NO: 234) and QQSYNTPIT (SEQ ID NO: 243).
  • the 3259 J06 antibody (also referred to herein as J06) includes a heavy chain variable region (SEQ ID NO: 148) encoded by the nucleic acid sequence shown below in SEQ ID NO: 147, and a light chain variable region (SEQ ID NO: 150) encoded by the nucleic acid sequence shown in SEQ ID NO: 149.
  • the heavy chain CDRs of the J06 antibody have the following sequences per Kabat definition: SDYWS (SEQ ID NO: 187), YIYNRGSTKYTPSLKS (SEQ ID NO: 237) and HVGGHTYGIDY (SEQ ID NO: 238).
  • the light chain CDRs of the J06 antibody have the following sequences per Kabat definition: RASQSISNYLN (SEQ ID NO: 241 ), AASSLQS (SEQ ID NO: 234) and QQSYNTPIT (SEQ ID NO: 243).
  • the heavy chain CDRs of the J06 antibody have the following sequences per Chothia definition: GASISS (SEQ ID NO: 239), YIYNRGSTK (SEQ ID NO: 240) and
  • HVGGHTYGIDY SEQ ID NO: 2378.
  • the light chain CDRs of the J06 antibody have the following sequences per Chothia definition: RASQSISNYLN (SEQ ID NO: 241 ), AASSLQS (SEQ ID NO: 234) and QQSYNTPIT (SEQ ID NO: 243).
  • the 3410_I23 antibody (also referred to herein as 123) includes a heavy chain variable region (SEQ ID NO: 152) encoded by the nucleic acid sequence shown below in SEQ ID NO: 151 , and a light chain variable region (SEQ ID NO: 1 54) encoded by the nucleic acid sequence shown in SEQ ID NO: 153.
  • the heavy chain CDRs of the 3410 123 antibody have the following sequences per Kabat definition: SYSWS (SEQ ID NO: 252), YLYYSGSTKYNPSLKS (SEQ ID NO: 253) and TGSESTTGYGMDV (SEQ ID NO: 254).
  • the light chain CDRs of the 3410J23 antibody have the following sequences per Kabat definition: RASQSISTYLN (SEQ ID NO: 192), AASSLHS (SEQ ID NO: 258) and QQSYSPPIT (SEQ ID NO: 259).
  • the heavy chain CDRs of the 3410 123 antibody have the following sequences per Chothia definition: GDSISS (SEQ ID NO: 255), YLYYSGSTK (SEQ ID NO: 256) and TGSESTTGYGMDV (SEQ ID NO: 254).
  • the light chain CDRs of the 3410J23 antibody have the following sequences per Chothia definition: RASQSISTYLN (SEQ ID NO: 192), AASSLHS (SEQ ID NO: 258) and QQSYSPPIT (SEQ ID NO: 259).
  • the 3139 P23 antibody (also referred to herein as P23) includes a heavy chain variable region (SEQ ID NO: 156) encoded by the nucleic acid sequence shown below in SEQ ID NO: 155, and a light chain variable region (SEQ ID NO: l 58) encoded by the nucleic acid sequence shown in SEQ ID NO: 157.
  • the heavy chain CDRs of the P23 antibody have the following sequences per Kabat definition: NSFWG (SEQ ID NO: 260), YVYNSGNTKYNPSLKS (SEQ ID NO: 261 ) and HDDASHGYSIS (SEQ ID NO: 262).
  • the light chain CDRs of the 3139_P23 antibody have the following sequences per Kabat definition: RASQTISTYLN (SEQ ID NO: 265), AASGLQS (SEQ ID NO: 61 ) and QQSYNTPLT (SEQ ID NO: 267).
  • the heavy chain CDRs of the 3139_P23 antibody have the following sequences per Chothia definition: GGSISN (SEQ ID NO: 263), YVYNSGNTK (SEQ ID NO: 264) and HDDASHGYSIS (SEQ ID NO: 262).
  • the light chain CDRs of the 3139_P23 antibody have the following sequences per Chothia definition: RASQTISTYLN (SEQ ID NO: 265), AASGLQS (SEQ ID NO: 61 ) and QQSYNTPLT (SEQ ID NO: 267).
  • the 3248 P18 antibody (also referred to herein as PI 8) includes a heavy chain variable region (SEQ ID NO: 160) encoded by the nucleic acid sequence shown below in SEQ ID NO: 159, and a light chain variable region (SEQ ID NO: 162) encoded by the nucleic acid sequence shown in SEQ ID NO: 161.
  • the heavy chain CDRs of the 3248_P18 antibody have the following sequences per Kabat definition: AYHWS (SEQ ID NO: 268), HIFDSGSTYYNPSLKS (SEQ ID NO: 269) and PLGSRYYYGMDV (SEQ ID NO: 270).
  • the light chain CDRs of the 3248_P18 antibody have the following sequences per Kabat definition: RASQSISRYLN (SEQ ID NO: 273), GASTLQN (SEQ ID NO: 274) and QQSYSVPA (SEQ ID NO: 275).
  • the heavy chain CDRs of the 3248 P18 antibody have the following sequences per Chothia definition: GGSISA (SEQ ID NO: 271), HIFDSGSTY (SEQ ID NO: 272) and PLGSRYYYGMDV (SEQ ID NO: 270).
  • the light chain CDRs of the 3248_P18 antibody have the following sequences per Chothia definition: RASQSISRYLN (SEQ ID NO: 273), GASTLQN (SEQ ID NO: 274) and QQSYSVPA (SEQ ID NO: 275).
  • the 3253 P 10 antibody (also referred to herein as P10) includes a heavy chain variable region (SEQ ID NO: 164) encoded by the nucleic acid sequence shown below in SEQ ID NO: 163, and a light chain variable region (SEQ ID NO: 166) encoded by the nucleic acid sequence shown in SEQ ID NO: 165.
  • the heavy chain CDRs of the 3253 P10 antibody have the following sequences per Kabat definition: SDYWS (SEQ ID NO: 187), FFYNGGSTKYNPSLKS (SEQ ID NO: 188) and HDAKFSGSYYVAS (SEQ ID NO: 189).
  • the light chain CDRs of the 3253_P10 antibody have the following sequences per Kabat definition: RASQSISTYLN (SEQ ID NO: 192), GATDLQS (SEQ ID NO: 282) and QQSYNTPLI (SEQ ID NO: 194).
  • the heavy chain CDRs of the 3253_P10 antibody have the following sequences per Chothia definition: GGSITS (SEQ ID NO: 190), FFYNGGSTK (SEQ ID NO: 191 ) and HDAKFSGSYYVAS (SEQ ID NO: 189).
  • the light chain CDRs of the 3253_P10 antibody have the following sequences per Chothia definition: RASQSISTYLN (SEQ ID NO: 192), GATDLQS (SEQ ID NO: 282) and QQSY TPLI (SEQ ID NO: 194).
  • the 3260 D19 antibody (also referred to herein as D19) includes a heavy chain variable region (SEQ ID NO: 168) encoded by the nucleic acid sequence shown below in SEQ ID NO: 167, and a light chain variable region (SEQ ID NO: 170) encoded by the nucleic acid sequence shown in SEQ ID NO: 169.
  • the heavy chain CDRs of the 3260 D19 antibody have the following sequences per Kabat definition: DNYIN (SEQ ID NO: 284), VFYSADRTSYADSVKG (SEQ ID NO: 285) and VQKSYYGMDV (SEQ ID NO: 286).
  • the light chain CDRs of the 3260_D19 antibody have the following sequences per Kabat definition: RASQSISRYLN (SEQ ID NO: 273), GASSLQS (SEQ ID NO: 226) and QQTFSIPL (SEQ ID NO: 291 ).
  • the heavy chain CDRs of the 3260 D19 antibody have the following sequences per Chothia definition: GFSVSD (SEQ ID NO: 287), VFYSADRTS (SEQ ID NO: 288) and VQKSYYGMDV (SEQ ID NO: 286).
  • the light chain CDRs of the 3260_D19 antibody have the following sequences per Chothia definition: RASQSISRYLN (SEQ ID NO: 273), GASSLQS (SEQ ID NO: 226) and QQTFSIPL (SEQ ID NO: 291 ).
  • the 3362_B1 1 antibody (also referred to herein as Bl 1 ) includes a heavy chain variable region (SEQ ID NO: 172) encoded by the nucleic acid sequence shown below in SEQ ID NO: 171 , and a light chain variable region (SEQ ID NO: 174) encoded by the nucleic acid sequence shown in SEQ ID NO: 173.
  • the heavy chain CDRs of the B l 1 antibody have the following sequences per Kabat definition: SGAYYWT (SEQ ID NO: 293), YIYYSGNTYYNPSLKS (SEQ ID NO: 294) and
  • AASTSVLGYGMDV (SEQ ID NO: 295).
  • the light chain CDRs of the B l 1 antibody have the following sequences per abat definition: RASQSISRYLN (SEQ ID NO: 273),
  • AASSLQS (SEQ ID NO: 234) and QQSYSTPLT (SEQ ID NO: 300).
  • the heavy chain CDRs of the B l 1 antibody have the following sequences per Chothia definition: GDSITSGA (SEQ ID NO: 296), YIYYSGNTY (SEQ ID NO: 297) and
  • AASTSVLGYGMDV (SEQ ID NO: 295).
  • the light chain CDRs of the B l 1 antibody have the following sequences per Chothia definition: RASQSISRYLN (SEQ ID NO: 273), AASSLQS (SEQ ID NO: 234) and QQSYSTPLT (SEQ ID NO: 300).
  • the 3242_P05 antibody (also referred to herein as P05) includes a heavy chain variable region (SEQ ID NO: 176) encoded by the nucleic acid sequence shown below in SEQ ID NO: 175, and a light chain variable region (SEQ ID NO: 178) encoded by the nucleic acid sequence shown in SEQ ID NO: 177.
  • SEQ ID NO: 176 encoded by the nucleic acid sequence shown below in SEQ ID NO: 175
  • SEQ ID NO: 178 encoded by the nucleic acid sequence shown in SEQ ID NO: 177.
  • the heavy chain CDRs of the 3242 P05 antibody have the following sequences per Kabat definition: VSDNYI (SEQ ID NO: 301), VFYSADRTSYADSVKG (SEQ ID NO: 285) and VQKSYYGMDV (SEQ ID NO: 286).
  • the light chain CDRs of the 3242_P05 antibody have the following sequences per Kabat definition: RASQSISRYLN (SEQ ID NO: 273), GASSLQS (SEQ ID NO: 226) and QQTFSIPL (SEQ ID NO: 291 ).
  • the heavy chain CDRs of the 3242 P05 antibody have the following sequences per Chothia definition: SGFSV (SEQ ID NO: 304), VFYSADRTS (SEQ ID NO: 288) and VQKSYYGMDV (SEQ ID NO: 286).
  • the light chain CDRs of the 3242_P05 antibody have the following sequences per Chothia definition:
  • the light chain CDRs of the 3242 P05 antibody have the following sequences per Kabat definition: RASQSISRYLN (SEQ ID NO: 273), GASSLQS (SEQ ID NO: 226) and QQTFSIPL (SEQ ID NO: 291).
  • HuM2e antibodies of the invention also include antibodies that include a heavy chain variable amino acid sequence that is at least 90%, 92%, 95%, 97% 98%, 99% or more identical the amino acid sequence of SEQ ID NO: 44, 277, 276, 50, 236, 235, 1 16, 120, 124, 128, 132, 136, 140, 14.4, 148, 152, 156, 160, 164, 168, 172, or 176.
  • a light chain variable amino acid that is at least 90%, 92%, 95%, 97% 98%, 99% or more identical the amino acid sequence of SEQ ID NO: 46, 52, 1 18, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178.
  • the monoclonal antibody is an antibody that binds to the same epitope as TCN-032 (8110), 21 B1 5, TCN-031 (23 12), 3241_G23, 3244_I10, 3243J07, 3259J21 , 3245_019, 3244_H04, 3136_G05, 3252_C13, 3255J06, 3420J23, 3139_P23, 3248_P18, 3253_P10, 3260_D19, 3362_B1 1 , or 3242 P05.
  • the heavy chain of a M2e antibody is derived from a germ line V (variable) gene such as, for example, the IgHV4 or the IgHV3 germline gene.
  • the M2e antibodies of the invention include a variable heavy chain (VH) region encoded by a human IgHV4 or the IgHV3 germline gene sequence.
  • VH variable heavy chain
  • An IgHV4 germline gene sequence is shown, e.g. , in Accession numbers LI 0088, M29812, M951 14, X56360 and M951 17.
  • An IgHV3 germline gene sequence is shown, e.g., in Accession numbers X92218, X70208, Z27504, M99679 and AB019437.
  • the M2e antibodies of the invention include a V H region that is encoded by a nucleic acid sequence that is at least 80% homologous to the IgHV4 or the IgHV3 germline gene sequence.
  • the nucleic acid sequence is at least 90%, 95%, 96%, 97% homologous to the IgHV4 or the IgHV3 germline gene sequence, and more preferably, at least 98%, 99% homologous to the IgHV4 or the IgHV3 germline gene sequence.
  • the V H region of the M2e antibody is at least 80% homologous to the amino acid sequence of the VH region encoded by the IgHV4 or the IgHV3 VH germline gene sequence.
  • the amino acid sequence of VH region of the M2e antibody is at least 90%, 95%, 96%, 97% homologous to the amino acid sequence encoded by the IgHV4 or the IgHV3 germline gene sequence, and more preferably, at least 98%, 99% homologous to the sequence encoded by the IgHV4 or the IgHV3 germline gene sequence.
  • the M2e antibodies of the invention also include a variable light chain (VL) region encoded by a human Ig V l germline gene sequence.
  • VL variable light chain
  • a human IgKVl VL germline gene sequence is shown, e.g. , Accession numbers X59315, X59312, X59318, J00248, and Y 14865.
  • the M2e antibodies include a VL region that is encoded by a nucleic acid sequence that is at least 80% homologous to the IgKVl germline gene sequence.
  • the nucleic acid sequence is at least 90%, 95%, 96%, 97% homologous to the Ig Vl germline gene sequence, and more preferably, at least 98%), 99% homologous to the IgKVl germline gene sequence.
  • the VL region of the M2e antibody is at least 80% homologous to the amino acid sequence of the VL region encoded the IgKVl germline gene sequence.
  • the amino acid sequence of VL region of the M2e antibody is at least 90%, 95%, 96%, 97% homologous to the amino acid sequence encoded by the IgKVl germline gene sequence, and more preferably, at least 98%, 99% homologous to the sequence encoded by e the IgKVl germline gene sequence.
  • the HA antibodies of the invention may also be capable of specifically binding to one or more fragments of influenza virus H5N1 , such as the surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), which are required for viral attachment and cellular release, or membrane proteins (M l and M2).
  • H5N1 hemagglutinin
  • NA neuraminidase
  • the HA antibodies of the invention are capable of specifically binding to the HA molecule of H5N1
  • strains They may be capable of specifically binding to the HA1 and/or HA2 subunit of the HA molecule. They may be capable of specifically binding to linear or structural and/or conformational epitopes on the HA1 and/or HA2 subunit of the HA molecule.
  • the HA molecule may be purified from viruses or recombinantly produced and optionally isolated before use. Alternatively, HA may be expressed on the surface of cells.
  • the HA antibodies may also be capable of specifically binding to proteins not present on the surface of H5N1 including the nucleoprotein, the nucleocapsid structural protein, polymerases (PA, PB and PB2), and non-structural proteins (NS1 and NS2).
  • the nucleotide and/or amino acid sequence of proteins of various H5N1 strains can be found in the GenBank-database, NCBI Influenza Virus Sequence Database, Influenza Sequence Database (ISD), EMBL-database and/or other databases. It is well within the reach of the skilled person to find such sequences in the respective databases.
  • the HA antibodies of the invention are capable of specifically binding to a fragment of the above-mentioned proteins and/or polypeptides, wherein the fragment at least includes an antigenic determinant recognized by the HA antibodies of the invention.
  • An "antigenic determinant” as used herein is a moiety that is capable of binding to an HA antibody of the invention with sufficiently high affinity to form a detectable antigen-antibody complex.
  • the terms “antigenic determinant” and “epitope” are equivalents.
  • the HA antibodies of the invention may or may not be capable of specifically binding to the extracellular part of HA (also called herein soluble HA (sHA)).
  • the HA antibodies of the invention can be intact immunoglobulin molecules such as polyclonal or monoclonal antibodies or the HA antibodies can be antigen-binding fragments including, but not limited to, Fab, F(ab'), F(ab')2, Fv, dAb, Fd, complementarity determining region (CDR) fragments, single-chain antibodies (scFv), bivalent single-chain antibodies, single-chain phage antibodies, diabodies, triabodies, tetrabodies, and (poly)peptides that contain at least a fragment of an immunoglobulin that is sufficient to confer specific antigen binding to influenza virus H5 1 strains or a fragment thereof.
  • the HA antibodies are human monoclonal antibodies.
  • HA antibodies can be used in non-isolated or isolated form. Furthermore, the HA antibodies can be used alone or in a mixture including at least one HA antibody (or variant or fragment thereof). Thus, HA antibodies can be used in combination, e.g., as a pharmaceutical composition comprising two or more antibodies of the invention, variants or fragments thereof. For example, antibodies having different, but complementary activities can be combined in a single therapy to achieve a desired prophylactic, therapeutic or diagnostic effect, but alternatively, antibodies having identical activities can also be
  • the mixture further includes at least one other therapeutic agent.
  • the therapeutic agent such as, e.g., M2 inhibitors (e.g., amantidine, rimantadine) and/or neuraminidase inhibitors (e.g., zanamivir, oseltamivir) is useful in the prophylaxis and/or treatment of an influenza virus H5N1 infection.
  • M2 inhibitors e.g., amantidine, rimantadine
  • neuraminidase inhibitors e.g., zanamivir, oseltamivir
  • HA antibodies can bind to their binding partners, i.e. influenza virus H5N1 or fragments thereof, with an affinity constant ( d-value) that is lower than 0.2x10 "4 M, l .Oxl O '5 M, l .Oxl O "6 M, l .Oxl O "7 M, preferably lower than l .Oxl O "8 M, more preferably lower than l .Oxl O "9 M, more preferably lower than 1.0x10 "l0 M, even more preferably lower than 1.0x10 ' " M, and in particular lower than l .Oxl O "12 M.
  • the affinity constants can vary for antibody isotypes.
  • affinity binding for an IgM isotype refers to a binding affinity of at least about 1.Oxl O "7 M.
  • Affinity constants can for instance be measured using surface plasmon resonance, for example using the BIACORE system (Pharmacia Biosensor AB, Uppsala, Sweden).
  • HA antibodies may bind to influenza virus H5N1 or a fragment thereof in soluble form such as for instance in a sample or in suspension or may bind to influenza virus H5N1 or a fragment thereof bound or attached to a carrier or substrate, e.g., microtiter plates, membranes and beads, etc.
  • Carriers or substrates may be made of glass, plastic (e.g., polystyrene), polysaccharides, nylon, nitrocellulose, or Teflon, etc.
  • the surface of such supports may be solid or porous and of any convenient shape.
  • the HA antibodies may bind to influenza virus H5N1 in purified/isolated or non purified/non-isolated form.
  • HA antibodies exhibit neutralizing activity.
  • Neutralizing activity can for instance be measured as described in International Patent Application PCT/EP2007/059356 (Publication No. WO 2008/028946, the contents of which are incorporated herein in their entirety).
  • the invention relates to an isolated human HA antibody that recognizes and binds to an epitope in the HA2 subunit of the influenza haemagglutinin protein (HA), characterized in that said HA antibody has neutralizing activity against an influenza virus, for instance, including HA of the H5 subtype.
  • influenza strains that contain such a HA of the H5 subtype and that are important strains in view of pandemic threats are H5N1 , H5N2, H5N8, and H5N9.
  • Particularly preferred are HA antibodies that at least neutralize the H5N1 influenza strain.
  • HA antibodies do not depend on an epitope in the HAI subunit of the HA protein for binding to said HA protein.
  • human HA antibody describes an intact immunoglobulin including monoclonal antibodies, such as chimeric, humanized or human monoclonal antibodies, or to an antigen-binding and/or variable domain comprising fragment of an immunoglobulin that competes with the intact immunoglobulin for specific binding to the binding partner of the immunoglobulin, e.g. H5N1 . Regardless of structure, the antigen binding fragment binds with the same antigen that is recognized by the intact immunoglobulin.
  • An antigen-binding fragment can comprise a peptide or polypeptide comprising an amino acid sequence of at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, or 250 contiguous amino acid residues of the amino acid sequence of the HA antibody.
  • HA antibody includes all immunoglobulin classes and subclasses known in the art. Depending on the amino acid sequence of the constant domain of their heavy chains, HA antibodies can be divided into the five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgAl, IgA2, IgGI, IgG2, IgG3 and IgG4.
  • Antigen-binding fragments include, inter alia, Fab, F(ab'), F(ab')2, Fv, dAb, Fd, complementarity determining region (CDR) fragments, single-chain antibodies (scFv), bivalent single-chain antibodies, single-chain phage antibodies, diabodies, triabodies, tetrabodies, (poly)peptides that contain at least a fragment of an immunoglobulin that is sufficient to confer specific antigen binding to the (poly)peptide, etc.
  • the above fragments may be produced synthetically or by enzymatic or chemical cleavage of intact
  • immunoglobulins or they may be genetically engineered by recombinant DNA techniques.
  • the methods of production are well known in the art and are described, for example, in Antibodies: A Laboratory Manual, Edited by: E. Harlow and D, Lane (1988), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, which is incorporated herein by reference.
  • An HA antibody or antigen-binding fragment thereof may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or they may be different.
  • CDR complementarity determining regions
  • the CDR regions of HA antibodies can be specific for linear epitopes, discontinuous epitopes, or conformational epitopes of proteins or protein fragments, either as present on the protein in its native conformation or, in some cases, as present on the proteins as denatured, e.g., by solubilization in SDS.
  • Epitopes of HA antibodies may also consist of posttranslational modifications of proteins.
  • the term "functional variant”, as used herein, refers to an HA antibody that includes a nucleotide and/or amino acid sequence that is altered by one or more nucleotides and/or amino acids compared to the nucleotide and/or amino acid sequences of the parental HA antibody and that is still capable of competing for binding to the binding partner, e.g. H5N1 , with the parental HA antibody.
  • the modifications in the amino acid and/or nucleotide sequence of the parental HA antibody do not significantly affect or alter the binding characteristics of the HA antibody encoded by the nucleotide sequence or containing the amino acid sequence, i.e. the antibody is still able to recognize and bind its target.
  • the functional variant may have conservative sequence modifications including nucleotide and amino acid substitutions, additions and deletions. These modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and random PCR- mediated mutagenesis, and may include natural as well as non-natural nucleotides and amino acids.
  • Conservative amino acid substitutions include the ones in which the amino acid residue is replaced with an amino acid residue having similar structural or chemical properties. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan).
  • basic side chains e.g.
  • a HA antibody functional variant may have non-conservative amino acid substitutions, e.g., replacement of an amino acid with an amino acid residue having different structural or chemical properties. Similar minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing immunological activity may be found using computer programs well known in the art.
  • a mutation in a nucleotide sequence can be a single alteration made at a locus (a point mutation), such as transition or transversion mutations, or alternatively, multiple nucleotides may be inserted, deleted or changed at a single locus. In addition, one or more alterations may be made at any number of loci within a nucleotide sequence.
  • the mutations may be performed by any suitable method known in the art.
  • HA antibodies when applied to HA antibodies, refers to molecules that are either directly derived from a human or based upon a human sequence.
  • HA antibody When an HA antibody is derived from or based on a human sequence and subsequently modified,
  • human when applied to HA antibodies is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences or based on variable or constant regions occurring in a human or human lymphocyte and modified in some form.
  • the human HA antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences, contain substitutions and/or deletions (e.g., mutations introduced by for instance random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • nucleic acid sequence may be exactly copied from a template, or with minor mutations, such as by error-prone PCR methods, or synthetically made matching the template exactly or with minor modifications.
  • Semi-synthetic molecules based on human sequences are also considered to be human as used herein.
  • the heavy chain of an HA antibody is derived from a germ line V (variable) gene such as, for example, the VH1 or VH3 germline gene (see, Tomlinson IM, Williams SC, Ignatovitch O, Corbett SJ, Winter G. V-BASE Sequence Directory. Cambridge, United Kingdom: MRC Centre for Protein Engineering (1997)).
  • the HA antibodies of the invention include a VH region that is encoded by a nucleic acid sequence that is at least 80% homologous to the VH1 or VH3 germline gene sequence.
  • the nucleic acid sequence is at least 90%, 95%, 96%, 97% homologous to the VH1 or VH3 germline gene sequence, and more preferably, at least 98%, 99% homologous to the VH1 or VH3 germline gene sequence.
  • the VH region of the HA antibody is at least 80% homologous to the amino acid sequence of the VH region encoded by the VH1 or VH3 VH germline gene sequence.
  • the amino acid sequence of VH region of the HA antibody is at least 90%, 95%, 96%, 97% homologous to the amino acid sequence encoded by the VH1 or VH3 germline gene sequence, and more preferably, at least 98%, 99% homologous to the sequence encoded by the VH1 or VH3 germline gene sequence.
  • VH1 germline gene is VH1 (1 -2), VH 1 (1 -18), VH1 (3-23), or VH 1 (1 -69).
  • VH3 germline gene is VH3 (3-21 )
  • the HA antibodies of the invention also include a variable light chain (VL) region encoded by a human germline gene sequence selected from the group consisting of VKI, VKII, VKIII, VKIV, VL1 , VL2, and VL3 (see, Tomlinson IM, Williams SC, Ignatovitch O, Corbett SJ, Winter G. V-BASE Sequence Directory. Cambridge, United Kingdom: MRC Centre for Protein Engineering (1997)).
  • the HA antibodies include a V L region that is encoded by a nucleic acid sequence that is at least 80% homologous to the germline gene sequence of VKI, VKII, VKIII, VKIV, VL1 , VL2, or VL3.
  • the nucleic acid sequence is at least 90%, 95%, 96%, 97% homologous to the germline gene sequence of VK1, VKII, VKIII, VKIV, VLl , VL2, or VL3, and more preferably, at least 98%, 99% homologous to the germline gene sequence of VKI, VKII, VKIII, VKIV, VLl , VL2, or VL3.
  • the VL region of the HA antibody is at least 80% homologous to the amino acid sequence of the V L region encoded the germline gene sequence of VKI, VKII, VKIII, VKIV, VLl , VL2, or VL3.
  • the amino acid sequence of VL region of the HA antibody is at least 90%, 95%, 96%, 97% homologous to the amino acid sequence encoded by the germline gene sequence of VKI, VKII, VKIII, VKIV, VLl, VL2, or VL3, and more preferably, at least 98%, 99% homologous to the sequence encoded by the germline gene sequence of VKI, VKII, VKIII, VKIV, VLl , VL2, or VL3.
  • the VKI germline gene is VKI (A20), the VKII germline gene is VKII (A3), the VKIII germline gene is VKIII (A27), and the VKIV germline gene is VKIV (B3).
  • the VLl germline gene is VLl (V l -13), VLl (Vl -16), VLl (V l -17), or. VLl (Vl-19).
  • the VL2 germline gene is VL2 (Vl -3) or VL2 (V l -4).
  • the VL3 germline gene is VL3 (V2-14).
  • HA antibodies contain two, three, four, five or all six CDR regions as disclosed herein.
  • HA antibodies contain at least two of the CDRs disclosed herein.
  • the SC06- 141 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 309) and a light chain variable region (SEQ ID NO: 310) encoded by the nucleic acid sequence shown in SEQ ID NO: 31 1 and the amino acid sequence shown in SEQ ID NO: 312.
  • the VH-locus is VH1 (1-18) and the VL locus is HKIV (B3).
  • the amino acids encompassing the CDRs are highlighted in bold in the sequences below.
  • the heavy chain CDRs of the SC06-141 antibody have the following CDR sequences: GYYVY (HCDR1 , SEQ ID NO: 566), WISAYNGNTNYAQKFQG (HCDR2, SEQ ID NO: 567) and SRSLDV (HCDR3, SEQ ID NO: 568).
  • the light chain CDRs of the SC06- 141 antibody have the following CDR sequences: KSSQSVLYSSNNKNYLA (LCDR1 , SEQ ID NO: 569), WASTRES (LCDR2, SEQ ID NO: 570) and QQYYSTPLT (LCDR3, SEQ ID NO: 200).
  • the SC06-255 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 313) and a light chain variable region (SEQ ID NO: 314) encoded by the nucleic acid sequence shown in SEQ ID NO: 315 and the amino acid sequence shown in SEQ ID NO: 316.
  • the VH-locus is VH1 (1 -69) and the VL locus is VL1 (V l-16).
  • the amino acids encompassing the CDRs are highlighted in bold in the sequences below.
  • the heavy chain CDRs of the SC06-255 antibody have the following CDR sequences: SYAIS (HCDR1 , SEQ ID NO: 571 ), GIIPIFGTTKYAPKFQG (HCDR2, SEQ ID NO: 572) and HMGYQVRETMDV (HCDR3, SEQ ID NO: 573).
  • the light chain CDRs of the SC06- 255 antibody have the following CDR sequences: SGSTFNIGSNAVD (LCDR1 , SEQ ID NO: 574), SNNQRPS (LCDR2, SEQ ID NO: 575) and AAWDDILNVPV (LCDR3, SEQ ID NO: 576).
  • the SC06-257 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 317) and a light chain variable region (SEQ ID NO: 318) encoded by the nucleic acid sequence shown in SEQ ID NO: 319 and the amino acid sequence shown in SEQ ID NO: 320.
  • the VH-locus is VH1 (1 -69) and the VL locus is VL2 (V I -4).
  • the amino acids encompassing the CDRs are highlighted in bold in the sequences below.
  • the heavy chain CDRs of the SC06-257 antibody have the following CDR sequences: SYAIS (HCDR1 , SEQ ID NO: 571 ), GIIPIFGTTKYAPKFQG (HCDR2, SEQ ID NO: 572) and HMGYQVRETMDV (HCDR3, SEQ ID NO: 573).
  • the light chain CDRs of the SC06- 257 antibody have the following CDR sequences: TGTSSDVGGY YVS (LCDR1 , SEQ ID NO: 577), EVSNRPS (LCDR2, SEQ ID NO: 578) and SSYTSSSTY (LCDR3, SEQ ID NO: 579).
  • the SC06-260 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 321 ) and a light chain variable region (SEQ ID NO: 322) encoded by the nucleic acid sequence shown in SEQ ID NO: 323 and the amino acid sequence shown in SEQ ID NO: 324.
  • the VH-locus is VH1 (1 -69) and the VL locus is VL1 (V I - 17).
  • the amino acids encompassing the CDRs are highlighted in bold in the sequences below.
  • the heavy chain CDRs of the SC06-260 antibody have the following CDR sequences: SYAIS (HCDR1 , SEQ ID NO: 571 ), GIIPIFGTTKYAPKFQG (HCDR2, SEQ ID NO: 572) and HMGYQVRETMDV (HCDR3, SEQ ID NO: 573).
  • the light chain CDRs of the SC06- 260antibody have the following CDR sequences: SGSRSNVGDNSVY (LCDR1 , SEQ ID NO: 580), NTQRPS (LCDR2, SEQ ID NO: 581 ) and VAWDDSVDGYV (LCDR3, SEQ ID NO: 582).
  • the SC06-261 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 325) and a light chain variable region (SEQ ID NO: 326) encoded by the nucleic acid sequence shown in SEQ ID NO: 327 and the amino acid sequence shown in SEQ ID NO: 328.
  • the VH-locus is VH1 (1 -69) and the VL locus is VL1 (Vl -19).
  • the amino acids encompassing the CDRs are highlighted in bold in the sequences below.
  • the heavy chain CDRs of the SC06-261 antibody have the following CDR sequences: SYAIS (HCDR1 , SEQ ID NO: 571 ), GIIPIFGTT YAPKFQG (HCDR2, SEQ ID NO: 572) and HMGYQVRETMDV (HCDR3, SEQ ID NO: 573).
  • the light chain CDRs of the SC06- 261 antibody have the following CDR sequences: SGSSSNIGNDYVS (LCDR1 , SEQ ID NO: 583), DNN RPS (LCDR2, SEQ ID NO: 584) and ATWDRRPTAYVV (LCDR3, SEQ ID NO: 585).
  • the SC06-262 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 329) and a light chain variable region (SEQ ID NO: 330) encoded by the nucleic acid sequence shown in SEQ ID NO: 331 and the amino acid sequence shown in SEQ ID NO: 332.
  • the VH-locus is VH1 (1 -69) and the VL locus is VKI (A20).
  • the amino acids encompassing the CDRs are highlighted in bold in the sequences below.
  • the heavy chain CDRs of the SC06-262 antibody have the following CDR sequences: GSAIS (HCDRl , SEQ ID NO: 586), GISPLFGTTNYAQKFQG (HCDR2, SEQ ID NO: 587) and GPKYYSEYMDV (HCDR3, SEQ ID NO: 588).
  • the light chain CDRs of the SC06-262 antibody have the following CDR sequences: RASQGISSYLA (LCDR1 , SEQ ID NO: 589), DASTLRS (LCDR2, SEQ ID NO: 590) and QRYNSAPPI (LCDR3, SEQ ID NO: 591 ).
  • the SC06-268 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 333) and a light chain variable region (SEQ ID NO: 334) encoded by the nucleic acid sequence shown in SEQ ID NO: 335 and the amino acid sequence shown in SEQ ID NO: 336.
  • the VH-locus is VH1 (1 -69) and the VL locus is VL3 (V2-14).
  • the amino acids encompassing the CDRs are highlighted in bold in the sequences below.
  • the heavy chain CDRs of the SC06-268 antibody have the following CDR sequences: SYAIS (HCDR1 , SEQ ID NO: 571 ), GIMGMFGTTNYAQKFQG (HCDR2, SEQ ID NO: 592) and SSGYYPEYFQD (HCDR3, SEQ ID NO: 593).
  • the light chain CDRs of the SC06- 268 antibody have the following CDR sequences: SGHKLGD YVS (LCDR1 , SEQ ID NO: 594), QDNRRPS (LCDR2, SEQ ID NO: 595) and QAWDSSTA (LCDR3, SEQ ID NO: 596).
  • the SC06-272 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 337) and a light chain variable region (SEQ ID NO: 338) encoded by the nucleic acid sequence shown in SEQ ID NO: 339 and the amino acid sequence shown in SEQ ID NO: 340.
  • the VH-locus is VH1 (1-69) and the VL locus is VL2 (VI -3).
  • the amino acids encompassing the CDRs are highlighted in bold in the sequences below.
  • the heavy chain CDRs of the SC06-272 antibody have the following CDR sequences: SYAIT (HCDR1, SEQ ID NO: 597), GIIGMFGSTNYAQNFQG (HCDR2, SEQ ID NO: 598) and STGYYPAYLHH (HCDR3, SEQ ID NO: 599).
  • the light chain CDRs of the SC06-272 antibody have the following CDR sequences: TGTSSDVGGYNYVS (LCDR1, SEQ ID NO: 577), DVSKRPS (LCDR2, SEQ ID NO: 601) and SSYTSSSTHV (LCDR3, SEQ ID NO: 602).
  • the SC06-296 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 341) and a light chain variable region (SEQ ID NO: 342) encoded by the nucleic acid sequence shown in SEQ ID NO: 343 and the amino acid sequence shown in SEQ ID NO: 344.
  • the VH-locus is VH1 (1-2) and the VL locus is V III (A27).
  • the amino acids encompassing the CDRs are highlighted in bold in the sequences below.
  • the heavy chain CDRs of the SC06-296 antibody have the following CDR sequences: SYYMH (HCDR1, SEQ ID NO: 603), WINPNSGGTNYAQ FQG (HCDR2, SEQ ID NO: 604) and EGKWGPQAAFDI (HCDR3, SEQ ID NO: 605).
  • the light chain CDRs of the SC06-296 antibody have the following CDR sequences: RASQSVSSSYLA (LCDR1 , SEQ ID NO: 646), DASSRAT (LCDR2, SEQ ID NO: 607) and QQYGSSLW (LCDR3, SEQ ID NO: 608).
  • the SC06-301 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 345) and a light chain variable region (SEQ ID NO: 346) encoded by the nucleic acid sequence shown in SEQ ID NO: 347 and the amino acid sequence shown in SEQ ID NO: 348.
  • the VH-locus is VH1 (3-23) and the VL locus is VKII (A3).
  • the amino acids encompassing the CDRs are highlighted in bold in the sequences below.
  • the heavy chain CDRs of the SC06-301 antibody have the following CDR sequences: IYAMS (HCDR1 , SEQ ID NO: 609), AISSSGDSTYYADSVKG (HCDR2, SEQ ID NO: 610) and AYGYTFDP (HCDR3, SEQ ID NO: 61 1).
  • the light chain CDRs of the SC06-301 antibody have the following CDR sequences: RSSQSLLHSNGYNYLD (LCDR1 , SEQ ID NO: 612), LGSNRAS (LCDR2, SEQ ID NO: 613) and MQALQTPL (LCDR3, SEQ ID NO: 614).
  • the SC06-307 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 349) and a light chain variable region (SEQ ID NO: 350) encoded by the nucleic acid sequence shown in SEQ ID NO: 351 and the amino acid sequence shown in SEQ ID NO: 352.
  • the VH-locus is VH3 (3-21 ) and the VL locus is V III (A27).
  • the amino acids encompassing the CDRs are highlighted in bold in the sequences below.
  • the heavy chain CDRs of the SC06-307 antibody have the following CDR sequences: SYSMN (HCDRl , SEQ ID NO: 615), SISSSSSYIYYVDSVKG (HCDR2, SEQ ID NO: 616) and GGGSYGAYEGFDY (HCDR3, SEQ ID NO: 617).
  • the light chain CDRs of the SC06- 307 antibody have the following CDR sequences: RASQRVSSYLA (LCDR1 , SEQ ID NO: 618), GASTRAA (LCDR2, SEQ ID NO: 619) and QQYGRTPLT (LCDR3, SEQ ID NO:
  • the SC06-310 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 353) and a light chain variable region (SEQ ID NO: 354) encoded by the nucleic acid sequence shown in SEQ ID NO: 355 and the amino acid sequence shown in SEQ ID NO: 356.
  • the VH-locus is VH1 (1 -69) and the VL locus is VL3 (V2- 14).
  • the amino acids encompassing the CDRs are highlighted in bold in the sequences below.
  • the heavy chain CDRs of the SC06-310 antibody have the following CDR sequences: SYAIS (HCDR1 , SEQ ID NO: 571 ), GIIPIFGTTKYAPKFQG (HCDR2, SEQ ID NO: 572) and HMGYQVRETMDV (HCDR3, SEQ ID NO: 573).
  • the light chain CDRs of the SC06- 310 antibody have the following CDR sequences: GGNNIGS SVH (LCDR1 , SEQ ID NO: 621 ), DDSDRPS (LCDR2, SEQ ID NO: 622) and QVWDSSSDHAV (LCDR3, SEQ ID NO: 623).
  • the SC06-314 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 357) and a light chain variable region (SEQ ID NO: 358) encoded by the nucleic acid sequence shown in SEQ ID NO: 359 and the amino acid sequence shown in SEQ ID NO: 360.
  • the VH-locus is VH1 (1-69) and the VL locus is VL1 (Vl -17).
  • the amino acids encompassing the CDRs are highlighted in bold in the sequences below.
  • the heavy chain CDRs of the SC06-314 antibody have the following CDR sequences: SYAIS (HCDR1 , SEQ ID NO: 571 ), GIIPIFGTT YAPKFQG (HCDR2, SEQ ID NO: 572) and H GYQVRETMDV (HCDR3, SEQ ID NO: 573).
  • the light chain CDRs of the SC06- 314 antibody have the following CDR sequences: SGSSSNIGSNYVY (LCDR1 , SEQ ID NO: 624), RDGQRPS (LCDR2, SEQ ID NO: 625) and ATWDDNLSGPV (LCDR3, SEQ ID NO: 626).
  • the SC06-323 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 361) and a light chain variable region (SEQ ID NO: 362) encoded by the nucleic acid sequence shown in SEQ ID NO: 363 and the amino acid sequence shown in SEQ ID NO: 364.
  • the VH-locus is VH1 (1-69) and the VL locus is VKIII (A27).
  • the amino acids encompassing the CDRs are highlighted in bold in the sequences below.
  • the heavy chain CDRs of the SC06-323 antibody have the following CDR sequences: SYGIS (HCDR1, SEQ ID NO: 627), DIIGMFGSTNYAQNFQG (HCDR2, SEQ ID NO: 628) and SSGYYPAYLPH (HCDR3, SEQ ID NO: 629).
  • the light chain CDRs of the SC06- 323 antibody have the following CDR sequences: RASQSVSSSYLA (LCDR1, SEQ ID NO: 646), GASSRAT (LCDR2, SEQ ID NO: 631) and QQYGSSPRT (LCDR3, SEQ ID NO: 632).
  • the SC06-325 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 365) and a light chain variable region (SEQ ID NO: 366) encoded by the nucleic acid sequence shown in SEQ ID NO: 367 and the amino acid sequence shown in SEQ ID NO: 368.
  • the VH-locus is VH1 (1 -69) and the VL locus is VL2 (Vl -4).
  • the amino acids encompassing the CDRs are highlighted in bold in the sequences below.
  • the heavy chain CDRs of the SC06-325 antibody have the following CDR sequences: FYSMS (HCDR1 , SEQ ID NO: 633), GIIPMFGTTNYAQ FQG (HCDR2, SEQ ID NO: 634) and GDKGIYYYYMDV (HCDR3, SEQ ID NO: 635).
  • the light chain CDRs of the SC06-325 antibody have the following CDR sequences: TGTSSDVGGY YVS (LCDR1 , SEQ ID NO: 577), EVSNRPS (LCDR2, SEQ ID NO: 578) and SSYTSSSTLV (LCDR3, SEQ ID NO: 636).
  • the SC06-327 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 369) and a light chain variable region (SEQ ID NO: 370) encoded by the nucleic acid sequence shown in SEQ ID NO: 371 and the amino acid sequence shown in SEQ ID NO: 372.
  • the VH-Iocus is VH1 (1-69) and the VL locus is VL3 (V2-14).
  • the amino acids encompassing the CDRs are highlighted in bold in the sequences below.
  • the heavy chain CDRs of the SC06-327 antibody have the following CDR sequences: THAIS (SEQ ID NO: 637), GIIAIFGTANYAQ FQG (SEQ ID NO: 638) and
  • GSGYHISTPFDN SEQ ID NO: 639.
  • the light chain CDRs of the SC06-327 antibody have the following CDR sequences: GGNNIGS GVH (SEQ ID NO: 640), DDSDRPS (SEQ ID NO: 622) and QVWDSSSDHVV (SEQ ID NO: 642).
  • the SC06-328 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 373) and a light chain variable region (SEQ ID NO: 374) encoded by the nucleic acid sequence shown in SEQ ID NO: 375 and the amino acid sequence shown in SEQ ID NO: 376.
  • the VH-locus is VH1 (1-69) and the VL locus is V III (A27).
  • the amino acids encompassing the CDRs are highlighted in bold in the sequences below.
  • the heavy chain CDRs of the SC06-328 antibody have the following CDR sequences: GYAIS (HCDR1, SEQ ID NO: 643), GIIPIFGTTNYAQKFQG (HCDR2, SEQ ID NO: 644) and VKDGYCTLTSCPVGWYFDL (HCDR3, SEQ ID NO: 645).
  • the light chain CDRs of the SC06-328 antibody have the following CDR sequences: RASQSVSSSYLA (LCDR1, SEQ ID NO: 646), GASSRAT (LCDR2, SEQ ID NO: 631) and QQYGSSLT (LCDR3, SEQ ID NO: 648).
  • the SC06-329 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 377) and a light chain variable region (SEQ ID NO: 378) encoded by the nucleic acid sequence shown in SEQ ID NO: 379 and the amino acid sequence shown in SEQ ID NO: 380.
  • the VH-locus is VH1 (1 -69) and the VL locus is VKIII (A27).
  • the amino acids encompassing the CDRs are highlighted in bold in the sequences below.
  • the heavy chain CDRs of the SC06-329 antibody have the following CDR sequences: SNSIS (HCDRl , SEQ ID NO: 649), GIFALFGTTDYAQKFQG (HCDR2, SEQ ID NO: 650) and GSGYTTRNYFDY (HCDR3, SEQ ID NO: 651 ).
  • the light chain CDRs of the SC06- 329 antibody have the following CDR sequences: RASQSVSSNYLG (LCDR1 , SEQ ID NO: 652), GASSRAS (LCDR2, SEQ ID NO: 653) and QQYGSSPLT (LCDR3, SEQ ID NO: 654).
  • the SC06-331 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 381 ) and a light chain variable region (SEQ ID NO: 382) encoded by the nucleic acid sequence shown in SEQ ID NO: 383 and the amino acid sequence shown in SEQ ID NO: 384.
  • the VH-locus is VH1 (1 -69) and the VL locus is VL3 (V2-14).
  • the amino acids encompassing the CDRs are highlighted in bold in the sequences below.
  • the heavy chain CDRs of the SC06-331 antibody have the following CDR sequences: SYAIS (HCDR1 , SEQ ID NO: 571 ), GIIGMFGTANYAQ FQG (HCDR2, SEQ ID NO: 655) and GNYYYESSLDY (HCDR3, SEQ ID NO: 656).
  • the light chain CDRs of the SC06-331 antibody have the following CDR sequences: GGNNIGSKSVH (LCDRl , SEQ ID NO: 621 ), DDSDRPS (LCDR2, SEQ ID NO: 622) and QVWDSSSDH (LCDR3, SEQ ID NO: 657).
  • the SC06-332 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 385) and a light chain variable region (SEQ ID NO: 386) encoded by the nucleic acid sequence shown in SEQ ID NO: 387 and the amino acid sequence shown in SEQ ID NO: 388.
  • the VH-locus is VH1 (1 -69) and the VL locus is VKI (A20).
  • the amino acids encompassing the CDRs are highlighted in bold in the sequences below.
  • the heavy chain CDRs of the SC06-332 antibody have the following CDR sequences: NFAIN (HCDR1 , SEQ ID NO: 658), GIIAVFGTT YAHKFQG (HCDR2, SEQ ID NO: 659) and GPHYYSSYMDV (HCDR3, SEQ ID NO: 660).
  • the light chain CDRs of the SC06-332 antibody have the following CDR sequences: RASQGISTYLA (LCDR1 , SEQ ID NO: 661 ), AASTLQS (LCDR2, SEQ ID NO: 662) and Q YNSAPS (LCDR3, SEQ ID NO: 663).
  • the SC06-334 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 389) and a light chain variable region (SEQ ID NO: 390) encoded by the nucleic acid sequence shown in SEQ ID NO: 391 and the amino acid sequence shown in SEQ ID NO: 392.
  • the VH-locus is VH1 (1-69) and the VL locus is VL3 (V2-14).
  • the amino acids encompassing the CDRs are highlighted in bold in the sequences below.
  • the heavy chain CDRs of the SC06-334 antibody have the following CDR sequences: SNAVS (HCDR1, SEQ ID NO: 664), GILGVFGSPSYAQ FQG (HCDR2, SEQ ID NO: 665) and GPTYYYSYMDV (HCDR3, SEQ ID NO: 666).
  • the light chain CDRs of the SC06-334 antibody have the following CDR sequences: GGNNIGRNSVH (LCDRl, SEQ ID NO: 667), DDSDRPS (LCDR2, SEQ ID NO: 622) and QVWHSSSDHYV (LCDR3, SEQ ID NO: 669).
  • the SC06-336 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 393) and a light chain variable region (SEQ ID NO: 394) encoded by the nucleic acid sequence shown in SEQ ID NO: 395 and the amino acid sequence shown in SEQ ID NO: 396.
  • the VH-Iocus is VH1 (1-69) and the VL locus is VKIII (A27).
  • the amino acids encompassing the CDRs are highlighted in bold in the sequences below.
  • the heavy chain CDRs of the SC06-336 antibody have the following CDR sequences: SYAIS (HCDR1, SEQ ID NO: 670), GIFGMFGTANYAQKFQG (HCDR2, SEQ ID NO: 671) and SSGYYPQYFQD (HCDR3, SEQ ID NO: 672).
  • the light chain CDRs of the SC06- 336 antibody have the following CDR sequences: RASQSVSSSYLA (LCDR1, SEQ ID NO: 646), GASSRAT (LCDR2, SEQ ID NO: 631) and QQYGSSSLT (LCDR3, SEQ ID NO: 308).
  • the SC06-339 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 397) and a light chain variable region (SEQ ID NO: 398) encoded by the nucleic acid sequence shown in SEQ ID NO: 399 and the amino acid sequence shown in SEQ ID NO: 400.
  • the VH-locus is VH1 (1-69) and the VL locus is VL3 (V2-14).
  • the amino acids encompassing the CDRs are highlighted in bold in the sequences below.
  • the heavy chain CDRs of the SC06-339 antibody have the following CDR sequences: SYAIS (HCDR1, SEQ ID NO: 303), GIIAIFHTPKYAQKFQG (HCDR2, SEQ ID NO: 306) and GSTYDFSSGLDY (HCDR3, SEQ ID NO: 725).
  • the light chain CDRs of the SC06-339 antibody have the following CDR sequences: GGNNIGSKSVH (LCDRl, SEQ ID NO: 621), DDSDRPS (LCDR2, SEQ ID NO: 622) and QVWDSSSDHVV (LCDR3, SEQ ID NO: 642).
  • the SC06-342 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 401) and a light chain variable region (SEQ ID NO: 402) encoded by the nucleic acid sequence shown in SEQ ID NO: 403 and the amino acid sequence shown in SEQ ID NO: 404.
  • the VH-locus is VH1 (1-69) and the VL locus is V IV (B3).
  • the amino acids encompassing the CDRs are highlighted in bold in the sequences below.
  • the heavy chain CDRs of the SC06-342 antibody have the following CDR sequences: SYAIS (HCDRl, SEQ ID NO: 251), GVIPIFRTANYAQNFQG (HCDR2, SEQ ID NO: 249) and LNYHDSGTYYNAPRGWFDP (HCDR3, SEQ ID NO: 246).
  • the light chain CDRs of the SC06-342 antibody have the following CDR sequences: SSQSILNSSNNKNYLA (LCDR1, SEQ ID NO: 245), WASTRES (LCDR2, SEQ ID NO: 570) and QQYYSSPPT (LCDR3, SEQ ID NO: 250).
  • the SC06-343 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 405) and a light chain variable region (SEQ ID NO: 406) encoded by the nucleic acid sequence shown in SEQ ID NO: 407 and the amino acid sequence shown in SEQ ID NO: 408.
  • the VH-locus is VH 1 (1 -69) and the VL locus is VL3 (V2-14).
  • the amino acids encompassing the CDRs are highlighted in bold in the sequences below.
  • the heavy chain CDRs of the SC06-343 antibody have the following CDR sequences: YYAMS (HCDR1 , SEQ ID NO: 242), GISPMFGTTTYAQKFQG (HCDR2, SEQ ID NO: 307) and SSNYYDSVYDY (HCDR3, SEQ ID NO: 290).
  • the light chain CDRs of the SC06-343 antibody have the following CDR sequences: GGHNIGSNSVH (LCDRl , SEQ ID NO: 224), DNSDRPS (LCDR2, SEQ ID NO: 223) and QVWGSSSDH (LCDR3, SEQ ID NO: 227).
  • the SC06-344 HA-specific single-chain Fv antibody includes a heavy chain variable region (SEQ ID NO: 409) and a light chain variable region (SEQ ID NO: 410) encoded by the nucleic acid sequence shown in SEQ ED NO: 41 1 and the amino acid sequence shown in SEQ ID NO: 412.
  • the VH-locus is VH1 (1 -69) and the VL locus is VL1 (Vl -13).
  • the amino acids encompassing the CDRs are highlighted in bold in the sequences below.
  • the heavy chain CDRs of the SC06-344 antibody have the following CDR sequences: NYAMS (HCDR1 , SEQ ID NO: 222), GIIAIFGTPKYAQ FQG (HCDR2, SEQ ID NO: 221 ) and IPHYNFGSGSYFDY (HCDR3, SEQ ID NO: 220).
  • the light chain CDRs of the SC06-344 antibody have the following CDR sequences: TGSSSNIGAGYDVH (LCDR1 , SEQ ID NO: 219), GNSNRPS (LCDR2, SEQ ID NO: 231 ) and GTWDSSLSAYV (LCDR3, SEQ ID NO: 280).
  • the CR6141 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 199) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 279 and the heavy chain amino acid sequence shown in SEQ ID NO: 413.
  • the CR6141 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 414) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 415 and the light chain amino acid sequence shown in SEQ ID NO: 416.
  • the CR6255 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 417) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 418 and the heavy chain amino acid sequence shown in SEQ ID NO: 419.
  • the CR6255 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 420) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 421 and the light chain amino acid sequence shown in SEQ ID NO: 422.
  • the CR6260 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 429) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 430 and the heavy chain amino acid sequence shown in SEQ ID NO: 431.
  • the CR6260 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 432) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 433 and the light chain amino acid sequence shown in SEQ ID NO: 434.
  • the CR6261 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 435) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 436 and the heavy chain amino acid sequence shown in SEQ ID NO: 437.
  • the CR6261 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 438) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 439 and the light chain amino acid sequence shown in SEQ ID NO: 440.
  • the CR6262 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 441 ) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 442 and the heavy chain amino acid sequence shown in SEQ ID NO: 443.
  • the CR6262 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 444) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 445 and the light chain amino acid sequence shown in SEQ ID NO: 446.
  • the CR6268 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 447) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 448 and the heavy chain amino acid sequence shown in SEQ ID NO: 449.
  • the CR6268 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 450) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 451 and the light chain amino acid sequence shown in SEQ ID NO: 452.
  • the CR6272 HA-spe " cific IgG antibody includes a heavy chain variable region (SEQ ID NO: 453) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 454 and the heavy chain amino acid sequence shown in SEQ ID NO: 455.
  • the CR6272 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 456) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 457 and the light chain amino acid sequence shown in SEQ ID NO: 458.
  • the CR696 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 459) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 460 and the heavy chain amino acid sequence shown in SEQ ID NO: 461.
  • the CR6296 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 462) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 463 and the light chain amino acid sequence shown in SEQ ID NO: 464.
  • the CR6307 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 471 ) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 472 and the heavy chain amino acid sequence shown in SEQ ID NO: 473.
  • the CR6307 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 474) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 475 and the light chain amino acid sequence shown in SEQ ID NO: 476.
  • the CR6310 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 477) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 478 and the heavy chain amino acid sequence shown in SEQ ID NO: 479.
  • the CR6310 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 480) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 481 and the light chain amino acid sequence shown in SEQ ID NO: 482.
  • the CR63 I 4 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 483) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 484 and the heavy chain amino acid sequence shown in SEQ ID NO: 485.
  • the CR6314 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 486) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 487 and the light chain amino acid sequence shown in SEQ ID NO: 488.
  • the CR6323 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 489) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 490 and the heavy chain amino acid sequence shown in SEQ ID NO: 491.
  • the CR6323 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 492) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 493 and the light chain amino acid sequence shown in SEQ ID NO: 494.
  • the CR6325 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 495) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 496 and the heavy chain amino acid sequence shown in SEQ ID NO: 497.
  • the CR6325 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 498) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 499 and the light chain amino acid sequence shown in SEQ ID NO: 500.
  • the CR6327 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 501 ) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 502 and the heavy chain amino acid sequence shown in SEQ ID NO: 503.
  • the CR6327 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 504) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 505 and the light chain amino acid sequence shown in SEQ ID NO: 506.
  • the CR6328 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 507) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 508 and the heavy chain amino acid sequence shown in SEQ ID NO: 509.
  • the CR6328 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 510) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 51 1 and the light chain amino acid sequence shown in SEQ ID NO: 512.
  • the CR6329 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 513) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 514 and the heavy chain amino acid sequence shown in SEQ ID NO: 515.
  • the CR6329 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 516) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 517 and the light chain amino acid sequence shown in SEQ ID NO: 518.
  • the CR6331 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 519) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 520 and the heavy chain amino acid sequence shown in SEQ ID NO: 521.
  • the CR6331 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 522) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 523 and the light chain amino acid sequence shown in SEQ ID NO: 524.
  • the CR6334 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 531 ) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 532 and the heavy chain amino acid sequence shown in SEQ ID NO: 533.
  • the CR6334 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 534) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 535 and the light chain amino acid sequence shown in SEQ ID NO: 536.
  • the CR6336 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 537) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 538 and the heavy chain amino acid sequence shown in SEQ ID NO: 539.
  • the CR6336 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 540) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 541 and the light chain amino acid sequence shown in SEQ ID NO: 542.
  • the CR6339 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 543) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 544 and the heavy chain amino acid sequence shown in SEQ ID NO: 545.
  • the CR6339 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 546) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 547 and the light chain amino acid sequence shown in SEQ ID NO: 548.
  • the CR6342 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 550) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 551 and the heavy chain amino acid sequence shown in SEQ ID NO: 552.
  • the CR6342 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 553) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 554 and the light chain amino acid sequence shown in SEQ ID NO: 555.
  • the CR6343 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 556) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 557 and the heavy chain amino acid sequence shown in SEQ ID NO: 558.
  • the CR6343 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 559) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 560 and the light chain amino acid sequence shown in SEQ ID NO: 561.
  • the CR6344 HA-specific IgG antibody includes a heavy chain variable region (SEQ ID NO: 562) encoded by the heavy chain nucleotide sequence shown in SEQ ID NO: 563 and the heavy chain amino acid sequence shown in SEQ ID NO: 564.
  • the CR6344 HA-specific IgG antibody also includes a light chain variable region (SEQ ID NO: 565) encoded by the light chain nucleotide sequence shown in SEQ ID NO: 566 and the light chain amino acid sequence shown in SEQ ID NO: 567.
  • the invention relates to an isolated human HA antibody that is able to recognize and bind to an epitope in the HA2 subunit of the influenza haemagglutinin protein (HA) (also known as hemagglutinin(HA)), characterized in that the HA antibody has neutralizing activity against an influenza virus 5 including HA of the H5 subtype.
  • influenza strains that contain such a HA of the H5 subtype and that are important strains in view of pandemic threats are H5N1 , H5N2, H5N8, and H5N9.
  • HA antibodies that at least neutralize the H5N1 influenza strain.
  • an HA antibody of the invention does not depend on an epitope in the HAl subunit of the HA protein for binding to said HA protein.
  • a number of the antibodies of the invention do not depend on conformational epitopes and recognize the HA2 epitope even in a reduced form (when used in western-blotting). This is an advantage over the antibodies from the art because when a conformational change is induced in the HA protein due to whatever mutation in another part of the protein, such conformational change will not most likely hamper the binding of the antibodies of the present invention to the HA2 epitope, whereas antibodies that do depend on conformation might very well be unable to bind when such mutations occur.
  • an HA antibody of the invention also serves as a preferred embodiment.
  • HA antibodies of the invention interact with an epitope present in the HA2 epitopes present in the H5, HI, H2, H6, and H9 subtypes (see, International Patent Application PCT/EP2007/059356, published as WO
  • an HA antibody of the invention binds to an epitope that is selected from the group consisting of the amino acid sequence: GVTNKVNSIID (SEQ ID NO: 198), GVTNKVNSIINK (SEQ ID NO: 283),
  • GVTNKENSIIDK (SEQ ID NO: 202), GVTNKVNRIIDK (SEQ ID NO: 201 ),
  • GITNKVNSVIE (SEQ ID NO: 281), GITNKENSVIE (SEQ ID NO: 257),
  • GITNKVNSIIDK SEQ ID NO: 225
  • KITSKVNNIVD SEQ ID NO: 216.
  • GVTNKVNSIIDK (SEQ ID NO: 198) epitope present in H5N1 , and are not hampered by a mutation in the TGLRN (SEQ ID NO: 200) epitope in HA 1 that do influence the binding of CI 79.
  • some HA antibodies, such as CR6307 and CR6323 are not even hampered by a escape mutant, as disclosed in Okuno etal. (1993) with a valine -> glutamic acid mutation at position 6 (exemplified by GVTNKENSIIDK (SEQ ID NO: 202)).
  • This epitope is part of an extended alpha helix in the HA2 region.
  • antibody as used herein includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity.
  • immunoglobulin immunoglobulin
  • Ig immunoglobulin
  • an "isolated antibody” is one that has been separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • the antibody is purified: (1 ) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
  • the basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.
  • An IgM antibody consists of 5 of the basic heterotetramer unit along with an additional polypeptide called J chain, and therefore contain 10 antigen binding sites, while secreted IgA antibodies can polymerize to form polyvalent assemblages comprising 2-5 of the basic 4-chain units along with J chain.
  • the 4-chain unit is generally about 150,000 daltons.
  • Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype.
  • Each H and L chain also has regularly spaced intrachain disulfide bridges.
  • Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the a and ⁇ chains and four CH domains for ⁇ and ⁇ isotypes.
  • Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end.
  • the VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CHI ).
  • Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
  • the pairing of a VH and VL together forms a single antigen-binding site.
  • immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated alpha (a), delta ( ⁇ ), epsilon ( ⁇ ), gamma ( ⁇ ) and mu (u), respectively.
  • the ⁇ and classes are further divided into subclasses on the basis of relatively minor differences in CH sequence and function, e.g. , humans express the following subclasses: IgGl , IgG2, IgG3, IgG4, IgAl , and IgA2.
  • variable refers to the fact that certain segments of the V domains differ extensively in sequence among antibodies.
  • the V domain mediates antigen binding and defines specificity of a particular antibody for its particular antigen.
  • variability is not evenly distributed across the 1 10-amino acid span of the variable domains.
  • the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called “hypervariable regions” that are each 9-12 amino acids long.
  • FRs framework regions
  • hypervariable regions that are each 9-12 amino acids long.
  • the variable domains of native heavy and light chains each comprise four FRs, largely adopting a ⁇ -sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
  • the hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991 )).
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).
  • hypervariable region when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding.
  • the hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" ⁇ e.g., around about residues 24-34 (LI ), 50-56 (L2) and 89-97 (L3) in the V L , and around about 31- 35 (HI ), 50-65 (H2) and 95-102 (H3) in the V H when numbered in accordance with the Kabat numbering system; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.
  • residues from a "hypervariable loop” ⁇ e.g., residues 24-34 (LI ), 50-56 (L2) and 89-97 (L3) in the VL, and 26-32 (HI ), 52-56 (H2) and 95-101 (H3) in the V H when numbered in accordance with the Chothia numbering system; Chothia and Lesk, J. ol. Biol. 196:901 -917 (1987)); and/or those residues from a "hypervariable loop'VCDR ⁇ e.g.
  • the antibody has symmetrical insertions at one or more of the following points 28, 36 (LI ), 63, 74-75 (L2) and 123 (L3) in the V L , and 28, 36 (HI ), 63, 74-75 (H2) and 123 (H3) in the V H when numbered in accordance with AHo; Honneger, A. and Plunkthun, A. J. Mol. Biol. 309:657-670 (2001 )).
  • germline nucleic acid residue is meant the nucleic acid residue that naturally occurs in a germline gene encoding a constant or variable region.
  • Germline gene is the DNA found in a germ cell (i.e. , a cell destined to become an egg or in the sperm).
  • germline mutation refers to a heritable change in a particular DNA that has occurred in a germ cell or the zygote at the single-cell stage, and when transmitted to offspring, such a mutation is incorporated in every cell of the body.
  • a germline mutation is in contrast to a somatic mutation which is acquired in a single body cell.
  • nucleotides in a germline DNA sequence encoding for a variable region are mutated (i.e., a somatic mutation) and replaced with a different nucleotide.
  • the term "monoclonal antibody.” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No.
  • the "monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991 ) and Marks et al, J. Mol. Biol., 222:581 -597 (1991), for example.
  • the monoclonal antibodies herein include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in. antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to
  • chimeric antibodies of primary interest herein include antibodies having one or more human antigen binding sequences (e.g., CDRs) and containing one or more sequences derived from a non-human antibody, e.g. , an FR or C region sequence.
  • chimeric antibodies of primary interest herein include those comprising a human variable domain antigen binding sequence of one antibody class or subclass and another sequence, e.g., FR or C region sequence, derived from another antibody class or subclass.
  • Chimeric antibodies of interest herein also include those containing variable domain antigen-binding sequences related to those described herein or derived from a different species, such as a non-human primate (e.g., Old World Monkey, Ape, etc).
  • Chimeric antibodies also include primatized and humanized antibodies.
  • chimeric antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. For further details, see Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
  • a "humanized antibody” is generally considered to be a human antibody that has one or more amino acid residues introduced into it from a source that is non-human. These non- human amino acid residues are often referred to as "import” residues, which are typically taken from an "import” variable domain. Humanization is traditionally performed following the method of Winter and co-workers (Jones et al., Nature, 321 :522-525 (1986); Reichmann et al, Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534-1 536 (1988)), by substituting import hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • human antibody is an antibody containing only sequences present in an antibody naturally produced by a human. However, as used herein, human antibodies may comprise residues or modifications not found in a naturally occurring human antibody, including those modifications and variant sequences described herein. These are typically made to further refine or enhance antibody performance.
  • an "intact" antibody is one that comprises an antigen-binding site as well as a C L and at least heavy chain constant domains, CH 1 , CH 2 and CH 3.
  • the constant domains may be native sequence constant domains (e.g. , human native sequence constant domains) or amino acid sequence variant thereof.
  • the intact antibody has one or more effector functions.
  • an "antibody fragment” comprises a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody.
  • antibody fragments include Fab, Fab', F(ab') 2 , and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641 ,870; Zapata et ai, Protein Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • a functional fragment or analog of an antibody is a compound having qualitative biological activity in common with a full-length antibody.
  • a functional fragment or analog of an anti-IgE antibody is one that can bind to an IgE immunoglobulin in such a manner so as to prevent or substantially reduce the ability of such molecule from having the ability to bind to the high affinity receptor, Fc E RI.
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, and a residual "Fc” fragment, a designation reflecting the ability to crystallize readily.
  • the Fab fragment consists of an entire L chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CH 1 ).
  • VH variable region domain of the H chain
  • CH 1 first constant domain of one heavy chain
  • Each Fab fragment is monovalent with respect to antigen binding, i.e. , it has a single antigen- binding site.
  • Pepsin treatment of an antibody yields a single large F(ab') 2 fragment that roughly corresponds to two disulfide linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen.
  • Fab' fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CHI domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab') 2 antibody fragments originally were produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • the "Fc” fragment comprises the carboxy-terminal portions of both H chains held together by disulfides.
  • the effector functions of antibodies are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells.
  • Fv is the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (three loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • Single-chain Fv also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain.
  • the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding.
  • a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding.
  • diabodies refers to small antibody fragments prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10 residues) between the VH and V L domains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e. , fragment having two antigen-binding sites.
  • Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains.
  • Diabodies are described more fully in, for example, EP 404,097; WO 93/1 1 161 ; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
  • an antibody that "internalizes" is one that is taken up by ' ⁇ i.e., enters) the cell upon binding to an antigen on a mammalian cell (e.g., a cell surface polypeptide or receptor).
  • the internalizing antibody will of course include antibody fragments, human or chimeric antibody, and antibody conjugates. For certain therapeutic applications, internalization in vivo is contemplated. The number of antibody molecules internalized will be sufficient or adequate to kill a cell or inhibit its growth, especially an infected cell.
  • the uptake of a single antibody molecule into the cell is sufficient to kill the target cell to which the antibody binds.
  • certain toxins are highly potent in killing such that internalization of one molecule of the toxin conjugated to the antibody is sufficient to kill the infected cell.
  • an antibody is said to be “immunospecific,” “specific for” or to “specifically bind” an antigen if it reacts at a detectable level with the antigen, preferably with an affinity constant, K A , of greater than or equal to about 1 0 4 M ⁇ ', or greater than or equal to about 1 0 5 M"', greater than or equal to about 1 0° M" 1 , greater than or equal to about 1 0 7 M " 1 , or greater than or equal to 1 0 8 M "1 .
  • K A affinity constant
  • HuM2e antibody specifically binds to M2e if it binds with a KD of less than or equal to 1 0 "4 M, less than or equal to about 1 0 "5 M, less than or equal to about 10 "6 M, less than or equal to 10 "7 M, or less than or equal to 1 0 "8 M.
  • Affinities of antibodies can be readily determined using conventional techniques, for example, those described by Scatchard et al. (Arm. N. Y. Acad. Sci. USA 5 1 :660 ( 1 949)).
  • Binding properties of an antibody to antigens, cells or tissues thereof may generally be determined and assessed using immunodetection methods including, for example, immunofluorescence-based assays, such as immuno-histochemistry (IHC) and/or fluorescence-activated cell sorting (F ACS).
  • immunodetection methods including, for example, immunofluorescence-based assays, such as immuno-histochemistry (IHC) and/or fluorescence-activated cell sorting (F ACS).
  • An antibody having a "biological characteristic" of a designated antibody is one that possesses one or more of the biological characteristics of that antibody which distinguish it from other antibodies.
  • an antibody with a biological characteristic of a designated antibody will bind the same epitope as that bound by the designated antibody and/or have a common effector function as the designated antibody.
  • antagonist antibody is used in the broadest sense, and includes an antibody that partially or fully blocks, inhibits, or neutralizes a biological activity of an epitope, polypeptide, or cell that it specifically binds.
  • Methods for identifying antagonist antibodies may comprise contacting a polypeptide or cell specifically bound by a candidate antagonist antibody with the candidate antagonist antibody and measuring a detectable change in one or more biological activities normally associated with the polypeptide or cell.
  • an "antibody that inhibits the growth of infected cells” or a “growth inhibitory” antibody is one that binds to and results in measurable growth inhibition of infected cells expressing or capable of expressing an M2e epitope bound by an antibody.
  • Preferred growth inhibitory antibodies inhibit growth of infected cells by greater than 20%, preferably from about 20% to about 50%, and even more preferably, by greater than 50% (e.g., from about 50% to about 100%>) as compared to the appropriate control, the control typically being infected cells not treated with the antibody being tested.
  • Growth inhibition can be measured at an antibody concentration of about 0.1 to 30 ⁇ g/ml or about 0.5 nM to 200 nM in cell culture, where the growth inhibition is determined 1 -10 days after exposure of the infected cells to the antibody. Growth inhibition of infected cells in vivo can be determined in various ways known in the art.
  • the antibody is growth inhibitory in vivo if administration of the antibody at about 1 ⁇ g/kg to about 100 mg/kg body weight results in reduction the percent of infected cells or total number of infected cells within about 5 days to 3 months from the first administration of the antibody, preferably within about 5 to 30 days.
  • An antibody that "induces apoptosis" is one which induces programmed cell death as determined by binding of annexin V, fragmentation of DNA, cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/or formation of membrane vesicles (called apoptotic bodies).
  • the cell is an infected cell.
  • phosphatidyl serine (PS) translocation can be measured by annexin binding; DNA fragmentation can be evaluated through DNA laddering; and nuclear/chromatin condensation along with DNA fragmentation can be evaluated by any increase in hypodiploid cells.
  • PS phosphatidyl serine
  • the antibody that induces apoptosis is one that results in about 2 to 50 fold, preferably about 5 to 50 fold, and most preferably about 10 to 50 fold, induction of annexin binding relative to untreated cell in an annexin binding assay.
  • Antibody effector functions refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: C l q binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcRs Fc receptors
  • cytotoxic cells e.g. , Natural Killer (NK) cells, neutrophils, and macrophages
  • NK cells Natural Killer cells
  • neutrophils neutrophils
  • macrophages cytotoxic cells
  • the antibodies “arm” the cytotoxic cells and are required for such killing.
  • the primary cells for mediating ADCC, NK cells express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII.
  • ADCC activity of a molecule of interest is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991 ).
  • an in vitro ADCC assay such as that described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821 ,337 may be performed.
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al, PNAS (USA) 95:652-656 (1998).
  • Fc receptor or “FcR” describes a receptor that binds to the Fc region of an antibody.
  • the FcR is a native sequence human FcR.
  • a preferred FcR is one that binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors.
  • FCyRII receptors include FcyRIIA (an "activating receptor") and FcyRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.
  • Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
  • Inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain, ⁇ see review M. in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)).
  • FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al, J. Lab. Clin. Med. 126:330-41 (1995).
  • FcR FcR
  • FcRn neonatal receptor
  • Human effector cells are leukocytes that express one or more FcRs and perform effector functions. Preferably, the cells express at least FcyRIII and perform ADCC effector function. Examples of human leukocytes that mediate ADCC include PBMC, NK cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being preferred. The effector cells may be isolated from a native source, e.g., from blood. [634] "Complement dependent cytotoxicity” or “CDC” refers to the lysis of a target cell in the presence of complement.
  • Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Cl q) to antibodies (of the appropriate subclass) that are bound to their cognate antigen.
  • a CDC assay e.g., as described in Gazzano-Santoro et al, J. Immunol. Methods 202: 163 (1996), may be performed.
  • influenza A and “Influenzavirus A” refer to a genus of the
  • Influenzavirus A includes only one species: influenza A virus which cause influenza in birds, humans, pigs, and horses. Strains of all subtypes of influenza A virus have been isolated from wild birds, although disease is uncommon. Some isolates of influenza A virus cause severe disease both in domestic poultry and, rarely, in humans.
  • a "mammal” for purposes of treating n infection refers to any mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc.
  • the mammal is human.
  • Treating” or “treatment” or “alleviation” refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder.
  • Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.
  • a subject or mammal is successfully "treated" for an infection if, after receiving a therapeutic amount of an antibody according to the methods of the present invention, the patient shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of infected cells or absence of the infected cells; reduction in the percent of total cells that are infected; and/or relief to some extent, one or more of the symptoms associated with the specific infection; reduced morbidity and mortality, and improvement in quality of life issues.
  • the above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.
  • terapéuticaally effective amount refers to an amount of an antibody or a drug effective to "treat” a disease or disorder in a subject or mammal. See preceding definition of “treating.”
  • “Chronic” administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time.
  • “Intermittent” administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.
  • Administration "in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.
  • Carriers as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution.
  • physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and or nonionic surfactants such as TWEENTM polyethylene glycol (PEG), and PLURONICSTM.
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • proteins such as serum albumin, gelatin
  • cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • the term is intended to include radioactive isotopes (e.g. , At 21 1 , 1 131 , 1 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 and radioactive isotopes of Lu), chemotherapeutic agents e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or
  • enzymatically active toxins of bacterial, fungal, plant or animal origin including fragments and/or variants thereof, and the various antitumor or anticancer agents disclosed below.
  • Other cytotoxic agents are described below.
  • a “growth inhibitory agent” when used herein refers to a compound or composition , which inhibits growth of a cell, either in vitro or in vivo.
  • growth inhibitory agents include agents that block cell cycle progression, such as agents that induce Gl arrest and M-phase arrest.
  • Classical M-phase blockers include the vinca alkaloids (vincristine, vinorelbine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
  • DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.
  • DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.
  • Docetaxel (TAXOTERETM, Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing
  • Label refers to a detectable compound or composition that is conjugated directly or indirectly to the antibody so as to generate a "labeled" antibody.
  • the label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable.
  • epitope tagged refers to a chimeric polypeptide comprising a polypeptide fused to a "tag polypeptide.”
  • the tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the polypeptide to which it is fused.
  • the tag polypeptide is also preferably fairly unique so that the antibody does not substantially cross-react with other epitopes.
  • Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues).
  • a "small molecule” is defined herein to have a molecular weight below about 500 Daltons.
  • nucleic acid and “polynucleotide” are used interchangeably herein to refer to single- or double-stranded RNA, DNA, or mixed polymers. Polynucleotides may include genomic sequences, extra-genomic and plasmid sequences, and smaller engineered gene segments that express, or may be adapted to express polypeptides.
  • An "isolated nucleic acid” is a nucleic acid that is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and
  • polymerases which naturally accompany a native sequence.
  • the term embraces a nucleic acid sequence that has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems.
  • a substantially pure nucleic acid includes isolated forms of the nucleic acid. Of course, this refers to the nucleic acid as originally isolated and does not exclude genes or sequences later added to the isolated nucleic acid by the hand of man.
  • polypeptide is used in its conventional meaning, i.e., as a sequence of amino acids.
  • the polypeptides are not limited to a specific length of the product.
  • Peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms may be used interchangeably herein unless specifically indicated otherwise.
  • This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
  • a polypeptide may be an entire protein, or a subsequence thereof.
  • Particular polypeptides of interest in the context of this invention are amino acid subsequences comprising CDRs and being capable of binding an antigen or Influenza A-infected cell.
  • an "isolated polypeptide” is one that has been identified and separated and/or recovered from a component of its natural environment.
  • the isolated polypeptide will be purified (1 ) to greater than 95% by weight of polypeptide as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated polypeptide includes the polypeptide in situ within recombinant cells since at least one component of the polypeptide's natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.
  • a “native sequence” polynucleotide is one that has the same nucleotide sequence as a polynucleotide derived from nature.
  • a “native sequence” polypeptide is one that has the same amino acid sequence as a polypeptide (e.g., antibody) derived from nature (e.g., from any species).
  • Such native sequence polynucleotides and polypeptides can be isolated from nature or can be produced by recombinant or synthetic means.
  • a polynucleotide "variant,” as the term is used herein, is a polynucleotide that typically differs from a polynucleotide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the
  • polynucleotide sequences of the invention and evaluating one or more biological activities of the encoded polypeptide as described herein and/or using any of a number of techniques well known in the art.
  • a polypeptide "variant,” as the term is used herein, is a polypeptide that typically differs from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the above polypeptide sequences of the invention and evaluating one or more biological activities of the polypeptide as described herein and/or using any of a number of techniques well known in the art.
  • amino acids may be substituted for other amino acids in a protein structure without appreciable loss of its ability to bind other polypeptides (e.g., antigens) or cells. Since it is the binding capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences that encode said peptides without appreciable loss of their biological utility or activity.
  • a polypeptide variant will contain one or more conservative substitutions.
  • a "conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged.
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art ( yte and Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
  • Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982).
  • threonine (-0.4); proline (-0.5 ⁇ 1 ); alanine (-0.5); histidine (-0.5); cysteine (-1.0);
  • methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
  • amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein.
  • substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those within ⁇ 1 are particularly preferred, and those within +0.5 are even more particularly preferred.
  • amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their
  • Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine.
  • variant polypeptides differ from a native sequence by substitution, deletion or addition of five amino acids or fewer.
  • Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure and hydropathic nature of the polypeptide.
  • Polypeptides may comprise a signal (or leader) sequence at the N-terminal end of the protein, which co-translationally or post-translationally directs transfer of the protein.
  • the polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support.
  • a polypeptide may be conjugated to an immunoglobulin Fc region.
  • comparison window refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, WI), using default parameters.
  • This program embodies several alignment schemes described in the following references: Dayhoff, M.O. (1978) A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol.
  • optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman ( ⁇ 9S ⁇ ) Add. APL. Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J Mol. Biol. 48:443, by the search for similarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by inspection.
  • BLAST and BLAST 2.0 are described in Altschul et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively.
  • BLAST and BLAST 2.0 can be used, for example with the parameters described herein, to determine percent sequence identity for the polynucleotides and polypeptides of the invention.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
  • cumulative scores can be calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0). Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the
  • the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
  • a scoring matrix can be used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
  • the "percentage of sequence identity” is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions ⁇ i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residues occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence ⁇ i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.
  • polynucleotide and polypeptide variants have at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% polynucleotide or polypeptide homology with a polynucleotide or polypeptide described herein.
  • Vector includes shuttle and expression vectors.
  • the plasmid construct will also include an origin of replication (e.g., the ColEl origin of replication) and a selectable marker (e.g., ampicillin or tetracycline resistance), for replication and selection, respectively, of the plasmids in bacteria.
  • An "expression vector” refers to a vector that contains the necessary control sequences or regulatory elements for expression of the antibodies including antibody fragment of the invention, in bacterial or eukaryotic cells.
  • Suitable vectors are disclosed below.
  • the present invention includes HuM2e antibodies comprising a polypeptide of the present invention, including those polypeptides encoded by a polynucleotide sequence set forth in Example 1 and amino acid sequences set forth in Example 1 and 2, and fragments and variants thereof.
  • the antibody is an antibody designated herein as TCN-032 (8110), 21 B 15, TCN-031 (23K12), 3241_G23, 3244 110, 3243_J07, 3259J21 , 3245_019, 3244 H04, 3136_G05, 3252_C13, 3255_J06, 3420J23, 3139_P23, 3248_P18, 3253_P10, 3260_D19, 3362_B1 1 , or 3242 P05.
  • TCN-032 8110
  • 21 B 15, TCN-031 (23K12)
  • 3241_G23, 3244 110 3243_J07, 3259J21 , 3245_019, 3244 H04, 3136_G05, 3252_C13, 3255_J06, 3420J23, 3139_P23, 3248_P18, 3253_P10, 3260_D19, 3362_B1 1 , or 3242 P05.
  • the antibodies of the present invention bind to the M2 protein.
  • the present invention provides HuM2e antibodies that bind to epitopes within M2e that are only present in the native conformation, i.e., as expressed in cells.
  • these antibodies fail to specifically bind to an isolated M2e polypeptide, e.g., the 23 amino acid residue M2e fragment. It is understood that these antibodies recognize non-linear (i.e. conformational) epitope(s) of the M2 peptide.
  • the antibodies of the present invention may be polyclonal or monoclonal antibodies. However, in preferred embodiments, they are monoclonal. In particular embodiments, antibodies of the present invention are fully human antibodies. Methods of producing polyclonal and monoclonal antibodies are known in the art and described generally, e.g., in U.S. Patent No. 6,824,780. Typically, the antibodies of the present invention are produced recombinantly, using vectors and methods available in the art, as described further below. Human antibodies may also be generated by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).
  • Human antibodies may also be produced in transgenic animals (e.g. , mice) that are capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production.
  • transgenic animals e.g. , mice
  • JH antibody heavy-chain joining region
  • Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge.
  • Jakobovits et al Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al, Nature, 362:255-258 (1993);
  • antibodies of the present invention are chimeric antibodies that comprise sequences derived from both human and non-human sources.
  • these chimeric antibodies are humanized or primatizedTM.
  • humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • chimeric antibodies also include fully human antibodies wherein the human hypervariable region or one or more CDRs are retained, but one or more other regions of sequence have been replaced by corresponding sequences from a non-human animal.
  • chimeric antibodies are prepared by a process of analysis of the parental sequences and various conceptual chimeric products using three-dimensional models of the parental human and non- human sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences.
  • antibodies can be divided into five different classes, based on differences in the amino acid sequences in the constant region of the heavy chains. All immunoglobulins within a given class have very similar heavy chain constant regions. These differences can be detected by sequence studies or more commonly by serological means (i.e. by the use of antibodies directed to these differences).
  • Antibodies, or fragments thereof, of the present invention may be any class, and may, therefore, have a gamma, mu, alpha, delta, or epsilon heavy chain.
  • a gamma chain may be gamma 1 , gamma 2, gamma 3, or gamma 4; and an alpha chain may be alpha 1 or alpha 2.
  • an antibody of the present invention, or fragment thereof is an IgG.
  • IgG is considered the most versatile immunoglobulin, because it is capable of carrying out all of the functions of immunoglobulin molecules.
  • IgG is the major Ig in serum, and the only class of Ig that crosses the placenta. IgG also fixes complement, although the IgG4 subclass does not. Macrophages, monocytes, PMN's and some lymphocytes have Fc receptors for the Fc region of IgG. Not all subclasses bind equally well; IgG2 and IgG4 do not bind to Fc receptors.
  • IgG is an opsonin that enhances phagocytosis. Binding of IgG to Fc receptors on other types of cells results in the activation of other functions.
  • Antibodies of the present invention may be of any IgG subclass.
  • an antibody, or fragment thereof, of the present invention is an IgE.
  • IgE is the least common serum Ig since it binds very tightly to Fc receptors on basophils and mast cells even before interacting with antigen. As a consequence of its binding to basophils an mast cells, IgE is involved in allergic reactions. Binding of the allergen to the IgE on the cells results in the release of various pharmacological mediators that result in allergic symptoms. IgE also plays a role in parasitic helminth diseases.
  • Eosinophils have Fc receptors for IgE and binding of eosinophils to IgE-coated helminths results in killing of the parasite. IgE does not fix complement.
  • antibodies of the present invention, and fragments thereof comprise a variable light chain that is either kappa or lambda.
  • the lamba chain may be any of subtype, including, e.g., lambda 1 , lambda 2, lambda 3, and lambda 4.
  • the present invention further provides antibody fragments comprising a polypeptide of the present invention.
  • antibody fragments comprising a polypeptide of the present invention.
  • the smaller size of the fragments allows for rapid clearance, and may lead to improved access to certain tissues, such as solid tumors.
  • antibody fragments include: Fab, Fab', F(ab') 2 and Fv fragments; diabodies; linear antibodies; single-chain antibodies; and multispecific antibodies formed from antibody fragments.
  • F(ab') 2 fragments can be isolated directly from recombinant host cell culture.
  • Fab and F(ab') 2 fragment with increased in vivo half-life comprising a salvage receptor binding epitope residues are described in U.S. Pat. No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.
  • the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571 ,894; and 5,587,458.
  • Fv and sFv are the only species with intact combining sites that are devoid of constant regions. Thus, they are suitable for reduced nonspecific binding during in vivo use.
  • sFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an sFv. See Antibody Engineering, ed. Borrebaeck, supra.
  • the antibody fragment may also be a "linear antibody", e.g. , as described in U.S. Pat. No. 5,641 ,870 for example. Such linear antibody fragments may be monospecific or bispecific.
  • antibodies of the present invention are bispecific or multi- specific.
  • Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes.
  • Exemplary bispecific antibodies may bind to two different epitopes of a single antigen.
  • Other such antibodies may combine a first antigen binding site with a binding site for a second antigen.
  • an anti-M2e arm may be combined with an arm that binds to a triggering molecule on a leukocyte, such as a T-cell receptor molecule (e.g., CD3), or Fc receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD16), so as to focus and localize cellular defense mechanisms to the infected cell.
  • a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g., CD3), or Fc receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD16), so as to focus and localize cellular defense mechanisms to the infected cell.
  • Bispecific antibodies may also be used to localize cytotoxic agents to infected cells.
  • bispecific antibodies possess an M2e-binding arm and an arm that binds the cytotoxic agent (e.g., saporin, anti- interferon-a, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten).
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab') 2 bispecific antibodies).
  • WO 96/16673 describes a bispecific anti-ErbB2/anti-FcYRIII antibody and U.S. Pat. No. 5,837,234 discloses a bispecific anti-ErbB2/anti-FcyRI antibody. A bispecific anti-ErbB2/Fccc antibody is shown in WO98/02463.
  • U.S. Pat. No. 5,821 ,337 teaches a bispecific anti-ErbB2/anti-CD3 antibody.
  • antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences.
  • the fusion is with an Ig heavy chain constant domain, comprising at least part of the hinge, (3 ⁇ 42, and (3 ⁇ 43 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light chain bonding, present in at least one of the fusions.
  • DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a suitable host cell.
  • the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121 :210 (1986).
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture.
  • the preferred interface comprises at least a part of the CH 3 domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan).
  • Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end- products such as homodimers.
  • Bispecific antibodies include cross-linked or "heteroconjugate" antibodies.
  • one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin.
  • Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089).
  • Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
  • bispecific antibodies can be prepared using chemical linkage.
  • Brennan et al, Science, 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab') 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent, sodium arsenite, to stabilize vicinal dithiols and prevent intermolecular disulfide formation.
  • the Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
  • One of the Fab'-TNB derivatives is then reconverted to the Fab' -thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody.
  • the bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
  • bispecific antibodies have been produced using leucine zippers. ostelny et ai, J. Immunol., 148(5): 1547-1553 (1992).
  • the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion.
  • the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
  • the fragments comprise a VH connected to a VL by a linker that is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.
  • Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).
  • Antibodies with more than two valencies are contemplated.
  • trispeciflc antibodies can be prepared. Tutt et ai, J. Immunol. 147: 60 (1991).
  • a multivalent antibody may be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind.
  • the antibodies of the present invention can be multivalent antibodies with three or more antigen binding sites ⁇ e.g. , tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody.
  • the multivalent antibody can comprise a dimerization domain and three or more antigen binding sites.
  • the preferred dimerization domain comprises (or consists of) an Fc region or a hinge region.
  • the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fc region.
  • the preferred multivalent antibody herein comprises (or consists of) three to about eight, but preferably four, antigen binding sites.
  • the multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable domains.
  • the polypeptide chain(s) may comprise VD1 -(X 1 ) folk -VD2-(X2) n -Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, XI and X2 represent an amino acid or polypeptide, and n is 0 or 1.
  • the polypeptide chain(s) may comprise: VH-CH1 -flexible linker-VH-CHl-Fc region chain; or VH-CHl -VH-CHl -Fc region chain.
  • the multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable domain polypeptides.
  • the multivalent antibody herein may, for instance, comprise from about two to about eight light chain variable domain polypeptides.
  • the light chain variable domain polypeptides contemplated here comprise a light chain variable domain and, optionally, further comprise a CL domain.
  • Antibodies of the present invention further include single chain antibodies.
  • antibodies of the present invention are internalizing antibodies.
  • Amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody.
  • Amino acid sequence variants of the antibody may be prepared by introducing appropriate nucleotide changes into a polynucleotide that encodes the antibody, or a chain thereof, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution may be made to arrive at the final antibody, provided that the final construct possesses the desired characteristics.
  • the amino acid changes also may alter post- translational processes of the antibody, such as changing the number or position of glycosylation sites. Any of the variations and modifications described above for polypeptides of the present invention may be included in antibodies of the present invention.
  • a useful method for identification of certain residues or regions of an antibody that are preferred locations for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells in Science, 244: 1081 -1085 (1989).
  • a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with PSCA antigen.
  • Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution.
  • the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at the target codon or region and the expressed anti- antibody variants are screened for the desired activity.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to a cytotoxic polypeptide.
  • Other insertional variants of an antibody include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. , for ADEPT) or a polypeptide that increases the serum half-life of the antibody.
  • variants are an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced by a different residue.
  • the sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative and non-conservative substitutions are contemplated.
  • substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • cysteine residue not involved in maintaining the proper conformation of the antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.
  • cysteine bond(s) may be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).
  • substitutional variant involves substituting one or more hypervariable region residues of a parent antibody.
  • the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated.
  • a convenient way for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino substitutions at each site.
  • the antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of Ml 3 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g. , binding affinity) as herein disclosed.
  • alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding.
  • Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein.
  • Another type of amino acid variant of the antibody alters the original glycosylation pattern of the antibody. By altering is meant deleting one or more carbohydrate moieties found in the antibody, and/or adding one or more glycosylation sites that are not present in the antibody.
  • Glycosylation of antibodies is typically either N-linked or O-linked.
  • N-Iinked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
  • the tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
  • X is any amino acid except proline
  • O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5- hydroxyproline or 5-hydroxylysine may also be used.
  • Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites).
  • the alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).
  • the antibody of the invention is modified with respect to effector function, e.g., so as to enhance antigen-dependent cell-mediated cyotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) of the antibody.
  • ADCC antigen-dependent cell-mediated cyotoxicity
  • CDC complement dependent cytotoxicity
  • This may be achieved by introducing one or more amino acid substitutions in an Fc region of the antibody.
  • cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody- dependent cellular cytotoxicity (ADCC). See Caron et al, J. Exp Med. 176: 1 191 -1 195 (1992) and Shopes, B. J.
  • Homodimeric antibodies with enhanced anti-infection activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al, Cancer Research 53:2560-2565 (1993).
  • an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al, Anti-Cancer Drug Design 3:219-230 (1989).
  • a salvage receptor binding epitope refers to an epitope of the Fc region of an IgG molecule (e.g., IgGi, IgG 2 , IgG 3 , or IgG 4 ) that is responsible for increasing the in vivo serum half-life of the IgG molecule.
  • Antibodies of the present invention may also be modified to include an epitope tag or label, e.g., for use in purification or diagnostic applications.
  • the invention also pertains to therapy with immunoconjugates comprising an antibody conjugated to an anti-cancer agent such as a cytotoxic agent or a growth inhibitory agent. Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above.
  • Conjugates of an antibody and one or more small molecule toxins such as a calicheamicin, maytansinoids, a trichothene, and CC1065, and the derivatives of these toxins that have toxin activity, are also contemplated herein.
  • an antibody (full length or fragments) of the invention is conjugated to one or more maytansinoid molecules.
  • Maytansinoids are mitototic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub Maytenus serrata (U.S. Pat. No. 3,896,1 1 1 ). Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,1 51 ,042). Synthetic maytansinol and derivatives and analogues thereof are disclosed, for example, in U.S. Pat. Nos.
  • maytansine and maytansinoids have been conjugated to antibodies specifically binding to tumor cell antigens.
  • Immunoconjugates containing maytansinoids and their therapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 Bl .
  • Liu et ai Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) described immunoconjugates comprising a maytansinoid designated DM 1 linked to the monoclonal antibody C242 directed against human colorectal cancer.
  • the conjugate was found to be highly cytotoxic towards cultured colon cancer cells, and showed antitumor activity in an in vivo tumor growth assay.
  • Antibody-maytansinoid conjugates are prepared by chemically linking an antibody to a maytansinoid molecule without significantly diminishing the biological activity of either the antibody or the maytansinoid molecule.
  • An average of 3-4 maytansinoid molecules conjugated per antibody molecule has shown efficacy in enhancing cytotoxicity of target cells without negatively affecting the function or solubility of the antibody, although even one molecule of toxin/antibody would be expected to enhance cytotoxicity over the use of naked antibody.
  • Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources. Suitable maytansinoids are disclosed, for example, in U.S. Pat. No.
  • Preferred maytansinoids are maytansinol and maytansinol analogues modified in the aromatic ring or at other positions of the maytansinol molecule, such as various maytansinol esters.
  • linking groups There are many linking groups known in the art for making antibody conjugates, including, for example, those disclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B l , and Chari et al. , Cancer Research 52: 127-131 (1992).
  • the linking groups include disufide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, or esterase labile groups, as disclosed in the above-identified patents, disulfide and thioether groups being preferred.
  • Immunoconjugates may be made using a variety of bifiinctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N- maleimidomethyl)cyclohexane-l -carboxylate, iminothiolane (IT), bifiinctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p- azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)- ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds
  • Particularly preferred coupling agents include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et ai , Biochem. J. 1 73:723-737 [1978]) and N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for a disulfide linkage.
  • SPDP N-succinimidyl-3-(2-pyridyldithio)propionate
  • SPP N-succinimidyl-4-(2-pyridylthio)pentanoate
  • a ricin immunotoxin can be prepared as described in Vitetta et ai, Science 238: 1098 (1987).
  • Carbon- 14-labeled l -isothidcyanatobenzyl-3-methyldiethylene triaminepentaacetic acid is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See W094/1 1026.
  • the linker may be a "cleavable linker" facilitating release of the cytotoxic drug in the cell.
  • an acid-labile linker Cancer Research 52: 127-131 (1992); U.S. Pat. No. 5,208,020 may be used.
  • Another immunoconjugate of interest comprises an antibody conjugated to one or more calicheamicin molecules.
  • the calicheamicin family of antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations.
  • For the preparation of conjugates of the calicheamicin family see U.S. Pat. Nos. 5,712,374, 5,714,586,
  • Another drug that the antibody can be conjugated is QFA which is an antifolate.
  • QFA is an antifolate.
  • calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Therefore, cellular uptake of these agents through antibody mediated internalization greatly enhances their cytotoxic effects.
  • agents that can be conjugated to the antibodies of the invention include BCNU, streptozoicin, vincristine and 5-fluorouracil, the family of agents known collectively LL-E33288 complex described in U.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No. 5,877,296).
  • Enzymatically active toxins and fragments thereof that can be used include, e.g. , diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPI1, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for example, WO 93/21232.
  • the present invention further includes an immunoconjugate formed between an antibody and a compound with nucleolytic activity (e.g., a ribonuclease or a
  • endonuclease such as a deoxyribonuclease; DNase).
  • the antibody includes a highly radioactive atom.
  • a variety of radioactive isotopes are available for the production of radioconjugated anti-PSCA antibodies. Examples include At 2 ", I 131 , 1 125 , Y 90 , Re 186 , Rc 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu.
  • the conjugate When used for diagnosis, it may comprise a radioactive atom for scintigraphic studies, for example tc 99m or I 123 , or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123, iodine- 131 , indium-1 1 1 , fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
  • NMR nuclear magnetic resonance
  • the radio- or other label is incorporated in the conjugate in known ways.
  • the peptide may be biosynthesized or may be synthesized by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of ' hydrogen.
  • Labels such as tc 99m or I 123 , Re 186 , Re 188 and In 1 1 1 can be attached via a cysteine residue in the peptide.
  • Yttrium-90 can be attached via a lysine residue.
  • the IODOGEN method (Fraker et al. (1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123. "Monoclonal Antibodies in Immunoscintigraphy" (Chatal,CRC Press 1989) describes other methods in detail.
  • a fusion protein comprising the antibody and cytotoxic agent is made, e.g. , by recombinant techniques or peptide synthesis.
  • the length of DNA may comprise respective regions encoding the two portions of the conjugate either adjacent one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate.
  • the antibodies of the present invention are also used in antibody dependent enzyme mediated prodrug therapy (ADET) by conjugating the antibody to a prodrug-activating enzyme which converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see
  • the enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such a way so as to covert it into its more active, cytotoxic form.
  • Enzymes that are useful in the method of this invention include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs;
  • cytosine deaminase useful for converting non-toxic 5-fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin,
  • carboxypeptidases and cathepsins that are useful for converting peptide-containing prodrugs into free drugs
  • D-alanylcarboxypeptidases useful for converting prodrugs that contain D-amino acid substituents
  • carbohydrate-cleaving enzymes such as ⁇ -galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs
  • ⁇ -lactamase useful for converting drugs derivatized with ⁇ -lactams into free drugs
  • penicillin amidases such as penicillin V amidase or penicillin G amidase, useful for converting drugs derivatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs.
  • antibodies with enzymatic activity can be used to convert the prodrugs of the invention into free active drugs (see, e.g., Massey, Nature 328: 457-458 (1987)).
  • Antibody-abzyme conjugates can be prepared as described herein for delivery of the abzyme to a infected cell population.
  • 729j The enzymes of this invention can be covalently bound to the antibodies by techniques well known in the art such as the use of the heterobifunctional crosslinking reagents discussed above.
  • fusion proteins comprising at least the antigen binding region of an antibody of the invention linked to at least a functionally active portion of an enzyme of the invention can be constructed using recombinant DNA techniques well known in the art ⁇ see, e.g., Neuberger et al , Nature, 312: 604-608 (1984).
  • the antibody may be linked to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol.
  • nonproteinaceous polymers e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol.
  • the antibody also may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly-
  • methylmethacylate microcapsules, respectively
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and
  • liposome is a small vesicle composed of various types of lipids, phospholipids and/or surfactant that is useful for delivery of a drug to a mammal.
  • the components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.
  • Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et ai, Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et ai, Proc. Natl Acad. Sci. USA, 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; and W097/38731 published Oct. 23, 1997. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.
  • Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG- derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired a diameter.
  • Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et ai, J. Biol. Chem. 257: 286-288 (1982) via a disulfide interchange reaction.
  • chemotherapeutic agent is optionally contained within the liposome. See Gabizon et ai, J. National Cancer Inst. 81(19)1484 (1989).
  • Antibodies of the present invention, or fragments thereof may possess any of a variety of biological or functional characteristics.
  • these antibodies are Influenza A specific or M2 protein specific antibodies, indicating that they specifically bind to or preferentially bind to Influenza A or the M2 protein thereof, respectively, as compared to a normal control cell.
  • the antibodies are HuM2e antibodies, indicating that they specifically bind to a M2e protein, preferably to an epitope of the M2e domain that is only present when the M2 protein is expressed in cells or present on a virus, as compared to a normal control cell.
  • an antibody of the present invention is an antagonist antibody, which partially or fully blocks or inhibits a biological activity of a polypeptide or cell to which it specifically or preferentially binds.
  • an antibody of the present invention is a growth inhibitory antibody, which partially or fully blocks or inhibits the growth of an infected cell to which it binds.
  • an antibody of the present invention induces apoptosis.
  • an antibody of the present invention induces or promotes antibody-dependent cell-mediated cytotoxicity or complement dependent cytotoxicity.
  • the present invention provides novel methods for the identification of HuM2e antibodies, as exemplified in Example 4. These methods may be readily adapted to identify antibodies specific for other polypeptides expressed on the cell surface by infectious agents, or even polypeptides expressed on the surface of an infectious agent itself.
  • the methods include obtaining serum samples from patients that have been infected with or vaccinated against an infectious agent. These serum samples are then screened to identify those that contain antibodies specific for a particular polypeptide associated with the infectious agent, such as, e.g., a polypeptide specifically expressed on the surface of cells infected with the infectious agent, but not uninfected cells.
  • the serum samples are screened by contacting the samples with a cell that has been transfected with an expression vector that expresses the polypeptide expressed on the surface of infected cells:
  • a patient is identified as having serum containing an antibody specific for the infectious agent polypeptide of interest is identified
  • mononuclear and/or B cells obtained from the same patient are used to identify a cell or clone thereof that produces the antibody, using any of the methods described herein or available in the art.
  • cDNAs encoding the variable regions or fragments thereof of the antibody may be cloned using standard RT-PCR vectors and primers specific for conserved antibody sequences, and subcloned in to expression vectors used for the recombinant production of monoclonal antibodies specific for the infectious agent polypeptide of interest.
  • the present invention provides a method of identifying an antibody that specifically binds influenza A-infected cells, comprising: contacting an Influenza A virus or a cell expressing the M2 protein with a biological sample obtained from a patient having been infected by Influenza A; determining an amount of antibody in the biological sample that binds to the cell; and comparing the amount determined with a control value, wherein if the value determined is at least two-fold greater than the control value, an antibody that specifically binds influenza A-infected cells is indicated.
  • the cells expressing an M2 protein are cells infected with an Influenza A virus or cells that have been transfected with a polynucleotide that expressed the M2 protein.
  • the cells may express a portion of the M2 protein that includes the M2e domain and enough additional M2 sequence that the protein remains associated with the cell and the M2e domain is presented on the cell surface in the same manner as when present within full length M2 protein.
  • the M2e-expressing cells or virus described above are used to screen the biological sample obtained from a patient infected with influenza A for the presence of antibodies that preferentially bind to the cell expressing the M2 polypeptide using standard biological techniques.
  • the antibodies may be labeled, and the presence of label associated with the cell detected, e.g., using FMAT or FACs analysis.
  • the biological sample is blood, serum, plasma, bronchial lavage, or saliva. Methods of the present invention may be practiced using high throughput techniques.
  • Identified human antibodies may then be characterized further. For example the particular conformational epitopes with in the M2e protein that are necessary or sufficient for binding of the antibody may be determined, e.g., using site-directed mutagenesis of expressed M2e polypeptides. These methods may be readily adapted to identify human antibodies that bind any protein expressed on a cell surface. Furthermore, these methods may be adapted to determine binding of the antibody to the virus itself, as opposed to a cell expressing recombinant M2e or infected with the virus.
  • Polynucleotide sequences encoding the antibodies, variable regions thereof, or antigen-binding fragments thereof may be subcloned into expression vectors for the recombinant production of HuM2e antibodies. In one embodiment, this is accomplished by obtaining mononuclear cells from the patient from the serum containing the identified HuM2e antibody was obtained; producing B cell clones from the mononuclear cells; inducing the B cells to become antibody-producing plasma cells; and screening the supernatants produced by the plasma cells to determine if it contains the HuM2e antibody.
  • RT-PCR reverse-transcription polymerase chain reaction
  • B cells isolated from peripheral blood or lymph nodes are sorted, e.g., based on their being CD 19 positive, and plated, e.g., as low as a single cell specificity per well, e.g., in 96, 384, or 1536 well configurations.
  • the cells are induced to differentiate into antibody-producing cells, e.g., plasma cells, and the culture supernatants are harvested and tested for binding to cells expressing the infectious agent polypeptide on their surface using, e.g. , FMAT or FACS analysis.
  • Positive wells are then subjected to whole well RT-PCR to amplify heavy and light chain variable regions of the IgG molecule expressed by the clonal daughter plasma cells.
  • the resulting PCR products encoding the heavy and light chain variable regions, or portions thereof, are subcloned into human antibody expression vectors for recombinant expression.
  • the resulting recombinant antibodies are then tested to confirm their original binding specificity and may be further tested for pan-specificity across various strains of isolates of the infectious agent.
  • a method of identifying HuM2e antibodies is practiced as follows. First, full length or approximately full length M2 cDNAs are transfected into a cell line for expression of M2 protein. Secondly, individual human plasma or sera samples are tested for antibodies that bind the cell-expressed M2. And lastly, MAbs derived from plasma- or serum-positive individuals are characterized for binding to the same cell-expressed M2. Further definition of the fine specificities of the MAbs can be performed at this point.
  • HuM2e antibodies including antibodies specific for (a) epitopes in a linear M2e peptide, (b) common epitopes in multiple variants of M2e, (c) conformational determinants of an M2 homotetramer, and (d) common conformational determinants of multiple variants of the M2 homotetramer.
  • T e last category is particularly desirable, as this specificity is perhaps specific for all A strains of influenza.
  • Polynucleotides that encode the HuM2e antibodies or portions thereof of the present invention may be isolated from cells expressing HuM2e antibodies, according to methods available in the art and described herein, including amplification by polymerase chain reaction using primers specific for conserved regions of human antibody polypeptides. For example, light chain and heavy chain variable regions may be cloned from the B cell according to molecular biology techniques described in WO 92/02551 ; U.S. Patent No. 5,627,052; or Babcook et al., Proc. Natl. Acad. Sci. USA 93:7843-48 (1996).
  • polynucleotides encoding all or a region of both the heavy and light chain variable regions of the IgG molecule expressed by the clonal daughter plasma cells expressing the HuM2e antibody are subcloned and sequenced.
  • the sequence of the encoded polypeptide may be readily determined from the polynucleotide sequence.
  • Isolated polynucleotides encoding a polypeptide of the present invention may be subcloned into an expression vector to recombinantly produce antibodies and polypeptides of the present invention, using procedures known in the art and described herein.
  • Binding properties of an antibody (or fragment thereof) to M2e or infected cells or tissues may generally be determined and assessed using immunodetection methods including, for example, immunofluorescence-based assays, such as immuno-histochemistry (IHC) and/or fluorescence-activated cell sorting (FACS). Immunoassay methods may include controls and procedures to determine whether antibodies bind specifically to 2e from one or more specific strains of Influenza A, and do not recognize or cross-react with normal control cells.
  • immunodetection methods including, for example, immunofluorescence-based assays, such as immuno-histochemistry (IHC) and/or fluorescence-activated cell sorting (FACS).
  • Immunoassay methods may include controls and procedures to determine whether antibodies bind specifically to 2e from one or more specific strains of Influenza A, and do not recognize or cross-react with normal control cells.
  • the methods of the present invention typically include the isolation or purification of B cells from a biological sample previously obtained from a patient or subject.
  • the patient or subject may be currently or previously diagnosed with or suspect or having a particular disease or infection, or the patient or subject may be considered free or a particular disease or infection.
  • the patient or subject is a mammal and, in particular embodiments, a human.
  • the biological sample may be any sample that contains B cells, including but not limited to, lymph node or lymph node tissue, pleural effusions, peripheral blood, ascites, tumor tissue, or cerebrospinal fluid (CSF).
  • B cells are isolated from different types of biological samples, such as a biological sample affected by a particular disease or infection.
  • any biological sample comprising B cells may be used for any of the embodiments of the present invention.
  • the B cells are induced to produce antibodies, e.g., by culturing the B cells under conditions that support B cell proliferation or development into a plasmacyte, plasmablast, or plasma cell.
  • the antibodies are then screened, typically using high throughput techniques, to identify an antibody that specifically binds to a target antigen, e.g., a particular tissue, cell, infectious agent, or polypeptide.
  • a target antigen e.g., a particular tissue, cell, infectious agent, or polypeptide.
  • the specific antigen, e.g., cell surface polypeptide bound by the antibody is not known, while in other embodiments, the antigen specifically bound by the antibody is known.
  • B cells may be isolated from a biological sample, e.g., a tumor, tissue, peripheral blood or lymph node sample, by any means known and available in the art.
  • B cells are typically sorted by FACS based on the presence on their surface of a B cell-specific marker, e.g., CD19, CD138, and/or surface IgG.
  • a B cell-specific marker e.g., CD19, CD138, and/or surface IgG.
  • other methods known in the art may be employed, such as, e.g., column purification using CD 19 magnetic beads or IgG-specific magnetic beads, followed by elution from the column.
  • the isolated cells are not sorted but, instead, phicol- purified mononuclear cells isolated from tumor are directly plated to the appropriate or desired number of specificities per well.
  • the B cells are typically plated at low density (e.g., a single cell specificity per well, 1-10 cells per well, 10- 100 cells per well, 1 -100 cells per well, less than 10 cells per well, or less than 100 cells per well) in multi-well or microtitre plates, e.g. , in 96, 384, or 1 536 well configurations.
  • low density e.g., a single cell specificity per well, 1-10 cells per well, 10- 100 cells per well, 1 -100 cells per well, less than 10 cells per well, or less than 100 cells per well
  • the methods of the present invention may include the step of subsequently diluting cells in a well identified as producing an antigen-specific antibody, until a single cell specificity per well is achieved, thereby facilitating the identification of the B cell that produces the antigen-specific antibody.
  • Cell supernatants or a portion thereof and/or cells may be frozen and stored for future testing and later recovery of antibody polynucleotides.
  • the B cells are cultured under conditions that favor the production of antibodies by the B cells.
  • the B cells may be cultured under conditions favorable for B cell proliferation and differentiation to yield antibody-producing plasmablast, plasmacytes, or plasma cells.
  • the B cells are cultured in the presence of a B cell mitogen, such as lipopolysaccharide (LPS) or CD40 ligand.
  • B cells are differentiated to antibody-producing cells by culturing them with feed cells and/or other B cell activators, such as CD40 ligand.
  • Cell culture supernatants or antibodies obtained therefrom may be tested for their ability to bind to a target antigen, using routine methods available in the art, including those described herein.
  • culture supernatants are tested for the presence of antibodies that bind to a target antigen using high- throughput methods.
  • B cells may be cultured in multi-well microtitre dishes, such that robotic plate handlers may be used to simultaneously sample multiple cell supernatants and test for the presence of antibodies that bind to a target antigen.
  • antigens are bound to beads, e.g. , paramagnetic or latex beads) to facilitate the capture of antibody/antigen complexes.
  • antigens and antibodies are fluorescently labeled (with different labels) and FACS analysis is performed to identify the presence of antibodies that bind to target antigen.
  • antibody binding is determined using FMATTM analysis and instrumentation (Applied Biosystems, Foster City, CA).
  • FMATTM is a fluorescence macro-confocal platform for high-throughput screening, which mix-and-read, non-radioactive assays using live cells or beads.
  • the antibody is considered to preferentially bind a particular target antigen if at least two-fold, at least three-fold, at least five-fold, or at least ten-fold more antibody binds to the particular target antigen as compared to the amount that binds a control sample.
  • Polynucleotides encoding antibody chains, variable regions thereof, or fragments thereof may be isolated from cells utilizing any means available in the art.
  • polynucleotides are isolated using polymerase chain reaction (PCR), e.g., reverse transcription-PCR (RT-PCR) using oligonucleotide primers that specifically bind to heavy or light chain encoding polynucleotide sequences or complements thereof using routine procedures available in the art.
  • PCR polymerase chain reaction
  • RT-PCR reverse transcription-PCR
  • positive wells are subjected to whole well RT-PCR to amplify the heavy and light chain variable regions of the IgG molecule expressed by the clonal daughter plasma cells. These PCR products may be sequenced.
  • nucleic acid molecules encoding a tumor-specific antibody or fragment thereof, as described herein, may be propagated and expressed according to any of a variety of well-known procedures for nucleic acid excision, ligation, transformation, and transfection.
  • expression of an antibody fragment may be preferred in a prokaryotic host cell, such as Escherichia coli ⁇ see, e.g., Pluckthun et al., Methods Enzymol. 1 78:497-515 (1989)).
  • expression of the antibody or an antigen-binding fragment thereof may be preferred in a eukaryotic host cell, including yeast (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris); animal cells (including mammalian cells); or plant cells.
  • yeast e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris
  • animal cells including mammalian cells
  • plant cells examples include, but are not limited to, myeloma, COS, CHO, or hybridoma cells.
  • nucleic acid vector may be designed for expressing foreign sequences in a particular host system, and then polynucleotide sequences encoding the tumor-specific antibody (or fragment thereof) may be inserted.
  • the regulatory elements will vary according to the particular host.
  • One or more replicable expression vectors containing a polynucleotide encoding a variable and/or constant region may be prepared and used to transform an appropriate cell line, for example, a non-producing myeloma cell line, such as a mouse NSO line or a bacteria, such as E.coli, in which production of the antibody will occur.
  • an appropriate cell line for example, a non-producing myeloma cell line, such as a mouse NSO line or a bacteria, such as E.coli, in which production of the antibody will occur.
  • the polynucleotide sequence in each vector should include appropriate regulatory sequences, particularly a promoter and leader sequence operatively linked to the variable domain sequence.
  • Particular methods for producing antibodies in this way are generally well known and routinely used. For example, molecular biology procedures are described by Sambrook et al.
  • regions of polynucleotides encoding the recombinant antibodies may be sequenced. DNA sequencing can be performed as described in Sanger et al. (Proc. Natl. Acad. Sci. USA 74:5463 (1977)) and the Amersham International pic sequencing handbook and including improvements thereto.
  • the resulting recombinant antibodies or fragments thereof are then tested to confirm their original specificity and may be further tested for pan- specificity, e.g. , with related infectious agents.
  • an antibody identified or produced according to methods described herein is tested for cell killing via antibody dependent cellular cytotoxicity (ADCC) or apoptosis, and/or well as its ability to internalize.
  • ADCC antibody dependent cellular cytotoxicity
  • polynucleotide compositions in other aspects, provides polynucleotide compositions.
  • these polynucleotides encode a polypeptide of the invention, e.g., a region of a variable chain of an antibody that binds to Influenza A, M2, or M2e.
  • Polynucleotides of the invention are single-stranded (coding or antisense) or double-stranded DNA (genomic, cDNA or synthetic) or RNA molecules.
  • RNA molecules include, but are not limited to, HnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns.
  • coding or non-coding sequences are present within a polynucleotide of the present invention.
  • a polynucleotide is linked to other molecules and/or support materials of the invention.
  • Polynucleotides of the invention are used, e.g., in hybridization assays to detect the presence of an Influenza A antibody in a biological sample, and in the recombinant production of polypeptides of the invention.
  • polynucleotide compositions include some or all of a polynucleotide sequence set forth in Example 1 , complements of a polynucleotide sequence set forth in Example 1 , and degenerate variants of a polynucleotide sequence set forth in Example 1.
  • the polynucleotide sequences set forth herein encode polypeptides capable of preferentially binding a Influenza A-infected cell as compared to a normal control uninfected cell, including a polypeptide having a sequence set forth in Examples 1 or 2.
  • the invention includes all polynucleotides that encode any polypeptide of the present invention.
  • the invention provides polynucleotide variants having substantial identity to the sequences set forth in Figure 1 , for example those comprising at least 70% sequence identity, preferably at least.75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher, sequence identity compared to a polynucleotide sequence of this invention, as determined using the methods described herein, (e.g., BLAST analysis using standard parameters).
  • BLAST analysis e.g., BLAST analysis using standard parameters.
  • polynucleotide variants typically contain one or more substitutions, additions, deletions and/or insertions, preferably such that the immunogenic binding properties of the polypeptide encoded by the variant polynucleotide is not substantially diminished relative to a polypeptide encoded by a polynucleotide sequence specifically set forth herein.
  • the present invention provides polynucleotide fragments comprising various lengths of contiguous stretches of sequence identical to or complementary to one or more of the sequences disclosed herein.
  • polynucleotides are provided by this invention that comprise at least about 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500 or 1000 or more contiguous nucleotides of one or more of the sequences disclosed herein as well as all intermediate lengths there between.
  • intermediate lengths is meant to describe any length between the quoted values, such as 16, 1 7, 18, 19, etc:; 2 ⁇ , 22, 23, etc.; 30, 31 , 32, etc. ; 50, 51 , 52, 53, etc. ; 100, 101 , 102, 103, etc. ; 150, 151 , 152, 153, etc. ; including all integers through 200-500; 500-1 ,000, and the like.
  • polynucleotide compositions are provided that are capable of hybridizing under moderate to high stringency conditions to a
  • suitable moderately stringent conditions for testing the hybridization of a polynucleotide of this invention with other polynucleotides include prewashing in a solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C-60°C, 5 X SSC, overnight; followed by washing twice at 65°C for 20 minutes with each of 2X, 0.5X and 0.2X SSC containing 0.1% SDS.
  • stringency of hybridization can be readily manipulated, such as by altering the salt content of the hybridization solution and/or the temperature at which the hybridization is performed.
  • suitable highly stringent hybridization conditions include those described above, with the exception that the temperature of hybridization is increased, e.g., to 60-65°C or 65-70°C.
  • the polypeptide encoded by the polynucleotide variant or fragment has the same binding specificity (i.e. , specifically or preferentially binds to the same epitope or Influenza A strain) as the polypeptide encoded by the native polynucleotide.
  • the polynucleotides described above, e.g., polynucleotide variants, fragments and hybridizing sequences encode polypeptides that have a level of binding activity of at least about 50%, preferably at least about 70%, and more preferably at least about 90% of that for a polypeptide sequence specifically set forth herein.
  • polynucleotides of the present invention may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably.
  • a nucleic acid fragment of almost any length is employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.
  • illustrative polynucleotide segments with total lengths of about 10,000, about 5000, about 3000, about 2,000, about 1 ,000, about 500, about 200, about 100, about 50 base pairs in length, and the like, (including all intermediate lengths) are included in many
  • mutagenesis of the disclosed polynucleotide sequences is performed in order to alter one or more properties of the encoded polypeptide, such as its binding specificity or binding strength.
  • Techniques for mutagenesis are well-known in the art, and are widely used to create variants of both polypeptides and polynucleotides.
  • a mutagenesis approach such as site-specific mutagenesis, is employed for the preparation of variants and/or derivatives of the polypeptides described herein. By this approach, specific modifications in a polypeptide sequence are made through mutagenesis of the underlying polynucleotides that encode them.
  • Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences include the nucleotide sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
  • Mutations are employed in a selected polynucleotide sequence to improve, alter, decrease, modify, or otherwise change the properties of the polynucleotide itself, and/or alter the properties, activity, composition, stability, or primary sequence of the encoded polypeptide.
  • the polynucleotide sequences provided herein are used as probes or primers for nucleic acid hybridization, e.g., as PCR primers.
  • the ability of such nucleic acid probes to specifically hybridize to a sequence of interest enable them to detect the presence of complementary sequences in a given sample.
  • other uses are also encompassed by the invention, such as the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions.
  • nucleic acid segments of the invention that include a sequence region of at least about 15 nucleotide long contiguous sequence that has the same sequence as, or is complementary to, a 15 nucleotide long contiguous sequence disclosed herein is particularly useful.
  • Longer contiguous identical or complementary sequences e.g., those of about 20, 30, 40, 50, 100, 200, 500, 1000 (including all intermediate lengths) including full length sequences, and all lengths in between, are also used in certain embodiments.
  • Polynucleotide molecules having sequence regions consisting of contiguous nucleotide stretches of 10-14, 1 5-20, 30, 50, or even of 100-200 nucleotides or so (including intermediate lengths as well), identical or complementary to a polynucleotide sequence disclosed herein, are particularly contemplated as hybridization probes for use in, e.g.,
  • PCR polymerase chain reaction
  • hybridization probe of about 15-25 nucleotides in length allows the formation of a duplex molecule that is both stable and selective.
  • Molecules having contiguous complementary sequences over stretches greater than 12 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained.
  • Nucleic acid molecules having gene-complementary stretches of 15 to 25 contiguous nucleotides, or even longer where desired, are generally preferred.
  • Hybridization probes are selected from any portion of any of the sequences disclosed herein. All that is required is to review the sequences set forth herein, or to any continuous portion of the sequences, from about 15-25 nucleotides in length up to and including the full length sequence, that one wishes to utilize as a probe or primer. The choice of probe and primer sequences is governed by various factors. For example, one may wish to employ primers from towards the termini of the total sequence.
  • Polynucleotide of the present invention are readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer. Also, fragments are obtained by application of nucleic acid reproduction technology, such as the PCRTM technology of U. S. Patent 4,683,202, by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.
  • the invention provides vectors and host cells comprising a nucleic acid of the present invention, as well as recombinant techniques for the production of a polypeptide of the present invention.
  • Vectors of the invention include those capable of replication in any type of cell or organism, including, e.g., plasmids, phage, cosmids, and mini chromosomes.
  • vectors comprising a polynucleotide of the present invention are vectors suitable for propagation or replication of the polynucleotide, or vectors suitable for expressing a polypeptide of the present invention. Such vectors are known in the art and commercially available.
  • Polynucleotides of the present invention are synthesized, whole or in parts that are then combined, and inserted into a vector using routine molecular and cell biology techniques, including, e.g., subcloning the polynucleotide into a linearized vector using appropriate restriction sites and restriction enzymes.
  • Polynucleotides of the present invention are amplified by polymerase chain reaction using oligonucleotide primers complementary to each strand of the polynucleotide. These primers also include restriction enzyme cleavage sites to facilitate subcloning into a vector.
  • the replicable vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, and one or more marker or selectable genes.
  • nucleotide sequences encoding the polypeptide, or functional equivalents are inserted into an appropriate expression vector, i.e., a vector that contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • an appropriate expression vector i.e., a vector that contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • a variety of expression vector/host systems are utilized to contain and express polynucleotide sequences. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g. , Ti or pBR322 plasmids); or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors insect cell systems infected with virus expression vectors (e.g., baculovirus)
  • plant cell systems transformed with virus expression vectors e.g., cauliflower mosaic virus, CaMV; tobacco mosaic
  • variable regions of a gene expressing a monoclonal antibody of interest are amplified from a hybridoma cell using nucleotide primers.
  • primers are synthesized by one of ordinary skill in the art, or may be purchased from commercially available sources (see, e.g., Stratagene (La Jolla, California), which sells primers for amplifying mouse and human variable regions.
  • the primers are used to amplify heavy or light chain variable regions, which are then inserted into vectors such as ImmunoZAPTM H or ImmunoZAPTM L (Stratagene), respectively. These vectors are then introduced into E. coli, yeast, or mammalian-based systems for expression. Large amounts of a single-chain protein containing a fusion of the V H and V L domains are produced using these methods (see Bird et al., Science 242:423-426 (1988)).
  • control elements or "regulatory sequences" present in an expression vector are those non-translated regions of the vector, e.g., enhancers, promoters, 5' and 3' untranslated regions, that interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, are used.
  • promoters suitable for use with prokaryotic hosts include the phoa promoter, ⁇ -lactamase and lactose promoter systems, alkaline phosphatase promoter, a tryptophan (tip) promoter system, and hybrid promoters such as the tac promoter.
  • phoa promoter alkaline phosphatase promoter
  • a tryptophan (tip) promoter system a tryptophan promoter system
  • hybrid promoters such as the tac promoter.
  • Other known bacterial promoters are suitable. Promoters for use in bacterial systems also usually contain a Shine-Dalgarno sequence operably linked to the DNA encoding the polypeptide.
  • Inducible promoters such as the hybrid lacZ promoter of the PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid (Gibco BRL, Gaithersburg, MD) and the like are used.
  • a variety of promoter sequences are known for eukaryotes and any are used according to the present invention.
  • Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated.
  • Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N may be any nucleotide.
  • N may be any nucleotide.
  • At the 3' end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.
  • promoters from mammalian genes or from mammalian viruses are generally preferred.
  • Polypeptide expression from vectors in mammalian host cells aer controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (e.g., Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV), a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g.
  • viruses such as polyoma virus, fowlpox virus, adenovirus (e.g., Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV), a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40
  • vectors based on SV40 or EBV may be advantageously used with an appropriate selectable marker.
  • a suitable expression vector is pcDNA-3.1 (Invitrogen, Carlsbad, CA), which includes a CMV promoter.
  • pcDNA-3.1 Invitrogen, Carlsbad, CA
  • a number of viral-based expression systems are available for mammalian expression of polypeptides. For example, in cases where an adenovirus is used as an expression vector, sequences encoding a polypeptide of interest may be ligated into an adenovirus
  • transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain a viable virus that is capable of expressing the polypeptide in infected host cells (Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81 :3655-3659).
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
  • RSV Rous sarcoma virus
  • any of a number of expression vectors are selected depending upon the use intended for the expressed polypeptide.
  • vectors that direct high level expression of fusion proteins that are readily purified are used.
  • Such vectors include, but are not limited to, the multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the sequence encoding the polypeptide of interest may be ligated into the vector in frame with sequences for the amino- terminal Met and the subsequent 7 residues of ⁇ -galactosidase, so that a hybrid protein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem.
  • pGEX Vectors are also used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione- agarose beads followed by elution in the presence of free glutathione.
  • Proteins made in such systems are designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
  • yeast Saccharomyces cerevisiae
  • a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH are used.
  • suitable promoter sequences for use with yeast hosts include the promoters for 3- phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3- phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
  • yeast promoters that are inducible promoters having the additional advantage of transcription controlled by growth conditions include the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657. Yeast enhancers also are advantageously used with yeast promoters.
  • the expression of sequences encoding polypeptides are driven by any of a number of promoters.
  • viral promoters such as the 35S and 19S promoters of CaMV are used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 3:17-31 1.
  • plant promoters such as the small subunit of RUBISCO or heat shock promoters are used (Coruzzi, G. et al. (1984) EMBO J. 3: 1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J., et al. (1991 ) Results Probl.
  • An insect system is also used to express a polypeptide of interest.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
  • the sequences encoding the polypeptide are cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter.
  • Successful insertion of the polypeptide-encoding sequence renders the polyhedrin gene inactive and produce recombinant virus lacking coat protein.
  • the recombinant viruses are then used to infect, for example, S.
  • Specific initiation signals are also used to achieve more efficient translation of sequences encoding a polypeptide of interest. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding the polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous translational control signals including the ATG initiation codon are provided. Furthermore, the initiation codon is in the correct reading frame to ensure correct translation of the inserted polynucleotide. Exogenous translational elements and initiation codons are of various origins, both natural and synthetic.
  • Enhancer sequences are known, including, e.g., those identified in genes encoding globin, elastase, albumin, a- fetoprotein, and insulin.
  • an enhancer from a eukaryotic cell virus is used. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • the enhancer is spliced into the vector at a position 5' or 3' to the polypeptide-encoding sequence, but is preferably located at a site 5' from the promoter.
  • Expression vectors used in eukaryotic host cells typically also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding anti-PSCA antibody.
  • One useful transcription termination component is the bovine growth hormone polyadenylation region. See W094/1 1026 and the expression vector disclosed therein.
  • Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, plant or higher eukaryote cells described above.
  • suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g. , Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B.
  • Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus
  • Salmonella e.g., Salmonella typhimurium
  • Serratia e.g.
  • E. coli cloning host is E. coli 294 (ATCC 31 ,446), although other strains such as E. coli B, E. coli X 1776 (ATCC 31 ,537), and E. coli W31 10 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting.
  • Saccharomyces cerevisiae or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms.
  • a number of other genera, species, and strains are commonly available and used herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K wickeramii (ATCC 24, 178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K.
  • a host cell strain is chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation. glycosylation, phosphorylation, lipidation, and acylation.
  • Post-translational processing that cleaves a "prepro" form of the protein is also used to facilitate correct insertion, folding and/or function.
  • Different host cells such as CHO, COS, HeLa, MDC , HE 293, and WI38, which have specific cellular machinery and characteristic mechanisms for such post-translational activities, are chosen to ensure the correct modification and processing of the foreign protein.
  • antibody heavy and light chains, or fragments thereof are expressed from the same or separate expression vectors. In one embodiment, both chains are expressed in the same cell, thereby facilitating the formation of a functional antibody or fragment thereof.
  • Full length antibody, antibody fragments, and antibody fusion proteins are produced in bacteria, in particular when glycosylation and Fc effector function are not needed, such as when the therapeutic antibody is conjugated to a cytotoxic agent (e.g. , a toxin) and the immunoconjugate by itself shows effectiveness in infected cell destruction.
  • a cytotoxic agent e.g. , a toxin
  • the immunoconjugate by itself shows effectiveness in infected cell destruction.
  • TIR translation initiation region
  • coli cell paste in a soluble fraction can be purified through, e.g., a protein A or G column depending on the isotype. Final purification can be carried out using a process similar to that used for purifying antibody expressed e.g., in CHO cells.
  • Suitable host cells for the expression of glycosylated polypeptides and antibodies are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells.
  • baculoviral strains and variants and corresponding permissive insect host t cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopicius (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified.
  • a variety of viral strains for transfection are publicly available, e.g. , the L-l variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses are used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.
  • Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco are also utilized as hosts.
  • [794J Methods of propagation of antibody polypeptides and fragments thereof in vertebrate cells in culture are encompassed by the invention.
  • mammalian host cell lines used in the methods of the invention are monkey kidney CV 1 line transformed by SV40 (COS-7, ATCC CRL 1651 ); human embryonic kidney line (293 or 293 cells subc!oned for growth in suspension culture, Graham et ai, J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BH , ATCC CCL 10); Chinese hamster ovary cellsADHFR (CHO, Urlaub et ai, Proc. Natl. Acad. Sci.
  • mice Sertoli cells TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (Wl 38, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51 ); TRl cells (Mather et ai, Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
  • Host cells are transformed with the above-described expression or cloning vectors for polypeptide production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • stable expression is generally preferred.
  • cell lines that stably express a polynucleotide of interest are transformed using expression vectors that contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells are allowed to grow for 1 -2 days in an enriched media before they are switched to selective media.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences. Resistant clones of stably transformed cells are proliferated using tissue culture techniques appropriate to the cell type.
  • a plurality of selection systems are used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell 77:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1990) Cell 22:817-23) genes that are employed in tk " or aprt " cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance is used as the basis for selection; for example, dhfr, which confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.
  • trpB allows cells to utilize indole in place of tryptophan
  • hisD allows cells to utilize histinol in place of histidine
  • marker gene expression suggests that the gene of interest is also present, its presence and expression is confirmed.
  • sequence encoding a polypeptide is inserted within a marker gene sequence, recombinant cells containing sequences are identified by the absence of marker gene function.
  • a marker gene is placed in tandem with a polypeptide-encoding sequence under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
  • host cells that contain and express a desired polynucleotide sequence are identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include, for example, membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescence activated cell sorting
  • a two- site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non- interfering epitopes on a given polypeptide is preferred for some applications, but a competitive binding assay may also be employed.
  • a competitive binding assay may also be employed.
  • RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
  • chemiluminescent or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • polypeptide produced by a recombinant cell is secreted or contained
  • Expression vectors containing polynucleotides of the invention are designed to contain signal sequences that direct secretion of the encoded polypeptide through a prokaryotic or eukaryotic cell membrane.
  • a polypeptide of the invention is produced as a fusion polypeptide further including a polypeptide domain that facilitates purification of soluble proteins.
  • purification-facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized
  • An exemplary expression vector provides for expression of a fusion protein containing a polypeptide of interest and a nucleic acid encoding 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification on IMIAC
  • a polypeptide of the present invention is fused with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • the heterologous signal sequence selected preferably is one that is recognized and processed ⁇ i.e., cleaved by a signal peptidase) by the host cell.
  • the signal sequence is selected, for example, from the group of the alkaline phosphatase, penicillinase, l pp, or heat-stable enterotoxin II leaders.
  • the signal sequence is selected from, e.g., the yeast invertase leader, a factor leader (including Saccharomyces and Kluyveromyces a factor leaders), or acid phosphatase leader, the C. albicans glucoamylase leader, or the signal described in WO 90/13646.
  • yeast invertase leader e.g., the yeast invertase leader, a factor leader (including Saccharomyces and Kluyveromyces a factor leaders), or acid phosphatase leader, the C. albicans glucoamylase leader, or the signal described in WO 90/13646.
  • mammalian signal sequences as well as viral secretory leaders for example, the herpes simplex gD signal, are available.
  • the polypeptide or antibody is produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the polypeptide or antibody is produced intracellularly, as a first step, the particulate debris, either host cells or ly ' sed fragments, are removed, for example, by centrifugation or ultrafiltration. Carter et al, Bio/Technology 10: 163-167 (1992) describe a procedure for isolating antibodies that are secreted to the periplasmic space of E. coli.
  • cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
  • PMSF phenylmethylsulfonylfluoride
  • Cell debris is removed by centrifugation.
  • supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
  • a protease inhibitor such as PMSF is included in any of the foregoing steps to inhibit proteolysis and antibiotics is included to prevent the growth of adventitious contaminants.
  • the polypeptide or antibody composition prepared from the cells are purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique.
  • affinity chromatography is the preferred purification technique.
  • the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the polypeptide or antibody. Protein A is used to purify antibodies or fragments thereof that are based on human ⁇ , ⁇ 2 , or ⁇ 4 heavy chains (Lindmark et ai, J. Immunol. Meth. 62: 1 -13 (1983)).
  • Protein G is recommended for all mouse isotypes and for human 73 (Guss et ai, EMBO J. 5: 15671575 (1986)).
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available.
  • Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
  • the polypeptide or antibody comprises a ⁇ 3 ⁇ 4 3 domain
  • the Bakerbond ABXTM resin J. T. Baker, Phillipsburg, N.J.
  • the mixture comprising the polypeptide or antibody of interest and contaminants are subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations ⁇ e.g., from about 0-0.25M salt).
  • the invention further includes pharmaceutical formulations including a polypeptide, antibody, or modulator of the present invention, at a desired degree of purity, and a pharmaceutically acceptable carrier, excipient, or stabilizer (Remingion's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)).
  • pharmaceutical formulations are prepared to enhance the stability of the polypeptide or antibody during storage, e.g., in the form of lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include, e.g., buffers such as acetate, Tris, phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
  • benzalkonium chloride benzethonium chloride
  • phenol butyl or benzyl alcohol
  • alkyl parabens such as methyl or propyl paraben
  • catechol resorcinol
  • cyclohexanol 3-pentanol
  • m-cresol low molecular weight (less than about 10 residues) polypeptides
  • proteins such as serum albumin, gelatin, or immunoglobulins
  • hydrophilic polymers such as polyvinylpyrrolidone
  • amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine
  • monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins chelating agents such as EDTA
  • tonicifiers such as trehalose and sodium chloride
  • sugars such as sucrose, mannitol, trehalose or sorbitol
  • surfactant such as polysorb
  • the therapeutic formulation preferably comprises the polypeptide or antibody at a concentration of between 5-200 mg/ml, preferably between 10- 100 mg ml.
  • the formulations herein also contain one or more additional therapeutic agents suitable for the treatment of the particular indication, e.g., infection being treated, or to prevent undesired side-effects.
  • the additional therapeutic agent has an activity complementary to the polypeptide or antibody of the resent invention, and the two do not adversely affect each other.
  • an additional or second antibody, anti-viral agent, anti-infective agent and/or cardioprotectant is added to the formulation.
  • Such molecules are suitably present in the pharmaceutical formulation in amounts that are effective for the purpose intended.
  • active ingredients e.g., polypeptides and antibodies of the invention and other therapeutic agents
  • microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example,
  • microcapsules respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in
  • sustained-release preparations are prepared. Suitable examples of sustained-release preparations include, but are not limited to, semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Nonlimiting examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or
  • poly(vinylalcohol) poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid- glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3- hydroxyburyric acid.
  • LUPRON DEPOTTM injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate
  • poly-D-(-)-3- hydroxyburyric acid poly(vinylalcohol)
  • Formulations to be used for in vivo administration are preferably sterile. This is readily accomplished by filtration through sterile filtration membranes.
  • Antibodies and fragments thereof, and therapeutic compositions, of the invention specifically bind or preferentially bind to infected cells or tissue, as compared to normal control cells and tissue.
  • these influenza A antibodies are used to detect infected cells or tissues in a patient, biological sample, or cell population, using any of a variety of diagnostic and prognostic methods, including those described herein.
  • the ability of an anti-M2e specific antibody to detect infected cells depends upon its binding specificity, which is readily determined by testing its ability to bind to infected cells or tissues obtained from different patients, and/or from patients infected with different strains of Influenza A.
  • Diagnostic methods generally involve contacting a biological sample obtained from a patient, such as, e.g., blood, serum, saliva, urine, sputum, a cell swab sample, or a tissue biopsy, with an Influenza A, e.g., HuM2e antibody and determining whether the antibody preferentially binds to the sample as compared to a control sample or predetermined cut-off value, thereby indicating the presence of infected cells.
  • at least two-fold, threefold, or five-fold more HuM2e antibody binds to an infected cell as compared to an appropriate control normal cell or tissue sample.
  • a pre-determined cut-off value is determined, e.g., by averaging the amount of HuM2e antibody that binds to several different appropriate control samples under the same conditions used to perform the diagnostic assay of the biological sample being tested.
  • Bound antibody is detected using procedures described herein and known in the art.
  • diagnostic methods of the invention are practiced using HuM2e antibodies that are conjugated to a detectable label, e.g., a fluorophore, to facilitate detection of bound antibody.
  • detectable label e.g., a fluorophore
  • methods of secondary detection of the HuM2e antibody include, for example, RIA, ELISA, precipitation, agglutination, complement fixation and immuno-fluorescence.
  • the HuM2e antibodies are labeled.
  • the label is detected directly.
  • Exemplary labels that are detected directly include, but are not limited to, radiolabels and fiuorochromes.
  • labels are moieties, such as enzymes, that must be reacted or derivatized to be detected.
  • isotope labels are 99 Tc, 1 C, 131 1, 125 1, 3 H, 32 P and 35 S.
  • Fluorescent materials that are used include, but are not limited to, for example, fluorescein and its derivatives, rhodamine and its derivatives, auramine, dansyl, umbelliferone, luciferia, 2,3-dihydrophthalazinediones, horseradish peroxidase, alkaline phosphatase, lysozyme, and glucose-6-phosphate dehydrogenase.
  • spectrophotometric fluorospectro-photometric or gasometric techniques.
  • Many enzymes which are used in these procedures are known and utilized by the methods of the invention.
  • Nonlimiting examples are peroxidase, alkaline phosphatase, ⁇ -glucuronidase, ⁇ -D- glucosidase, ⁇ -D-galactosidase, urease, glucose oxidase plus peroxidase, galactose oxidase plus peroxidase and acid phosphatase.
  • the antibodies are tagged with such labels by known methods. For instance, coupling agents such as aldehydes, carbodiimides, dimaleimide, imidates, succinimides, bid-diazotized benzadine and the like are used to tag the antibodies with the above-described fluorescent, chemiluminescent, and enzyme labels.
  • An enzyme is typically combined with an antibody using bridging molecules such as carbodiimides, periodate, diisocyanates, glutaraldehyde and the like.
  • bridging molecules such as carbodiimides, periodate, diisocyanates, glutaraldehyde and the like.
  • HuM2e antibodies of the present invention are capable of differentiating between patients with and patients without an Influenza A infection, and determining whether or not a patient has an infection, using the representative assays provided herein.
  • a biological sample is obtained from a patient suspected of having or known to have an Influenza A infection.
  • the biological sample includes cells from the patient.
  • the sample is contacted with an HuM2e antibody, e.g., for a time and under conditions sufficient to allow the HuM2e antibody to bind to infected cells present in the sample.
  • the sample is contacted with an HuM2e antibody for 10 seconds, 30 seconds, 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 3 days or any point in between.
  • the amount of bound HuM2e antibody is determined and compared to a control value, which may be, e.g. , a pre-determined value or a value determined from normal tissue sample.
  • a control value which may be, e.g. , a pre-determined value or a value determined from normal tissue sample.
  • An increased amount of antibody bound to the patient sample as compared to the control sample is indicative of the presence of infected cells in the patient sample.
  • a biological sample obtained from a patient is contacted with an HuM2e antibody for a time and under conditions sufficient to allow the antibody to bind to infected cells. Bound antibody is then detected, and the presence of bound antibody indicates that the sample contains infected cells.
  • This embodiment is particularly useful when the HuM2e antibody does not bind normal cells at a detectable level.
  • HuM2e antibodies are used to detect the presence of one or more strains of Influenza A.
  • certain antibodies bind specifically to only one or several strains of Influenza virus, whereas others bind to all or a majority of different strains of Influenza virus.
  • Antibodies specific for only one strain of Influenza A are used to identify the strain of an infection.
  • antibodies that bind to an infected cell preferably generate a signal indicating the presence of an infection in at least about 20% of patients with the infection being detected, more preferably at least about 30% of patients. Alternatively, or in addition, the antibody generates a negative signal indicating the absence of the infection in at least about 90% of individuals without the infection being detected.
  • Each antibody satisfies the above criteria; however, antibodies of the present invention are used in combination to improve sensitivity.
  • kits useful in performing diagnostic and prognostic assays using the antibodies of the present invention include a suitable container comprising a HuM2e antibody of the invention in either labeled or unlabeled form.
  • the kit further includes reagents for performing the appropriate indirect assay.
  • the kit includes one or more suitable containers including enzyme substrates or derivatizing agents, depending on the nature of the label. Control samples and/or instructions are also included.
  • Passive immunization has proven to be an effective and safe strategy for the prevention and treatment of viral diseases. (See Keller et al., Clin. Microbiol. Rev. 13:602-14 (2000); Casadevail, Nat. Biotechnol. 20: 1 14 (2002); Shibata et al., Nat. Med. 5:204- 10 (1999); and Igarashi et al., Nat. Med. 5:21 1 - 16 ( 1999), each of which are incorporated herein by reference)). Passive immunization using human monoclonal antibodies provide an immediate treatment strategy for emergency prophylaxis and treatment of influenza
  • HuM2e antibodies and fragments thereof, and therapeutic compositions, of the invention specifically bind or preferentially bind to infected cells, as compared to normal control uninfected cells and tissue.
  • these HuM2e antibodies are used to selectively target infected cells or tissues in a patient, biological sample, or cell population.
  • the present invention provides methods of regulating (e.g., inhibiting) the growth of infected cells, methods of killing infected cells, and methods of inducing apoptosis of infected cells. These methods include contacting an infected cell with an HuM2e antibody of the invention. These methods are practiced in vitro, ex vivo, and in vivo.
  • antibodies of the invention are intrinsically therapeutically active.
  • antibodies of the invention are conjugated to a cytotoxic agent or growth inhibitory agent, e.g., a radioisotope or toxin, that is used in treating infected cells bound or contacted by the antibody.
  • a cytotoxic agent or growth inhibitory agent e.g., a radioisotope or toxin
  • the invention provides methods of treating or preventing infection in a patient, including the steps of providing an HuM2e antibody of the invention to a patient diagnosed with, at risk of developing, or suspected of having an Influenza A infection.
  • the methods of the invention are used in the first-line treatment of the infection, follow-on treatment, or in the treatment of a relapsed or refractory infection.
  • Treatment with an antibody of the invention is a stand alone treatment.
  • treatment with an antibody of the invention is one component or phase of a combination therapy regime, in which one or more additional therapeutic agents are also used to treat the patient.
  • Subjects at risk for an influenza virus -related diseases or disorders include patients who have come into contact with an infected person or who have been exposed to the influenza virus in some other way. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the influenza virus -related disease or disorder, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
  • the huM2e is administered substantially contemporaneously with or following infection of the subject, i.e., therapeutic treatment.
  • the antibody provides a therapeutic benefit.
  • a therapeutic benefit includes reducing or decreasing progression, severity, frequency, duration or probability of one or more symptoms or complications of influenza infection, virus titer, virus replication or an amount of a viral protein of one or more influenza strains, still another aspect, a therapeutic benefit includes hastening or accelerating a subject's recovery from influenza infection.
  • a method includes administering to the subject an amount of a huM2e antibody effective to prevent an increase in influenza virus titer, virus replication or an amount of an influenza viral protein of one or more influenza strains or isolates in the subject.
  • a method includes administering to the subject an amount of huM2e antibody that specifically binds influenza M2 effective to protect the subject from infection, or effective to decrease susceptibility of the subject to infection, by one or more influenza strains/isolates or subtypes.
  • the subject is further administered with a second agent such as, but not limited to, an influenza virus antibody, an anti-viral drug such as a neuraminidase inhibitor, a HA inhibitor, a sialic acid inhibitor or an M2 ion channel inhibitor, a viral entry inhibitor or a viral attachment inhibitor.
  • a second agent such as, but not limited to, an influenza virus antibody, an anti-viral drug such as a neuraminidase inhibitor, a HA inhibitor, a sialic acid inhibitor or an M2 ion channel inhibitor, a viral entry inhibitor or a viral attachment inhibitor.
  • the M2 ion channel inhibitor is for example amantadine or rimantadine.
  • the neuraminidase inhibitor for example zanamivir, or oseltamivir phosphate.
  • Symptoms or complications of influenza infection that can be reduced or decreased include, for example, chills, fever, cough, sore throat, nasal congestion, sinus congestion, nasal infection, sinus infection, body ache, head ache, fatigue, pneumonia, bronchitis, ear infection, ear ache or death.
  • the patient is usually administered or provided a pharmaceutical formulation including a HuM2e antibody of the invention.
  • the antibodies of the invention are administered to the patient in therapeutically effective amounts (i.e., amounts that eliminate or reduce the patient's viral burden).
  • the antibodies are administered to a human patient, in accord with known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
  • the antibodies may be administered parenterally, when possible, at the target cell site, or intravenously. Intravenous or subcutaneous administration of the antibody is preferred in certain
  • compositions of the invention are administered to a patient or subject systemically, parenterally, or locally.
  • the antibodies are formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable, parenteral vehicle.
  • a pharmaceutically acceptable, parenteral vehicle examples include water, saline, Ringer's solution, dextrose . solution, and 5% human serum albumin.
  • Nonaqueous vehicles such as fixed oils and ethyl oleate are also used.
  • Liposomes are used as carriers.
  • the vehicle contains minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives.
  • the antibodies are typically formulated in such vehicles at concentrations of about 1 mg/ml to 10 mg/ml.
  • the dose and dosage regimen depends upon a variety of factors readily determined by a physician, such as the nature of the infection and the characteristics of the particular cytotoxic agent or growth inhibitory agent conjugated to the antibody (when used), e.g., its therapeutic index, the patient, and the patient's history.
  • a therapeutically effective amount of an antibody is administered to a patient.
  • the amount of antibody administered is in the range of about 0.1 mg kg to about 50 mg kg of patient body weight.
  • about 0.1 mg kg to about 50 mg/kg body weight (e.g. , about 0.1 -15 mg/kg/dose) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate
  • an immunoconjugate including the antibody conjugated with a cytotoxic agent is administered to the patient.
  • the antibody conjugated with a cytotoxic agent is administered to the patient.
  • the immunoconjugate is internalized by the cell, resulting in increased therapeutic efficacy of the immunoconjugate in killing the cell to which it binds.
  • the cytotoxic agent targets or interferes with the nucleic acid in the infected cell. Examples of such cytotoxic agents are described above and include, but are not limited to, maytansinoids, calicheamicins, ribonucleases and DNA endonucleases.
  • Other therapeutic regimens are combined with the administration of the HuM2e antibody of the present invention.
  • the combined administration includes co-administration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities.
  • Preferably such combined therapy results in a synergistic therapeutic effect.
  • the invention provides methods of administration of the antibody by gene therapy.
  • administration of nucleic acid encoding the antibody is encompassed by the expression "administering a therapeutically effective amount of an antibody”. See, for example, PCT Patent Application Publication WO96/07321 concerning the use of gene therapy to generate intracellular antibodies.
  • anti-M2e antibodies of the invention are used to determine the structure of bound antigen, e.g., conformational epitopes, the structure of which is then used to develop a vaccine having or mimicking this structure, e.g., through chemical modeling and SAR methods. Such a vaccine could then be used to prevent Influenza A infection.
  • Example 1 Screening and Characterization of M2e-specific Antibodies Present in Human Plasma Using Cells Expressing Recombinant M2e Protein
  • the M2 cDNA is encoded by the following polynucleotide sequence and SEQ ID NO: 53:
  • V 1203/2004 V 1203
  • M2e-specific monoclonal antibodies of the present invention since 14C2 binding may be abrogated by the various amino acid substitutions in M2e.
  • the human MAbs identified through this process proved to bind to conformational epitopes on the M2 homotetramer. They bound to the original 293-M2 transfectant, as well as to the two other cell-expressed M2 variants.
  • the 14C2 MAb in addition to binding the M2e peptide, proved to be more sensitive to the M2 variant sequences. Moreover, 14C2 does not readily bind influenza virions, while the conformation specific anti-M2 MAbs did.
  • M2 MAbs from normal human immune responses to influenza without a need for specific immunization of M2. If used for immunotherapy, these fully human MAbs have the potential to be better tolerated by patients that humanized mouse antibodies.
  • M2e peptide the MAbs of the invention bind to conformational epitopes of M2, and are specific not only for cells infected with A strain influenza, but also for the virus itself.
  • Antibody containing supernatants were screened for binding to 293 FT cells stably transfected with the full length M2E protein from influenza strain Influenza subtype H3N2. Supernatants which showed positive staining/binding were re-screened again on 293 FT cells stably transfected with the full length M2E protein from influenza strain Influenza subtype H3N2 and on vector alone transfected cells as a control.
  • variable regions of the antibodies were then rescue cloned from the B cell wells whose supernatants showed positive binding.
  • Transient transfections were performed in 293 FT cells to reconstitute and produce these antibodies.
  • Reconstituted antibody supernatants were screened for binding to 293 FT cells stably transfected with the full length M2E protein as detailed above to identify the rescued anti-M2E antibodies.
  • Three different antibodies were identified: 8i 10, 21 B15 and 23 12.
  • a fourth additional antibody clone was isolated by the rescue screens, 4C2. However, it was not unique and had the exact same sequence as clone 8 ⁇ 0 even though it came from a different donor than clone 8i l 0.
  • the Kappa LC variable region of the anti M2 clone 8 ⁇ 0 was cloned as Hind III to BsiW l fragment (see below), and is encoded by the following polynucleotide sequences, and SEQ ID NO: 54 (top) and SEQ ID NO: 55 (bottom):
  • the 8i 10 Gamma HC variable region was cloned as a Hind III to Xho 1 fragment, and is encoded the following polynucleotide sequences, and SEQ ID NO: 67 (top) and SEQ ID NO: 67 (top) and SEQ ID NO: 67 (top) and SEQ ID NO: 67 (top) and SEQ ID NO: 67 (top) and SEQ ID NO: 67 (top) and SEQ ID NO: 67 (top) and SEQ ID NO: 67 (top) and SEQ ID NO: 67 (top) and SEQ ID NO: 67 (top) and SEQ ID NO: 67 (top) and SEQ ID NO: 67 (top) and SEQ ID NO: 67 (top) and SEQ ID NO: 67 (top) and SEQ ID NO: 67 (top) and SEQ ID NO: 67 (top) and SEQ ID NO: 67 (top) and SEQ ID NO: 67 (top) and SEQ ID NO: 67 (
  • the framework 4 (FR4) region of the Gamma HC normally ends with two serines (SS), so that the full framework 4 region should be W G Q G T L V T V S S (SEQ ID NO: 80).
  • the original vector did not adjust for the silent mutation made when the Xhol site (CTCGAG, SEQ ID NO: 81) was created and contained an "A" nucleotide downstream of the Xhol site, which caused an amino acid change at the end of framework 4: a serine to arginine (S to R) substitution present in all the working Gamma HC clones.
  • the full framework 4 region reads W G Q G T L V T V S R (SEQ ID NO: 82) .
  • Future constructs are being created wherein the base downstream of the Xho 1 site is a "C" nucleotide.
  • the creation of the Xho 1 site used for cloning of the Gamma HC variable region sequences in alternative embodiments is a silent mutation and restores the framework 4 amino acid sequence to its proper W G Q G T L V T V S S (SEQ ID NO: 80). This is true for all M2 Gamma HC clones described herein.
  • the translation of the 21 B 15 Kappa LC variable region is as follows, polynucleotide sequence (above, SEQ ID NO: 83, top) and amino acid sequence (below, corresponding to SEQ ID NO: 298):
  • the primer used to clone the Kappa LC variable region extended across a region of diversity and had wobble base position in its design.
  • a D or E amino acid could occur in the framework 4 region.
  • the amino acid in this position in the rescued antibody may not be the original parental amino acid that was produced in the B cell.
  • the position is an E.
  • D I K R T D in framework 4
  • the native antibody from the B cell may have had an E in this position.
  • the 21B15 Gamma HC variable region was cloned as a Hind III to Xho 1 fragment, and is encoded by the following polynucleotide sequences and SEQ ID NO: 85 (top), and SEQ ID NO: 86 (bottom):
  • the Kappa LC variable region of the anti M2 clone 23K12 was cloned as Hind III to BsiW l fragment (see below), and is encoded by the following polynucleotide sequences SEQ ID NO: 88 (top) and SEQ ID NO: 89 (below).
  • the translation of the 23 12 Gamma HC variable region is as follows, polynucleotide sequence (above, SEQ ID NO: 99, top), and amino acid sequence (below, corresponding to SEQ ID NO: 100):
  • Clones 8110 and 21 B15 came from two different donors, yet they have the same exact Gamma HC and differ in the Kappa LC by only one amino acid at position 4 in the framework 1 region (amino acids M versus V, see above), (excluding the D versus E wobble position in framework 4 of the Kappa LC).
  • the antibodies were purified over protein A columns from the supematants. FACs analysis was performed using purified antibodies at a concentration of 1 ug per ml to examine the binding of the antibodies to transiently transfected 293 PEAK cells expressing the M2 proteins on the cell surface. Binding was measured testing binding to mock transfected cells and cells transiently transfected with the Influenza subtype H3N2, A/Vietnam/ 1203/2004 (VN1203), or A/Hong Kong/483/1997 HK483 M2 proteins. As a positive control the antibody 14C2 was used. Unstained and secondary antibody alone controls helped determined background. Specific staining for cells transfected with the M2 protein was observed for all three clones. Furthermore, all three clones bound to the high path strains A/Vietnam/ 1203/2004 and A/Hong
  • Antibodies 21 B15, 23K12, and 8110 bound to the surface of 293-HEK cells stably expressing the M2 protein, but not to vector transfected cells (see Figure 1 ). In addition, binding of these antibodies was not competed by the presence of 5 mg/ml 24-mer M2 peptide, whereas the binding of the control chimeric mouse V-region human IgGl kappa 14C2 antibody (hul4C2) generated against the linear M2 peptide was completely inhibited by the M2 peptide (see Figure 1 ). These data confirm that these antibodies bind to conformational epitopes present in M2e expressed on the cell or virus surface, as opposed to the linear M2e peptide.
  • UV-inactivated influenza A virus (A/PR/8/34) (Applied Biotechnologies) was plated in 384-well MaxiSorp plates (Nunc) at 1.2 ⁇ g/ml in PBS, with 25 ⁇ /well, and was incubated at 4 ° C overnight. The plates were then washed three times with PBS, and blocked with 1% Nonfat dry milk in PBS, 50 ⁇ /well, and then were incubated at room temp for 1 hr. After a second wash with PBS, MAbs were added at the indicated concentrations in triplicate, and the plates were incubated at room temp for 1 hour.
  • M2 variants were selected for analysis. See Figure 3A for sequences.
  • M2 cDNA constructs were transiently transfected in HE 293 cells and analyzed as follows: To analyze the transient transfectants by FACS, cells on 10 cm tissue culture plates were treated with 0.5 ml Cell Dissociation Buffer (Invitrogen), and harvested. Cells were washed in PBS containing 1 % FBS, 0.2% NaN 3 (FACS buffer), and resuspended in 0.6 ml FACS buffer supplemented with 100 pg/ml rabbit IgG.
  • Each transfectant was mixed with the indicated MAbs at 1 ⁇ g/ml in 0.2 ml FACS buffer, with 5 x 10 5 to 10 6 cells per sample. Cells were washed three times with FACS buffer, and each sample was resuspended in 0.1 ml containing 1 ⁇ g/ml alexafluor (AF) 647-anti human IgG H&L (Invitrogen). Cells were again washed and flow cytometry was performed on a FACSCanto device (Becton-Dickenson). The data is expressed as a percentage of the mean fluorescence of the M2-D20 transient transfectant. Data for variant binding are representative of 2 experiments. Data for alanine mutants are average readouts from 3 separate experiments with standard error. Results are shown in Figure 3B and 3C.
  • M2 cDNA constructs were transiently transfected in HE 293 cells and analyzed as described above in Example 6. Results are shown in Figure 4A and 4B.
  • Figure 8 shows that the epitope is in a highly conserved region of the amino terminus of the M2 polypeptide. As shown in Figures 4A, 4B and Figure 8, the epitope includes the serine at position 2, the threonine at position 5 and the glutamic acid at position 6 of the M2 polypeptide.
  • M2 protein representing influenza strain A/H /483/1997 sequence was stably expressed in the CHO (Chinese Hamster Ovary) cell line DG44.
  • Cells were treated with Cell Dissociation Buffer (Invitrogen), and harvested.
  • Cells were washed in PBS containing 1% FBS, 0.2% NaN 3 (FACS buffer), and resuspended at 10 7 cells/ml in FACS buffer supplemented with 100 ⁇ g/ml rabbit IgG.
  • the cells were pre-bound by either MAb (or the 2N9 control) at 10 ⁇ g/ml for 1 hr at 4 °C, and were then washed with FACS buffer.
  • each peptide was coated at 2 ⁇ g/mL to a flat bottom 384 well plate (Nunc) in 25 ⁇ _7 well of PBS buffer overnight at 4°C. Plates were washed three times and blocked with 1 % Milk/PBS for one hour at room temperature. After washing three times, MAb titers were added and incubated for one hour at room temperature. Diluted HRP conjugated goat anti-human immunoglobulin FC specific (Pierce) was added to each well after washing three times. Plates were incubated for one hour at room temperature and washed three times.
  • Example 10 In Vivo Evaluation of the Ability of Human Anti -Influenza Monoclonal Antibodies to Protect From Lethal Viral Challenge
  • mice Female BALB/c mice were randomized into 5 groups of 10. One day prior (Day -1 (minus one)) and two days post infection (Day +2 (plus two), 200 ug of antibody was given via 200 ul intra-peritoneal injection. On Day 0 (zero), an approximate LD90 (lethal dose 90) of A/Vietnam/1203/04 influenza virus, in a volume of 30 ⁇ was given intra-nasally. Survival rate was observed from Day 1 through Day 28 post-infection. Results are shown in Figure 7.
  • Example 1 1 Characterization of M2 Antibodies TCN-032 (8110), 21 B15. TCN-031 (23 12). 3241 G23, 3244 110. 3243 J07, 3259 J21. 3245 Q19. 3244 H04. 3136 G05.
  • Positive control supernatant from tranisent transfection with the IgG heavy and light chain combination of mAb 8110
  • Negative control supernatant from tranisent transfection with the IgG heavy and light chain combination of mAb 2N9
  • Example 12 Human Antibodies Reveal a Protective Epitope That is Highly conserveed Among Human and Non-Human Influenza A Viruses
  • Influenza remains a serious public health threat throughout the world. Vaccines and antivirals are available that can provide protection from infection. However, new viral strains emerge continuously because of the plasticity of the influenza genome which necessitates annual reformulation of vaccine antigens, and resistance to antivirals can appear rapidly and become entrenched in circulating virus populations. In addition, the spread of new pandemic strains is difficult to contain due to the time required to engineer and manufacture effective vaccines. Monoclonal antibodies that target highly conserved viral epitopes might offer an alternative protection paradigm.
  • Antiviral drugs include oseltamivir and zanamivir which inhibit the function of the viral protein neuraminidase (NA), and adamantanes which inhibit the ion channel function of the viral M2 protein (3, 4). Antiviral agents are effective for sensitive virus strains but viral resistance can develop quickly and has the potential to render these drugs ineffective.
  • M2e ectodomain of the viral M2 protein
  • Monoclonal antibodies to M2e have been shown to be protective in vivo ( 1 1 - 13, 40, 43), and several groups have demonstrated protection against infection with vaccine strategies based on M2e (14-19).
  • purified M2 protein or peptides derived from M2e sequence have been used as immunogens to generate anti-M2e antibodies in animals or as vaccine candidates.
  • mAbs directly from human B cells that bind to the M2 protein displayed on virus particles and on virus- infected cells.
  • IgG + memory B cells Serum samples from 140 healthy adult, United States-sourced donors were tested for reactivity with M2e expressed on the surface of HE 293 cells that were transfected with a viral M2 gene (derived from A/Fort Worth/50 H lNl). IgG + memory B cells from 5 of the 23 M2e-seropositive subjects were cultured under conditions where they proliferated and differentiated into IgG-secreting plasma cells.
  • VH and VL immunoglobulin heavy and light chain variable region
  • the two more distantly related mAbs 62B1 1 and 41G23 utilize the germline V gene segment IGHV4- 3 1 *03 which has only 5 amino acid residue differences from the germline V gene segment IGHV4-59*01 of group A. All of these mAbs utilize the same light chain V gene, IGKV 1-39*01 or its allele IGKV 1 D-39*01 and show evidence of somatic hypermutation from the germline heavy or kappa chain sequence (Fig. 12).
  • Competitive binding experiments showed that all of these human mAbs appear to bind similar sites on native M2e expressed on the surface of Chinese hamster ovary (CHO) cells (Fig. 13).
  • CHO Chinese hamster ovary
  • a chimeric derivative of the murine anti-M2e mAb 14C2 (chl4C2), which was originally generated by immunization with purified M2 (20), exhibited the opposite behavior to that observed with the human mAbs, with little binding to virus but robust binding to the isolated 23mer M2e peptide with half-maximal binding to peptide at 10 ng/mL (Figs. 14a and 14b).
  • both the human mAbs and chl4C2 bound to the surface of Madin-Darby canine kidney (MDCK) cells infected with H 1N 1 virus (A/Puerto Rico/8/34) with similar avidities (Fig. ) 4c).
  • mice that were subjected to similar treatment regimens with a subclass-matched, irrelevant control mAb 2N9, which targets the AD2 epitope of the gpl 16 portion of the human cytomegalovirus gB, or with a vehicle control were protected to a lesser extent, or not at all, resulting in 70-80% survival for mice treated with human mAbs versus 20% survival for control mAb and 0% survival for vehicle (Fig. 15a).
  • the anti-M2e mAb ch 14C2 did not confer substantial protection in this model (20% survival; Figure 15a), though this mAb has been shown to reduce the titer of virus in the lungs of mice infected with other strains of influenza virus (40).
  • the human anti-M2e mAbs and ch 14C2 bound to cell surface-expressed M2e from A/Vietnam 1203/04 and A/Puerto Rico/8/34 viruses (Fig 19b, Table 6) and cells infected with A/Puerto Rico/8/34 (Fig. 14c).
  • Mechanisms for antibody-mediated protection could include killing of infected host cells by antibody-dependent cell-mediated cytotoxicity or complement- dependent cytotoxicity ( 1 1 , 21).
  • We found in vitro evidence for both of these mechanisms with the human anti-M2e mAbs and ch l4C2 (Fig. 17 and 6).
  • TCN-031 and TCN-032 recognize a core sequence of SLLTE at positions 1 -5 of the N-terminus of mature M2e. This is supported by data which show that these mAbs compete effectively with each other for binding to M2e expressed on the surface of CHO cells (Fig. 20).
  • ch l4C2 binds to a site that is spatially distinct and downstream of the SLLTE core that is recognized by the human anti-M2e mAbs.
  • 14C2 binds a relatively broad, linear epitope with the sequence EVERTPIRNEW at positions 5-14 of processed M2e (1 1 ).
  • TCN-031 and TCN-032 While the epitopes recognized by TCN-031 and TCN-032 are likely very similar, there were some differences between these human mAbs in their binding to several of the M2e mutants. For instance, TCN-03 1 appears to have a greater dependence than TCN-032 on residues 2 (L) and 3 (L) of the mature M2e sequence (Fig. 19a).
  • the VH regions of these two human mAbs utilize different variable, diversity, and joining gene segments which may explain the minor differences in binding observed between these mAbs.
  • Despite the differences in their VH make-up these human mAbs utilize the same germline kappa chain V gene segments, albeit with distinct kappa chain joining segments.
  • the linear M2e epitopes recognized by peptide-elicited antibodies may be more sensitive to escape mutations and natural substitutions that are present in some viral isolates.
  • P10L and PI OH escape mutations to mAb 14C2 have been mapped to the central portion of M2e (27) and those same substitutions also occur in M2e variants from some highly pathogenic H5N1 strains.
  • monoclonal antibodies with specificities similar to that of 14C2 are likely to have limited utility as broad spectrum therapeutic agents.
  • mice immunized with M2e-derived peptides produced antibodies with a range of specificities within M2e, including the conserved N-terminus and also downstream regions (13).
  • Table 7 Conservation of the viral binding site for human anti-M2e mAbs compared with those for mAbs derived from immunized mice, in influenza A.
  • PBMC peripheral blood mononuclear cells
  • B cell cultures were set up using PBMC, B cells enriched by selection with M2-expressing cells, or IgG + memory B cells enriched from PBMC via negative depletion of nonIgG + cells with antibodies to CD3, CD14, CD16, Ig , IgA, and IgD on magnetic beads (Miltenyi, Aubum, CA) as previously described (35).
  • cells were seeded in 384-well microtiter plates in the presence of feeder cells and conditioned media generated from mitogen-stimulated human T cells from healthy donors.
  • the culture supematants were collected 8 days later and screened in a high throughput format for binding reactivity to M2 protein expressed on HEK 293 cells stably transfected with influenza virus M2 (A/Fort Worth/50 HINI) using fluorescent imaging (FMAT system, Applied Biosystems).
  • VH pool was combined with the corresponding VK, or Vk pools from individual BCC wells and was transiently transfected in 293- 6E cells to generate recombinant antibody.
  • Conditioned media was harvested 3-5 days after transfection and assayed for antibody binding to M2 protein expressed on HEK 293 cells.
  • Individual clones were isolated from positive pools and unique VH and VL genes were identified by sequencing. From these, monoclonal antibodies were subsequently expressed and re-assayed for binding activity.
  • Binding of mAbs at the indicated concentrations was detected with HRP-conjugated goat anti-human Fc antibody (Pierce) and visualized with TMB substrate (ThermoFisher).
  • the M2e peptide, SLLTEVETPIRNEWGCRCNDSSD (SEQ ID NO: 680) (Genscript) was passively adsorbed at 1 ug/mL and antibody binding to the peptide was detected by the same method.
  • HEK293 cells were treated with 1 ⁇ g/mL of the indicated mAbs in PBS supplemented with 1% fetal bovine serum and 0.2% NaN3 (FACS buffer). Bound anti-M2 mAbs were visualized on viable cells with Alexafluor 647-conjugated goat anti-Human IgG H&L antibody (Invitrogen). Flow cytometry was performed with FACSCanto equipped with the FACSDiva software (Becton Dickenson).
  • the relative binding to the naturally occurring variants was expressed as the percentage of the respective mAb staining of the D20 transiently transfected cells, using the formula of Normalized MFI (%) 100 x (MFIexperimental ' MFImock transfected)/(MFID20 ⁇ MFImock transfected).
  • mice [924J Therapeutic efficacy studies in mice. Animal studies were conducted under Institutional Animal Care and Use Committee protocols. We inoculated 6 groups of 10 mice (female 6-8 week old BALB/C) intranasally with 5 x LD50 of A/Vietnam/ 1203/04 (Fig 15a and b) or 6 groups of 5 mice intranasally with 5 x LD50 A/Puerto Rico/8/34 (Fig 15c and d).
  • mice received intraperitoneal injections of 400 ⁇ g200 ⁇ L dose of the anti-M2e mAbs TCN-031 TCN-032, control human mAb 2N9, control chimeric mAb chl4C2, PBS, or were left untreated.
  • Mice were weighed daily for 2 weeks and were euthanized when weight loss exceeded 20% (H5N 1 study shown in Fig 15a and 15b and HlNl study shown in Fig 15c and 15d) of the pre-infection body weight.

Abstract

La présente invention concerne des compositions, des vaccins et des méthodes de diagnostic, de traitement et de prévention de l'infection grippale au moyen d'une combinaison d'anticorps dirigés contre l'hémagglutinine et les polypeptides de l'ectodomaine de la protéine matricielle M2.
PCT/US2010/045346 2009-08-14 2010-08-12 Compositions et méthodes utilisables dans le cadre du traitement et du diagnostic de la grippe WO2011019932A2 (fr)

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US20110033476A1 (en) * 2007-11-12 2011-02-10 Theraclone Sciences Inc. Compositions and methods for the therapy and diagnosis of influenza
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KR20140012131A (ko) * 2011-03-15 2014-01-29 테라클론 사이언시스, 아이엔씨. 인플루엔자의 치료 및 진단을 위한 조성물 및 방법
US9969794B2 (en) * 2012-05-10 2018-05-15 Visterra, Inc. HA binding agents
CA2909802A1 (fr) * 2013-04-22 2014-10-30 Theraclone Sciences, Inc. Compositions et methodes pour le traitement et le diagnostic de la grippe
EP3076993A1 (fr) * 2013-12-05 2016-10-12 Janssen Vaccines & Prevention B.V. Procédé pour préparer des vaccins contre la grippe
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